Andrographolide ameliorates bleomycin-induced pulmonary fibrosis by suppressing cell proliferation and myofibroblast differentiation of fibroblasts via the TGF-β1-mediated Smad-dependent and -independent pathways

Journal Pre-proof Andrographolide ameliorates bleomycin-induced pulmonary fibrosis by suppressing cell proliferation and myofibroblast differentiation of fibroblasts via the TGF-␤1-mediated Smad-dependent and -independent pathways Jingpei Li, Mingxiang Feng, Ruiting Sun, Zhuoyi Li, Lei Hu, Guilin Peng, Xin Xu, Wei Wang, Fei Cui, Weifeng Yue, Jianxing He, Jun Liu

PII:

S0378-4274(19)30354-6

DOI:

https://doi.org/10.1016/j.toxlet.2019.11.003

Reference:

TOXLET 10614

To appear in:

Toxicology Letters

Received Date:

30 July 2019

Revised Date:

1 November 2019

Accepted Date:

5 November 2019

Please cite this article as: Li J, Feng M, Sun R, Li Z, Hu L, Peng G, Xu X, Wang W, Cui F, Yue W, He J, Liu J, Andrographolide ameliorates bleomycin-induced pulmonary fibrosis by suppressing cell proliferation and myofibroblast differentiation of fibroblasts via the TGF-␤1-mediated Smad-dependent and -independent pathways, Toxicology Letters (2019), doi: https://doi.org/10.1016/j.toxlet.2019.11.003

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier.

Title: Andrographolide ameliorates bleomycin-induced pulmonary fibrosis by suppressing cell proliferation and myofibroblast differentiation of fibroblasts via the TGF-β1-mediated Smaddependent and -independent pathways

Authors: Jingpei Li1,2*, Mingxiang Feng3*, Ruiting Sun2*, Zhuoyi Li1,2, Lei Hu4, Guilin Peng1,2,

of

Xin Xu1,2, Wei Wang1,2, Fei Cui1,2, Weifeng Yue2, Jianxing He1,2†, Jun Liu 1, 2†

1

ro

Affiliations:

Department of Thoracic Surgery, The First Affiliated Hospital of Guangzhou Medical University,

State Key Lab of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, Guangzhou

re

2

-p

Guangzhou, Guangdong 510120, China;

Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University,

3

lP

Guangzhou, Guangdong 510120, China;

Department of Thoracic Surgery, Zhongshan Hospital, Fudan University, Shanghai 200032,

ur na

China; 4

Department of Pharmacy, Peking University People's Hospital, Beijing 10004, China;

†

Corresponding to: Dr. Jun Liu, Department of Thoracic Surgery, The First Affiliated Hospital of

Jo

Guangzhou Medical University, Guangzhou 510120, China. Email: [email protected] Dr. Jianxing He, Department of Thoracic Surgery, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, China. Email: [email protected] *These authors contributed equally to this work

Highlights: 

Andrographolide improves pulmonary function of BLM-treated rats.



Andrographolide prevents BLM-induced fibroblast proliferation and differentiation in the lungs.



Andrographolide inhibits cell proliferation and promotes apoptosis in TGF-β1-stimulated



of

fibroblasts. Andrographolide reduced TGF-β1-induced myofibroblast differentiation and ECM synthesis

Andrographolide inhibits TGF-β1-activated Smad2/3 and Erk1/2 signaling pathways in

-p



ro

in fibroblasts.

re

fibroblasts.

ABSTRACT

lP

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with no effective medication. Andrographolide (Andro), extracted from Chinese herbal Andrographis paniculata, could

ur na

attenuate bleomycin (BLM)-induced pulmonary fibrosis via inhibition of inflammation and oxidative stress, however, the anti-fibrotic mechanisms have not been clarified. Myofibroblasts are the primary cell types responsible for the accumulation of extracellular matrix (ECM) in

Jo

fibrotic diseases, and targeting fibroblast proliferation and differentiation is an important therapeutic strategy for the treatment of IPF. Hence, this study aimed to investigate the effects of Andro on the fibroblast proliferation and differentiation in the in vivo and in vitro models. The results showed that Andro improved pulmonary function and inhibited BLM-induced fibroblast proliferation and differentiation and ECM deposition in the lungs. In vitro, Andro inhibited proliferation and induced apoptosis of TGF-β1-stimulated NIH 3T3 fibroblasts and primary lung

fibroblasts (PLFs). Andro also inhibited TGF-β1-induced myofibroblast differentiation and ECM deposition in both cells. We also found that Andro suppressed TGF-β1-induced Smad2/3 and Erk1/2 activation, suggesting that Smad2/3 and Erk1/2 inactivation mediates Andro-induced effects on TGF-β1-induced fibroblast proliferation and differentiation. These results indicated that Andro has novel and potent anti-fibrotic effects in lung fibroblasts via inhibition of the

of

proliferation and myofibroblast differentiation of fibroblasts and subsequent ECM deposition, which are modulated by TGF-β1-mediated Smad-dependent and -independent pathways.

ro

Key words: Andrographolide; Pulmonary fibrosis; Bleomycin; Fibroblasts; Myofibroblasts;

-p

Extracellular matrix deposition; Smad2/3; Erk1/2

re

Abbreviations

Andro, andrographolide; α-SMA, α-smooth muscle actin; BLM, bleomycin; CCK-8, cell counting

lP

kit-8; CMC-Na, sodium carboxymethyl cellulose; ECM, extracellular matrix; Exp. resistance, expiratory resistance; FSP-1, fibroblast-specific protein-1; Insp. Resistance, inspiratory resistance;

ur na

IPF, idiopathic pulmonary fibrosis; MMPs, metalloproteinases; PCNA, proliferating cell nuclear antigen; PLFs, primary lung fibroblasts; TGF-β1, transforming growth factor-β1; TIMPs; tissue inhibitor of metalloproteinases 1. Introduction

Jo

Idiopathic pulmonary fibrosis (IPF) is a chronic, aggressive and fatal lung disease that occurs primarily in middle-aged and elderly adults and is defined histopathologically by the development of usual interstitial pneumonia (King et al., 2011). The survival time of patients with IPF is likely to be only 3-5 years and most patients eventually died of irreversible loss of lung function with subsequent respiratory failure (Raghu et al., 2011; Xaubet et al., 2014). Up to date, there is no

known cause or real cure for this deadly disease (Raghu et al., 2011). IPF still remains a major cause of morbidity and mortality (Crystal et al., 2002). Thus, novel agents with improved efficacy and low toxicity are urgently desired for the prevention and treatment of pulmonary fibrosis. IPF is characterized by patchy fibrotic areas, with exorbitant extracellular matrix (ECM) remodeling leading to scarring and destruction of lung architecture (Selman et al., 2001). Although

of

the pathogenesis of IPF is not fully understood, the proliferation and differentiation of fibroblasts into myofibroblasts is considered to play a critical role (Hinz et al., 2007). Myofibroblasts are the

ro

primary cell type responsible for the secretion of exaggerated amounts of ECM proteins such as collagens (Darby and Hewitson, 2007). Targeting excessive myofibroblast proliferation and

-p

differentiation has become an effective method for therapies for IPF.

re

Transforming growth factor (TGF)-β1 is thought of as the primary profibrotic cytokine responsible for the induction of proliferation and myofibroblast differentiation of fibroblasts and

lP

has been found to be a central factor in the development of pulmonary fibrosis (Guan et al., 2016; Khalil et al., 1991; Kottmann et al., 2015). Increased levels of TGF-β1 have been observed in the

ur na

lungs of animals and patients with pulmonary fibrosis (Kakugawa et al., 2004). In addition to promoting proliferation, fibroblasts treated with TGF-β1 also differentiate to a myofibroblast phenotype that generates exorbitant ECM proteins, and exhibits stronger contractile activity (Burgess et al., 2005; Zhao et al., 2016). Therefore, the suppression of profibrotic properties of

Jo

TGF-β1 has become a promising therapeutic target in IPF (Guan et al., 2016). Andrographolide (Andro) (see Supplementary Fig. S1), a bicyclic diterpenoid lactone isolated

from the plant Andrographis paniculata, was reported to have multiple pharmacological properties such as antiviral, antioxidant, anti-cancer, anti-inflammatory and immunomodulatory activities (Ji et al., 2016; Lin et al., 2008; Lin et al., 2011; Sheeja and Kuttan, 2007). Andro was also reported

to attenuate bile duct ligation-induced liver fibrosis via suppression of hepatic apoptosis, and alleviate high glucose-induced fibrosis and apoptosis in murine renal mesangeal cells (Lee et al., 2010; Lee et al., 2010). Moreover, Andro was also demonstrated to ameliorate bleomycin (BLM)induced oxidative stress and inflammation in mice (Yin et al., 2015; Zhu et al., 2013). Karkale et al also found that Andro could suppress silica-induced inflammation, oxidative stress, and

of

epithelial-mesenchymal transition in the lungs (Karkale et al., 2018). However, to the best of our knowledge, there was no report on the role of Andro in the BLM-induced fibroblast proliferation

ro

and differentiation in the lungs, as well as in the TGF-β1-induced cell proliferation and myofibroblast differentiation in fibroblasts.

-p

In this study, we investigated the effects of Andro on the proliferation and myofibroblast

re

differentiation of fibroblasts in vivo and in vitro. The results showed that Andro induced apoptosis and inhibited cell proliferation in TGF-β1-stimulated NIH 3T3 fibroblasts and primary lung

lP

fibroblasts (PLFs). Andro also inhibited TGF-β1-induced myofibroblast differentiation and ECM deposition in both cells. We also demonstrated that Andro suppressed TGF-β1-induced fibroblast

ur na

activation and differentiation via Smad and non-Smad (Erk1/2) pathways. Moreover, we provided in vivo evidence demonstrating that Andro improved the pulmonary function and inhibited the BLM-induced fibroblast proliferation and differentiation and ECM deposition in the lungs,

Jo

suggesting a rationale for the treatment of IPF.

2. Materials and methods 2.1. Chemicals and reagents Andro was purchased from Chengdu Herbpurify Co., Ltd. (Chengdu, China). BLM was purchased from Hisun Pfizer Pharmaceuticals Co., Ltd. (Hangzhou, China). Recombinant TGF-β1 was

purchased from novoprotein (SinoBio, Shanghai, China). The trizol reagent was obtained from Invitrogen (Carlsbad, USA). The PrimeScript RT reagent Kit with gDNA Eraser was purchased from Takara Bio Inc. (Shiga, Japan), and the SsoFast EvaGreen Supermix was from Bio-Rad Laboratories, Inc. (CA, USA). Antibodies to Cleaved caspase 3 (Cat# 9661), proliferating cell nuclear antigen (PCNA, Cat# 13110), p-Smad2 (Cat# 3108), Smad2 (Cat# 5339), p-Smad3 (Cat#

of

9520), Smad3 (Cat# 9523), p-Erk1/2 (Cat# 9101), and Erk1/2 (Cat# 4695) were from Cell Signaling Technology (MA, USA). Antibodies to fibronectin (Cat# 15613-1-AP) and TGF-β1

ro

(Cat# 21898-1-AP) were from Proteintech (Chicago, USA). Antibodies to α-smooth muscle actin (α-SMA, Cat# A5228) were from Sigma-Aldrich (St. Louis, MO, USA). Antibodies to GAPDH

-p

(Cat# AC001) and collagen 1 (Cat# GB11022-1, GB11022-2) were respectively purchased from

re

ABclonal (Wuhan, China) and Servicebio (Wuhan, China); Antibodies to anti- fibroblast-specific protein (FSP)-1 (Cat# ab41532), and collagen 3 (Cat# ab7778), and all secondary antibodies were

lP

from Abcam Biotechnology (Cambridge, MA, USA). All other chemicals and reagents used in the experiment were of analytical grade. Other reagents were all from Beyotime Institute of

ur na

Biotechnology unless otherwise indicated.

2.2. Animals and treatments

Forty adult male Sprague-Dawley rats weighing 190–210 g were purchased from Guangdong

Jo

Medical Experimental Animal Center (Guangzhou, China), and housed in a room at controlled temperature of 25°C with 12/12 light/dark cycle, where they were allowed free access to food and water ad libitum. All of the animal procedures including housing, care and experimental protocols were approved by the Animal Care and Use Committee of the First Affiliated Hospital of Guangzhou Medical University (Acceptance number: 2017-346). All procedures on the mice were

performed in accordance with the guidelines from the National Institutes of Health. All the animals were randomly divided into one of the following four groups: (a) intratracheal saline plus 0.5% sodium carboxymethyl cellulose (CMC-Na) intragastrically (Con group); (b) intratracheal saline plus 10 mg/kg of Andro in 0.5% CMC-Na intragastrically (Andro group); (c) intratracheal BLM plus 0.5% CMC-Na intragastrically (BLM group); and (d) intratracheal BLM plus 10 mg/kg of

of

Andro in 0.5% CMC-Na intragastrically (BLM + Andro group). BLM in saline or saline alone was given intratracheally to the rats on day 0. One day after BLM stimulation, Andro or 0.5% CMC-

ro

Na was given by gavage daily for 21 days. After all the rats were euthanized with 1% pentobarbital

2.3. Masson’s trichrome staining

re

animal experiment, there were five mice per group.

-p

sodium in saline (50 mg/kg i.p.), lungs were harvested for the assays described below. In each

lP

The lung tissues were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin and cut into 5 μm-thick slices. The slices were then prepared and stained with Masson trichrome staining for

ur na

the assessment of pathological changes in the lung.

2.4. Pulmonary function measurement

Pulmonary function measurement was performed according to the descriptions by Guan et al

Jo

(Guan et al., 2016). Briefly, rats were anesthetized with 1% pentobarbital sodium in saline (50 mg/kg i.p.), tracheotomized below the larynx, and intubated with a trachea cannula. Whereafter, the rat was transferred into a plethysmographic chamber to examine the pulmonary function, according to the manufacture’s instruction, using the Anires2005 system (Beijing Biolab, Beijing, China).

2.5. Immunohistochemistry Tissue sections were deparaffinized using xylene, rehydrated in a graded ethanol series, and then used for immumohistochemical staining. After antigen retrieval, eliminating endogenous peroxidase and pre-incubated with 3% bull serum albumin (BSA) to block nonspecific binding,

of

lung sections were incubated with primary antibodies [rabbit anti-FSP1 antibody (1:100 dilution) or rabbit anti-α-SMA antibody (1:500 dilution), rabbit anti-Collagen 1 (1:200 dilution) or rabbit

ro

anti-TGF-β1 antibody (1:100 dilution)] at 4 °C overnight. Then, the slices were incubated with goat anti-rabbit IgG secondary antibody for 1 h at 37 °C. The color reaction was then made with

re

-p

HRP-linked polymer detection system and counterstained with hematoxylin.

2.6. Cell culture

lP

NIH 3T3 fibroblasts were also obtained from Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and grown in DMEM containing 10% FBS.

ur na

PLFs were isolated from the lungs of adult mice by combining trypsin digestion and tissue adherent methods, according to previously described methods (Lv et al., 2018). The freshly isolated PLFs were then collected by centrifugation and seeded in DMEM containing 10% FBS. The viability of the cells was examined by trypan blue exclusion, and the cell samples with a purity greater than

Jo

95% were used in the subsequent experiments.

2.7. Morphological analysis

NIH 3T3 fibroblasts were seeded into 6-well plates and treated with culture medium alone (Con) or with 2, 5, and 10 μM of Andro for 24 h, in the presence and absence of TGF-β1 (5 ng/mL). Cell morphology was observed and randomly captured under an inverted microscope (Leica, Germany).

2.8. Cell viability analysis NIH 3T3 cells were seeded in a 96-well plate and treated with different doses of Andro in the

of

presence and absence of TGF-β1 (5 ng/mL) as indicated. Cell viability was determined according

ro

to the manufacturer's instructions of Cell Counting Kit-8 (CCK-8) Kit (Dojindo, Japan).

-p

2.9. Immunofluorescence staining

re

PLFs were seeded on glass slides in 12-well plates and incubated as indicated for 24 h. The slides were washed three times with PBS, fixed in 4% paraformaldehyde for 10 min and then

lP

permeabilized with 0.1% Triton X-100 for 20 min. Subsequently, the cells were blocked with 3% BSA at room temperature for 1 h and then incubated with primary antibody (α-SMA, 1:100;

ur na

Collagen 1, 1:100) at 4°C overnight. After incubation, slices were washed three times with PBS and incubated with corresponding secondary antibodies (1:200, Beyotime Institute of Biotechnology, Haimen, China). The nuclei were stained with DAPI (Beyotime Institute of Biotechnology, Haimen, China) for 5 min. Samples were washed three times with PBS and

Jo

mounted in antifade mounting medium, and fluorescence were detected using a confocal laser scanning microscope (Nikon D-Eclipse C1si, Nikon Corporation, Japan) or a general fluorescence microscope (OLYMPUS, BX53).

2.10. Cell apoptotic determination

NIH 3T3 cells were plated in a six-well plate and treated with Andro in the presence and absence of TGF-β1 (5 ng/mL) for 24 h. The cellular apoptotic rate was determined using Annexin V- 7AAD, Apoptosis Detection Kit (Dojindo, Japan).

2.11. Real-time PCR

of

Total RNA was extracted from NIH 3T3 fibroblasts and PLFs using TRIzol Reagent (Invitrogen Corporation, CA, USA) and reverse-transcribed into first-strand cDNA using the PrimeScript RT

ro

reagent Kit with gDNA Eraser (Takara, Shiga, Japan). The mRNA levels were analyzed with an iCyler iQ Real-time PCR Detection System (Bio-Rad Laboratories Inc., USA) using SsoFast

-p

EvaGreen Supermix (Bio-Rad Laboratories, Inc., CA, USA) in a total volume of 15 μL. Relative

re

levels of mRNA expression were normalized to 18s expression for each gene. The primers for real-

2.12. Western blot

lP

time PCR assays are listed in Supplementary Table 1.

ur na

The samples of NIH 3T3 fibroblasts were homogenized in radioimmune precipitation assay (RIPA) lysis buffer with protease or phosphatase inhibitor (Roche) and centrifuged to obtain supernatants. The total protein concentration was measured by bicinchoninic acid (BCA). Equal amounts (30 μg) of protein extracts from lung tissues or cells were loaded into each lane onto 10% SDS-

Jo

polyacrylamide gels, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA) and incubated with appropriate primary antibodies overnight at 4°C. After reacting with horseradish peroxidase (HRP)-labeled secondary antibodies, the immunoreactive bands were visualized using an ECL chemiluminescent kit (Tiangen Biotech Co. Ltd, Beijing, China) and then scanned with Tanon-5200 (Tanon Science & Technology Co., Ltd., Shanghai, China). The results

were analyzed by Image J. The primary and second antibodies used were as follows: rabbit antifibronectin antibody (1:500 dilution) , mouse anti-α-SMA antibody (1:3000 dilution), rabbit antiTGF-β1 antibody (1:600 dilution), rabbit anti-PCNA antibody (1:1000 dilution), rabbit antiCleaved caspase 3 antibody (1:1000 dilution), rabbit anti-p-Smad2 antibody (1:1000 dilution), rabbit anti-Smad2 antibody (1:1000 dilution), rabbit anti-p-Smad3 antibody (1:1000 dilution),

of

rabbit anti-Smad3 antibody (1:1000 dilution), rabbit anti-p-Erk1/2 antibody (1:2000 dilution), rabbit anti-Erk1/2 antibody (1:2000 dilution), rabbit anti-collagen 1 antibody (1:1000 dilution),

ro

rabbit anti-collagen 3 antibody (1:1000 dilution), and rabbit anti-GAPDH antibody (1:3000 dilution). HRP-labeled Goat Anti-Rabbit IgG (H+L) (1:5000 dilution) and HRP-labeled Goat Anti-

2.13. Data and statistical analysis

re

-p

Mouse IgG (H+L) (1:5000 dilution) antibodies were used as secondary antibodies.

lP

Data analysis was performed using SigmaPlot 12.5 (Systat Software, Inc., Chicago, IL, USA), and expressed as means ± SEM. One-way ANOVA were used to compare differences between more

0.05.

3. Results

ur na

than two treatment groups. Differences between means were considered to be significant at P <

Jo

3.1. Andrographolide (Andro) alleviates BLM-induced pulmonary fibrosis in rats To determine the anti-fibrotic effects of Andro in vivo, rats challenged with BLM as described previously (Guan et al., 2016) were treated with 10 mg/kg Andro (daily, i.g.) for 21 days and pulmonary function were monitored using Anires2005 system. Our results showed that the lung function of Andro-treated rats was markedly improved, as indicated by decreased inspiratory

resistance (Insp. resistance), expiratory resistance (Exp. resistance) and increased compliance, when compared with BLM-treated rats (Fig. 1A-C). Andro was well tolerated by the rats as described by no weight loss or acute or delayed toxicity. Further, we examined the changes in pulmonary pathology after Andro treatment. As shown in Fig. 1D, Andro treatment observably reduced the damage to normal lung architecture and excessive collage deposition due to BLM, as

of

measured by Masson’ trichrome staining. Andro in non-BLM-treated rats generated no alterations in pulmonary pathology or lung function. The attenuated fibrosis in Andro-treated rats was further

ro

supported by decreased protein level for collagen 1 (Fig. 1E). Moreover, we demonstrated that the rat lungs treated with Andro presented a reduced protein level of TGF-β1, when compared with

-p

BLM-treated lungs, which have extensive TGF-β1 (Fig. 1F). Collectively, these results suggest an

Jo

ur na

lP

re

anti-fibrotic effect of Andro on pulmonary fibrosis in vivo.

of ro -p re lP ur na

Jo

Figure 1. Andrographolide (Andro) alleviates bleomycin (BLM)-induced pulmonary fibrosis in rats. Rats were intratracheally injected with a single dose of BLM (3.0 mg/kg body weight) and subsequently received vehicle and Andro (10 mg/kg body weight), via oral gavage daily for 21 days. (A-C) Pulmonary function parameters including inspiratory (Insp.) resistance, expiratory (Insp.) resistance and compliance among different groups were compared three weeks after BLM instillation. (D) Lung tissue sections were prepared and stained with Masson’s trichrome staining.

Original magnification, ×200 (E, F) The levels of collagen 1 and TGF-β1 were visualized using immunohistologic analysis in the lungs. Original magnification, ×200. Data are expressed as mean ± SEM, n = 5, *P<0.05 versus Con group. #P<0.05 versus BLM group.

3.2. Andro prevents fibroblast proliferation and differentiation in BLM-induced pulmonary

of

fibrosis in rats Since fibroblasts and myofibroblasts have emerged as key players in this disease (Sakai and Tager,

ro

2013), we assessed the roles of Andro in fibroblast proliferation and differentiation during BLMinduced pulmonary fibrosis in rats. In conformity with our histological findings above, the

-p

proliferation of fibroblasts was distinctly observed after BLM administration on day 21, as

re

demonstrated by immunohistochemistry ofFSP1, also known as S100A4, a marker of fibroblast proliferation (Fig. 2A). In Andro-treated rats, the accumulation of FSP-1-positive cells was

lP

reduced, indicating that Andro inhibits the BLM-induced fibroblast proliferation in vivo. Additionally, lung sections were also immunostained for α-SMA, a dependable marker of activated

ur na

fibroblasts/myofibroblasts. As shown in Fig. 2B, α-SMA was restricted to the vessel walls in the control rats. Three weeks after BLM administration, the α-SMA-positive cells were remarkably increased and initiate to largely expressed in the interstitium. While treatment with Andro reduced the myofibroblasts expressing α-SMA. Meanwhile, the FSP-1/α-SMA double-positive cells in the

Jo

pulmonary mesenchyme are also significantly decreased by Andro, as determined by immunofluorescent double staining, further implying the inhibitory effect of Andro on the differentiation of fibroblast to myofibroblasts (Fig. 3C). Taken together, all these findings provide in vivo evidence that Andro suppresses fibroblast proliferation and differentiation in BLM-induced pulmonary fibrosis in rats.

of ro -p re lP

ur na

Figure 2. Andrographolide (Andro) prevents fibroblast proliferation and differentiation in BLM-induced pulmonary fibrosis in rats. Rats were intratracheally injected with a single dose of BLM (3.0 mg/kg body weight) and subsequently received vehicle and Andro (10 mg/kg body weight), via oral gavage daily for 21 days. (A, B) The levels of FSP-1 and α-SMA were visualized

Jo

using immunohistologic analysis in the lungs. Original magnification, ×200. (C) The levels of FSP-1 and α-SMA were visualized using immunofluorescence double staining in the lungs. Original magnification, ×200. Brown coloration indicates positive staining and white arrows point to representative double-positive cells. Data are expressed as mean ± SEM, n = 5, *P<0.05 versus Con group. #P<0.05 versus BLM group.

3.3. Andro inhibits cell proliferation and promotes apoptosis in both TGF-β1-stimulated NIH 3T3 fibroblasts and primary lung fibroblasts (PLFs) To assess the effects of Andro on the proliferation of fibroblasts/activated fibroblasts, NIH 3T3 fibroblasts were cultured with and without 5 ng/mL TGF-β1 and/or 2, 5 or 10 μM Andro for 24 h. As shown in Fig. 3A, B, treatment with TGF-β1 resulted in an increased number of viable

of

fibroblasts, whereas Andro dose-dependently reduced the cell viability caused by TGF-β1. In line with this finding, the Western blot result showed that the TGF-β1-induced upregulation of PCNA,

ro

a cell proliferation protein, was also significantly reduced by Andro treatment (Fig. 3E, F). Likewise, Andro at the concentrations of 2, 5, 10 μM induced no significant cell cytotoxicity in

-p

NIH 3T3 cells (Supplementary Fig. S2). These data reveals that Andro could inhibit TGF-β1-

re

induced fibroblast/myofibroblast proliferation. We next examined whether the anti-proliferative effects of Andro on fibroblasts was mediated via apoptosis. The FACS analysis showed that

lP

treatment with Andro increased cell apoptosis in the basal and TGF-β1-stimulated NIH 3T3 fibroblasts (Fig. 3C, D). Since apoptosis is induced principally via caspase-mediated signal

ur na

transduction, we then explored whether Andro induced apoptosis via caspase pathway. Western blot analysis demonstrated that Andro significantly increased the expression level of Cleaved caspase-3 in TGF-β1-stimulated fibroblasts (Fig. 3E, F). This auxo-action of Andro on fibroblast/myofibroblast apoptosis is in a dose-dependent manner, and was conformed in PLFs, as

Jo

illustrated by the Cleaved caspase-3 immunofluorescent staining-positive cells (Fig. 3G). These results showed that Andro promotes apoptosis of TGF-β1-stimulated fibroblasts, revealing that apoptosis may be an important mechanism underlying the anti-proliferative effect.

of ro -p re lP

Figure 3. Andrographolide (Andro) inhibits cell proliferation and promotes apoptosis in

ur na

mouse NIH 3T3 fibroblasts and primary lung fibroblasts (PLFs). NIH 3T3 fibroblasts were treated with TGF-β1 (5 ng/mL) and Andro for 24 h. Cell proliferation was performed by examining (A) the viabilities of fibroblasts with CCK-8 assay. All the values were normalized to the control,

Jo

representing 100% cell viability. (B) the morphological changes. Original magnification, ×400. (C) The cells were double-stained with Annexin V-PE and 7-AAD, and then the cellular apoptosis was measured by flow cytometry. (D) The ratio of apoptotic cells (Annexin V+ 7-AAD-, Annexin V7-AAD+ and Annexin V+ 7-AAD+) was statistically analyzed. (E) The effects of Andro on cell proliferation and apoptosis were further determined by Western blot analysis on related genes. (F) Densitometric analysis of proliferating cell nuclear antigen (PCNA) and Cleaved caspase 3 in the

immunoblots using GAPDH as the internal reference. (G) PLFs were treated with TGF-β1 (5 ng/mL) and Andro (10μM) for 24 h, and Cleaved caspase 3 was examined by immunofluorescence. The presented figures are representative data from at least three independent experiments. Data are presented as mean ± SEM, *P<0.05 versus Con [TGF-β1 (-) and Andro (-)]. #P<0.05 versus TGF-

of

β1 only.

3.4. Andro suppresses TGF-β1-induced myofibroblast differentiation and ECM synthesis in both

ro

NIH 3T3 fibroblasts and PLFs

In injured lung tissues, fibroblasts are activated and differentiate into myofibroblasts, which are

-p

the key effector cells and the principal contributors of ECM deposition in the lung (Zhao et al.,

re

2016). Hence, we examined the effect of Andro on TGF-β1-induced myofibroblast differentiation in NIH 3T3 cells. The Real-time PCR and Western blot results showed that TGF-β1 significantly

lP

increased the mRNA and protein levels of α-SMA, a marker of myofibroblast differentiation, when compared with control cells. Nevertheless, Andro treatment reduced the TGF-β1-induced

ur na

upregulation of α-SMA levels, as compared to TGF-β1-treated cells (Fig. 4). Next, we corroborated the roles of Andro in PLFs. In agreement with the results observed in NIH 3T3 cells, Andro could also downregulate the TGF-β1-induced upregulation of α-SMA in PLFs (Fig. 5). Collectively, these data provide in vitro evidence that Andro inhibits TGF-β1-induced

Jo

myofibroblast differentiation. Afterwards, we investigated whether Andro treatment could reduce ECM synthesis in fibroblasts. As shown in Fig. 4, 5, TGF-β1 treatment significantly increased the production of fibrotic matrix components, such as fibronectin, types I and III collagens in both NIH 3T3 cells and PLFs. Interestingly, such changes were substantially attenuated by Andro treatment, demonstrating an anti-fibrotic role of Andro in contradicting ECM deposition. These

results were further confirmed by testing for the level of matrix metalloproteinases (MMPs) and the tissue inhibitors of MMPs (TIMPs), which have been found upregulation and contribute to maintain ECM turnover and homeostasis (Xiang et al., 2016). The result showed that the levels of TIMP-1 mRNA were significantly increased in response to TGF-β1 and were partially reversed by Andro treatment (Fig. 4A). Furthermore, the TGF-β1-induced increase of MMP-2 and MMP-9 was also down-regulated by Andro (Fig. 4B). Accordingly, these results indicated that Andro

Andrographolide (Andro) reduces myofibroblast differentiation and ECM

Jo

Figure 4.

ur na

lP

re

-p

ro

of

inhibits TGF-β1-induced myofibroblast differentiation and ECM accumulation in fibroblasts.

accumulation in NIH 3T3 fibroblasts. After treatment with TGF-β1 (5 ng/mL) and/or Andro for 24 h, mouse NIH 3T3 fibroblasts were subjected to real-time PCR for detecting the effects of Andro on the transcriptional levels of (A) TIMP-1 and (B) α-SMA (a marker of myofibroblast differentiation), collagens and MMPs. (C) Western blot was used to analyze the α-SMA, fibronectin, collagen 1 and collagen 3. (D) Densitometric analysis of α-SMA, fibronectin, collagen

1 and collagen 3 in the immunoblots using GAPDH as the internal reference. The presented figures are representative data from at least three independent experiments. Data are presented as mean ±

re

-p

ro

of

SEM, *P<0.05 versus Con [TGF-β1 (-) and Andro (-)]. #P<0.05 versus TGF-β1 only.

lP

Figure 5. Andrographolide (Andro) inhibits myofibroblast differentiation and collagen production in PLFs. PLFs were incubated with TGF-β1 (5 ng/ml) in the absence or presence of Andro (10μM) for 24 h. (A, B) The mRNA levels of α-SMA and collagen 1 were evaluated by

ur na

real-time PCR analysis. (C) The α-SMA protein level was analyze with Western blot assay. (D) Immunofluorescence staining of α-SMA and collagen 1 was performed. Original magnification, ×400. The presented figures are representative data from at least three independent experiments.

Jo

Data are presented as mean ± SEM, *P<0.05 versus Con [TGF-β1 (-) and Andro (-)]. #P<0.05 versus TGF-β1 only.

3.5. Andro antagonizes TGF-β1-activated Smad2 and Smad3 signaling pathways in both NIH 3T3 fibroblasts and PLFs

Having established the inhibitory role of Andro in TGF-β1-induced fibroblast proliferation and differentiation, we next investigated the underlying mechanism. The ability of TGF-β1/Smad signaling stimulate myofibroblast proliferation and differentiation is well-documented (Castelino and Varga, 2010; Santibanez et al., 2011). Therefore, we investigated whether the Andro suppressed TGF-β1-induced fibroblast proliferation and differentiation through inactivation of

of

TGF-β1/Smad signaling pathway. The results showed that TGF-β1 significantly stimulated Smad2/3 phosphorylation, and treatment with Andro evidently reduced TGF-β1-induced Smad2/3

ro

phosphorylation in both NIH 3T3 fibroblasts and PLFs, indicating the regulation of Smad2/3 by

ur na

lP

re

-p

Andro is involved in the alleviation of pulmonary fibrosis (Fig. 6).

Jo

Figure 6. Andrographolide (Andro) antagonizes TGF-β1-activated Smad2/3 signaling pathways in vitro. (A) NIH 3T3 fibroblasts were cultured with and without TGF-β1 and/or 2, 5, or 10μM Andro for 24 h. Western blot analysis was used to assess the phosphorylation of Smad2 and Smad3. (B) Scanning densitometry of western blot on different samples was analyzed quantitatively. Expression of p-Smad2 and p-Smad3 was normalized to Smad2 and Smad3 level,

respectively. (C) PLFs were incubated with TGF-β1 (5 ng/ml) in the absence or presence of Andro (10μM) for 24 h. Western blot analysis was used to assess the phosphorylation of Smad2 and Smad3. (D) Scanning densitometry of western blot on different samples was analyzed quantitatively. Expression of p-Smad2 and p-Smad3 was normalized to Smad2 and Smad3 level, respectively. The presented figures are representative data from three independent experiments.

of

Data are presented as mean ± SEM, *P<0.05 versus Con [TGF-β1 (-) and Andro (-)]. #P<0.05

ro

versus TGF-β1 only.

3.6 Andro suppresses TGF-β1-activated Erk1/2 signaling in both NIH 3T3 fibroblasts and PLFs

-p

TGF-β1 is also known to induce fibroblast proliferation, activation and differentiation by Smad-

re

independent pathways including Erk1/2 (Caraci et al., 2008; Hu et al., 2006; Sakai and Tager, 2013). We next observe whether Andro mediated TGF-β1-induced myofibroblast proliferation and

lP

differentiation via the regulation of Erk1/2 activation. Western blot analysis showed that p-Erk1/2 was significantly increased in TGF-β1-stimulated fibroblasts, when compared to control cells.

ur na

Nevertheless, Andro treatment significantly suppressed the TGF-β1-induced phosphorylation of Erk1/2, compared to TGF-β1 only treated cells (Fig. 7), implying the modulation of Erk1/2 by

Jo

Andro is also involved in the attenuation of pulmonary fibrosis.

of ro

-p

Figure 7. Andrographolide (Andro) suppresses TGF-β1-activated Erk1/2 signaling in vitro.

re

(A) NIH 3T3 fibroblasts were cultured with and without TGF-β1 and/or 2, 5, or 10μM Andro for 24 h. Western blot analysis was used to assess the phosphorylation of Erk1/2. Expression of p-

lP

Erk1/2 was normalized to Erk1/2. (B) PLFs were incubated with TGF-β1 (5 ng/ml) in the absence or presence of Andro (10μM) for 24 h. Western blot analysis was used to assess the

ur na

phosphorylation of Erk1/2. Expression of p-Erk1/2 was normalized to Erk1/2. The presented figures are representative data from three independent experiments. Data are presented as mean ± SEM, *P<0.05 versus Con [TGF-β1 (-) and Andro (-)]. #P<0.05 versus TGF-β1 only.

Jo

3.7 TGF-β1–induced fibroblast proliferation and differentiation are inhibited by both SB431542 and SCH 772984

To further explore the role of Smad2/3 and Erk1/2 in TGF-β1-induced fibroblast proliferation and differentiation, both SB431542 (a ALK5 inhibitor) and SCH 772984 (a novel specific inhibitor of Erk1/2) and were used in our study. As expected, the results showed that the induction of α-SMA

and vimentin by TGF-β1 in primary lung fibroblasts was significantly inhibited by SB431542 and

ur na

lP

re

-p

ro

of

SCH 772984 (Fig. 8).

Figure 8. TGF-β1–induced fibroblast proliferation and differentiation are inhibited by both SB431542 and SCH 772984. Pulmonary lung fibroblasts were treated with TGF-β1 in the presence or absence of SCH772984 (a novel specific inhibitor of Erk1/2) or ALK5 inhibitor

Jo

SB431542 for 24h. The expressions of a-SMA and vimentin were visualized using fluorescence microscope. The presented figures are representative data from at least three independent experiments.

4. Discussion The aim of this study was to investigate whether Andro attenuates BLM-induced pulmonary

fibrosis via the regulation of proliferation and myofibroblast differentiation of fibroblasts in vivo and in vitro. Our result showed that Andro apparently induced apoptosis and inhibited cell proliferation, differentiation and ECM synthesis in TGF-β1-stimulated fibroblasts. We also provided in vivo evidence demonstrating that Andro improved the pulmonary function and inhibited the BLM-induced fibroblast proliferation, differentiation and ECM deposition in the

of

lungs. Moreover, we established that Andro counteracted TGF-β1 canonical Smad2/3 signaling and non-canonical Erk1/2 signaling pathway to repress fibroblast proliferation and differentiation.

ro

IPF is a refractory disease of unknown origin, with an estimated 5-year survival rates of approximately 20% (Kreuter et al., 2015). However, treatment of pulmonary fibrosis is badly

-p

limited and often ineffective or only marginally effective. Lung transplantation is the most

re

effective treatment option for end-stage pulmonary fibrosis. Andro, a frequently prescribed drug for the treatment of fever, cold, laryngitis, diarrhea and other infectious diseases in Asian countries,

lP

has recently gained attention for its pleiotropic effects including the ability of reducing pulmonary fibrosis (Cabrera et al., 2014; Karkale et al., 2018; Roy et al., 2011; Zhu et al., 2013). Yet, its

ur na

underlying mechanism remains largely elusive. In this study, we analyzed the effect and underlying mechanisms of Andro on pulmonary fibrosis. Establishment of an appropriate model of IPF is therefore a central step in the deciphering the pathogenesis of IPF, and for the drug screening. BLM, an antibiotic, is a cytotoxic agent used for various cancers. Its cytotoxicity occurs

Jo

principally in the lung, for example, causing inflammatory and fibrotic reactions. Likewise, following single dose intratracheal BLM treatment, the murine lung also undergoes obvious biochemical, histological and physiological alterations, which resembles those of humans, resulting in IPF (Song et al., 2015; Thrall et al., 1979; Zhao et al., 2015). The “switch” between inflammation and fibrosis appears to occur around day 9 after BLM (Moeller et al., 2008). And by

the second and third week post-BLM there is definitive formation of fibrosis with prominent deposition of ECM components including fibronectin and collagens (Degryse and Lawson, 2011). In the present study, we also used this animal model to clarify the roles of Andro in BLM-induced pulmonary fibrosis. And, we found that Andro significantly prevented lung architecture destruction, reduced collagen deposition and improved lung function in rats with BLM-induced

of

pulmonary fibrosis. Moreover, no obvious poisonous side effects were found in our present study, implying the efficacy and safety of Andro, in conformity to earlier studies, indicating tolerance,

ro

with no reported poisonousness. Based on these observations, we speculate that this traditional Chinese medicine may be used as a novel anti-fibrotic drug clinically.

-p

IPF are characterized by the progressive, irreversible and ultimately lethal accumulation of

re

fibroblasts/myofibroblasts and ECM in the lung that damages its architecture and impairs its function (Sakai and Tager, 2013). Although the pathogenesis of IPF remains to be largely

lP

illustrated, fibroblasts, in particular myofibroblasts, have been thought of as primary players in this disease. Fibroblasts and myofibroblasts activate and cluster in IPF lungs in “fibroblastic foci”

ur na

that, as the principal sites of excess ECM generation, has emerged as the major edge of active fibrosis (Scotton and Chambers, 2007). Myofibroblasts, key effector cells in multiple fibrotic diseases in which they have stronger contractile activity and cause exaggerated ECM deposition, are largely derived from the differentiation of resident lung fibroblasts (Hinz et al., 2012; Zhao et

Jo

al., 2016). Reports have shown that around 50% of myofibroblasts originate from resident mesenchymal cells (King et al., 2011). Thus, we examined whether Andro could reduce myofibroblast differentiation and ECM deposition in vivo and in vitro. Interestingly, we found a less infiltration of FSP-1-positive, α-SMA-positive and double FSP-1- and α-SMA-positive myofibroblasts, as well as decreased infiltration of collagen 1-positive cells, following Andro

treatment, demonstrating the inhibitory role of Andro in myofibroblast differentiation and ECM production in vivo. Additionally, fibroblasts challenged with TGF-β1 differentiate to a myofibroblast phenotype that can produce excess ECM proteins and exhibit stronger contractile activity (Burgess et al., 2005). Hence, we next observed the effects of Andro on TGF-β1-induced myofibroblast differentiation and ECM synthesis in vitro. Consistent with the observations in vivo,

of

Andro also significantly inhibited the upregulation of α-SMA levels in both TGF-β1-treated NIH 3T3 fibroblasts and PLFs in vitro, suggesting Andro inhibits TGF-β1-mediated myofibroblast

ro

differentiation. Further, Andro reduced the levels of TIMP-1, MMP-2, MMP-9 and ECM molecules, and thus consequently promoted ECM degradation to reverse the established fibrosis.

-p

These results are noteworthy considering that prevention of myofibroblast differentiation and

re

ECM deposition may represent a crucial target for therapies aimed at limiting fibrosis formation. Dysregulated fibroblast proliferation in response to alveolar epithelial injury has recently

lP

gained extensive attention in pulmonary fibrosis (Gao et al., 2015; Ryu et al., 2014). In this study, we demonstrated that Andro significantly inhibited the TGF-β1-induced cell proliferation in NIH

ur na

3T3 fibroblasts. In accordance with our study, similar results were observed in 3T3-L1 preadipocytes, mesangial cells and rheumatoid arthritis fibroblast-like synoviocytes (Chen et al., 2016; Lan et al., 2013; Yan et al., 2012). Likewise, the BLM-induced fibroblast proliferation was also attenuated by Andro treatment, as demonstrated by decreased infiltration of FSP-1-positive

Jo

cells. Additionally, emerging evidence indicates that decreased apoptosis in activated fibroblasts/myofibroblasts is closely related to the formation of fibrotic lesions and induction of their apoptosis reverses the pulmonary fibrosis (Fattman, 2008). Given this, we also investigated the effects of Andro on fibroblast apoptosis. Here, we found that Andro, in addition to the inhibition of fibroblast proliferation, also induced their apoptosis, which is in accordance with

previous observations on the activity of Andro in a variety of tumor cells (Liu and Chu, 2018; Monger et al., 2017). We also found that the TGF-β1-induced downregulation of Cleaved caspase 3 in NIH 3T3 cells and PLFs was both significantly increased by Andro treatment, implying that Andro induces caspase-dependent apoptosis in fibroblasts. These findings established that Andro alleviates BLM-induced pulmonary fibrosis partially by regulating fibroblast/activated fibroblast activities.

of

The mechanism by which Andro inhibits fibroblast proliferation and differentiation, and

ro

eventual ECM production in the lungs remains obscure. TGF-β1 has been implicated in a broad spectrum of activities in the pathogenesis of pulmonary fibrosis such as the proliferation and

-p

diff erentiation of fibroblasts into myofibroblasts and inhibition of fibroblast apoptosis, as well as

re

the synthesis of collagen and ECM molecules by myofibroblasts (Ignotz and Massague, 1986; Lee et al., 2006). And, lung fibroblasts undergo proliferation and differentiation when exposed to TGF-

lP

β1. The inhibitory effect of Andro on TGF-β1-mediated fibroblast proliferation and differentiation, and ECM production indicates the possibility that Andro directly or indirectly modulates the

ur na

critical molecules in the TGF-β1 signaling pathways. Smads are important regulators in TGF-β1induced proliferation and differentiation of fibroblasts to myofibroblast (Guan et al., 2016). In this study, we identified that Andro inhibits the production of TGF-β1 and specifically targets Smad2/3 to suppress TGF-β1-induced pro-fibrotic activities as the activations of Smad2/3 were significantly

Jo

repressed by Andro treatment in TGF-β1 primed fibroblasts. In addition, mitogen-activated protein kinases (MAPKs) are also involved in non-canonical TGF-β1 signaling. Erk1/2, one of the MAPK family of serine-threonine protein kinases, also mediate a wide variety of cellular responses, including proliferation, differentiation, and apoptosis (Caraci et al., 2008; Hu et al., 2006). For instance, in lung fibroblasts, TGF-β1-induced α-SMA and collagen expression has been shown to

be dependent on Erk1/2. Here, we found that treatment of fibroblasts with Andro also suppressed TGF-β1-induced activation of Erk1/2. Moreover, the inhibition of Smad2/3 and Erk1/2 significantly attenuated TGF-β1-induced fibroblast proliferation and differentiation in fibroblasts, further emphasize the importance of Andro in antagonizing TGF-β1 signaling. Based on these encouraging data generated from cellular and animal models of lung fibrosis in this study,

of

considerable therapeutic benefits of Andro could be expected to improve IPF care. In conclusion, Andro inhibited TGF-β1-mediated Smad2/3 and non-Smad Erk1/2 signaling

ro

and subsequently suppressed the proliferation and myofibroblast differentiation of fibroblasts, as well as ECM deposition, thereby improves pulmonary function and prevents BLM-induced

re

-p

pulmonary fibrosis in rats.

lP

Declaration of interests

ur na

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Conflict of interest

Jo

The authors declare that they have no conflicts of interest.

Acknowledgement This research was supported by the National Natural Science Foundation of China (grant 81703792), Guangzhou Science and Technology Programs for Science Study (grant 201804010052), and Project of State Key Laboratory of Respiratory Disease (grant SKLRD-QN-

of

ro

-p

re

lP

ur na

Jo 201706).

References: Burgess, H.A., Daugherty, L.E., Thatcher, T.H., Lakatos, H.F., Ray, D.M., Redonnet, M., Phipps, R.P., Sime, P.J., 2005. PPARgamma agonists inhibit TGF-beta induced pulmonary myofibroblast differentiation and collagen production: implications for therapy of lung fibrosis. Am J Physiol Lung Cell Mol Physiol 288, L1146-L1153. Cabrera, D., Gutierrez, J., Cabello-Verrugio, C., Morales, M.G., Mezzano, S., Fadic, R., Casar, J.C., Hancke, J.L.,

of

Brandan, E., 2014. Andrographolide attenuates skeletal muscle dystrophy in mdx mice and increases efficiency of cell therapy by reducing fibrosis. SKELET MUSCLE 4, 6.

ro

Caraci, F., Gili, E., Calafiore, M., Failla, M., La Rosa, C., Crimi, N., Sortino, M.A., Nicoletti, F., Copani, A., Vancheri, C., 2008. TGF-beta1 targets the GSK-3beta/beta-catenin pathway via ERK activation in the transition of human lung

-p

fibroblasts into myofibroblasts. PHARMACOL RES 57, 274-282.

Castelino, F.V., Varga, J., 2010. Interstitial lung disease in connective tissue diseases: evolving concepts of

re

pathogenesis and management. ARTHRITIS RES THER 12, 213.

Chen, W., Su, H., Feng, L., Zheng, X., 2016. Andrographolide suppresses preadipocytes proliferation through

lP

glutathione antioxidant systems abrogation. LIFE SCI 156, 21-29.

Crystal, R.G., Bitterman, P.B., Mossman, B., Schwarz, M.I., Sheppard, D., Almasy, L., Chapman, H.A., Friedman, S.L., King, T.J., Leinwand, L.A., Liotta, L., Martin, G.R., Schwartz, D.A., Schultz, G.S., Wagner, C.R., Musson, R.A.,

ur na

2002. Future research directions in idiopathic pulmonary fibrosis: summary of a National Heart, Lung, and Blood Institute working group. Am J Respir Crit Care Med 166, 236-246. Darby, I.A., Hewitson, T.D., 2007. Fibroblast differentiation in wound healing and fibrosis. Int Rev Cytol 257, 143179.

Jo

Degryse, A.L., Lawson, W.E., 2011. Progress toward improving animal models for idiopathic pulmonary fibrosis. AM J MED SCI 341, 444-449. Fattman, C.L., 2008. Apoptosis in pulmonary fibrosis: too much or not enough? Antioxid Redox Signal 10, 379-385. Gao, L., Tang, H., He, H., Liu, J., Mao, J., Ji, H., Lin, H., Wu, T., 2015. Glycyrrhizic acid alleviates bleomycininduced pulmonary fibrosis in rats. FRONT PHARMACOL 6, 215. Guan, R., Wang, X., Zhao, X., Song, N., Zhu, J., Wang, J., Wang, J., Xia, C., Chen, Y., Zhu, D., Shen, L., 2016. Emodin ameliorates bleomycin-induced pulmonary fibrosis in rats by suppressing epithelial-mesenchymal transition

and fibroblast activation. Sci Rep 6, 35696. Guan, R., Zhao, X., Wang, X., Song, N., Guo, Y., Yan, X., Jiang, L., Cheng, W., Shen, L., 2016. Emodin alleviates bleomycin-induced pulmonary fibrosis in rats. TOXICOL LETT 262, 161-172. Hinz, B., Phan, S.H., Thannickal, V.J., Galli, A., Bochaton-Piallat, M.L., Gabbiani, G., 2007. The myofibroblast: one function, multiple origins. AM J PATHOL 170, 1807-1816. Hinz, B., Phan, S.H., Thannickal, V.J., Prunotto, M., Desmouliere, A., Varga, J., De Wever, O., Mareel, M., Gabbiani, G., 2012. Recent developments in myofibroblast biology: paradigms for connective tissue remodeling. AM J

of

PATHOL 180, 1340-1355.

Hu, Y., Peng, J., Feng, D., Chu, L., Li, X., Jin, Z., Lin, Z., Zeng, Q., 2006. Role of extracellular signal-regulated kinase,

ro

p38 kinase, and activator protein-1 in transforming growth factor-beta1-induced alpha smooth muscle actin expression in human fetal lung fibroblasts in vitro. LUNG 184, 33-42.

-p

Ignotz, R.A., Massague, J., 1986. Transforming growth factor-beta stimulates the expression of fibronectin and

re

collagen and their incorporation into the extracellular matrix. J BIOL CHEM 261, 4337-4345. Ji, X., Li, C., Ou, Y., Li, N., Yuan, K., Yang, G., Chen, X., Yang, Z., Liu, B., Cheung, W.W., Wang, L., Huang, R.,

lP

Lan, T., 2016. Andrographolide ameliorates diabetic nephropathy by attenuating hyperglycemia-mediated renal oxidative stress and inflammation via Akt/NF-kappaB pathway. MOL CELL ENDOCRINOL 437, 268-279. Kakugawa, T., Mukae, H., Hayashi, T., Ishii, H., Abe, K., Fujii, T., Oku, H., Miyazaki, M., Kadota, J., Kohno, S.,

J 24, 57-65.

ur na

2004. Pirfenidone attenuates expression of HSP47 in murine bleomycin-induced pulmonary fibrosis. EUR RESPIR

Karkale, S., Khurana, A., Saifi, M.A., Godugu, C., Talla, V., 2018. Andrographolide ameliorates silica induced pulmonary fibrosis. INT IMMUNOPHARMACOL 62, 191-202.

Jo

Khalil, N., O'Connor, R.N., Unruh, H.W., Warren, P.W., Flanders, K.C., Kemp, A., Bereznay, O.H., Greenberg, A.H., 1991. Increased production and immunohistochemical localization of transforming growth factor-beta in idiopathic pulmonary fibrosis. Am J Respir Cell Mol Biol 5, 155-162. King, T.J., Pardo, A., Selman, M., 2011. Idiopathic pulmonary fibrosis. LANCET 378, 1949-1961. Kottmann, R.M., Trawick, E., Judge, J.L., Wahl, L.A., Epa, A.P., Owens, K.M., Thatcher, T.H., Phipps, R.P., Sime, P.J., 2015. Pharmacologic inhibition of lactate production prevents myofibroblast differentiation. Am J Physiol Lung Cell Mol Physiol 309, L1305-L1312.

Kreuter, M., Bonella, F., Wijsenbeek, M., Maher, T.M., Spagnolo, P., 2015. Pharmacological Treatment of Idiopathic Pulmonary Fibrosis: Current Approaches, Unsolved Issues, and Future Perspectives. BIOMED RES INT 2015, 329481. Lan, T., Wu, T., Gou, H., Zhang, Q., Li, J., Qi, C., He, X., Wu, P., Wang, L., 2013. Andrographolide suppresses high glucose-induced fibronectin expression in mesangial cells via inhibiting the AP-1 pathway. J CELL BIOCHEM 114, 2562-2568. Lee, C.G., Kang, H.R., Homer, R.J., Chupp, G., Elias, J.A., 2006. Transgenic modeling of transforming growth factor-

of

beta(1): role of apoptosis in fibrosis and alveolar remodeling. Proc Am Thorac Soc 3, 418-423.

Lee, M.J., Rao, Y.K., Chen, K., Lee, Y.C., Chung, Y.S., Tzeng, Y.M., 2010. Andrographolide and 14-deoxy-11,12-

murine renal mesangeal cell lines. J ETHNOPHARMACOL 132, 497-505.

ro

didehydroandrographolide from Andrographis paniculata attenuate high glucose-induced fibrosis and apoptosis in

-p

Lee, T.Y., Lee, K.C., Chang, H.H., 2010. Modulation of the cannabinoid receptors by andrographolide attenuates

re

hepatic apoptosis following bile duct ligation in rats with fibrosis. APOPTOSIS 15, 904-914. Lin, H.H., Tsai, C.W., Chou, F.P., Wang, C.J., Hsuan, S.W., Wang, C.K., Chen, J.H., 2011. Andrographolide down-

250, 336-345.

lP

regulates hypoxia-inducible factor-1alpha in human non-small cell lung cancer A549 cells. Toxicol Appl Pharmacol

Lin, T.P., Chen, S.Y., Duh, P.D., Chang, L.K., Liu, Y.N., 2008. Inhibition of the epstein-barr virus lytic cycle by

ur na

andrographolide. BIOL PHARM BULL 31, 2018-2023.

Liu, G., Chu, H., 2018. Andrographolide inhibits proliferation and induces cell cycle arrest and apoptosis in human melanoma cells. ONCOL LETT 15, 5301-5305.

Lv, X.M., Li, M.D., Cheng, S., Liu, B.L., Liu, K., Zhang, C.F., Xu, X.H., Zhang, M., 2018. Neotuberostemonine

Jo

inhibits the differentiation of lung fibroblasts into myofibroblasts in mice by regulating HIF-1alpha signaling. ACTA PHARMACOL SIN.

Moeller, A., Ask, K., Warburton, D., Gauldie, J., Kolb, M., 2008. The bleomycin animal model: a useful tool to investigate treatment options for idiopathic pulmonary fibrosis? Int J Biochem Cell Biol 40, 362-382. Monger, A., Boonmuen, N., Suksen, K., Saeeng, R., Kasemsuk, T., Piyachaturawat, P., Saengsawang, W., Chairoungdua, A., 2017. Inhibition of Topoisomerase IIalpha and Induction of Apoptosis in Gastric Cancer Cells by 19-Triisopropyl Andrographolide. Asian Pac J Cancer Prev 18, 2845-2851.

Raghu, G., Collard, H.R., Egan, J.J., Martinez, F.J., Behr, J., Brown, K.K., Colby, T.V., Cordier, J.F., Flaherty, K.R., Lasky, J.A., Lynch, D.A., Ryu, J.H., Swigris, J.J., Wells, A.U., Ancochea, J., Bouros, D., Carvalho, C., Costabel, U., Ebina, M., Hansell, D.M., Johkoh, T., Kim, D.S., King, T.J., Kondoh, Y., Myers, J., Muller, N.L., Nicholson, A.G., Richeldi, L., Selman, M., Dudden, R.F., Griss, B.S., Protzko, S.L., Schunemann, H.J., 2011. An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management. Am J Respir Crit Care Med 183, 788-824. Roy, D.N., Sen, G., Chowdhury, K.D., Biswas, T., 2011. Combination therapy with andrographolide and d-

of

penicillamine enhanced therapeutic advantage over monotherapy with d-penicillamine in attenuating fibrogenic

54-68.

ro

response and cell death in the periportal zone of liver in rats during copper toxicosis. Toxicol Appl Pharmacol 250,

Ryu, J.H., Moua, T., Daniels, C.E., Hartman, T.E., Yi, E.S., Utz, J.P., Limper, A.H., 2014. Idiopathic pulmonary

-p

fibrosis: evolving concepts. MAYO CLIN PROC 89, 1130-1142.

re

Sakai, N., Tager, A.M., 2013. Fibrosis of two: Epithelial cell-fibroblast interactions in pulmonary fibrosis. Biochim Biophys Acta 1832, 911-921.

lP

Santibanez, J.F., Quintanilla, M., Bernabeu, C., 2011. TGF-beta/TGF-beta receptor system and its role in physiological and pathological conditions. Clin Sci (Lond) 121, 233-251. Scotton, C.J., Chambers, R.C., 2007. Molecular targets in pulmonary fibrosis: the myofibroblast in focus. CHEST 132,

ur na

1311-1321.

Selman, M., King, T.E., Pardo, A., 2001. Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. ANN INTERN MED 134, 136-151. Sheeja, K., Kuttan, G., 2007. Activation of cytotoxic T lymphocyte responses and attenuation of tumor growth in vivo

Jo

by Andrographis paniculata extract and andrographolide. Immunopharmacol Immunotoxicol 29, 81-93. Song, N., Liu, J., Shaheen, S., Du L, Proctor, M., Roman, J., Yu, J., 2015. Vagotomy attenuates bleomycin-induced pulmonary fibrosis in mice. Sci Rep 5, 13419. Thrall, R.S., McCormick, J.R., Jack, R.M., McReynolds, R.A., Ward, P.A., 1979. Bleomycin-induced pulmonary fibrosis in the rat: inhibition by indomethacin. AM J PATHOL 95, 117-130. Xaubet, A., Serrano-Mollar, A., Ancochea, J., 2014. Pirfenidone for the treatment of idiopathic pulmonary fibrosis. Expert Opin Pharmacother 15, 275-281.

Xiang, J., Cheng, S., Feng, T., Wu, Y., Xie, W., Zhang, M., Xu, X., Zhang, C., 2016. Neotuberostemonine attenuates bleomycin-induced pulmonary fibrosis by suppressing the recruitment and activation of macrophages. INT IMMUNOPHARMACOL 36, 158-164. Yan, J., Chen, Y., He, C., Yang, Z.Z., Lu, C., Chen, X.S., 2012. Andrographolide induces cell cycle arrest and apoptosis in human rheumatoid arthritis fibroblast-like synoviocytes. CELL BIOL TOXICOL 28, 47-56. Yin, J.N., Li, Y.N., Gao, Y., Li, S.B., Li, J.D., 2015. Andrographolide plays an important role in bleomycin-induced pulmonary fibrosis treatment. INT J CLIN EXP MED 8, 12374-12381.

of

Zhao, H., Bian, H., Bu, X., Zhang, S., Zhang, P., Yu, J., Lai, X., Li, D., Zhu, C., Yao, L., Su, J., 2016. Targeting of Discoidin Domain Receptor 2 (DDR2) Prevents Myofibroblast Activation and Neovessel Formation During

ro

Pulmonary Fibrosis. MOL THER 24, 1734-1744.

Zhao, H., Qin, H.Y., Cao, L.F., Chen, Y.H., Tan, Z.X., Zhang, C., Xu, D.X., 2015. Phenylbutyric acid inhibits

-p

epithelial-mesenchymal transition during bleomycin-induced lung fibrosis. TOXICOL LETT 232, 213-220.

re

Zhu, T., Zhang, W., Xiao, M., Chen, H., Jin, H., 2013. Protective role of andrographolide in bleomycin-induced

Jo

ur na

lP

pulmonary fibrosis in mice. INT J MOL SCI 14, 23581-23596.