- Email: [email protected]
Slit2 signaling contributes to cholestatic fibrosis in mice by activation of hepatic stellate cells
Journal Pre-proof Slit2 signaling contributes to cholestatic fibrosis in mice by activation of hepatic stellate cells Changzheng Li, Guizhi Yang, Liteng Lin, Yuanyuan Xuan, Sishan Yan, Xiaoqian Ji, Fenyun Song, Minqiang Lu, Tian Lan PII:
S0014-4827(19)30488-4
DOI:
https://doi.org/10.1016/j.yexcr.2019.111626
Reference:
YEXCR 111626
To appear in:
Experimental Cell Research
Received Date: 16 May 2019 Revised Date:
14 September 2019
Accepted Date: 15 September 2019
Please cite this article as: C. Li, G. Yang, L. Lin, Y. Xuan, S. Yan, X. Ji, F. Song, M. Lu, T. Lan, Slit2 signaling contributes to cholestatic fibrosis in mice by activation of hepatic stellate cells, Experimental Cell Research (2019), doi: https://doi.org/10.1016/j.yexcr.2019.111626. 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 Inc.
Slit2 signaling contributes to cholestatic fibrosis in mice by activation of hepatic stellate cells
Changzheng Li¶1, Guizhi Yang¶1, Liteng Lin¶3, Yuanyuan Xuan1, Sishan Yan1, Xiaoqian Ji1, Fenyun Song1, Minqiang Lu2**, Tian Lan1*
1
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006,
China. 2
Department of HBP SURGERY II, Guangzhou First People's Hospital, School of
Medicine, South China University of Technology, Guangzhou, Guangdong, China. 3
Laboratory of Interventional Radiology, Department of Minimally Invasive
Interventional Radiology, and Department of Radiology, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, China.
*
Corresponding authors:
Tian Lan, 280 Wai Huan Dong Road, Department of Pharmacology, School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou Higher Education Mega Center, Guangzhou 510006, China. Email: [email protected] **
Corresponding authors:
Minqiang Lu, No.1 Panfu Road, Yuexiu, Guangzhou 510180, China. Email: [email protected]
Author’s contributions Changzheng Li, Guizhi Yang, Liteng Lin, Sishan Yan, Yuanyuan Xuan, Xiaoqian Ji and Fenyun Song performed the acquisition, analysis and interpretation of data. Changzheng Li and Guizhi Yang drafted the paper. Tian Lan, Minqiang Lu and Liteng Lin designed the research and revised the paper.
Abstract
Liver Cholestasis is a widespread disease of broad etiologies and ultimately results in fibrosis, which is still lacking effective therapeutic strategies. Activation of hepatic stellate cells (HSCs) is the key event of liver fibrosis. Here, we aimed to investigate the effect and mechanism of the Slit2 signaling in cholestasis-induced liver fibrosis. Our findings revealed that the serum levels and hepatic expression of Slit2 were significantly increased in patients with primary biliary cirrhosis (PBC). Additionally, Slit2-Tg mice were much more vulnerable to BDL-induced liver injury and fibrosis compared to WT control. Slit2 up-regulation by Slit2 recombinant protein induced proliferation, and inhibited apoptosis of human HSCs cell line LX-2 via p38 and ERK signaling pathway, resulting in the activation of HSCs. In contrast, Slit2 down-regulation by siRNA silencing inhibit the activation of HSCs. In conclusion, Slit2 is involved in the activation of HSCs and liver fibrogenesis, highlighting Slit2 as a potential therapeutic target for liver fibrosis.
Keywords: Cholestatic liver fibrosis; HSCs; Slit2; apoptosis; proliferation; p38 MAPK; ERK1/2.
INTRODUCTION Hepatic fibrosis, a reversible wound healing response that follows various chronic liver injury, is characterized by accumulation of abundant extracellular matrix (ECM)[1]. Advanced fibrosis often progressed into cirrhosis, which is accompanied by architectural distortion of the hepatic vasculature and sets the stage for hepatic decompensation, primary liver cancer, and death. Hepatic stellate cells (HSCs) activation and transformation into active myofibroblasts are key steps in liver fibrogenesis[1-3]. The primary causes of liver fibrosis include cholestatic liver disease, viral hepatitis, alcoholic liver disease, and nonalcoholic steatohepatitis. As the clinically proven therapy of liver fibrosis is still lacking, understanding the molecular mechanism of fibrogenesis remains an urgent goal for the development of effective antifibrotic agents. Activated HSCs are the principal cell type responsible for the synthesis of ECM during hepatic fibrogenesis[4]. Upon the stimulation of a variety of cytokines and growth factors induced by liver injury, HSCs activate and transform into α-smooth muscle actin (α-SMA)-positive myofibroblasts, leading to liver fibrosis by producing excess ECM[5]. The activated HSCs display a distinct phenotype with increased resistance to apoptosis and enhanced ability of proliferation, contraction, and chemotaxis. The MAPK signaling pathway, mainly comprised of ERK p44/p42, JNK, and p38, is a fundamental mechanism modulating the cellular proliferation and apoptosis during multiple diseases (e.g., liver fibrosis and cancer)[6-9]. Currently, medicinal candidates specifically targeting the MAPK signaling pathway have 1
granted a profound amelioration in hepatic fibrogenesis via suppressing proliferation and inducing apoptosis of HSCs[10, 11]. The detailed pro-fibrogenic mechanism and clinical translational potential of MAPK-related proliferation and apoptosis of HSCs deserve further investigation. The Slit-Robo signaling was originally discovered during development of the nervous system and includes the Slit family of secreted proteins (Slit1, 2 and 3) and their specific receptors (Robo1, 2, 3 and 4) [12, 13]. Recently, aberrant expression of the Slit-Robo genes in a wide variety of cancers has been uncovered.[14-16]. For instance, hepatocellular carcinoma (HCC) tissues presented overexpression of Robo1 and Slit2, which was associated with the pathological parameters such as tumor staging and differentiation [17]. Furthermore, our previous study expanded the vision on the role of Slit2-Robo1 signaling in promoting liver fibrosis through activation of HSCs in CCl4-induced mice [18]. Blocking Slit2-Robo1 signaling by Robo1 neutralizing antibody R5 could reduce liver injury and collagen deposition in fibrotic mice induced by CCl4, suggesting that targeting Slit2-Robo1 signaling might be a novel therapy in the treatment of liver fibrosis. Herein, we extended our exploration on the regulatory role of Slit2-Robo1 signaling in bile duct ligation (BDL)-induced mice, a classical rodent model mimicking liver fibrosis resulted from cholestatic liver disease. Additionally, we also aimed at determining the effects of Slit2 on phenotypic activation, as well as MAPK-related proliferation and apoptosis in HSCs.
2
MATERIALS AND METHODS Human serum and liver samples Serum samples were provided by 30 liver biliary atresia (PBC) patients with clinically diagnosed liver fibrosis and 30 healthy volunteers. Liver tissues were taken from 20 PBC patients with liver fibrosis. Liver tissues with normal histology (n=20) obtained from patients with various benign liver conditions or transplant donors were used as controls. All clinical samples were from the third affiliated hospital of Sun Yat-sen University, Guangzhou, China. Written informed consent was obtained from each patient and volunteer, and the study was approved by the Clinical Ethics Committee of the third affiliated hospital of Sun Yat-sen University. Animals Slit2 transgenic (Slit2-Tg) mice were donated by Dr. Geng Jianguo of the University of Michigan, and the detailed procedures for animal generation and breeding were described [16], age-matched specific pathogen-free C57BL/6J mice obtained from the Medical Laboratory Animal Center, Guangdong, China were used as wild-type (WT). In brief, the transgene was constructed by cloning the full-length human Slit2 cDNA into the pCEP4F vector, which contains the CMV promoter, and was injected into the pronuclei of fertilized C57×CBA F1oocytes. Genotypes were confirmed by dot blotting and Southern blotting. PCR screening of Slit2 heterozygotes was performed using the following primers: Slit2 (Forward): 5’-CCCTCCGGATCCTTTACCTGTCAAGGTCCT-3’;
Slit2
(Reverse),
5’-TGGAGAGAGCTC ACAGAACAAGCCACTGTA-3’. Transgenic founders were 3
maintained by breeding with C57 mice. All mice received humane care and animal experiments were approved by the University Committee on Use and Care of Animals (UCUCA) of the Guangdong Pharmaceutical University, Guangzhou, China. Induction of hepatic fibrosis by BDL To create animal models of hepatic fibrosis, Slit2-Tg (C57 background) and C57 mice (12 male, 8-week-old, respectively; 24 mice in total), were randomly allocated to two experimental groups (n =6 per group). After anesthesia with isoflurane and abdominal disinfection, BDL in mice was produced by ligating the common bile duct with 5-0 silk, and an abdominal incision without a ligation served as the sham operation [19, 20]. All surgical procedures were performed under aseptic conditions. Animals were allowed to recover from anesthesia and surgery under a warming lamp. At the end of 15th day after surgery, all mice were anesthetized with isoflurane and sacrificed for further analyses. Serum biochemistry Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and gamma-glutamyl transferase (γ-GT) were measured using standard enzymatic procedures according to the manufactures’ instruction (NanJing JianCheng, NanJing, China). Liver histopathology Liver specimens fixed in 10% buffered formalin were embedded in paraffin blocks. Paraffin embedded liver tissues were sectioned at thickness of 5 µm and stained with hematoxylin and eosin (H&E) and Sirius red. Collagen deposition in Sirius 4
red-positive stained area was measured on each slide and quantified using Image J software. Immunohistochemistry and immunofluorescent staining Liver specimens fixed in 10% buffered formalin were embedded in paraffin blocks. Liver sections (5 µm thick) were processed using a standard immunostaining protocol. After routine deparaffinization, hydration and blockage of endogenous peroxidase, sections were pretreated by microwave for 20 min in 10 mmol/L sodium citrate buffer (pH 6.0) for antigen retrieval, followed by incubation sequentially with blocking agent, rabbit CD68 (1:100, Boster, China) and α-SMA antibodies (1:100, Boster, China) and secondary antibody (1:200, Promega, Madison, WI, USA). Slides were incubated with diaminobenzidine, followed by a brief counterstaining with hematoxylin, and mounted with Vectashield (Vector Labs, Burlingame, CA, USA). The areas of interest were photographed and converted to a digital image using light microscopy
equipped
with
camera
(Olympus
BX51,
NY,
USA).
For
immunofluorescent staining, liver specimens were fixed in 10% buffered formalin and sequentially exposed to 10% and 30% sucrose in PBS for 10 h each and then embedded in Tissue Tek OTC compound (Sakura Finetek, Torance, CA). The liver sections were permeabilized by 0.25% Triton X-100 and incubated with the primary antibody against Slit2 (1:200, Abcam, Cambridge, MA), α-SMA (1:200, Abcam, Cambridge, MA). Then, the liver sections were incubated with corresponding Alexa Fluor 594- and Alexa Fluor 488-conjugated secondary antibodies (1:200, Invitrogen, Carlsbad, CA) for 1 h at room temperature and stained with DAPI (1 µg/mL) for 10 5
min. Finally, the stained sections were viewed and photographed under the confocal microscopy (Leica TCS SP5, Leica, Mannheim, Germany). Cell Culture The well-characterized cell line derived from human HSCs, LX-2, was used in in vitro studies. LX-2 was generously provided as a gift by Professor Qi Zhang (the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China). Cells were cultured in DMEM (high glucose) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a 5% CO2 incubator at 37°C. Reverse-transcription (RT) PCR Total RNA was extracted from mouse liver tissues and cells using TRIzol reagent followed by treatment with RNase-free DNase (Takara, Dalian, China) for 30 min at 37°C. RNA were subjected to reverse transcription using a first-strand cDNA kit (Takara, Dalian, China) according to the manufacturer’s protocol. RT-PCR was conducted using the PrimeScript RT-PCR Kit (Takara, Dalian, China) according to the manufacturer’s instructions. The PCR was run on CFX96 real-time PCR system (Bio-Rad, Hercules, CA). The PCR reactions were carried out at: 95°C for 30s; and 40 cycles of 95°C/5 s, 60°C/34 s. PCR primer sequences were listed in supplementary Table1. The relative abundance of the target genes was obtained by calculating against the standard curve and normalized to β-actin as an internal control. Western blot analysis Equal amounts of total proteins (30µg) from each sample was subjected to 10% SDS-PAGE by electrophoresis under reducing conditions and transferred to PVDF 6
membrane (Millipore Corporation, Billerica, MA, USA). The blotted membrane was then blocked with 5% skim milk for 1 h at room temperature and incubated respectively with anti-Slit2 (1:1000, CST, USA), anti-α-SMA (1:1000, Boster, China), anti-Bcl-2 (1:1000, CST, USA), anti-ERK1/2 (1:1000, CST, USA), anti-p-ERK1/2 (1:1000, CST, USA), anti-p38 (1:1000, CST, USA), anti-p-p38 (1:1000, CST, USA), anti-caspase3 (1:1000, CST, USA), anti-Cleaved Caspase3 (1:1000, CST, USA) and anti-GAPDH (1:5000, Beyotime Biotechnology, China) overnight at 4°C. The membranes were further incubated with horseradish peroxidase (HRP) conjugated anti-mouse/rabbit secondary antibodies (1:5000 dilutions of each antibody) for 1h at room temperature and detected by enhanced chemiluminescent (ECL) method and captured on X-ray film. The light signal was captured by a charge-coupled device (CCD) camera (GE Healthcare, UK). The densitometric measurement of each band was analyzed using Quantity-One Protein Analysis Software (Bio-Rad Laboratories, USA). Knockdown of Slit2 by RNA interference LX-2, a well-characterized cell line derived from human HSCs, was used in in vitro studies. Recombinant human Slit2 (rh Slit2) were used at doses indicated in respective figure legends. Cells were cultured in DMEM supplemented with 10% fetal bovine serum, 1mmol/L L-glutamine, and 100 IU/mL streptomycin/penicillin. To silence Slit2 expression, specific predesigned small interfering RNA (siRNAs) from SMARTpool Slit2 siRNA kit (Dharmacon, Fisher Scientific, Pittsbrugh, USA) was used according to the manufacture’s instructions. For transfection, LX-2 cells were 7
plated at 1×105 cells/well in 6-well plates and cultured overnight. Cells were transfected with Slit2 siRNA (final concentration of 100 nM) using Lipofectamine 3000 (Invitrogen, Shanghai, China). Verification of transfection efficiency and the expression of target proteins in the transfected LX-2 cells were analyzed by immunoblotting 72 h after transfection. The non-targeted siRNA SMARTpool (Dharmacon, Fisher Scientific, Pittsbrugh, USA) was used as a negative control. Flow cytometric analyses of cell cycle and apoptosis The quantitative analysis of cell cycle and apoptosis was conducted by flow cytometry. LX-2 cells (8 × 104/well) were seeded in six-well plates in complete culture medium. After incubating for 12 h, cells were treated with H2O2 for 30 min (200µmol/L) and then Slit2 recombinant protein 10ng/mL for 24 h or only treated with Slit2 recombinant protein 10ng/mL for 24 h. Cells were harvested separately at 27 and 24 h later, and immediately fixed with 75% ethanol. For the cell cycle progression analysis, cells were stained with PI staining buffer (10 mg/mL PI and 100 mg/mL RNase A) for 30 min, and fluorescence intensity was measured by BD FACS Calibur (BD Biosciences, San Jose, CA, United States). For apoptosis analysis, cells were stained with the Annexin V-FITC/PI apoptosis detection kit. The rate of apoptotic cells was analyzed using a dual laser flow cytometer and estimated using the ModFit software (BD Biosciences). Apoptotic cell Hoechst 33258 detecting In addition to the flow cytometric analyses, the role of slit2 on the LX-2 cell apoptosis induced by H2O2 (200µmol/L) for 30min was further confirmed by Hoechst 8
33258 detecting. Firstly, the cells were washed sufficiently by PBS twice, and fixed by 1 mL 4% formaldehyde solution at 4°C. After 10min, the 4% formaldehyde solution was discarded and the cells were washed sufficiently by PBS twice. Then the cells were stained by 100 µL Hoechst 33258 working solution at room temperature for 10 min. After washed by PBS twice, the cells were viewed and photographed under the fluorescent microscopy (Olympus Co., Tokyo, Japan). Statistical analysis Values were expressed as mean ± S.D. Statistical differences between two groups were analyzed by the unpaired Student’s t test and differences between multiple groups of data were analyzed by one-way ANOVA with Bonferroni correction (GraphPad Prism 7.0, San Diego, CA, USA). P<0.05 was considered statistically significant.
RESULTS The Slit2 was highly expressed in liver biliary atresia (PBC) patients. Though Slit2 signaling has been demonstrated to promote liver injury and fibrosis in CCl4-induced mice in our previous work, its role in hepatic fibrosis caused by cholestatic liver damage is still unclear[18]. In present study, we initially examined Slit2 levels in human liver fibrotic tissue of PBC patients by immunohistochemistry staining. Compared to healthy control, the liver tissues of PBC patients had larger fibrotic areas represented by Sirius red staining and higher levels of HSCs activation represented by α-SMA positive staining (Fig. 1A, B, C). 9
Interestingly,
immunohistochemical analyses revealed that the hepatic Slit2 expression in PBC patients was significantly higher than that in healthy control (Fig. 1D). Next, we investigated the serum levels of Slit2 in 30 PBC patients with liver fibrosis and 30 healthy controls. The serum levels of Slit2 were significantly higher in PBC patients than that in healthy controls (Fig. 1H). These results raised the hypothesis that Slit2 signaling might be involved in HSCs activation and the pathogenesis of liver fibrosis. Slit2 aggravated the progression of BDL-induced liver fibrosis. On the basis of increased Slit2 expression in clinical fibrotic liver, we further explored the role of Slit2 signaling in hepatic fibrogenesis in BDL-induced mice, a classical rodent model mimicking liver fibrosis resulted from PBC patients. As shown in Fig. 2A, B, BDL-induced Slit2-Tg mice exhibited more fibrous hyperplasia and collagen deposition than BDL-induced WT mice as presented by H&E and Sirius red staining, implying that Slit2 significantly exacerbated hepatic injury and fibrosis. Consistently, immunochemical staining revealed higher hepatic expression of α-SMA and CD68 in BDL-induced Slit2-Tg mice, which respectively suggested the HSCs activation and inflammatory macrophages infiltration upon Slit2 overexpression (Fig. 2C, D). Additionally, immunoblotting assessment of α-SMA expression also confirmed a higher level of HSCs activation in BDL-induced Slit2-Tg mice (Fig. 2P). Furthermore, hepatic mRNA levels of fibrogenic genes, including α-SMA, Col-α1, TGF-β1, and TIMP1, were remarkably increased in BDL-induced Slit2-Tg mice compared to the BDL-induced WT mice. (Fig. 2L, M, N, O). To validate the association between the higher level of fibrosis observed in BDL-induced Slit2-Tg 10
mice with the severer injury process, serum indicators of liver damage including ALT, AST, ALP and γ-GT were detected by commercial kits. As expected, BDL induction brought about more serious liver injury in Slit2-Tg mice in comparison to the WT mice (Fig. 2H, I, J, K). Furthermore, we used Robo1 knockout (Robo1-/-) mice subjected to BDL to further clarify the role of the Slit2-Robo1 signaling in the pathogenesis of cholestatic liver fibrosis. As shown by Sirius Red and α-SMA immumohistochemical staining (Figure S1), BDL-induced liver fibrosis and HSC activation were significantly attenuated in Robo1-/- mice. Together, the overexpression of Slit2-Robo1 signaling was likely to contribute to the hepatic fibrogenesis resulted from cholestatic liver disease. Slit2 was involved in HSCs activation To illuminate the mechanism by which Slit2 facilitates liver fibrosis, we next investigated the cellular source of Slit2 in the fibrotic liver of BDL induced Slit2-Tg mice. By immunofluorescence co-staining of Slit2 with α-SMA (a marker for activated HSCs), we observed that Slit2 was highly expressed in activated HSCs, suggesting that the pro-fibrogenic effects of Slit2 were probably dependent of acting on HSCs (Fig. 3A). Next, qRT-PCR assays demonstrated that overexpression of Slit2 in LX-2 cells (a human HSCs line) treated with Slit2 recombinant protein dramatically enhanced the mRNA levels of pro-fibrogenic genes (a-SMA, Col1a1, TIMP-1). In contrast, Slit2 silencing via siRNA obtained almost 60% downregulation of Slit2 mRNA and successfully repressed these pro-fibrogenic mRNA levels of a-SMA, Col1a1 and TIMP-1 in LX-2 cells. These results made it clear that Slit2 11
promoted liver fibrosis through the regulation of HSCs activation. Slit2 promoted proliferation and protected against apoptosis in HSCs In addition to the determination of Slit2 mediated HSCs activation, we attempted to clarify the regulation of Slit2 signaling on the transformation of HSCs phenotypes (proliferation and apoptosis). Herein, an in vitro model of cell apoptosis was induced by H2O2. Initially, flow cytometry analysis was performed to investigate the effects of Slit2 recombinant protein and (or) H2O2 on the cell cycle of LX-2 cells. As shown in figure 3F, the proportions of G2/M and S-phase cells were significantly increased upon Slit2 stimulation. Whereas, cells challenged by H2O2 showed significantly decreased proportions of G2/M and S-phase cells. Noteworthily, such effect of H2O2 on the cell cycle of LX-2 cells was partially abolished by Slit2 recombinant protein. These results proved preliminarily that Slit2 not only promoted proliferation, but also protected against apoptosis induced by H2O2, which was further confirmed by both Hochest (Fig. 3G) and AnnexinV/PI (Fig. 3H) staining. Next, we conducted immunoblotting assay to disclose the changes of protein expression beneath the above effects of Slit2 on LX-2 cells. Accordingly, the expression of Slit2, anti-apoptotic protein (Bcl-2), and apoptosis regulating factor (Caspase3) were significantly increased, while Cleaved-caspase3 was significantly decreased in LX-2 cells treated with Slit2 recombinant protein. In contrast, LX-2 cells challenged by H2O2 exhibited a sharp decline in Bcl-2 and Caspase3 expression and a remarkable elevation in Cleaved-caspase3 expression. As expected, such pro-apoptotic effect of H2O2 in protein levels was partially eliminated in LX-2 cells treated with the combination of 12
H2O2 and Slit2 recombinant protein. Taken together, these data demonstrated strongly the role of Slit2 as a regulator in the proliferation and apoptosis of HSCs. Slit2 affected HSCs proliferation and apoptosis through MAPK pathway To shed light on the molecular mechanism involved in the regulation of Slit2 on HSCs phenotypes, we performed the immunoblotting analysis of MAPK pathway which was closely related to cell proliferation and apoptosis as firmly established in the literature. We provided evidences that Slit2 stimulation via Slit2 recombinant protein remarkably up-regulated p-P38 and p-ERK1/2 while blockade of Slit2 signaling via siRNA silencing significantly down-regulated p-P38 and p-ERK1/2 in LX-2 cells (Fig. 4A). Besides, in H2O2 induced LX-2 cells, we observed reduced expressions of both p-P38 and p-ERK1/2, which were reactivated by the exogenous supplementation of Slit2 recombinant protein (Fig. 4B). These results demonstrated that Slit2 intervened in HSCs proliferation and apoptosis through p-P38 and p-ERK1/2 pathway.
DISCUSSION Our previous study has demonstrated that Slit2 signaling promoted liver injury and fibrosis in CCl4-induced mice[18]. Nevertheless, the role of Slit2 signaling in hepatic fibrosis caused by cholestatic liver damage is still unclear. In cholestatic liver diseases (e.g., PBC), biliary obstruction makes the bile acid to accumulate within bile ducts and leak into the liver and blood circulation, leading to ductular reaction, hepatocyte damage, hepatic oxidative stress, inflammation, and fibrosis [21-23]. In the current 13
study, we first verified that Slit2 contributed to liver injury and liver fibrosis in bile duct ligation (BDL)-induced mice, a classical rodent model mimicking cholestatic liver disease. Initially, we found that PBC patients had much higher hepatic Slit2 expression and serum levels of Slit2 than healthy controls. This pivotal clinical clue triggered our further investigation in BDL-induced mice as a model of cholestatic hepatic fibrosis. Our research revealed that overexpression of Slit2 exacerbated liver injury and fibrosis in response to BDL induction. Thus, both clinical and preclinical data verified the definite pro-fibrogenic effects of Slit2 during cholestatic liver damage. Next, on the basis of defining HSCs as the one of main cellular source of Slit2 in the fibrotic liver of Slit2-Tg mice induced by BDL, HSCs debuted in the focus of our research on the pro-fibrogenic effects of Slit2. Upon various stimuli during liver damage, quiescent HSCs activate into collagen-producing myofibroblasts and play a paramount role in the pathogenesis of hepatic fibrosis [19, 24]. We verified that Slit2 overexpression was accompanied by a higher hepatic expression of α-SMA in BDL-induced Slit2-Tg mice, strongly suggesting that Slit2 signaling is implicated in HSC activation. To verify the above finding, in the in vitro studies, we up-regulated Slit2 by the exogenous supplementation of Slit2 recombinant protein and down-regulated Slit2 by siRNA silencing in LX-2 cells (a well-characterized HSCs line derived from human). Not surprisingly, Slit2 overexpression dramatically enhanced the mRNA levels of a-SMA (marker for activated HSCs) and Col1a1 (marker for collagen production), whereas Slit2 silencing successfully repressed the 14
above pro-fibrogenic mRNA levels in LX-2 cells. The above data collectively demonstrated that Slit2 signaling mediates HSC activation and the pathogenesis of liver fibrosis. Enhanced ability of survival and proliferation represent the two major distinct phenotypes of activated HSCs distinguishing from the quiescent ones. In the present study, evidences from LX-2 cells were provided that Slit2 could effectively promote proliferation and protect against H2O2 induced apoptosis in HSCs by affecting the cell cycle, expanding our work to exploring the involved molecular mechanism. MAPKs are a class of mitogen-activated protein kinases, including p38, ERK and JNK. MAPKs are involved in cell proliferation, differentiation, survival, apoptosis and autophagy in response to stress stimuli, such as cytokines, ultraviolet irradiation, heat shock and osmotic shock [25-27]. Specifically, JNK is involved in cell apoptosis, and p38 is related to cell proliferation and apoptosis, while ERK is associated with cell proliferation,
apoptosis,
migration,
and
senescence[28-31].
Herein,
the
phosphorylated status of the MAPK family members (ERK1/2, JNK and p38) of LX-2 cells were detected by western blotting. We made it clear that Slit2 exerted its proliferation-promoting and anti-apoptotic effects by inducing the phosphorylated activation of ERK1/2 and p38 but not JNK (JNK data were not shown). Despite the significant findings revealed by this investigation, limitations still exist. Whether Slit2 inhibition could alleviate liver fibrosis in BDL-induced mice was not addressed
in
the
current
study
since
suppressing
Slit2
signaling
by
systemic administration with non-targeted inhibitors was not feasible because of its 15
vital physiological function such as regulating neural development and leukocyte migration[32]. Hence the further clinical transformation of Slit2 signaling as therapeutic target of liver fibrosis remains an urgent challenge. Recently, the newly-developed nanomedicine has emerged as a promising strategy for targeted drug delivery or gene regulation with both high efficiency and low side effects[33]. For instance, in our previous study, we developed hyaluronate-graft-polyethylenimine to specifically deliver cyclooxygenase-1 siRNA to liver sinusoidal endothelial cells, achieving the successful amelioration of cirrhotic portal hypertension in CCl4-induced mice[34]. Thus, for the treatment of liver fibrosis, it will be taken into consideration in our future study to develop novel nanomedicine for targeted Slit2 regulation in HSCs. In conclusion, our studies elucidate that Slit2 promoted HSCs activation, and contributed to the survival and proliferation of HSCs via activation of ERK1/2 and p38 signaling pathways, pushing forward the pathogenesis of hepatic fibrosis. Though whether Slit2 inhibition could alleviate liver fibrosis in BDL-induced mice was not addressed in the current study, further investigations are warranted to shed new light on the potential of Slit2 as therapeutic target for liver fibrosis treatment.
ACKNOWLEDGEMENTS This study was supported by the Key Project of Natural Science Foundation of Guangdong
Province
(2016A030311014),
China;
National
Natural
Science
Foundation of China (81870420); and the Guangzhou Science and Technology 16
Programs (201704020153), China.
CONFLICT OF INTEREST STATEMENT The authors declare that they have no conflicts of interest.
17
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liver injury[J]. The Journal of Nutritional Biochemistry, 2014, 25(11):1183-1195. 25. Lui G Y L, Kovacevic Z, Richardson V, et al. Targeting cancer by binding iron: Dissecting cellular signaling pathways[J]. Oncotarget, 2015, 6(22). 26. Zaballos M A, Santisteban P. Key signaling pathways in thyroid cancer[J]. Journal of Endocrinology, 2017, 235(2):R43-R61. 27. Kamiyama M, Naguro I, Ichijo H. In vivo\r, gene manipulation reveals the impact of stress-responsive MAPK pathways on tumor progression[J]. Cancer Science, 2015, 106(7):785-796. 28. Chang L, Karin M. Mammalian MAP kinase signalling cascades.[J]. Nature, 2001, 410(6824):37-40. 29. Boutros T, Chevet E, Metrakos P. Mitogen-activated protein (MAP) kinase/MAP kinase phosphatase regulation: roles in cell growth, death, and cancer[J]. Pharmacological Reviews, 2008, 60(3):261-310. 30. Chen M J , Chou C H , Shun C T . Iron suppresses ovarian granulosa cell proliferation and arrests cell cycle through regulating p38 MAPK/p53/p21 pathway.[J]. Biology of Reproduction, 2017, 106(3):e249-e249. 31. Gao H , Zhang Y , Dong L , et al. Triptolide induces autophagy and apoptosis through ERK activation in human breast cancer MCF-7 cells[J]. Experimental & Therapeutic Medicine, 2018:3413-3419. 32. Chaturvedi S, Robinson L A. Slit2-Robo signaling in inflammation and kidney injury[J]. Pediatric Nephrology, 2015, 30(4):561-566. 33. Pelaz B, Alexiou C, Alvarezpuebla R A, et al. Diverse Applications of 21
Nanomedicine.[J]. Acs Nano, 2017, 11(3):2313-2381. 34. Lin L, Cai M, Deng S, et al. Amelioration of cirrhotic portal hypertension by targeted cyclooxygenase-1 siRNA delivery to liver sinusoidal endothelium with polyethylenimine grafted hyaluronic acid[J]. Nanomedicine Nanotechnology Biology & Medicine, 2017, 13(7).
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Figure Legends
Fig.1. Hepatic expression of Slit2 in PBC patients. Paraffin-embedded sections of liver tissues from healthy controls (Control, n=20) and PBC patients (Fibrosis, n=20) were stained with H&E (A), Sirius Red (B), and immunohistochemical staining of α-SMA (C) and Slit2 (D). The positive staining areas were measured by ImageJ software (E, F and G). Serum level of Slit2 was measured in the healthy control (n=30) and PBC patients with liver fibrosis (n=30) by ELISA (H). The horizontal lines mark the mean values of triplicate measurements. Data are presented as means ± S.D. ** P<0.01; **** P<0.0001 vs. Control.
Fig.2. Slit2 contributes to BDL-induced liver fibrosis in mice. C57 and Slit2-Tg mice were induced by BDL for 15days. Representative histology staining of H&E (A) and Sirius Red (B), and immunohistochemical staining of α-SMA (C) and CD68 (D). (E, F, G) Quantification of positive staining areas was measured by ImageJ software. (H, I, J, K) Serum levels of ALT, AST, ALP and γ-GT. (L-O) RT-PCR of α-SMA, Col-α1, TGF-β1 and TIMP1. (P) Immunoblotting analyses of hepatic α-SMA expression in the mouse. **P<0.01, ***P<0.001, ****P<0.0001 vs. WT mice induced by BDL.
Fig.3. Slit2 promotes the activation, proliferation and suppresses the apoptosis of HSCs in vitro. (A) Representative immunostaining images showed that the expression of Slit2 colocalized with activated HSCs marker, α-SMA in liver from the Slit2-Tg mice induced by BDL for 15 days. DAPI staining was used to identify the nuclei. (B, C, D, E) mRNA levels of Slit2, α-SMA, Col-α1 and TIMP1 in LX-2 cells transfected with Slit2 siRNA or treated with Slit2 recombinant protein. (F, G, H) Cell cycle, Hoechst and AnnexinV/PI staining of LX-2 cells treated with Slit2 recombinant protein and (or) H2O2. (I) Analysis of cell cycle by ModFit software. (J) Quantification of Hoechst positive staining areas was measured by ImageJ software. (K) Immunoblotting analyses of Slit2, Bcl-2, Caspase3 and Cleaved-Caspase3 23
expression of LX-2 cells treated with Slit2 recombinant protein and (or) H2O2.
Fig.4. Slit2 promotes the proliferation and suppresses the apoptosis of HSCs via MAPKs activation. (A) Immunoblotting analyses of Slit2, ERK1/2, p-ERK1/2, P38 and p-P38 expression of LX-2 cells transfected with Slit2 siRNA or treated with Slit2 recombinant protein. (B) Immunoblotting analyses of ERK1/2，p-ERK1/2, P38 and p-P38 expression of LX-2 treated with Slit2 recombinant protein and (or) H2O2.
Fig. S1. Robo1-/- mice are less readily to develop HSC activation and liver fibrosis in response to BDL induction. WT and Robo1-/- mice were induced by BDL for 15 days. Representative histology staining of Sirius Red, and immunohistochemical staining of α-SMA.
24
Figures
Fig. 1
25
Fig. 2
26
Fig. 3
27
Fig.4
28
Fig. S1
29
Table 1: Primer Sequences used for qRT-PCR Gene
Primer Sequence
Mouse α-SMA Col1α1 TIMP-1 TGF-β1 Slit2 β-actin
F 5’- GTCCCAGACATCAGGGAGTAA-3’ R 5’- TCGGATACTTCAGCGTCAGGA-3’ F 5’- GCTCCTCTTAGGGGCCACT-3’ R 5’- CCACGTCTCACCATTGGGG-3’ F 5’- GCAACTCGGACCTGGTCATAA-3’ R 5’- CGGCCCGTGATGAGAAACT-3’ F 5’- CTCCCGTGGCTTCTAGTGC-3’ R 5’- GCCTTAGTTTGGACAGGATCTG-3’ F 5’- GGCAGACACTGTCCCTATCG-3’ R 5’- ATCTATCTTCGTGATCCTCGTGA-3’ F 5’- GGCTGTATTCCCCTCCATCG-3’ R 5’- CCAGTTGGTAACAATGCCATGT-3’
Human α-SMA Col1α1 TIMP-1 TGF-β1 Slit2 β-actin
F 5’- GTGTTGCCCCTGAAGAGCAT-3’ R 5’- GCTGGGACATTGAAAGTCTCA-3’ F 5’- GTGCGATGACGTGATCTGTGA-3’ R 5’- CGGTGGTTTCTTGGTCGGT-3’ F 5’- CTTCTGCAATTCCGACCTCGT-3’ R 5’- ACGCTGGTATAAGGTGGTCTG-3’ F 5’- GGCCAGATCCTGTCCAAGC-3’ R 5’- GTGGGTTTCCACCATTAGCAC-3’ F 5’- GCGAAGCTATACAGGCTTGAT-3’ R 5’- TGCAGTCGAAAAGTCCTAAGTTT-3’ F 5’- GGCATTCACGAGACCACCTAC-3’ R 5’- CGACATGACGTTGTTGGCATAC-3’
30
Highlights: 1. Liver fibrosis is a reversible wound healing response for liver injury. Advanced liver fibrosis might progress into irreversible cirrhosis, which is the leading cause of liver-related mortality worldwide. In our study, we observed that hepatic Slit2 expression was significantly increased in liver biliary atresia (PBC) patients with liver fibrosis, and Slit2-Tg mice were much more vulnerable to BDL-induced liver injury and fibrosis when compared to WT mice. 2. During liver fibrogenesis, the key pro-fibrogenic HSCs activate and display a distinct phenotype with increased resistance to apoptosis and enhanced ability of proliferation, contraction, and chemotaxis. We demonstrated that Slit2 up-regulation promoted the activation and proliferation, whereas inhibited the apoptosis of human HSCs cell line LX-2. 3. We also provided evidences that Slit2 promoted HSCs activation, and contributed to the survival and proliferation of HSCs likely via activation of ERK1/2 and p38 signaling pathways.
Declarations of interest: none
S0014-4827(19)30488-4
DOI:
https://doi.org/10.1016/j.yexcr.2019.111626
Reference:
YEXCR 111626
To appear in:
Experimental Cell Research
Received Date: 16 May 2019 Revised Date:
14 September 2019
Accepted Date: 15 September 2019
Please cite this article as: C. Li, G. Yang, L. Lin, Y. Xuan, S. Yan, X. Ji, F. Song, M. Lu, T. Lan, Slit2 signaling contributes to cholestatic fibrosis in mice by activation of hepatic stellate cells, Experimental Cell Research (2019), doi: https://doi.org/10.1016/j.yexcr.2019.111626. 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 Inc.
Slit2 signaling contributes to cholestatic fibrosis in mice by activation of hepatic stellate cells
Changzheng Li¶1, Guizhi Yang¶1, Liteng Lin¶3, Yuanyuan Xuan1, Sishan Yan1, Xiaoqian Ji1, Fenyun Song1, Minqiang Lu2**, Tian Lan1*
1
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006,
China. 2
Department of HBP SURGERY II, Guangzhou First People's Hospital, School of
Medicine, South China University of Technology, Guangzhou, Guangdong, China. 3
Laboratory of Interventional Radiology, Department of Minimally Invasive
Interventional Radiology, and Department of Radiology, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, China.
*
Corresponding authors:
Tian Lan, 280 Wai Huan Dong Road, Department of Pharmacology, School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou Higher Education Mega Center, Guangzhou 510006, China. Email: [email protected] **
Corresponding authors:
Minqiang Lu, No.1 Panfu Road, Yuexiu, Guangzhou 510180, China. Email: [email protected]
Author’s contributions Changzheng Li, Guizhi Yang, Liteng Lin, Sishan Yan, Yuanyuan Xuan, Xiaoqian Ji and Fenyun Song performed the acquisition, analysis and interpretation of data. Changzheng Li and Guizhi Yang drafted the paper. Tian Lan, Minqiang Lu and Liteng Lin designed the research and revised the paper.
Abstract
Liver Cholestasis is a widespread disease of broad etiologies and ultimately results in fibrosis, which is still lacking effective therapeutic strategies. Activation of hepatic stellate cells (HSCs) is the key event of liver fibrosis. Here, we aimed to investigate the effect and mechanism of the Slit2 signaling in cholestasis-induced liver fibrosis. Our findings revealed that the serum levels and hepatic expression of Slit2 were significantly increased in patients with primary biliary cirrhosis (PBC). Additionally, Slit2-Tg mice were much more vulnerable to BDL-induced liver injury and fibrosis compared to WT control. Slit2 up-regulation by Slit2 recombinant protein induced proliferation, and inhibited apoptosis of human HSCs cell line LX-2 via p38 and ERK signaling pathway, resulting in the activation of HSCs. In contrast, Slit2 down-regulation by siRNA silencing inhibit the activation of HSCs. In conclusion, Slit2 is involved in the activation of HSCs and liver fibrogenesis, highlighting Slit2 as a potential therapeutic target for liver fibrosis.
Keywords: Cholestatic liver fibrosis; HSCs; Slit2; apoptosis; proliferation; p38 MAPK; ERK1/2.
INTRODUCTION Hepatic fibrosis, a reversible wound healing response that follows various chronic liver injury, is characterized by accumulation of abundant extracellular matrix (ECM)[1]. Advanced fibrosis often progressed into cirrhosis, which is accompanied by architectural distortion of the hepatic vasculature and sets the stage for hepatic decompensation, primary liver cancer, and death. Hepatic stellate cells (HSCs) activation and transformation into active myofibroblasts are key steps in liver fibrogenesis[1-3]. The primary causes of liver fibrosis include cholestatic liver disease, viral hepatitis, alcoholic liver disease, and nonalcoholic steatohepatitis. As the clinically proven therapy of liver fibrosis is still lacking, understanding the molecular mechanism of fibrogenesis remains an urgent goal for the development of effective antifibrotic agents. Activated HSCs are the principal cell type responsible for the synthesis of ECM during hepatic fibrogenesis[4]. Upon the stimulation of a variety of cytokines and growth factors induced by liver injury, HSCs activate and transform into α-smooth muscle actin (α-SMA)-positive myofibroblasts, leading to liver fibrosis by producing excess ECM[5]. The activated HSCs display a distinct phenotype with increased resistance to apoptosis and enhanced ability of proliferation, contraction, and chemotaxis. The MAPK signaling pathway, mainly comprised of ERK p44/p42, JNK, and p38, is a fundamental mechanism modulating the cellular proliferation and apoptosis during multiple diseases (e.g., liver fibrosis and cancer)[6-9]. Currently, medicinal candidates specifically targeting the MAPK signaling pathway have 1
granted a profound amelioration in hepatic fibrogenesis via suppressing proliferation and inducing apoptosis of HSCs[10, 11]. The detailed pro-fibrogenic mechanism and clinical translational potential of MAPK-related proliferation and apoptosis of HSCs deserve further investigation. The Slit-Robo signaling was originally discovered during development of the nervous system and includes the Slit family of secreted proteins (Slit1, 2 and 3) and their specific receptors (Robo1, 2, 3 and 4) [12, 13]. Recently, aberrant expression of the Slit-Robo genes in a wide variety of cancers has been uncovered.[14-16]. For instance, hepatocellular carcinoma (HCC) tissues presented overexpression of Robo1 and Slit2, which was associated with the pathological parameters such as tumor staging and differentiation [17]. Furthermore, our previous study expanded the vision on the role of Slit2-Robo1 signaling in promoting liver fibrosis through activation of HSCs in CCl4-induced mice [18]. Blocking Slit2-Robo1 signaling by Robo1 neutralizing antibody R5 could reduce liver injury and collagen deposition in fibrotic mice induced by CCl4, suggesting that targeting Slit2-Robo1 signaling might be a novel therapy in the treatment of liver fibrosis. Herein, we extended our exploration on the regulatory role of Slit2-Robo1 signaling in bile duct ligation (BDL)-induced mice, a classical rodent model mimicking liver fibrosis resulted from cholestatic liver disease. Additionally, we also aimed at determining the effects of Slit2 on phenotypic activation, as well as MAPK-related proliferation and apoptosis in HSCs.
2
MATERIALS AND METHODS Human serum and liver samples Serum samples were provided by 30 liver biliary atresia (PBC) patients with clinically diagnosed liver fibrosis and 30 healthy volunteers. Liver tissues were taken from 20 PBC patients with liver fibrosis. Liver tissues with normal histology (n=20) obtained from patients with various benign liver conditions or transplant donors were used as controls. All clinical samples were from the third affiliated hospital of Sun Yat-sen University, Guangzhou, China. Written informed consent was obtained from each patient and volunteer, and the study was approved by the Clinical Ethics Committee of the third affiliated hospital of Sun Yat-sen University. Animals Slit2 transgenic (Slit2-Tg) mice were donated by Dr. Geng Jianguo of the University of Michigan, and the detailed procedures for animal generation and breeding were described [16], age-matched specific pathogen-free C57BL/6J mice obtained from the Medical Laboratory Animal Center, Guangdong, China were used as wild-type (WT). In brief, the transgene was constructed by cloning the full-length human Slit2 cDNA into the pCEP4F vector, which contains the CMV promoter, and was injected into the pronuclei of fertilized C57×CBA F1oocytes. Genotypes were confirmed by dot blotting and Southern blotting. PCR screening of Slit2 heterozygotes was performed using the following primers: Slit2 (Forward): 5’-CCCTCCGGATCCTTTACCTGTCAAGGTCCT-3’;
Slit2
(Reverse),
5’-TGGAGAGAGCTC ACAGAACAAGCCACTGTA-3’. Transgenic founders were 3
maintained by breeding with C57 mice. All mice received humane care and animal experiments were approved by the University Committee on Use and Care of Animals (UCUCA) of the Guangdong Pharmaceutical University, Guangzhou, China. Induction of hepatic fibrosis by BDL To create animal models of hepatic fibrosis, Slit2-Tg (C57 background) and C57 mice (12 male, 8-week-old, respectively; 24 mice in total), were randomly allocated to two experimental groups (n =6 per group). After anesthesia with isoflurane and abdominal disinfection, BDL in mice was produced by ligating the common bile duct with 5-0 silk, and an abdominal incision without a ligation served as the sham operation [19, 20]. All surgical procedures were performed under aseptic conditions. Animals were allowed to recover from anesthesia and surgery under a warming lamp. At the end of 15th day after surgery, all mice were anesthetized with isoflurane and sacrificed for further analyses. Serum biochemistry Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and gamma-glutamyl transferase (γ-GT) were measured using standard enzymatic procedures according to the manufactures’ instruction (NanJing JianCheng, NanJing, China). Liver histopathology Liver specimens fixed in 10% buffered formalin were embedded in paraffin blocks. Paraffin embedded liver tissues were sectioned at thickness of 5 µm and stained with hematoxylin and eosin (H&E) and Sirius red. Collagen deposition in Sirius 4
red-positive stained area was measured on each slide and quantified using Image J software. Immunohistochemistry and immunofluorescent staining Liver specimens fixed in 10% buffered formalin were embedded in paraffin blocks. Liver sections (5 µm thick) were processed using a standard immunostaining protocol. After routine deparaffinization, hydration and blockage of endogenous peroxidase, sections were pretreated by microwave for 20 min in 10 mmol/L sodium citrate buffer (pH 6.0) for antigen retrieval, followed by incubation sequentially with blocking agent, rabbit CD68 (1:100, Boster, China) and α-SMA antibodies (1:100, Boster, China) and secondary antibody (1:200, Promega, Madison, WI, USA). Slides were incubated with diaminobenzidine, followed by a brief counterstaining with hematoxylin, and mounted with Vectashield (Vector Labs, Burlingame, CA, USA). The areas of interest were photographed and converted to a digital image using light microscopy
equipped
with
camera
(Olympus
BX51,
NY,
USA).
For
immunofluorescent staining, liver specimens were fixed in 10% buffered formalin and sequentially exposed to 10% and 30% sucrose in PBS for 10 h each and then embedded in Tissue Tek OTC compound (Sakura Finetek, Torance, CA). The liver sections were permeabilized by 0.25% Triton X-100 and incubated with the primary antibody against Slit2 (1:200, Abcam, Cambridge, MA), α-SMA (1:200, Abcam, Cambridge, MA). Then, the liver sections were incubated with corresponding Alexa Fluor 594- and Alexa Fluor 488-conjugated secondary antibodies (1:200, Invitrogen, Carlsbad, CA) for 1 h at room temperature and stained with DAPI (1 µg/mL) for 10 5
min. Finally, the stained sections were viewed and photographed under the confocal microscopy (Leica TCS SP5, Leica, Mannheim, Germany). Cell Culture The well-characterized cell line derived from human HSCs, LX-2, was used in in vitro studies. LX-2 was generously provided as a gift by Professor Qi Zhang (the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China). Cells were cultured in DMEM (high glucose) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a 5% CO2 incubator at 37°C. Reverse-transcription (RT) PCR Total RNA was extracted from mouse liver tissues and cells using TRIzol reagent followed by treatment with RNase-free DNase (Takara, Dalian, China) for 30 min at 37°C. RNA were subjected to reverse transcription using a first-strand cDNA kit (Takara, Dalian, China) according to the manufacturer’s protocol. RT-PCR was conducted using the PrimeScript RT-PCR Kit (Takara, Dalian, China) according to the manufacturer’s instructions. The PCR was run on CFX96 real-time PCR system (Bio-Rad, Hercules, CA). The PCR reactions were carried out at: 95°C for 30s; and 40 cycles of 95°C/5 s, 60°C/34 s. PCR primer sequences were listed in supplementary Table1. The relative abundance of the target genes was obtained by calculating against the standard curve and normalized to β-actin as an internal control. Western blot analysis Equal amounts of total proteins (30µg) from each sample was subjected to 10% SDS-PAGE by electrophoresis under reducing conditions and transferred to PVDF 6
membrane (Millipore Corporation, Billerica, MA, USA). The blotted membrane was then blocked with 5% skim milk for 1 h at room temperature and incubated respectively with anti-Slit2 (1:1000, CST, USA), anti-α-SMA (1:1000, Boster, China), anti-Bcl-2 (1:1000, CST, USA), anti-ERK1/2 (1:1000, CST, USA), anti-p-ERK1/2 (1:1000, CST, USA), anti-p38 (1:1000, CST, USA), anti-p-p38 (1:1000, CST, USA), anti-caspase3 (1:1000, CST, USA), anti-Cleaved Caspase3 (1:1000, CST, USA) and anti-GAPDH (1:5000, Beyotime Biotechnology, China) overnight at 4°C. The membranes were further incubated with horseradish peroxidase (HRP) conjugated anti-mouse/rabbit secondary antibodies (1:5000 dilutions of each antibody) for 1h at room temperature and detected by enhanced chemiluminescent (ECL) method and captured on X-ray film. The light signal was captured by a charge-coupled device (CCD) camera (GE Healthcare, UK). The densitometric measurement of each band was analyzed using Quantity-One Protein Analysis Software (Bio-Rad Laboratories, USA). Knockdown of Slit2 by RNA interference LX-2, a well-characterized cell line derived from human HSCs, was used in in vitro studies. Recombinant human Slit2 (rh Slit2) were used at doses indicated in respective figure legends. Cells were cultured in DMEM supplemented with 10% fetal bovine serum, 1mmol/L L-glutamine, and 100 IU/mL streptomycin/penicillin. To silence Slit2 expression, specific predesigned small interfering RNA (siRNAs) from SMARTpool Slit2 siRNA kit (Dharmacon, Fisher Scientific, Pittsbrugh, USA) was used according to the manufacture’s instructions. For transfection, LX-2 cells were 7
plated at 1×105 cells/well in 6-well plates and cultured overnight. Cells were transfected with Slit2 siRNA (final concentration of 100 nM) using Lipofectamine 3000 (Invitrogen, Shanghai, China). Verification of transfection efficiency and the expression of target proteins in the transfected LX-2 cells were analyzed by immunoblotting 72 h after transfection. The non-targeted siRNA SMARTpool (Dharmacon, Fisher Scientific, Pittsbrugh, USA) was used as a negative control. Flow cytometric analyses of cell cycle and apoptosis The quantitative analysis of cell cycle and apoptosis was conducted by flow cytometry. LX-2 cells (8 × 104/well) were seeded in six-well plates in complete culture medium. After incubating for 12 h, cells were treated with H2O2 for 30 min (200µmol/L) and then Slit2 recombinant protein 10ng/mL for 24 h or only treated with Slit2 recombinant protein 10ng/mL for 24 h. Cells were harvested separately at 27 and 24 h later, and immediately fixed with 75% ethanol. For the cell cycle progression analysis, cells were stained with PI staining buffer (10 mg/mL PI and 100 mg/mL RNase A) for 30 min, and fluorescence intensity was measured by BD FACS Calibur (BD Biosciences, San Jose, CA, United States). For apoptosis analysis, cells were stained with the Annexin V-FITC/PI apoptosis detection kit. The rate of apoptotic cells was analyzed using a dual laser flow cytometer and estimated using the ModFit software (BD Biosciences). Apoptotic cell Hoechst 33258 detecting In addition to the flow cytometric analyses, the role of slit2 on the LX-2 cell apoptosis induced by H2O2 (200µmol/L) for 30min was further confirmed by Hoechst 8
33258 detecting. Firstly, the cells were washed sufficiently by PBS twice, and fixed by 1 mL 4% formaldehyde solution at 4°C. After 10min, the 4% formaldehyde solution was discarded and the cells were washed sufficiently by PBS twice. Then the cells were stained by 100 µL Hoechst 33258 working solution at room temperature for 10 min. After washed by PBS twice, the cells were viewed and photographed under the fluorescent microscopy (Olympus Co., Tokyo, Japan). Statistical analysis Values were expressed as mean ± S.D. Statistical differences between two groups were analyzed by the unpaired Student’s t test and differences between multiple groups of data were analyzed by one-way ANOVA with Bonferroni correction (GraphPad Prism 7.0, San Diego, CA, USA). P<0.05 was considered statistically significant.
RESULTS The Slit2 was highly expressed in liver biliary atresia (PBC) patients. Though Slit2 signaling has been demonstrated to promote liver injury and fibrosis in CCl4-induced mice in our previous work, its role in hepatic fibrosis caused by cholestatic liver damage is still unclear[18]. In present study, we initially examined Slit2 levels in human liver fibrotic tissue of PBC patients by immunohistochemistry staining. Compared to healthy control, the liver tissues of PBC patients had larger fibrotic areas represented by Sirius red staining and higher levels of HSCs activation represented by α-SMA positive staining (Fig. 1A, B, C). 9
Interestingly,
immunohistochemical analyses revealed that the hepatic Slit2 expression in PBC patients was significantly higher than that in healthy control (Fig. 1D). Next, we investigated the serum levels of Slit2 in 30 PBC patients with liver fibrosis and 30 healthy controls. The serum levels of Slit2 were significantly higher in PBC patients than that in healthy controls (Fig. 1H). These results raised the hypothesis that Slit2 signaling might be involved in HSCs activation and the pathogenesis of liver fibrosis. Slit2 aggravated the progression of BDL-induced liver fibrosis. On the basis of increased Slit2 expression in clinical fibrotic liver, we further explored the role of Slit2 signaling in hepatic fibrogenesis in BDL-induced mice, a classical rodent model mimicking liver fibrosis resulted from PBC patients. As shown in Fig. 2A, B, BDL-induced Slit2-Tg mice exhibited more fibrous hyperplasia and collagen deposition than BDL-induced WT mice as presented by H&E and Sirius red staining, implying that Slit2 significantly exacerbated hepatic injury and fibrosis. Consistently, immunochemical staining revealed higher hepatic expression of α-SMA and CD68 in BDL-induced Slit2-Tg mice, which respectively suggested the HSCs activation and inflammatory macrophages infiltration upon Slit2 overexpression (Fig. 2C, D). Additionally, immunoblotting assessment of α-SMA expression also confirmed a higher level of HSCs activation in BDL-induced Slit2-Tg mice (Fig. 2P). Furthermore, hepatic mRNA levels of fibrogenic genes, including α-SMA, Col-α1, TGF-β1, and TIMP1, were remarkably increased in BDL-induced Slit2-Tg mice compared to the BDL-induced WT mice. (Fig. 2L, M, N, O). To validate the association between the higher level of fibrosis observed in BDL-induced Slit2-Tg 10
mice with the severer injury process, serum indicators of liver damage including ALT, AST, ALP and γ-GT were detected by commercial kits. As expected, BDL induction brought about more serious liver injury in Slit2-Tg mice in comparison to the WT mice (Fig. 2H, I, J, K). Furthermore, we used Robo1 knockout (Robo1-/-) mice subjected to BDL to further clarify the role of the Slit2-Robo1 signaling in the pathogenesis of cholestatic liver fibrosis. As shown by Sirius Red and α-SMA immumohistochemical staining (Figure S1), BDL-induced liver fibrosis and HSC activation were significantly attenuated in Robo1-/- mice. Together, the overexpression of Slit2-Robo1 signaling was likely to contribute to the hepatic fibrogenesis resulted from cholestatic liver disease. Slit2 was involved in HSCs activation To illuminate the mechanism by which Slit2 facilitates liver fibrosis, we next investigated the cellular source of Slit2 in the fibrotic liver of BDL induced Slit2-Tg mice. By immunofluorescence co-staining of Slit2 with α-SMA (a marker for activated HSCs), we observed that Slit2 was highly expressed in activated HSCs, suggesting that the pro-fibrogenic effects of Slit2 were probably dependent of acting on HSCs (Fig. 3A). Next, qRT-PCR assays demonstrated that overexpression of Slit2 in LX-2 cells (a human HSCs line) treated with Slit2 recombinant protein dramatically enhanced the mRNA levels of pro-fibrogenic genes (a-SMA, Col1a1, TIMP-1). In contrast, Slit2 silencing via siRNA obtained almost 60% downregulation of Slit2 mRNA and successfully repressed these pro-fibrogenic mRNA levels of a-SMA, Col1a1 and TIMP-1 in LX-2 cells. These results made it clear that Slit2 11
promoted liver fibrosis through the regulation of HSCs activation. Slit2 promoted proliferation and protected against apoptosis in HSCs In addition to the determination of Slit2 mediated HSCs activation, we attempted to clarify the regulation of Slit2 signaling on the transformation of HSCs phenotypes (proliferation and apoptosis). Herein, an in vitro model of cell apoptosis was induced by H2O2. Initially, flow cytometry analysis was performed to investigate the effects of Slit2 recombinant protein and (or) H2O2 on the cell cycle of LX-2 cells. As shown in figure 3F, the proportions of G2/M and S-phase cells were significantly increased upon Slit2 stimulation. Whereas, cells challenged by H2O2 showed significantly decreased proportions of G2/M and S-phase cells. Noteworthily, such effect of H2O2 on the cell cycle of LX-2 cells was partially abolished by Slit2 recombinant protein. These results proved preliminarily that Slit2 not only promoted proliferation, but also protected against apoptosis induced by H2O2, which was further confirmed by both Hochest (Fig. 3G) and AnnexinV/PI (Fig. 3H) staining. Next, we conducted immunoblotting assay to disclose the changes of protein expression beneath the above effects of Slit2 on LX-2 cells. Accordingly, the expression of Slit2, anti-apoptotic protein (Bcl-2), and apoptosis regulating factor (Caspase3) were significantly increased, while Cleaved-caspase3 was significantly decreased in LX-2 cells treated with Slit2 recombinant protein. In contrast, LX-2 cells challenged by H2O2 exhibited a sharp decline in Bcl-2 and Caspase3 expression and a remarkable elevation in Cleaved-caspase3 expression. As expected, such pro-apoptotic effect of H2O2 in protein levels was partially eliminated in LX-2 cells treated with the combination of 12
H2O2 and Slit2 recombinant protein. Taken together, these data demonstrated strongly the role of Slit2 as a regulator in the proliferation and apoptosis of HSCs. Slit2 affected HSCs proliferation and apoptosis through MAPK pathway To shed light on the molecular mechanism involved in the regulation of Slit2 on HSCs phenotypes, we performed the immunoblotting analysis of MAPK pathway which was closely related to cell proliferation and apoptosis as firmly established in the literature. We provided evidences that Slit2 stimulation via Slit2 recombinant protein remarkably up-regulated p-P38 and p-ERK1/2 while blockade of Slit2 signaling via siRNA silencing significantly down-regulated p-P38 and p-ERK1/2 in LX-2 cells (Fig. 4A). Besides, in H2O2 induced LX-2 cells, we observed reduced expressions of both p-P38 and p-ERK1/2, which were reactivated by the exogenous supplementation of Slit2 recombinant protein (Fig. 4B). These results demonstrated that Slit2 intervened in HSCs proliferation and apoptosis through p-P38 and p-ERK1/2 pathway.
DISCUSSION Our previous study has demonstrated that Slit2 signaling promoted liver injury and fibrosis in CCl4-induced mice[18]. Nevertheless, the role of Slit2 signaling in hepatic fibrosis caused by cholestatic liver damage is still unclear. In cholestatic liver diseases (e.g., PBC), biliary obstruction makes the bile acid to accumulate within bile ducts and leak into the liver and blood circulation, leading to ductular reaction, hepatocyte damage, hepatic oxidative stress, inflammation, and fibrosis [21-23]. In the current 13
study, we first verified that Slit2 contributed to liver injury and liver fibrosis in bile duct ligation (BDL)-induced mice, a classical rodent model mimicking cholestatic liver disease. Initially, we found that PBC patients had much higher hepatic Slit2 expression and serum levels of Slit2 than healthy controls. This pivotal clinical clue triggered our further investigation in BDL-induced mice as a model of cholestatic hepatic fibrosis. Our research revealed that overexpression of Slit2 exacerbated liver injury and fibrosis in response to BDL induction. Thus, both clinical and preclinical data verified the definite pro-fibrogenic effects of Slit2 during cholestatic liver damage. Next, on the basis of defining HSCs as the one of main cellular source of Slit2 in the fibrotic liver of Slit2-Tg mice induced by BDL, HSCs debuted in the focus of our research on the pro-fibrogenic effects of Slit2. Upon various stimuli during liver damage, quiescent HSCs activate into collagen-producing myofibroblasts and play a paramount role in the pathogenesis of hepatic fibrosis [19, 24]. We verified that Slit2 overexpression was accompanied by a higher hepatic expression of α-SMA in BDL-induced Slit2-Tg mice, strongly suggesting that Slit2 signaling is implicated in HSC activation. To verify the above finding, in the in vitro studies, we up-regulated Slit2 by the exogenous supplementation of Slit2 recombinant protein and down-regulated Slit2 by siRNA silencing in LX-2 cells (a well-characterized HSCs line derived from human). Not surprisingly, Slit2 overexpression dramatically enhanced the mRNA levels of a-SMA (marker for activated HSCs) and Col1a1 (marker for collagen production), whereas Slit2 silencing successfully repressed the 14
above pro-fibrogenic mRNA levels in LX-2 cells. The above data collectively demonstrated that Slit2 signaling mediates HSC activation and the pathogenesis of liver fibrosis. Enhanced ability of survival and proliferation represent the two major distinct phenotypes of activated HSCs distinguishing from the quiescent ones. In the present study, evidences from LX-2 cells were provided that Slit2 could effectively promote proliferation and protect against H2O2 induced apoptosis in HSCs by affecting the cell cycle, expanding our work to exploring the involved molecular mechanism. MAPKs are a class of mitogen-activated protein kinases, including p38, ERK and JNK. MAPKs are involved in cell proliferation, differentiation, survival, apoptosis and autophagy in response to stress stimuli, such as cytokines, ultraviolet irradiation, heat shock and osmotic shock [25-27]. Specifically, JNK is involved in cell apoptosis, and p38 is related to cell proliferation and apoptosis, while ERK is associated with cell proliferation,
apoptosis,
migration,
and
senescence[28-31].
Herein,
the
phosphorylated status of the MAPK family members (ERK1/2, JNK and p38) of LX-2 cells were detected by western blotting. We made it clear that Slit2 exerted its proliferation-promoting and anti-apoptotic effects by inducing the phosphorylated activation of ERK1/2 and p38 but not JNK (JNK data were not shown). Despite the significant findings revealed by this investigation, limitations still exist. Whether Slit2 inhibition could alleviate liver fibrosis in BDL-induced mice was not addressed
in
the
current
study
since
suppressing
Slit2
signaling
by
systemic administration with non-targeted inhibitors was not feasible because of its 15
vital physiological function such as regulating neural development and leukocyte migration[32]. Hence the further clinical transformation of Slit2 signaling as therapeutic target of liver fibrosis remains an urgent challenge. Recently, the newly-developed nanomedicine has emerged as a promising strategy for targeted drug delivery or gene regulation with both high efficiency and low side effects[33]. For instance, in our previous study, we developed hyaluronate-graft-polyethylenimine to specifically deliver cyclooxygenase-1 siRNA to liver sinusoidal endothelial cells, achieving the successful amelioration of cirrhotic portal hypertension in CCl4-induced mice[34]. Thus, for the treatment of liver fibrosis, it will be taken into consideration in our future study to develop novel nanomedicine for targeted Slit2 regulation in HSCs. In conclusion, our studies elucidate that Slit2 promoted HSCs activation, and contributed to the survival and proliferation of HSCs via activation of ERK1/2 and p38 signaling pathways, pushing forward the pathogenesis of hepatic fibrosis. Though whether Slit2 inhibition could alleviate liver fibrosis in BDL-induced mice was not addressed in the current study, further investigations are warranted to shed new light on the potential of Slit2 as therapeutic target for liver fibrosis treatment.
ACKNOWLEDGEMENTS This study was supported by the Key Project of Natural Science Foundation of Guangdong
Province
(2016A030311014),
China;
National
Natural
Science
Foundation of China (81870420); and the Guangzhou Science and Technology 16
Programs (201704020153), China.
CONFLICT OF INTEREST STATEMENT The authors declare that they have no conflicts of interest.
17
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Figure Legends
Fig.1. Hepatic expression of Slit2 in PBC patients. Paraffin-embedded sections of liver tissues from healthy controls (Control, n=20) and PBC patients (Fibrosis, n=20) were stained with H&E (A), Sirius Red (B), and immunohistochemical staining of α-SMA (C) and Slit2 (D). The positive staining areas were measured by ImageJ software (E, F and G). Serum level of Slit2 was measured in the healthy control (n=30) and PBC patients with liver fibrosis (n=30) by ELISA (H). The horizontal lines mark the mean values of triplicate measurements. Data are presented as means ± S.D. ** P<0.01; **** P<0.0001 vs. Control.
Fig.2. Slit2 contributes to BDL-induced liver fibrosis in mice. C57 and Slit2-Tg mice were induced by BDL for 15days. Representative histology staining of H&E (A) and Sirius Red (B), and immunohistochemical staining of α-SMA (C) and CD68 (D). (E, F, G) Quantification of positive staining areas was measured by ImageJ software. (H, I, J, K) Serum levels of ALT, AST, ALP and γ-GT. (L-O) RT-PCR of α-SMA, Col-α1, TGF-β1 and TIMP1. (P) Immunoblotting analyses of hepatic α-SMA expression in the mouse. **P<0.01, ***P<0.001, ****P<0.0001 vs. WT mice induced by BDL.
Fig.3. Slit2 promotes the activation, proliferation and suppresses the apoptosis of HSCs in vitro. (A) Representative immunostaining images showed that the expression of Slit2 colocalized with activated HSCs marker, α-SMA in liver from the Slit2-Tg mice induced by BDL for 15 days. DAPI staining was used to identify the nuclei. (B, C, D, E) mRNA levels of Slit2, α-SMA, Col-α1 and TIMP1 in LX-2 cells transfected with Slit2 siRNA or treated with Slit2 recombinant protein. (F, G, H) Cell cycle, Hoechst and AnnexinV/PI staining of LX-2 cells treated with Slit2 recombinant protein and (or) H2O2. (I) Analysis of cell cycle by ModFit software. (J) Quantification of Hoechst positive staining areas was measured by ImageJ software. (K) Immunoblotting analyses of Slit2, Bcl-2, Caspase3 and Cleaved-Caspase3 23
expression of LX-2 cells treated with Slit2 recombinant protein and (or) H2O2.
Fig.4. Slit2 promotes the proliferation and suppresses the apoptosis of HSCs via MAPKs activation. (A) Immunoblotting analyses of Slit2, ERK1/2, p-ERK1/2, P38 and p-P38 expression of LX-2 cells transfected with Slit2 siRNA or treated with Slit2 recombinant protein. (B) Immunoblotting analyses of ERK1/2，p-ERK1/2, P38 and p-P38 expression of LX-2 treated with Slit2 recombinant protein and (or) H2O2.
Fig. S1. Robo1-/- mice are less readily to develop HSC activation and liver fibrosis in response to BDL induction. WT and Robo1-/- mice were induced by BDL for 15 days. Representative histology staining of Sirius Red, and immunohistochemical staining of α-SMA.
24
Figures
Fig. 1
25
Fig. 2
26
Fig. 3
27
Fig.4
28
Fig. S1
29
Table 1: Primer Sequences used for qRT-PCR Gene
Primer Sequence
Mouse α-SMA Col1α1 TIMP-1 TGF-β1 Slit2 β-actin
F 5’- GTCCCAGACATCAGGGAGTAA-3’ R 5’- TCGGATACTTCAGCGTCAGGA-3’ F 5’- GCTCCTCTTAGGGGCCACT-3’ R 5’- CCACGTCTCACCATTGGGG-3’ F 5’- GCAACTCGGACCTGGTCATAA-3’ R 5’- CGGCCCGTGATGAGAAACT-3’ F 5’- CTCCCGTGGCTTCTAGTGC-3’ R 5’- GCCTTAGTTTGGACAGGATCTG-3’ F 5’- GGCAGACACTGTCCCTATCG-3’ R 5’- ATCTATCTTCGTGATCCTCGTGA-3’ F 5’- GGCTGTATTCCCCTCCATCG-3’ R 5’- CCAGTTGGTAACAATGCCATGT-3’
Human α-SMA Col1α1 TIMP-1 TGF-β1 Slit2 β-actin
F 5’- GTGTTGCCCCTGAAGAGCAT-3’ R 5’- GCTGGGACATTGAAAGTCTCA-3’ F 5’- GTGCGATGACGTGATCTGTGA-3’ R 5’- CGGTGGTTTCTTGGTCGGT-3’ F 5’- CTTCTGCAATTCCGACCTCGT-3’ R 5’- ACGCTGGTATAAGGTGGTCTG-3’ F 5’- GGCCAGATCCTGTCCAAGC-3’ R 5’- GTGGGTTTCCACCATTAGCAC-3’ F 5’- GCGAAGCTATACAGGCTTGAT-3’ R 5’- TGCAGTCGAAAAGTCCTAAGTTT-3’ F 5’- GGCATTCACGAGACCACCTAC-3’ R 5’- CGACATGACGTTGTTGGCATAC-3’
30
Highlights: 1. Liver fibrosis is a reversible wound healing response for liver injury. Advanced liver fibrosis might progress into irreversible cirrhosis, which is the leading cause of liver-related mortality worldwide. In our study, we observed that hepatic Slit2 expression was significantly increased in liver biliary atresia (PBC) patients with liver fibrosis, and Slit2-Tg mice were much more vulnerable to BDL-induced liver injury and fibrosis when compared to WT mice. 2. During liver fibrogenesis, the key pro-fibrogenic HSCs activate and display a distinct phenotype with increased resistance to apoptosis and enhanced ability of proliferation, contraction, and chemotaxis. We demonstrated that Slit2 up-regulation promoted the activation and proliferation, whereas inhibited the apoptosis of human HSCs cell line LX-2. 3. We also provided evidences that Slit2 promoted HSCs activation, and contributed to the survival and proliferation of HSCs likely via activation of ERK1/2 and p38 signaling pathways.
Declarations of interest: none