- Email: [email protected]
Astilbin ameliorates pulmonary fibrosis via blockade of Hedgehog signaling pathway
Accepted Manuscript Astilbin ameliorates pulmonary fibrosis via blockade of Hedgehog signaling pathway Jie Zhang, Hanchen Liu, Chenguang Song, Jinjin Zhang, Youlei Wang, Changjun Lv, Xiaodong Song PII:
S1094-5539(18)30030-0
DOI:
10.1016/j.pupt.2018.03.006
Reference:
YPUPT 1718
To appear in:
Pulmonary Pharmacology & Therapeutics
Received Date: 31 January 2018 Revised Date:
25 March 2018
Accepted Date: 31 March 2018
Please cite this article as: Zhang J, Liu H, Song C, Zhang J, Wang Y, Lv C, Song X, Astilbin ameliorates pulmonary fibrosis via blockade of Hedgehog signaling pathway, Pulmonary Pharmacology & Therapeutics (2018), doi: 10.1016/j.pupt.2018.03.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Astilbin ameliorates pulmonary fibrosis via blockade
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of Hedgehog signaling pathway
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a Department of Cellular and Genetic Medicine, School of Pharmaceutical Sciences,
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b Department of Respiratory Medicine, Affiliated Hospital to Binzhou Medical University, Binzhou 256602, China.
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Binzhou Medical University, Yantai 264003, China.
c Department of Medical Oncology, People`s Liberation Army 107th Hospital, Yantai,264003, China.
d Department of Respiratory Medicine, Zouping Chinese Medicine Hospital, Binzhou 256602, China.
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Jie Zhang△a,b, Hanchen Liu△c, Chenguang Song△d, Jinjin Zhanga,b, Youlei Wang a, Changjun Lv *a,b, Xiaodong Song* a,b
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Running Title: Astilbin ameliorates pulmonary fibrosis
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△These authors contributed equally to this work.
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*Correspondence author: Prof. Xiaodong Song and Changjun Lv, School of
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Pharmaceutical Sciences of Binzhou Medical University, No. 346, Guanhai Road,
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Laishan District, Yantai City, 264003, China. Email: [email protected];
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[email protected]
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Tel: +86-535-6913985; Fax: +86-535-6913985
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ABSTRACT
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Background and objective: The nature of pulmonary fibrosis involves inadequate
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repair of the epithelial cell barrier accompanied by impaired regulation of the
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fibroblast. Moreover, pulmonary fibrosis currently lacks an effective therapeutic drug.
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This study targets the protection of the epithelial cell and fibroblast to identify a novel,
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potentially therapeutic drug (i.e., astilbin).
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Methods: In this study, the cytotoxicity of astilbin was firstly detected using CCK-8.
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A real-time proliferation/migration analysis system was used to test the inhibitory
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proliferation and migration of astilbin in vitro. The expression of mesenchymal
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markers and the loss of epithelial cell markers were analyzed to evaluate the
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antifibrotic activity of astilbin on TGF-β1-treated AEC-II and L929 cells and
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bleomycin-treated mice. Then, in fibrosis-associated signaling pathways, the
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regulation of astilbin was tested using RNA sequencing and Cignal Finder
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45-Pathway system. Rescue and other experiments were used to confirm this pathway
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regulation further.
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Results: The data showed that astilbin inhibited proliferation and migration of cell
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samples. Its treatment resulted in the reduction of pathological score and collagen
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deposition, with a decrease in α-SMA and Snail and an increase in E-cadherin and
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notable components of the Hedgehog signaling pathway.
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Conclusions: Astilbin ameliorates pulmonary fibrosis via blockade of Hedgehog
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signaling pathway and has potential therapeutic value for lung fibrosis treatment.
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Key Words: Astilbin; Anti-pulmonary fibrosis; Hedgehog pathway
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1. Introduction
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Pulmonary fibrosis results from abnormal tissue repair and is associated with
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persistent and/or severe tissue damage and cellular stress [1]. The nature of this
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aberrance involves inadequate repair of the epithelial cell barrier accompanied by
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impaired regulation of the fibroblast [2], which results in profound changes to
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alveolar epithelial cell and fibroblast, leading to uncontrolled fibrosis [3]. Thus,
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targeting the protection of the epithelial cell and fibroblast is an attractive approach to
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treat pulmonary fibrosis [4]. Pirfenidone and nintedanib were approved for the
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treatment of pulmonary fibrosis in 2014 [5,6]. However, differences in adverse event
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frequency, such as photosensitivity, gastrointestinal symptoms, and liver function test
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abnormalities, were observed [7,8]. Gene therapy is a potential treatment; however,
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successfully applying it to clinical practice has so far been a formidable challenge
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[9,10]. Therefore, the identification of a novel therapeutic drug in preclinical study is
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highly desired.
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Astilbin, (2R,3R)-3,3′,4′,5,7-pentahydroxyflavanon-3-α-L-rhamnopyranoside, is a
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dihydroflavonol rhamnoside, which is significant in the treatment of immunologically 3
ACCEPTED MANUSCRIPT related diseases. Several studies have demonstrated that astilbin ameliorates
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experimental autoimmune myasthenia gravis and imiquimod-induced psoriasis-like
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skin lesions [11,12]. Astilbin has also shown many other bioactivities, such as
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anti-oxidative, anti-inflammation, and 3-hydroxy-3-methylglutaryl coenzyme A
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reductase inhibitor [13,14]. Astilbin is currently used to ameliorate cisplatin-induced
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nephrotoxicity through reducing oxidative stress and inflammation [15]. In the
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treatment of lung injury, astilbin can alleviate sepsis-induced acute lung injury by
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inhibiting the expression of macrophage inhibitory factor in rats [16]. However, the
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anti-pulmonary fibrosis of astilbin remains unknown. Pulmonary fibrosis is actually a
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kind of lung injury, except that this is a specific form of chronic, progressive injury.
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This study aims to investigate the effects of astilbin against pulmonary fibrosis and its
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potential anti-pulmonary fibrosis mechanism in vivo and in vitro.
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2. Methods
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2.1. Cell culture
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Mouse type II alveolar epithelial cell (AEC-II) and mouse lung fibroblast (L929)
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cell lines were purchased from Cell Bank of Chinese Academy of Sciences.
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Transforming growth factor beta 1 (TGF-β1), the most important regulatory factor
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among fibrogenic cytokines, was used to stimulate AEC-II and L929 cells to establish
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the pulmonary fibrosis model in vitro [17,18]. Cells were maintained in advanced
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minimum essential medium and supplemented with 10% newborn calf serum, 100
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U/mL penicillin and 100 µg/mL streptomycin at 37℃ under a humidified atmosphere
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ACCEPTED MANUSCRIPT of 5% CO2 and 95% air. AEC-II and L929 cells were first administered with 5 ng/mL
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TGF-β1 for 6 h and then co-treated with astilbin (Dalian Meilun Biotech Co., Ltd) for
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48 h.
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2.2. Cell counting kit-8 assay (CCK-8)
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Cell proliferation was determined using CCK-8 kit (DOJINDO, Japan) in
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accordance with the manufacturer’s instructions. In brief, 1 × 106 cells/well were
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seeded in a 96-well and flat-bottomed plate and then grown at 37 °C for 24 h. After 10
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µL of WST-8 dye was added to each well, the cells were incubated at 37 °C for 2 h.
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Absorbance was determined at 450 nm by using a microplate reader. Cell proliferation
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was calculated on the basis of the coloration depth by using the following formula:
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Cell proliferation (%) = (Measurement tube absorbance − Absorbance of blank) /
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(Standard pipe of absorbance − Absorbance of blank) × 100%.
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2.3. Real-time cellular proliferation and migration analysis
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Cell proliferation was performed in E-Plate (Proliferation plate) and CIM-Plate
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(Migration plate) in a real-time cellular proliferation/migration analysis DPlus
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instrument (ACEA Biosciences, Inc., Hangzhou, China), which can automatically
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record proliferation/migration curves. The cell index representing the amount of
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proliferation/migration cells was calculated with RTCA Software from ACEA
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Biosciences.
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2.4. Animal model and ethics statement
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Research Center of Nanjing University (Nanjing, China). All animal experiments
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were performed following regulations of Committee on the Ethics of Animal
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Experiments of Binzhou Medical University. Mice were housed under 12 h light/dark
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cycle, allowed free access to food and water, and randomly divided into four groups
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(10 mice per group), namely, sham group, bleomycin-treated group (BLM group), 20
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mg/kg astilbin (AST I group), and 40 mg/kg AST II group. BLM animal model was
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administered with 5 mg/kg BLM dissolved in saline via single intratracheal
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instillation under anesthesia as previously described. At day 14, astilbin was orally
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administered every day. At day 30, all mice were killed, and lung tissue sections were
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collected and immediately frozen in liquid nitrogen for further studies.
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2.5. Western blot
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20 µg of protein sample was subjected to 10% sodium dodecyl sulfate
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polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride
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membranes, and blocked with 7% non-fat milk in Tris-buffered saline and Tween-20
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(TBST; 50 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.1% Tween-20). Membranes were
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washed thrice with TBST buffer and incubated at 4°C overnight with specific
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antibodies. After washing with TBST, membranes were incubated with horseradish
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peroxidase-labeled IgG for 1.5 h. Membranes were then washed with TBST,
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incubated with ECL reagent, and exposed. Then, membranes were subsequently
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stripped and re-probed with glyceraldehyde 3-phosphate dehydrogenase antibody,
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which served as loading control.
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2.6. RNA-Sequencing
A total amount of 2 µg RNA per sample was used as input material for the RNA
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sample preparations. Sequencing libraries were generated using NEBNext® Ultra™
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RNA Library Prep Kit for Illumina® (#E7530L, NEB, USA) following the
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manufacturer’s recommendations and index codes were added to attribute sequences
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to each sample. Briefly, mRNA was purified from total RNA using poly-T
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oligo-attached magnetic beads. Fragmentation was carried out using divalent cations
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under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X).
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First strand cDNA was synthesized using random hexamer primer and RNase H.
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Second strand cDNA synthesis was subsequently performed using buffer, dNTPs,
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DNA polymerase I and RNase H. The library fragments were purified with QiaQuick
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PCR kits and elution with EB buffer, then terminal repair、A-tailing and adapter added
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were implemented. The aimed products were retrieved by agarose gel electrophoresis
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and PCR was performed, then the library was completed. RNA concentration of
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library was measured using Qubit® RNA Assay Kit in Qubit® 3.0 to preliminary
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quantify and then dilute to 1 ng/µL. Insert size was assessed using the Agilent
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Bioanalyzer 2100 system (Agilent Technologies, CA, USA), and qualified insert size
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was accurately quantified using StepOnePlus™ Real-Time PCR System (Library valid
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concentration＞10 nM). The clustering of the index-coded samples was performed on
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a cBot cluster generation system using HiSeq PE Cluster Kit v4-cBot-HS (Illumina)
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were sequenced on an Illumina Hiseq 4000 platform and 150 bp paired-end reads
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were generated.
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2.7. Cignal Finder 45-Pathway reporter array
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The signaling pathway was evaluated using the Cignal Finder 45-Pathway
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reporter array [19]. All the techniques were performed using standard protocols
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established according to the manufacturer’s protocol (Qiagen Co Ltd, Germany).
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Briefly, 50 µL of
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Array plate. 0.6 µL of Attractene used in 50 µL of Opti-MEM® per well for each
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individual transfection. After the 5 minute incubation, 50 µL of diuted Attractene was
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added into each well containing 50 µL of the diluted nucleic acids. Then cells were
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washed in a culture dish once with Dulbecco's PBS without calcium and magnesium,
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and treated with 1-3 mL trypsin-EDTA for 2-5 minutes at 37℃ in a humidified
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atmosphere containing 5% CO2. Cells were suspended in 7-9 mL of Opti-MEM®
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containing 5% of fetal bovine serum, then centrifuged, removed the superatant, and
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resuspended to 8 x 105 cells/mL in Opti-MEM® containing 10% of fetal bovine
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serum and 1% NEAA. 50 µL of prepared cell suspension was added to each well
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containing constructes-Attractene complexes. A final volume in each well was 150 µL.
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After 16-24 h incubation, the medium was changed to complete growth medium.
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50 µL of Dual-Glo Luciferase reagent was added and the fluorescence intensity was
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determined using a microplate reader (SpectraMax M2).
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2.8. Patients and healthy volunteers
Lung tissue samples were obtained by open lung biopsy. IPF was diagnosed in
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accordance with the American Thoracic Society/European Respiratory Society
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consensus criteria [20], which include clinical, radiographic, and characteristic
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histopathological features (n=4). Control non-pulmonary fibrosis lung tissue samples
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were obtained from smokers who underwent thoracic surgery for localized primary
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lung carcinoma. As stated in agreement, written informed consent was obtained by
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doctors from each participant. This study was approved by ethics committee of
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Binzhou Medical University.
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2.9. Immunofluorescence, Immunohistochemistry, H&E and Masson staining
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laboratory [21].
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2.10. Statistical analysis
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Statistical analysis was performed with SPSS 17 software. All data were
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generated from minimum of three independent experiments and expressed as mean ±
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standard deviation (SD). Different groups were compared using student’s t-test (for
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two groups) or one-way ANOVA (for more than two groups). P < 0.05 was considered
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statistically significant.
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3. Results
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3.1. Cytotoxicity of astilbin in vitro
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and L929 were incubated for 48 h under increasing concentrations of astilbin. Then,
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astilbin toxicity was evaluated. As compared with untreated cells, the half maximal
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inhibitory concentration (IC50) in AEC-II and L929 cell lines were 800 µg/mL <
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IC50 < 1 mg/mL and 1 mg/mL < IC50 < 1.2 mg/mL, respectively (Fig. 1A and B).
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Then, the inhibitory effects of astilbin on TGF-β1-treated cell viability were tested.
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Astilbin inhibited cell viability in a dose-dependent manner when the cells were
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exposed to graded doses (2.5–80 µg/mL) for 48 h. This inhibition was pronounced in
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vitro. The most pronounced inhibition was at the beginning of 5 and 20 µg/mL
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concentrations in AEC-II and L929, respectively. Thus, 5, 10, and 20 µg/mL in
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AEC-II and 20 and 40 µg/mL in L929 were used for further in vitro studies (Fig. 1C
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and D).
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3.2. Anti-pulmonary fibrosis of astilbin in vitro
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Fundamental pathogenic hallmarks of pulmonary fibrosis exhibited uncontrolled
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proliferation and high migration rates. Thus, a real-time proliferation/migration
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analysis system was used to test the inhibitory proliferation and high migration of
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astilbin on TGF-β1-treated AEC-II and L929 cells. As compared with TGF-β1-treated
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cells, astilbin inhibited proliferation and migration in a dose-dependent manner when
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the cells were exposed to graded doses (Fig. 2A and B). Then, lung fibrosis and
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epithelial cell markers were assessed using Western blot analysis. The analysis
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showed that astilbin increased the epithelial cell marker expressions of E-cadherin and
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and collagen I and III compared with the TGF-β1-treated group (Fig. 2C and D).
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3.3. Anti-pulmonary fibrosis of astilbin in vivo
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The anti-pulmonary fibrosis of astilbin in mice was tested to evaluate the
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therapeutic value of astilbin further. First, astilbin increased the force vital capacity
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(FVC) compared with the BLM-treated group (Fig. 3A). Thin alveolar walls were
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observed in tissue sections, and the number of fibroblasts and collagen matrices
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decreased significantly in astilbin-treated groups (Fig. 3B and C). The indicators of
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pulmonary fibrosis, such as E-cadherin, SP-C, vimentin, α-SMA, Snail, and collagen I
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and III, were investigated using Western blot analysis and immunofluorescence
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staining (Fig 4A and B). The data showed that astilbin increased the epithelial cell
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markers of E-cadherin and SP-C and decreased the mesenchymal markers of vimentin,
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α-SMA, Snail, and collagen I and III.
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3.4. Regulation of astilbin in pulmonary fibrosis-associated aberrant mRNAs
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The differentially expressed RNAs were evaluated using RNA sequencing to
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elucidate the anti-pulmonary fibrosis mechanism of astilbin. From the RNA
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sequencing data, the mRNA expression profiles of the astilbin-treated group were
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compared with that of the BLM-treated group. Enrichment analysis was applied to
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explore the functions of the mRNAs identified in this study. Genes were organized
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into hierarchical categories to uncover gene regulatory networks on the basis of
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biological processes, cellular components, and molecular functions. The analysis
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pathway (Fig. 5), such as Boc, Lrp, Rab23, Gli, Gsk3b, Csnk, Prkaca, Sufu, Stk, Zic2
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and so on.
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3.5. Confirmation of the Hedgehog pathway involved in the antifibrotic activity of
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astilbin
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The Cignal Finder 45-Pathway Reporter Array confirmed the RNA sequencing
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results which showed that astilbin significantly inhibited glioma-associated oncogene
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homolog (Gli) reporter luciferase activities compared with other reporters (Fig. 6A).
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Then, Gli1 was selected as a representative gene to confirm the in vivo and in vitro
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results using Western blot analysis and immunofluorescence staining. The data
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showed that Gli1 increased in the pulmonary fibrosis model in vivo and in vitro, and
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astilbin remarkably decreased Gli1 expression (Fig. 6B-E). Gli1 expression in patients
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with pulmonary fibrosis was tested using immunohistochemistry to evaluate
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therapeutic value of astilbin. Gli1 expression was also increased in patients with
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pulmonary fibrosis, supporting the in vivo and in vitro results (Fig. 6F). The rescue
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experiment was used to evaluate and verify that the Hedgehog pathway is involved in
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the antifibrotic activity of astilbin further. Chemical activator smoothened agonist
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(SAG) and inhibitor vismodegib (GDC-0449) that are specific to the Hedgehog
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pathway were used, which showed that the activator can promote the expression of
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α-SMA and collagen I and III, but the inhibitor can block the expression of these
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fibrotic indicators. And astilbin reversed these changes caused by SAG and
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GDC-0449 (Fig. 7).
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3.6. Analysis of co-expressed lncRNA with Gli1
Long noncoding RNA (lncRNA) is the upstream factor of mRNA that regulates
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its function. From the RNA sequencing data, Gli1 and its co-expressed lncRNA were
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analyzed in the astilbin-treated group compared with the BLM-treated group. The
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degree of mRNA–lncRNA co-expression was analyzed. An increased degree indicated
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an
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lncRNA-NUNMM028949.2 has the highest degree (Figures 8A and 8B).
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lncRNA-NUNMM028949.2 was selected to determine its expression levels through
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qRT-PCR to validate the data. As shown in Figure 8C, the lncRNA decreased in the
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astilbin-treated group compared with the BLM-treated group.
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4. Discussion
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TGF-β1/BLM-induced lung fibrogenesis in vivo and in vitro, and its mechanism of
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Hedgehog pathway signaling pathway in astilbin treatment. Our results showed that
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astilbin significantly decreased alveolar wall thickness and collagen fiber formation,
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and these anti-fibrotic effects of astilbin may be mediated through blockade of
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Hedgehog signaling pathway.
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Similar to cancer, pulmonary fibrosis show functional features such as
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low adhesion, uncontrolled proliferation and high migration rates [22，23]. Snail is the
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transcriptional repressors of adheren that is a direct repressor of E-cadherin 13
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expression. Meanwhile, astilbin also could inhibit the proliferation and migration of
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TGF-β1-treated AEC-II and L929 cells. One of the phenomena during the process of
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pulmonary fibrosis is the manifestation of mesenchymal characteristics, including the
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expression of mesenchymal markers and the loss of epithelial cell markers [26]. Our
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data showed that astilbin increased levels of epithelial cell markers such as E-cadherin
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and SP-C, and decreased levels of mesenchymal markers such as a-SMA, collagen
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and vimentin. All these finding showed that the anti-pulmonary fibrosis of astilbin is
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significant.
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The pathogenesis of pulmonary fibrosis indicates that the fibrotic response is
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driven by abnormally activated alveolar epithelial cells, which produce mediators that
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induce the formation of fibroblast and myofibroblast foci through the proliferation of
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resident mesenchymal cells. The fibroblast and myofibroblast foci secrete excess
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amounts of extracellular matrix, including collagen, α-SMA, and vimentin, resulting
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in scarring and destruction of the lung architecture. However, the pathways leading to
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the secretion of excess amounts of extracellular matrix that produce fibroblasts are
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uncertain [27]. To address these uncertainties, RNA sequencing was used to explore
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the pathways in this study. RNA sequencing is a powerful technique to investigate the
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complexity of gene expression [28]. The findings showed that astilbin can ameliorate
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pulmonary fibrosis through multigene, multi-pathway. These genes are some of the
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most notable components of the Hedgehog signaling pathway, such as Hedgehog
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ligand (Boc), Hedgehog receptors and cofactors (Lrp and Rab23), transcription factors
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genes (Ptch, Bcl-2, Vegfa, Disp, Mapk, Runx2, and Trp53), and pathways crosstalking
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with Hedgehog (Wnt, Bmp, Mob1b, Nf2, and Wif1) [29]. Based on these results, the
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influence of astilbin on Hedgehog pathways was further detected.
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Aberrant activation of the Hedgehog signaling pathway, which has a crucial role
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in the development and homeostasis of many organs and tissues, has been linked to
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tumorigenesis [30]. The role of the Hedgehog signaling pathway in pulmonary
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fibrosis pathogenesis has not been thoroughly investigated. In this study, we analyzed
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the function and mechanism of this pathway in pulmonary fibrosis. Gli is one of
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components of the Hedgehog signaling pathway including Gli1, Gli2, and Gli3
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[31,32], and its expression level is widely accepted to reflect Hedgehog pathway
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activity [33]. It acts as a zinc finger transcription factor initially characterized in
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human glioblastoma to control cell differentiation and proliferation [34]. Although the
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involvement of Gli in a variety of cancers is well established [35], its role in
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pulmonary fibrosis remains poorly understood. Genetic lineage tracing analysis
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indicates that Gli1+ cells proliferate after kidney, lung, liver, or heart injury to
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generate myofibroblasts. Genetic ablation of these cells substantially ameliorates
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kidney and heart fibrosis and preserves ejection fraction in a model of induced heart
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failure [36]. Our results showed that Gli1 increased in lung fibrosis, which supports
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the notion that Gli1 is a key contributor to organ fibrosis and indicates the possible
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pathways implicated in lung fibrosis as targets of therapeutic attempts. Astilbin can
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block the progression of pulmonary fibrosis via blockade of Gli expression.
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High-throughput technologies such as RNA sequencing have revealed that only 2% of the transcribed genome codes are attributed to proteins. The remaining part of the
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transcribed genome is known as noncoding RNA. Among the various types of
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noncoding RNA, a class referred to as long noncoding RNA (lncRNA) has attracted
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increasing attention [37]. Emerging evidence suggests lncRNA plays important roles
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in disease development. These noncoding RNAs are the upstream factors of mRNAs
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that directly or indirectly regulate the biological function of mRNAs [38,39]. Here,
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we demonstrate the lncRNAs in targeting Gli1 to regulate the anti-pulmonary fibrosis
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mechanism of astilbin. We will be designing experiments to determine the
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relationship between astilbin and lncRNAs -mediated pulmonary fibrosis for future
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research.
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5. Conclusions
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In summary, our study showed that astilbin can attenuate lung fibrosis by
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suppressing the Hedgehog pathway, which provided a rationale for exploring the
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therapeutic and chemoprotective potentials of astilbin for the treatment and prevention
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of pathologic conditions involving pulmonary fibrosis in future studies.
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Acknowledgements
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This study was supported by National Natural Science Foundation of China
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(31470415, 31670365, 81670064, 81741170), Natural Science Foundation of
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Shandong Province (ZR2016HP34).
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Conflicts of Interest: The authors declare no conflict of interest. 16
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References
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[1] A.L. Mora1, M. Rojas, A. Pardo, M. Selman, Emerging therapies for idiopathic
339
pulmonary fibrosis, a progressive age-related disease, Nature Reviews Drug
340
Discovery 170 (2017) 1-18.
Reviews Drug Discovery 9 (2010) 129-140.
SC
342
[2] R.M. du Bois, Strategies for treating idiopathic pulmonary fibrosis, Nature
M AN U
341
RI PT
337
343
[3] M. Selman, A. Pardo, Revealing the pathogenic and aging-related mechanisms of
344
the enigmatic idiopathic pulmonary fibrosis. an integral model, Am. J. Respir. Crit.
345
Care Med. 189 (2014) 1161–1172.
347
[4] B.D. Uhal, The role of apoptosis in pulmonary fibrosis, Eur. Respir. Rev. 17 (2008) 138–144.
TE D
346
[5] T.E. King Jr, W.Z. Bradford, S. Castro-Bernardini, E. A. Fagan, I. Glaspole, M. K.
349
Glassberg, et al., A phase 3 trial of pirfenidone in patients with idiopathic
AC C
350
EP
348
pulmonary fibrosis, N. Engl. J. Med. 370 (2014 ) 2083–2092.
351
[6] L. Richeldi, R.M. du Bois , G. Raghu, A. Azuma, K.K. Brown, U. Costabel , et al.,
352
Efficacy and safety of nintedanib in idiopathic pulmonary fibrosis, N. Engl. J.
353
Med. 370 (2014) 2071–2082.
354
[7] P.W. Noble, C. Albera, W. Z. Bradford, U. Costabel , R.M. du Bois, E.A. Fagan, et
355
al., Pirfenidone for idiopathic pulmonary fibrosis: analysis of pooled data from 17
ACCEPTED MANUSCRIPT 356
three multinational phase 3 trials, Eur. Respir. J. 47 (2016) 243–253.
[8] L. Richeldi, V. Cottin, R.M. du Bois, M. Selman, T. Kimura, Z. Bailes, et al.,
358
Nintedanib in patients with idiopathic pulmonary fibrosis: Combined evidence
359
from the TOMORROW and INPULSIS® trials, Respiratory Medicine 113 (2016)
360
74-79.
RI PT
357
[9] L. Naldini, Gene therapy returns to centre stage, Nature 526 (2015) 351–360.
362
[10] N. Somia, I.M. Verma, Gene therapy: trials and tribulations, Nat. Rev. Genet. 1
M AN U
363
SC
361
(2000) 91–99.
[11] Q.F. Meng, Z. Zhang, Y.J. Wang, W. Chen, F.F. Li, L.T. Yue, et al., Astilbin
365
ameliorates experimental autoimmune myasthenia gravis by decreased Th17
366
cytokines and up-regulated T regulatory cells, J. Neuroimmunol. 298 (2016)
367
138-45.
TE D
364
[12] T.T. Di, Z.T. Ruan, J.X. Zhao, Y. Wang, X. Liu, Y. Wang, et al., Astilbin inhibits
369
Th17 cell differentiation and ameliorates imiquimod-induced psoriasis-like skin
370
lesions in BALB/c mice via Jak3/Stat3 signaling pathway, Int. Immunopharmacol.
AC C
371
EP
368
32 (2016) 32-38.
372
[13] Q.F. Zhang, Y.J. Fu, Z.W. Huang, X.C. Shangguang, Y.X. Guo, Aqueous Stability
373
of Astilbin: Effects of pH, Temperature, and Solvent, J. Agric. Food Chem. 61
374
(2013) 12085−12091.
375
[14] Q.F. Zhang, Z.R. Zhang, H.Y. Cheung, Antioxidant activity of Rhizoma Smilacis 18
ACCEPTED MANUSCRIPT 376
Glabrae extracts and its key constituent − astilbin, Food Chem. 115 (2009)
377
297−303.
[15] S.W. Wang, Y. Xu, Y.Y. Weng, X.Y. Fan, Y.F. Bai, X.Y. Zheng, et al., Astilbin
379
ameliorates cisplatin-induced nephrotoxicity through reducing oxidative stress and
380
inflammation, Food Chem. Toxicol. 114 (2018) 227-236.
RI PT
378
[16] H.B. Zhang, L.C. Sun, L.D. Zhi, Q.K. Wen, Z.W. Qi, S.T. Yan, et al., Astilbin
382
alleviates sepsis-induced acute lung injury by inhibiting the expression of
383
macrophage inhibitory factor in rats, Arch. Pharm. Res. 40 (2017) 1176-1185.
384
[17] Y. Shi, B.R. Gochuico, G. Yu, X. Tang, J.C. Osorio, I.E. Fernandez, et al.,
385
Syndecan-2 exerts antifibrotic effects by promoting caveolin-1-mediated
386
transforming growth factor-β receptor I internalization and inhibiting transforming
387
growth factor-β1 signaling, Am. J. Respir. Crit. Care Med. 188 (2013) 831–841.
388
[18] H. Liu, B. Wang, J. Zhang, S. Zhang, Y. Wang, J. Zhang, et al., A novel lnc-PCF
389
promotes the proliferation of epithelial cell activated with TGF-β1 by targeting
390
miR-344a-5p to regulate map3k11 in pulmonary fibrosis, Cell death and disease 8
M AN U
TE D
EP
AC C
391
SC
381
(2017) e3137.
392
[19] Z. Xing, A. Lin, C. Li, K. Liang, S. Wang, Y. Liu, et al., lncRNA directs
393
cooperative epigenetic regulation downstream of chemokine signals, Cell 159
394
(2014) 1110-1125.
395
[20] G. Raghu, H.R. Collard, J.J. Egan, F.J. Martinez, J. Behr, K.K. Brown, et al., An 19
ACCEPTED MANUSCRIPT 396
Official
397
Evidence-based Guidelines for Diagnosis and Management, Am. J. Respir. Crit.
398
Care Med. 183 (2011) 788-824.
Statement:
Idiopathic
Pulmonary
Fibrosis:
[21] X.D. Song, W.L. Liu, S.Y. Xie, M.R. Wang, G.H. Cao, C.P. Mao, et al.,
400
All-transretinoic
ameliorates
bleomycin-induced
lung
fibrosis
by
401
downregulating the TGF-b1/Smad3 signaling pathway in rats, Lab. Invest. 93
402
(2013) 1219–1231.
SC
acid
RI PT
399
ATS/ERS/JRS/ALAT
[22] C. Vancheri, M. Failla, N. Crimi, G. Raghu, Idiopathic pulmonary fibrosis: a
404
disease with similarities and links to cancer biology, Eur. Respir. J. 35 (2010)
405
496-504.
407
[23] C. Vancheri, Idiopathic pulmonary fibrosis: An altered fibroblast proliferation
TE D
406
M AN U
403
linked to cancer biology, Proc. Am. Thorac. Soc. 9 (2012) 153-157.
[24] A. Cano, M.A. Pérez-Moreno, I. Rodrigo, A. Locascio, M.J. Blanco, M.G. del
409
Barrio,et al., The transcription factor Snail controls epithelial–mesenchymal
410
transitions by repressing E-cadherin expression, Nat. Cell Biol. 2 (2000) 76-83.
411
[25] E. Batlle, E. Sancho, C. Francí, D. Domínguez, M. Monfar, J. Baulida, et al., The
412
transcription factor Snail is a repressor of E-cadherin gene expression in epithelial
413
AC C
EP
408
tumour cells, Nat. Cell Biol. 2 (2000) 84-89.
414
[26] K.V. Pandit, D. Corcoran, H. Yousef, M. Yarlagadda, A. Gibson, K.F.
415
Tzouvelekis, et al., Inhibition and Role of let-7d in Idiopathic Pulmonary Fibrosis, 20
ACCEPTED MANUSCRIPT 416
Am. J. Respir. Crit. Care Med. 182 (2010) 220–229.
[27] K.K. Kim, M.C. Kugler, P.J. Wolters, L. Robillard, M.G. Galvez, A.N. Brumwell,
418
et al., Alveolar epithelial cell mesenchymal transition develops in vivo during
419
pulmonary fibrosis and is regulated by the extracellular matrix, PNAS 103 (2006 )
420
13180–13185.
RI PT
417
[28] T.K. Doktor, Y. Hua, H.S. Andersen, S. Brøner, Y.H. Liu, A. Wieckowska, et al.,
422
RNA-sequencing of a mouse-model of spinal muscular atrophy reveals
423
tissue-wide changes in splicing of U12-dependent introns, Nucleic Acids Research
424
45 (2017) 395-416.
M AN U
425
SC
421
[29] S.J. Scales, F. J. de Sauvage, Mechanisms of Hedgehog pathway activation in cancer and implications for therapy, Trends in Pharmacological Sciences 30 (2009)
427
303-312.
430 431
EP
429
[30] D. Amakye, Z. Jagani, M. Dorsch, Unraveling the therapeutic potential of the Hedgehog pathway in cancer, Nature Medicine 19 (2013) 1410-1422.
[31] L. Lum, P.A. Beachy, The Hedgehog response network: sensors, switches, and
AC C
428
TE D
426
routers, Science 304 (2004) 1755–1759.
432
[32] A. Ruiz i Altaba, C. Mas , B. Stecca, The Gli code: an information nexus
433
regulating cell fate, stemness and cancer, Trends Cell Biol. 17 (2007) 438–447.
434
[33] S.J. Scales, F.J. de Sauvage, Mechanisms of Hedgehog pathway activation in
435
cancer and implications for therapy, Trends Pharmacol. Sci. 30 (2009) 303–312. 21
ACCEPTED MANUSCRIPT 436
[34] S. Sheybani-Deloui, L. Chi, M.V. Staite, J.E. Cain, B.J. Nieman, R.M.
437
Henkelman, et al., Activated Hedgehog-GLI Signaling Causes Congenital
438
Ureteropelvic Junction Obstruction, J. Am. Soc. Nephrol. (2017).
[35] L. Huang, V. Walter, D.N. Hayes, M. Onaitis, Hedgehog–GLI Signaling
440
Inhibition Suppresses Tumor Growth in Squamous Lung Cancer, Clin. Cancer Res.
441
20 (2014) 1566-1575.
SC
RI PT
439
[36] R. Kramann, R.K. Schneider, D.P. DiRocco, F. Machado, S. Fleig, P.A. Bondzie,
443
et al., Perivascular Gli1+ Progenitors Are Key Contributors to Injury-Induced
444
Organ Fibrosis, Cell Stem Cell 16 (2015) 51–66.
446
[37] F. Kopp, J.T. Mendell, Functional Classification and Experimental Dissection of Long Noncoding RNAs, Cell 172 (2018) 393-407.
TE D
445
M AN U
442
[38] L. Meng, A.J. Ward, S. Chun, C.F. Bennett, A.L. Beaudet, F. Rigo, Towards a
448
therapy for Angelman syndrome by targeting a long non-coding RNA, Nature 518
449
(2015) 409–412.
EP
447
[39] Y. Liu, R. Guo, G. Hao, J. Xiao, Y. Bao, J. Zhou, et al., The expression profiling
451
and ontology analysis of noncoding RNAs in peritoneal fibrosis induced by
452
AC C
450
peritoneal dialysis fluid, Gene 564 (2015) 210–219.
453
454
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Figure legends
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Fig. 1. Astilbin cytotoxicity in AEC-II and L929 using CCK-8. (A) Astilbin
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cytotoxicity in the normal AEC-II cells (800 µg/mL < IC50 < 1 mg/mL). (B) Astilbin
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cytotoxicity in the normal L929 cells (1 mg/mL < IC50 < 1.2 mg/mL). (C) and (D)
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AEC-II and L929 cells were initially administered with 5 ng/mL TGF-β1 for 6 h and 23
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the most pronounced inhibition was at the beginning of 5 and 20 µg/mL
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concentrations in AEC-II and L929, respectively. Each bar represents the mean ± SD,
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with n = 6 and *p < 0.05, **p < 0.01.
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Fig. 2. Anti-pulmonary fibrosis of astilbin in AEC-II and L929 cells. (A) The
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real-time proliferation analysis system showed that astilbin inhibited TGF-β1-treated
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cell proliferation. (B) The real-time migration analysis system showed that astilbin
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inhibited TGF-β1-treated cell migration. (C) Astilbin increased the expressions of
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E-cadherin and SP-C and decreased the expressions of vimentin, α-SMA, Snail, and
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collagen I and III in AEC-II. (D) Astilbin decreased the expressions of vimentin,
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α-SMA, Snail, and collagen I and III in L929. *p < 0.05, **p < 0.01 vs. the normal
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group.
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Fig. 3. Anti-pulmonary fibrosis of astilbin in mice. (A) Astilbin increased FVC
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compared with the BLM-treated group. (B) Astilbin improved the alveolar structure
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through H&E staining. (C) Astilbin inhibited the collagen fibers through Masson’s
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staining. The color blue represents collagen fibers.
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Fig. 4. Astilbin inhibited the indicators of pulmonary fibrosis in mice. (A) Western
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blot analysis showed that astilbin promoted E-cadherin expression and inhibited
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vimentin, α-SMA, Snail, and collagen I and III expressions. (B) Immunofluorescence
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staining showed that astilbin promoted SP-C expression and inhibited α-SMA
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expression. The nuclei and SP-C/α-SMA were counterstained with DAPI (blue) and
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Fig. 5. Heatmap of the expression profiles of the differentially expressed mRNAs
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in vivo. The colors blue to yellow indicate the low to high expression levels. The
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column labels at the right represent the differentially expressed mRNAs in the
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Hedgehog signaling pathway. M represents the BLM-treated group; R represents the
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astilbin-treated group.
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Fig. 6. The aberrant signaling pathway of astilbin was tested in vivo and in vitro,
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and in patients with pulmonary fibrosis. (A) Identification of signaling pathways
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affected by astilbin in AEC-II cells. The x- and y-axes represent the normalized ratio
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of firefly/Renilla luciferase activities, respectively. Most significant changes were
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observed in the Hedgehog signaling pathways. (B) Western blot analysis showed that
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astilbin
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Immunofluorescence staining in L929 showed that astilbin blocked Gli1 expression.
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The nuclei and Gli1 were counterstained with DAPI (blue) and Gli1 (green)
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antibodies. (D) Western blot analysis showed that astilbin inhibited Gli1 expression in
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mice. (E) Immunohistochemistry in mice showed that astilbin blocked Gli1
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expression. (F) Gli1 expression increased in patients with pulmonary fibrosis.
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Fig. 7. Rescue experiment was used to detect the Hedgehog pathway involved in
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the antifibrotic mechanism of astilbin with chemical activator smoothened
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agonist (SAG) and inhibitor vismodegib (GDC-0449) in L929. Astilbin further
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reduced the expression of α-SMA and collagen I and III which increase or decrease
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expression
levels
of
Gli1
protein
in
cells.
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Fig. 8. Analysis of co-expressed lncRNAs with Gli1. (A) Co-expression degrees of
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lncRNAs with Gli1. The list only showed the top 10 degrees of lncRNAs with Gli1.
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(B) lncRNA-NUNMM028949.2 expression decreased in the astilbin-treated group
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compared with the BLM-treated group using RNA sequencing. (C) qRT-PCR was
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used to validate that lncRNA-NUNMM028949.2 expression was inhibited in the
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astilbin-treated group compared with the BLM-treated group. *p < 0.05, **p < 0.01.
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S1094-5539(18)30030-0
DOI:
10.1016/j.pupt.2018.03.006
Reference:
YPUPT 1718
To appear in:
Pulmonary Pharmacology & Therapeutics
Received Date: 31 January 2018 Revised Date:
25 March 2018
Accepted Date: 31 March 2018
Please cite this article as: Zhang J, Liu H, Song C, Zhang J, Wang Y, Lv C, Song X, Astilbin ameliorates pulmonary fibrosis via blockade of Hedgehog signaling pathway, Pulmonary Pharmacology & Therapeutics (2018), doi: 10.1016/j.pupt.2018.03.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Astilbin ameliorates pulmonary fibrosis via blockade
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of Hedgehog signaling pathway
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a Department of Cellular and Genetic Medicine, School of Pharmaceutical Sciences,
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b Department of Respiratory Medicine, Affiliated Hospital to Binzhou Medical University, Binzhou 256602, China.
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Binzhou Medical University, Yantai 264003, China.
c Department of Medical Oncology, People`s Liberation Army 107th Hospital, Yantai,264003, China.
d Department of Respiratory Medicine, Zouping Chinese Medicine Hospital, Binzhou 256602, China.
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Jie Zhang△a,b, Hanchen Liu△c, Chenguang Song△d, Jinjin Zhanga,b, Youlei Wang a, Changjun Lv *a,b, Xiaodong Song* a,b
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Running Title: Astilbin ameliorates pulmonary fibrosis
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△These authors contributed equally to this work.
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*Correspondence author: Prof. Xiaodong Song and Changjun Lv, School of
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Pharmaceutical Sciences of Binzhou Medical University, No. 346, Guanhai Road,
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Laishan District, Yantai City, 264003, China. Email: [email protected];
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[email protected]
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Tel: +86-535-6913985; Fax: +86-535-6913985
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ABSTRACT
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Background and objective: The nature of pulmonary fibrosis involves inadequate
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repair of the epithelial cell barrier accompanied by impaired regulation of the
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fibroblast. Moreover, pulmonary fibrosis currently lacks an effective therapeutic drug.
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This study targets the protection of the epithelial cell and fibroblast to identify a novel,
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potentially therapeutic drug (i.e., astilbin).
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Methods: In this study, the cytotoxicity of astilbin was firstly detected using CCK-8.
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A real-time proliferation/migration analysis system was used to test the inhibitory
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proliferation and migration of astilbin in vitro. The expression of mesenchymal
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markers and the loss of epithelial cell markers were analyzed to evaluate the
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antifibrotic activity of astilbin on TGF-β1-treated AEC-II and L929 cells and
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bleomycin-treated mice. Then, in fibrosis-associated signaling pathways, the
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regulation of astilbin was tested using RNA sequencing and Cignal Finder
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45-Pathway system. Rescue and other experiments were used to confirm this pathway
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regulation further.
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Results: The data showed that astilbin inhibited proliferation and migration of cell
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samples. Its treatment resulted in the reduction of pathological score and collagen
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deposition, with a decrease in α-SMA and Snail and an increase in E-cadherin and
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ACCEPTED MANUSCRIPT SP-C in vivo and in vitro. The fibrosis-associated aberrant genes are some of the most
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notable components of the Hedgehog signaling pathway.
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Conclusions: Astilbin ameliorates pulmonary fibrosis via blockade of Hedgehog
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signaling pathway and has potential therapeutic value for lung fibrosis treatment.
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Key Words: Astilbin; Anti-pulmonary fibrosis; Hedgehog pathway
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1. Introduction
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Pulmonary fibrosis results from abnormal tissue repair and is associated with
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persistent and/or severe tissue damage and cellular stress [1]. The nature of this
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aberrance involves inadequate repair of the epithelial cell barrier accompanied by
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impaired regulation of the fibroblast [2], which results in profound changes to
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alveolar epithelial cell and fibroblast, leading to uncontrolled fibrosis [3]. Thus,
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targeting the protection of the epithelial cell and fibroblast is an attractive approach to
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treat pulmonary fibrosis [4]. Pirfenidone and nintedanib were approved for the
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treatment of pulmonary fibrosis in 2014 [5,6]. However, differences in adverse event
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frequency, such as photosensitivity, gastrointestinal symptoms, and liver function test
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abnormalities, were observed [7,8]. Gene therapy is a potential treatment; however,
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successfully applying it to clinical practice has so far been a formidable challenge
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[9,10]. Therefore, the identification of a novel therapeutic drug in preclinical study is
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highly desired.
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Astilbin, (2R,3R)-3,3′,4′,5,7-pentahydroxyflavanon-3-α-L-rhamnopyranoside, is a
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dihydroflavonol rhamnoside, which is significant in the treatment of immunologically 3
ACCEPTED MANUSCRIPT related diseases. Several studies have demonstrated that astilbin ameliorates
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experimental autoimmune myasthenia gravis and imiquimod-induced psoriasis-like
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skin lesions [11,12]. Astilbin has also shown many other bioactivities, such as
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anti-oxidative, anti-inflammation, and 3-hydroxy-3-methylglutaryl coenzyme A
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reductase inhibitor [13,14]. Astilbin is currently used to ameliorate cisplatin-induced
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nephrotoxicity through reducing oxidative stress and inflammation [15]. In the
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treatment of lung injury, astilbin can alleviate sepsis-induced acute lung injury by
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inhibiting the expression of macrophage inhibitory factor in rats [16]. However, the
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anti-pulmonary fibrosis of astilbin remains unknown. Pulmonary fibrosis is actually a
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kind of lung injury, except that this is a specific form of chronic, progressive injury.
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This study aims to investigate the effects of astilbin against pulmonary fibrosis and its
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potential anti-pulmonary fibrosis mechanism in vivo and in vitro.
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2. Methods
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2.1. Cell culture
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Mouse type II alveolar epithelial cell (AEC-II) and mouse lung fibroblast (L929)
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cell lines were purchased from Cell Bank of Chinese Academy of Sciences.
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Transforming growth factor beta 1 (TGF-β1), the most important regulatory factor
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among fibrogenic cytokines, was used to stimulate AEC-II and L929 cells to establish
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the pulmonary fibrosis model in vitro [17,18]. Cells were maintained in advanced
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minimum essential medium and supplemented with 10% newborn calf serum, 100
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U/mL penicillin and 100 µg/mL streptomycin at 37℃ under a humidified atmosphere
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TGF-β1 for 6 h and then co-treated with astilbin (Dalian Meilun Biotech Co., Ltd) for
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48 h.
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2.2. Cell counting kit-8 assay (CCK-8)
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Cell proliferation was determined using CCK-8 kit (DOJINDO, Japan) in
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accordance with the manufacturer’s instructions. In brief, 1 × 106 cells/well were
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seeded in a 96-well and flat-bottomed plate and then grown at 37 °C for 24 h. After 10
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µL of WST-8 dye was added to each well, the cells were incubated at 37 °C for 2 h.
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Absorbance was determined at 450 nm by using a microplate reader. Cell proliferation
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was calculated on the basis of the coloration depth by using the following formula:
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Cell proliferation (%) = (Measurement tube absorbance − Absorbance of blank) /
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(Standard pipe of absorbance − Absorbance of blank) × 100%.
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2.3. Real-time cellular proliferation and migration analysis
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Cell proliferation was performed in E-Plate (Proliferation plate) and CIM-Plate
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(Migration plate) in a real-time cellular proliferation/migration analysis DPlus
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instrument (ACEA Biosciences, Inc., Hangzhou, China), which can automatically
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record proliferation/migration curves. The cell index representing the amount of
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proliferation/migration cells was calculated with RTCA Software from ACEA
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Biosciences.
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2.4. Animal model and ethics statement
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Research Center of Nanjing University (Nanjing, China). All animal experiments
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were performed following regulations of Committee on the Ethics of Animal
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Experiments of Binzhou Medical University. Mice were housed under 12 h light/dark
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cycle, allowed free access to food and water, and randomly divided into four groups
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(10 mice per group), namely, sham group, bleomycin-treated group (BLM group), 20
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mg/kg astilbin (AST I group), and 40 mg/kg AST II group. BLM animal model was
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administered with 5 mg/kg BLM dissolved in saline via single intratracheal
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instillation under anesthesia as previously described. At day 14, astilbin was orally
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administered every day. At day 30, all mice were killed, and lung tissue sections were
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collected and immediately frozen in liquid nitrogen for further studies.
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2.5. Western blot
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20 µg of protein sample was subjected to 10% sodium dodecyl sulfate
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polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride
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membranes, and blocked with 7% non-fat milk in Tris-buffered saline and Tween-20
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(TBST; 50 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.1% Tween-20). Membranes were
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washed thrice with TBST buffer and incubated at 4°C overnight with specific
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antibodies. After washing with TBST, membranes were incubated with horseradish
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peroxidase-labeled IgG for 1.5 h. Membranes were then washed with TBST,
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incubated with ECL reagent, and exposed. Then, membranes were subsequently
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stripped and re-probed with glyceraldehyde 3-phosphate dehydrogenase antibody,
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which served as loading control.
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2.6. RNA-Sequencing
A total amount of 2 µg RNA per sample was used as input material for the RNA
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sample preparations. Sequencing libraries were generated using NEBNext® Ultra™
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RNA Library Prep Kit for Illumina® (#E7530L, NEB, USA) following the
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manufacturer’s recommendations and index codes were added to attribute sequences
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to each sample. Briefly, mRNA was purified from total RNA using poly-T
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oligo-attached magnetic beads. Fragmentation was carried out using divalent cations
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under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X).
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First strand cDNA was synthesized using random hexamer primer and RNase H.
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Second strand cDNA synthesis was subsequently performed using buffer, dNTPs,
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DNA polymerase I and RNase H. The library fragments were purified with QiaQuick
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PCR kits and elution with EB buffer, then terminal repair、A-tailing and adapter added
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were implemented. The aimed products were retrieved by agarose gel electrophoresis
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and PCR was performed, then the library was completed. RNA concentration of
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library was measured using Qubit® RNA Assay Kit in Qubit® 3.0 to preliminary
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quantify and then dilute to 1 ng/µL. Insert size was assessed using the Agilent
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Bioanalyzer 2100 system (Agilent Technologies, CA, USA), and qualified insert size
143
was accurately quantified using StepOnePlus™ Real-Time PCR System (Library valid
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concentration＞10 nM). The clustering of the index-coded samples was performed on
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a cBot cluster generation system using HiSeq PE Cluster Kit v4-cBot-HS (Illumina)
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were sequenced on an Illumina Hiseq 4000 platform and 150 bp paired-end reads
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were generated.
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2.7. Cignal Finder 45-Pathway reporter array
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The signaling pathway was evaluated using the Cignal Finder 45-Pathway
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reporter array [19]. All the techniques were performed using standard protocols
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established according to the manufacturer’s protocol (Qiagen Co Ltd, Germany).
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Briefly, 50 µL of
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Array plate. 0.6 µL of Attractene used in 50 µL of Opti-MEM® per well for each
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individual transfection. After the 5 minute incubation, 50 µL of diuted Attractene was
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added into each well containing 50 µL of the diluted nucleic acids. Then cells were
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washed in a culture dish once with Dulbecco's PBS without calcium and magnesium,
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and treated with 1-3 mL trypsin-EDTA for 2-5 minutes at 37℃ in a humidified
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atmosphere containing 5% CO2. Cells were suspended in 7-9 mL of Opti-MEM®
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containing 5% of fetal bovine serum, then centrifuged, removed the superatant, and
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resuspended to 8 x 105 cells/mL in Opti-MEM® containing 10% of fetal bovine
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serum and 1% NEAA. 50 µL of prepared cell suspension was added to each well
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containing constructes-Attractene complexes. A final volume in each well was 150 µL.
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After 16-24 h incubation, the medium was changed to complete growth medium.
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50 µL of Dual-Glo Luciferase reagent was added and the fluorescence intensity was
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determined using a microplate reader (SpectraMax M2).
added
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2.8. Patients and healthy volunteers
Lung tissue samples were obtained by open lung biopsy. IPF was diagnosed in
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accordance with the American Thoracic Society/European Respiratory Society
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consensus criteria [20], which include clinical, radiographic, and characteristic
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histopathological features (n=4). Control non-pulmonary fibrosis lung tissue samples
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were obtained from smokers who underwent thoracic surgery for localized primary
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lung carcinoma. As stated in agreement, written informed consent was obtained by
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doctors from each participant. This study was approved by ethics committee of
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Binzhou Medical University.
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2.9. Immunofluorescence, Immunohistochemistry, H&E and Masson staining
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laboratory [21].
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2.10. Statistical analysis
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Statistical analysis was performed with SPSS 17 software. All data were
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generated from minimum of three independent experiments and expressed as mean ±
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standard deviation (SD). Different groups were compared using student’s t-test (for
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two groups) or one-way ANOVA (for more than two groups). P < 0.05 was considered
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statistically significant.
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3. Results
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3.1. Cytotoxicity of astilbin in vitro
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and L929 were incubated for 48 h under increasing concentrations of astilbin. Then,
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astilbin toxicity was evaluated. As compared with untreated cells, the half maximal
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inhibitory concentration (IC50) in AEC-II and L929 cell lines were 800 µg/mL <
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IC50 < 1 mg/mL and 1 mg/mL < IC50 < 1.2 mg/mL, respectively (Fig. 1A and B).
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Then, the inhibitory effects of astilbin on TGF-β1-treated cell viability were tested.
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Astilbin inhibited cell viability in a dose-dependent manner when the cells were
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exposed to graded doses (2.5–80 µg/mL) for 48 h. This inhibition was pronounced in
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vitro. The most pronounced inhibition was at the beginning of 5 and 20 µg/mL
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concentrations in AEC-II and L929, respectively. Thus, 5, 10, and 20 µg/mL in
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AEC-II and 20 and 40 µg/mL in L929 were used for further in vitro studies (Fig. 1C
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and D).
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3.2. Anti-pulmonary fibrosis of astilbin in vitro
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Fundamental pathogenic hallmarks of pulmonary fibrosis exhibited uncontrolled
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proliferation and high migration rates. Thus, a real-time proliferation/migration
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analysis system was used to test the inhibitory proliferation and high migration of
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astilbin on TGF-β1-treated AEC-II and L929 cells. As compared with TGF-β1-treated
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cells, astilbin inhibited proliferation and migration in a dose-dependent manner when
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the cells were exposed to graded doses (Fig. 2A and B). Then, lung fibrosis and
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epithelial cell markers were assessed using Western blot analysis. The analysis
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showed that astilbin increased the epithelial cell marker expressions of E-cadherin and
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and collagen I and III compared with the TGF-β1-treated group (Fig. 2C and D).
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3.3. Anti-pulmonary fibrosis of astilbin in vivo
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The anti-pulmonary fibrosis of astilbin in mice was tested to evaluate the
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therapeutic value of astilbin further. First, astilbin increased the force vital capacity
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(FVC) compared with the BLM-treated group (Fig. 3A). Thin alveolar walls were
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observed in tissue sections, and the number of fibroblasts and collagen matrices
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decreased significantly in astilbin-treated groups (Fig. 3B and C). The indicators of
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pulmonary fibrosis, such as E-cadherin, SP-C, vimentin, α-SMA, Snail, and collagen I
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and III, were investigated using Western blot analysis and immunofluorescence
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staining (Fig 4A and B). The data showed that astilbin increased the epithelial cell
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markers of E-cadherin and SP-C and decreased the mesenchymal markers of vimentin,
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α-SMA, Snail, and collagen I and III.
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3.4. Regulation of astilbin in pulmonary fibrosis-associated aberrant mRNAs
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The differentially expressed RNAs were evaluated using RNA sequencing to
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elucidate the anti-pulmonary fibrosis mechanism of astilbin. From the RNA
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sequencing data, the mRNA expression profiles of the astilbin-treated group were
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compared with that of the BLM-treated group. Enrichment analysis was applied to
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explore the functions of the mRNAs identified in this study. Genes were organized
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into hierarchical categories to uncover gene regulatory networks on the basis of
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biological processes, cellular components, and molecular functions. The analysis
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pathway (Fig. 5), such as Boc, Lrp, Rab23, Gli, Gsk3b, Csnk, Prkaca, Sufu, Stk, Zic2
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and so on.
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3.5. Confirmation of the Hedgehog pathway involved in the antifibrotic activity of
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astilbin
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The Cignal Finder 45-Pathway Reporter Array confirmed the RNA sequencing
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results which showed that astilbin significantly inhibited glioma-associated oncogene
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homolog (Gli) reporter luciferase activities compared with other reporters (Fig. 6A).
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Then, Gli1 was selected as a representative gene to confirm the in vivo and in vitro
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results using Western blot analysis and immunofluorescence staining. The data
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showed that Gli1 increased in the pulmonary fibrosis model in vivo and in vitro, and
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astilbin remarkably decreased Gli1 expression (Fig. 6B-E). Gli1 expression in patients
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with pulmonary fibrosis was tested using immunohistochemistry to evaluate
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therapeutic value of astilbin. Gli1 expression was also increased in patients with
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pulmonary fibrosis, supporting the in vivo and in vitro results (Fig. 6F). The rescue
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experiment was used to evaluate and verify that the Hedgehog pathway is involved in
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the antifibrotic activity of astilbin further. Chemical activator smoothened agonist
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(SAG) and inhibitor vismodegib (GDC-0449) that are specific to the Hedgehog
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pathway were used, which showed that the activator can promote the expression of
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α-SMA and collagen I and III, but the inhibitor can block the expression of these
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fibrotic indicators. And astilbin reversed these changes caused by SAG and
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GDC-0449 (Fig. 7).
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3.6. Analysis of co-expressed lncRNA with Gli1
Long noncoding RNA (lncRNA) is the upstream factor of mRNA that regulates
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its function. From the RNA sequencing data, Gli1 and its co-expressed lncRNA were
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analyzed in the astilbin-treated group compared with the BLM-treated group. The
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degree of mRNA–lncRNA co-expression was analyzed. An increased degree indicated
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an
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lncRNA-NUNMM028949.2 has the highest degree (Figures 8A and 8B).
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lncRNA-NUNMM028949.2 was selected to determine its expression levels through
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qRT-PCR to validate the data. As shown in Figure 8C, the lncRNA decreased in the
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astilbin-treated group compared with the BLM-treated group.
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4. Discussion
in
their
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interaction.
Among
these
lncRNAs,
In
the
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present
study,
we
investigated
the
effect
of
astilbin
on
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TGF-β1/BLM-induced lung fibrogenesis in vivo and in vitro, and its mechanism of
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Hedgehog pathway signaling pathway in astilbin treatment. Our results showed that
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astilbin significantly decreased alveolar wall thickness and collagen fiber formation,
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and these anti-fibrotic effects of astilbin may be mediated through blockade of
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Hedgehog signaling pathway.
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Similar to cancer, pulmonary fibrosis show functional features such as
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low adhesion, uncontrolled proliferation and high migration rates [22，23]. Snail is the
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transcriptional repressors of adheren that is a direct repressor of E-cadherin 13
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expression. Meanwhile, astilbin also could inhibit the proliferation and migration of
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TGF-β1-treated AEC-II and L929 cells. One of the phenomena during the process of
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pulmonary fibrosis is the manifestation of mesenchymal characteristics, including the
275
expression of mesenchymal markers and the loss of epithelial cell markers [26]. Our
276
data showed that astilbin increased levels of epithelial cell markers such as E-cadherin
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and SP-C, and decreased levels of mesenchymal markers such as a-SMA, collagen
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and vimentin. All these finding showed that the anti-pulmonary fibrosis of astilbin is
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significant.
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The pathogenesis of pulmonary fibrosis indicates that the fibrotic response is
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driven by abnormally activated alveolar epithelial cells, which produce mediators that
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induce the formation of fibroblast and myofibroblast foci through the proliferation of
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resident mesenchymal cells. The fibroblast and myofibroblast foci secrete excess
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amounts of extracellular matrix, including collagen, α-SMA, and vimentin, resulting
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in scarring and destruction of the lung architecture. However, the pathways leading to
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the secretion of excess amounts of extracellular matrix that produce fibroblasts are
287
uncertain [27]. To address these uncertainties, RNA sequencing was used to explore
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the pathways in this study. RNA sequencing is a powerful technique to investigate the
289
complexity of gene expression [28]. The findings showed that astilbin can ameliorate
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pulmonary fibrosis through multigene, multi-pathway. These genes are some of the
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most notable components of the Hedgehog signaling pathway, such as Hedgehog
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ligand (Boc), Hedgehog receptors and cofactors (Lrp and Rab23), transcription factors
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genes (Ptch, Bcl-2, Vegfa, Disp, Mapk, Runx2, and Trp53), and pathways crosstalking
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with Hedgehog (Wnt, Bmp, Mob1b, Nf2, and Wif1) [29]. Based on these results, the
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influence of astilbin on Hedgehog pathways was further detected.
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Aberrant activation of the Hedgehog signaling pathway, which has a crucial role
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in the development and homeostasis of many organs and tissues, has been linked to
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tumorigenesis [30]. The role of the Hedgehog signaling pathway in pulmonary
300
fibrosis pathogenesis has not been thoroughly investigated. In this study, we analyzed
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the function and mechanism of this pathway in pulmonary fibrosis. Gli is one of
302
components of the Hedgehog signaling pathway including Gli1, Gli2, and Gli3
303
[31,32], and its expression level is widely accepted to reflect Hedgehog pathway
304
activity [33]. It acts as a zinc finger transcription factor initially characterized in
305
human glioblastoma to control cell differentiation and proliferation [34]. Although the
306
involvement of Gli in a variety of cancers is well established [35], its role in
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pulmonary fibrosis remains poorly understood. Genetic lineage tracing analysis
308
indicates that Gli1+ cells proliferate after kidney, lung, liver, or heart injury to
309
generate myofibroblasts. Genetic ablation of these cells substantially ameliorates
310
kidney and heart fibrosis and preserves ejection fraction in a model of induced heart
311
failure [36]. Our results showed that Gli1 increased in lung fibrosis, which supports
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the notion that Gli1 is a key contributor to organ fibrosis and indicates the possible
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pathways implicated in lung fibrosis as targets of therapeutic attempts. Astilbin can
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block the progression of pulmonary fibrosis via blockade of Gli expression.
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High-throughput technologies such as RNA sequencing have revealed that only 2% of the transcribed genome codes are attributed to proteins. The remaining part of the
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transcribed genome is known as noncoding RNA. Among the various types of
318
noncoding RNA, a class referred to as long noncoding RNA (lncRNA) has attracted
319
increasing attention [37]. Emerging evidence suggests lncRNA plays important roles
320
in disease development. These noncoding RNAs are the upstream factors of mRNAs
321
that directly or indirectly regulate the biological function of mRNAs [38,39]. Here,
322
we demonstrate the lncRNAs in targeting Gli1 to regulate the anti-pulmonary fibrosis
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mechanism of astilbin. We will be designing experiments to determine the
324
relationship between astilbin and lncRNAs -mediated pulmonary fibrosis for future
325
research.
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5. Conclusions
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In summary, our study showed that astilbin can attenuate lung fibrosis by
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suppressing the Hedgehog pathway, which provided a rationale for exploring the
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therapeutic and chemoprotective potentials of astilbin for the treatment and prevention
330
of pathologic conditions involving pulmonary fibrosis in future studies.
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Acknowledgements
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This study was supported by National Natural Science Foundation of China
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(31470415, 31670365, 81670064, 81741170), Natural Science Foundation of
334
Shandong Province (ZR2016HP34).
335
Conflicts of Interest: The authors declare no conflict of interest. 16
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References
338
[1] A.L. Mora1, M. Rojas, A. Pardo, M. Selman, Emerging therapies for idiopathic
339
pulmonary fibrosis, a progressive age-related disease, Nature Reviews Drug
340
Discovery 170 (2017) 1-18.
Reviews Drug Discovery 9 (2010) 129-140.
SC
342
[2] R.M. du Bois, Strategies for treating idiopathic pulmonary fibrosis, Nature
M AN U
341
RI PT
337
343
[3] M. Selman, A. Pardo, Revealing the pathogenic and aging-related mechanisms of
344
the enigmatic idiopathic pulmonary fibrosis. an integral model, Am. J. Respir. Crit.
345
Care Med. 189 (2014) 1161–1172.
347
[4] B.D. Uhal, The role of apoptosis in pulmonary fibrosis, Eur. Respir. Rev. 17 (2008) 138–144.
TE D
346
[5] T.E. King Jr, W.Z. Bradford, S. Castro-Bernardini, E. A. Fagan, I. Glaspole, M. K.
349
Glassberg, et al., A phase 3 trial of pirfenidone in patients with idiopathic
AC C
350
EP
348
pulmonary fibrosis, N. Engl. J. Med. 370 (2014 ) 2083–2092.
351
[6] L. Richeldi, R.M. du Bois , G. Raghu, A. Azuma, K.K. Brown, U. Costabel , et al.,
352
Efficacy and safety of nintedanib in idiopathic pulmonary fibrosis, N. Engl. J.
353
Med. 370 (2014) 2071–2082.
354
[7] P.W. Noble, C. Albera, W. Z. Bradford, U. Costabel , R.M. du Bois, E.A. Fagan, et
355
al., Pirfenidone for idiopathic pulmonary fibrosis: analysis of pooled data from 17
ACCEPTED MANUSCRIPT 356
three multinational phase 3 trials, Eur. Respir. J. 47 (2016) 243–253.
[8] L. Richeldi, V. Cottin, R.M. du Bois, M. Selman, T. Kimura, Z. Bailes, et al.,
358
Nintedanib in patients with idiopathic pulmonary fibrosis: Combined evidence
359
from the TOMORROW and INPULSIS® trials, Respiratory Medicine 113 (2016)
360
74-79.
RI PT
357
[9] L. Naldini, Gene therapy returns to centre stage, Nature 526 (2015) 351–360.
362
[10] N. Somia, I.M. Verma, Gene therapy: trials and tribulations, Nat. Rev. Genet. 1
M AN U
363
SC
361
(2000) 91–99.
[11] Q.F. Meng, Z. Zhang, Y.J. Wang, W. Chen, F.F. Li, L.T. Yue, et al., Astilbin
365
ameliorates experimental autoimmune myasthenia gravis by decreased Th17
366
cytokines and up-regulated T regulatory cells, J. Neuroimmunol. 298 (2016)
367
138-45.
TE D
364
[12] T.T. Di, Z.T. Ruan, J.X. Zhao, Y. Wang, X. Liu, Y. Wang, et al., Astilbin inhibits
369
Th17 cell differentiation and ameliorates imiquimod-induced psoriasis-like skin
370
lesions in BALB/c mice via Jak3/Stat3 signaling pathway, Int. Immunopharmacol.
AC C
371
EP
368
32 (2016) 32-38.
372
[13] Q.F. Zhang, Y.J. Fu, Z.W. Huang, X.C. Shangguang, Y.X. Guo, Aqueous Stability
373
of Astilbin: Effects of pH, Temperature, and Solvent, J. Agric. Food Chem. 61
374
(2013) 12085−12091.
375
[14] Q.F. Zhang, Z.R. Zhang, H.Y. Cheung, Antioxidant activity of Rhizoma Smilacis 18
ACCEPTED MANUSCRIPT 376
Glabrae extracts and its key constituent − astilbin, Food Chem. 115 (2009)
377
297−303.
[15] S.W. Wang, Y. Xu, Y.Y. Weng, X.Y. Fan, Y.F. Bai, X.Y. Zheng, et al., Astilbin
379
ameliorates cisplatin-induced nephrotoxicity through reducing oxidative stress and
380
inflammation, Food Chem. Toxicol. 114 (2018) 227-236.
RI PT
378
[16] H.B. Zhang, L.C. Sun, L.D. Zhi, Q.K. Wen, Z.W. Qi, S.T. Yan, et al., Astilbin
382
alleviates sepsis-induced acute lung injury by inhibiting the expression of
383
macrophage inhibitory factor in rats, Arch. Pharm. Res. 40 (2017) 1176-1185.
384
[17] Y. Shi, B.R. Gochuico, G. Yu, X. Tang, J.C. Osorio, I.E. Fernandez, et al.,
385
Syndecan-2 exerts antifibrotic effects by promoting caveolin-1-mediated
386
transforming growth factor-β receptor I internalization and inhibiting transforming
387
growth factor-β1 signaling, Am. J. Respir. Crit. Care Med. 188 (2013) 831–841.
388
[18] H. Liu, B. Wang, J. Zhang, S. Zhang, Y. Wang, J. Zhang, et al., A novel lnc-PCF
389
promotes the proliferation of epithelial cell activated with TGF-β1 by targeting
390
miR-344a-5p to regulate map3k11 in pulmonary fibrosis, Cell death and disease 8
M AN U
TE D
EP
AC C
391
SC
381
(2017) e3137.
392
[19] Z. Xing, A. Lin, C. Li, K. Liang, S. Wang, Y. Liu, et al., lncRNA directs
393
cooperative epigenetic regulation downstream of chemokine signals, Cell 159
394
(2014) 1110-1125.
395
[20] G. Raghu, H.R. Collard, J.J. Egan, F.J. Martinez, J. Behr, K.K. Brown, et al., An 19
ACCEPTED MANUSCRIPT 396
Official
397
Evidence-based Guidelines for Diagnosis and Management, Am. J. Respir. Crit.
398
Care Med. 183 (2011) 788-824.
Statement:
Idiopathic
Pulmonary
Fibrosis:
[21] X.D. Song, W.L. Liu, S.Y. Xie, M.R. Wang, G.H. Cao, C.P. Mao, et al.,
400
All-transretinoic
ameliorates
bleomycin-induced
lung
fibrosis
by
401
downregulating the TGF-b1/Smad3 signaling pathway in rats, Lab. Invest. 93
402
(2013) 1219–1231.
SC
acid
RI PT
399
ATS/ERS/JRS/ALAT
[22] C. Vancheri, M. Failla, N. Crimi, G. Raghu, Idiopathic pulmonary fibrosis: a
404
disease with similarities and links to cancer biology, Eur. Respir. J. 35 (2010)
405
496-504.
407
[23] C. Vancheri, Idiopathic pulmonary fibrosis: An altered fibroblast proliferation
TE D
406
M AN U
403
linked to cancer biology, Proc. Am. Thorac. Soc. 9 (2012) 153-157.
[24] A. Cano, M.A. Pérez-Moreno, I. Rodrigo, A. Locascio, M.J. Blanco, M.G. del
409
Barrio,et al., The transcription factor Snail controls epithelial–mesenchymal
410
transitions by repressing E-cadherin expression, Nat. Cell Biol. 2 (2000) 76-83.
411
[25] E. Batlle, E. Sancho, C. Francí, D. Domínguez, M. Monfar, J. Baulida, et al., The
412
transcription factor Snail is a repressor of E-cadherin gene expression in epithelial
413
AC C
EP
408
tumour cells, Nat. Cell Biol. 2 (2000) 84-89.
414
[26] K.V. Pandit, D. Corcoran, H. Yousef, M. Yarlagadda, A. Gibson, K.F.
415
Tzouvelekis, et al., Inhibition and Role of let-7d in Idiopathic Pulmonary Fibrosis, 20
ACCEPTED MANUSCRIPT 416
Am. J. Respir. Crit. Care Med. 182 (2010) 220–229.
[27] K.K. Kim, M.C. Kugler, P.J. Wolters, L. Robillard, M.G. Galvez, A.N. Brumwell,
418
et al., Alveolar epithelial cell mesenchymal transition develops in vivo during
419
pulmonary fibrosis and is regulated by the extracellular matrix, PNAS 103 (2006 )
420
13180–13185.
RI PT
417
[28] T.K. Doktor, Y. Hua, H.S. Andersen, S. Brøner, Y.H. Liu, A. Wieckowska, et al.,
422
RNA-sequencing of a mouse-model of spinal muscular atrophy reveals
423
tissue-wide changes in splicing of U12-dependent introns, Nucleic Acids Research
424
45 (2017) 395-416.
M AN U
425
SC
421
[29] S.J. Scales, F. J. de Sauvage, Mechanisms of Hedgehog pathway activation in cancer and implications for therapy, Trends in Pharmacological Sciences 30 (2009)
427
303-312.
430 431
EP
429
[30] D. Amakye, Z. Jagani, M. Dorsch, Unraveling the therapeutic potential of the Hedgehog pathway in cancer, Nature Medicine 19 (2013) 1410-1422.
[31] L. Lum, P.A. Beachy, The Hedgehog response network: sensors, switches, and
AC C
428
TE D
426
routers, Science 304 (2004) 1755–1759.
432
[32] A. Ruiz i Altaba, C. Mas , B. Stecca, The Gli code: an information nexus
433
regulating cell fate, stemness and cancer, Trends Cell Biol. 17 (2007) 438–447.
434
[33] S.J. Scales, F.J. de Sauvage, Mechanisms of Hedgehog pathway activation in
435
cancer and implications for therapy, Trends Pharmacol. Sci. 30 (2009) 303–312. 21
ACCEPTED MANUSCRIPT 436
[34] S. Sheybani-Deloui, L. Chi, M.V. Staite, J.E. Cain, B.J. Nieman, R.M.
437
Henkelman, et al., Activated Hedgehog-GLI Signaling Causes Congenital
438
Ureteropelvic Junction Obstruction, J. Am. Soc. Nephrol. (2017).
[35] L. Huang, V. Walter, D.N. Hayes, M. Onaitis, Hedgehog–GLI Signaling
440
Inhibition Suppresses Tumor Growth in Squamous Lung Cancer, Clin. Cancer Res.
441
20 (2014) 1566-1575.
SC
RI PT
439
[36] R. Kramann, R.K. Schneider, D.P. DiRocco, F. Machado, S. Fleig, P.A. Bondzie,
443
et al., Perivascular Gli1+ Progenitors Are Key Contributors to Injury-Induced
444
Organ Fibrosis, Cell Stem Cell 16 (2015) 51–66.
446
[37] F. Kopp, J.T. Mendell, Functional Classification and Experimental Dissection of Long Noncoding RNAs, Cell 172 (2018) 393-407.
TE D
445
M AN U
442
[38] L. Meng, A.J. Ward, S. Chun, C.F. Bennett, A.L. Beaudet, F. Rigo, Towards a
448
therapy for Angelman syndrome by targeting a long non-coding RNA, Nature 518
449
(2015) 409–412.
EP
447
[39] Y. Liu, R. Guo, G. Hao, J. Xiao, Y. Bao, J. Zhou, et al., The expression profiling
451
and ontology analysis of noncoding RNAs in peritoneal fibrosis induced by
452
AC C
450
peritoneal dialysis fluid, Gene 564 (2015) 210–219.
453
454
455 22
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Fig. 1. Astilbin cytotoxicity in AEC-II and L929 using CCK-8. (A) Astilbin
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cytotoxicity in the normal AEC-II cells (800 µg/mL < IC50 < 1 mg/mL). (B) Astilbin
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cytotoxicity in the normal L929 cells (1 mg/mL < IC50 < 1.2 mg/mL). (C) and (D)
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AEC-II and L929 cells were initially administered with 5 ng/mL TGF-β1 for 6 h and 23
ACCEPTED MANUSCRIPT subsequently co-treated with astilbin for 48 h. As compared with TGF-β1-treated cells,
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the most pronounced inhibition was at the beginning of 5 and 20 µg/mL
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concentrations in AEC-II and L929, respectively. Each bar represents the mean ± SD,
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with n = 6 and *p < 0.05, **p < 0.01.
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Fig. 2. Anti-pulmonary fibrosis of astilbin in AEC-II and L929 cells. (A) The
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real-time proliferation analysis system showed that astilbin inhibited TGF-β1-treated
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cell proliferation. (B) The real-time migration analysis system showed that astilbin
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inhibited TGF-β1-treated cell migration. (C) Astilbin increased the expressions of
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E-cadherin and SP-C and decreased the expressions of vimentin, α-SMA, Snail, and
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collagen I and III in AEC-II. (D) Astilbin decreased the expressions of vimentin,
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α-SMA, Snail, and collagen I and III in L929. *p < 0.05, **p < 0.01 vs. the normal
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group.
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Fig. 3. Anti-pulmonary fibrosis of astilbin in mice. (A) Astilbin increased FVC
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compared with the BLM-treated group. (B) Astilbin improved the alveolar structure
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through H&E staining. (C) Astilbin inhibited the collagen fibers through Masson’s
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staining. The color blue represents collagen fibers.
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Fig. 4. Astilbin inhibited the indicators of pulmonary fibrosis in mice. (A) Western
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blot analysis showed that astilbin promoted E-cadherin expression and inhibited
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vimentin, α-SMA, Snail, and collagen I and III expressions. (B) Immunofluorescence
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staining showed that astilbin promoted SP-C expression and inhibited α-SMA
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expression. The nuclei and SP-C/α-SMA were counterstained with DAPI (blue) and
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Fig. 5. Heatmap of the expression profiles of the differentially expressed mRNAs
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in vivo. The colors blue to yellow indicate the low to high expression levels. The
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column labels at the right represent the differentially expressed mRNAs in the
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Hedgehog signaling pathway. M represents the BLM-treated group; R represents the
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astilbin-treated group.
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Fig. 6. The aberrant signaling pathway of astilbin was tested in vivo and in vitro,
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and in patients with pulmonary fibrosis. (A) Identification of signaling pathways
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affected by astilbin in AEC-II cells. The x- and y-axes represent the normalized ratio
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of firefly/Renilla luciferase activities, respectively. Most significant changes were
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observed in the Hedgehog signaling pathways. (B) Western blot analysis showed that
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astilbin
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Immunofluorescence staining in L929 showed that astilbin blocked Gli1 expression.
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The nuclei and Gli1 were counterstained with DAPI (blue) and Gli1 (green)
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antibodies. (D) Western blot analysis showed that astilbin inhibited Gli1 expression in
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mice. (E) Immunohistochemistry in mice showed that astilbin blocked Gli1
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expression. (F) Gli1 expression increased in patients with pulmonary fibrosis.
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Fig. 7. Rescue experiment was used to detect the Hedgehog pathway involved in
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the antifibrotic mechanism of astilbin with chemical activator smoothened
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agonist (SAG) and inhibitor vismodegib (GDC-0449) in L929. Astilbin further
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reduced the expression of α-SMA and collagen I and III which increase or decrease
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Fig. 8. Analysis of co-expressed lncRNAs with Gli1. (A) Co-expression degrees of
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lncRNAs with Gli1. The list only showed the top 10 degrees of lncRNAs with Gli1.
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(B) lncRNA-NUNMM028949.2 expression decreased in the astilbin-treated group
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compared with the BLM-treated group using RNA sequencing. (C) qRT-PCR was
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used to validate that lncRNA-NUNMM028949.2 expression was inhibited in the
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astilbin-treated group compared with the BLM-treated group. *p < 0.05, **p < 0.01.
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