Abstract
Aim
To testify the hypothesis that endostatin exerts antifibrotic effects in hepatic stellate cells (HSCs) by modulating RhoA (ras homolog gene family, member A)/ROCK 1 (Rho-associated protein kinase 1) signal pathways.
Materials and methods
HSCs-T6 of passages 3–5 were cultured in DMEM and serum starved for 48 hours. HSCs were grouped as follows: control group, TGF-β1 (transforming growth factor β1) group, endostatin+TGF-β1 group, PDGF-BB (platelet-derived growth factor-BB) group, and endostatin+PDGF-BB group. In the PDGF-BB group, HSCs were treated with PDGF-BB (200 ng/mL) for 72 hours; in the TGF-β1 group, they were treated with TGF-β1 (10 ng/mL) for 72 hours. In the Endostatin+TGF-β1 group or Endostatin+PDGF-BB group, HSCs were treated with TGF-β1 (10 ng/mL) or PDGF-BB (200 ng/mL) for 72 hours after pretreatment with endostatin (5 µg/mL) for 1 hour. In the control group, HSCs were only treated with serum-free DMEM for 72 hours. Collagen I was analyzed with ELISA. F-actin was detected with immunofluorescent staining. The mRNAs and proteins of α-smooth muscle actin, RhoA, and ROCK1 were analyzed by using real-time PCR and Western blot, respectively.
Results
TGF-β1 and PDGF-BB promote the proliferation of HSCs significantly at 48 and 72 hours. Endostatin inhibits the proliferation effect induced by TGF-β1 or PDGF-BB significantly (P<0.01). The expression of collagen I and F-actin was significantly upregulated in both TGF-β1 and PDGF-BB groups than in the control group (P<0.01). Both the collagen I and F-actin expression were downregulated significantly in the endostatin-treated groups (P<0.05). Endostatin significantly inhibited the upregulated expression of α-smooth muscle actin, RhoA, and ROCK1 induced by TGF-β1 or PDGF-BB (P<0.01).
Conclusion
These results suggested that endostatin inhibited TGF-β1- or PDGF-BB-induced fibrosis in HSCs by modulating RhoA/ROCK signal pathways.
Introduction
Hepatic fibrosis is a worldwide health care burden with excessive synthesis and deposition of extracellular matrix (ECM),Citation1 which results in high mortality and morbidity. Unfortunately, no ideal therapies are effective in clinical application Therefore, the research for treating liver fibrosis is highly urgent. Hepatic stellate cells (HSCs) are recognized as liver-specific type of pericytes and can be activated by injury or inflammation. The activated HSCs differentiate into myofibroblasts and produce excessive ECM.
Endostatin is a peptide involved in multiple functions of physiological and pathological processes including angiogenesis, fibrosis, sepsis, and acute kidney injury.Citation2–Citation5 The antifibrotic activity has emerged as a newly attractive function. For example, endostatin was proved to have protective effects against hepatic fibrosis.Citation6 However, the precise molecular mechanisms remain unclear.
Multiple signal pathways have relation to liver fibrosis. For example, RhoA (ras homolog gene family, member A)/ROCK (Rho-associated protein kinase) pathways play important roles in the process of liver fibrosis;Citation7 therefore, it is hypothesized that endostatin inhibits fibrosis by modulating RhoA/ROCK1 pathways. This study was designed to investigate whether endostatin has an effect on RhoA/ROCK1 pathways in a transforming growth factor (TGF)-β1- or platelet-derived growth factor (PDGF)-induced fibrosis cell model.
Materials and methods
Reagents and antibodies
Primary antibodies against α-smooth muscle actin (α-SMA), RhoA, ROCK1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Affinity Biosciences (Cincinnati, OH, USA). The recombinant endostatin was purchased from the Simcere Pharmaceutical Company (Nanjing, P.R. China). Both PDGF-BB and TGF-β1 were purchased from the ProSpec Bio (Rehovot, Israel). The ELISA kits of human collagen I were purchased from the Abbexa Company (Cambridge, UK).
Culture of rat HSCs
The rat HSCs line, HSC-T6, was obtained from ATCC (Manassas, VA, USA). Cell passages 3–5 were cultured in DMEM with 10% fetal bovine serum (Wisent, Canada), and seeded in six-well plates at a density of 5×105 cells per well for 24 hours, then serum-starved for 48 hours before treatment.
Treatments and groups
HSCs were divided into five groups: control group, TGF-β1 group, endostatin+TGF-β1 group, PDGF-BB group, and endostatin+PDGF-BB group. In the TGF-β1 group, HSCs were treated with 10 ng/mL TGF-β1; in the PDGF-BB group, the HSCs were treated with 200 ng/mL PDGF-BB for 72 hours. In the endostatin+TGF-β1 group or endostatin+PDGF-BB group, HSCs were pretreated with endostatin (5 µg/mL) for 1 hour and then treated with TGF-β1 (10 ng/mL) or PDGF-BB (200 ng/mL) for 72 hours. Endostatin was removed before treatment with TGF-β1 or PDGF-BB. In the control group, HSCs were treated only with serum-free DMEM for 72 hours. All cells were cultured in serum-free DMEM.
Cell viability assay
Based on our previous studies of concentration determination, the aim of the cell viability assay was to choose the best working time of treatments. The cell viability was measured by CCK-8 assay. HSC-T6 cells were cultured and seeded in the 96-well microplates at a density of 1×104 per 100 mL, then treated according to the manufacturer’s guidance. The cell viability was measured at 0, 24, 48, and 72 hours. The optical density was measured by using the microplate reader at 450 nm. An additional endostatin-alone treatment group was used to analyze the potential effect of endostatin on the viability of HSCs. HSCs were incubated with endostatin (5 µg/mL) for 1 hour, and then cultured in serum-free DMEM for 72 hours. All experiments were repeated three times.
ELISA assay
HSC-T6 cells were seeded in six-well plates at a density of 5×105/mL, and treated as described above in section “Treatments and groups.” The concentrations of collagen I in the supernatants were detected using ELISA kits according to the manufacturer’s guidance. Absorbance value was measured at 450 nm with the microplate reader (Bio-Rad Laboratories Inc., Hercules, CA, USA). The concentration of type I collagen was measured consistent with the corresponding standard curves. The OD value was measured at 450 nm on a microplate reader (Bio-Rad). The concentrations of collagen I were quantified according to the standard curves. The supernatant fluid was harvested and measured three times.
Immunofluorescent staining
HSCs were treated with 4% (0.04 g/mL) paraformaldehyde solution followed by 0.2% (v/v) Triton X-100. Immunofluorescent staining with the antibody against F-actin was performed according to the manufacturer’s instruction as described previously.Citation8 DAPI reagent (4′,6-diamidino-2-phenylindole, MP Biomedicals, 1:7,500) was used to stain the nucleus.
Real-time PCR analysis (RT-qPCR)
The total RNA was isolated with TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. The expression of target genes was assayed by using the SYBR Green Real-Time PCR system (Thermo Fisher Scientific). The target gene primer sequences are displayed in . All experiments were repeated three times.
Table 1 Primer probe sequences used for real-time PCR
Western blot analysis
The procedure was performed according to our previous study.Citation3 The proteins were transferred to a polyvinylidene fluoride membrane and then incubated overnight at 4°C with the following primary antibodies: α-SMA (1:10,000 dilution), RhoA (1:1,000 dilution), and ROCK1 (1:1,000 dilution). GAPDH (1:2,000 dilution) was used as an internal control. Secondary antibodies (goat anti-rabbit IgG, 1:2,000 dilution) were then added. The quantification of target protein bands was analyzed with ImageJ software (v.1.60) and normalized to GAPDH. All experiments were repeated three times.
Statistics
The data were analyzed with Prism 6.0 software by using the one-way analysis of variance. The differences with P<0.05 were considered significant.
Results
Endostatin suppressed PDGF-BB- or TGF-β1-induced HSC proliferation
There is no significant difference among these groups at 0 and 24 hours. TGF-β1, 10 ng/mL, or PDGF-BB, 200 ng/mL, significantly promoted HSC-T6 proliferation at 48 and 72 hours (P<0.01, ). Endostatin significantly suppressed the proliferation effect at 48 and 72 hours in both endostatin-pretreated and endostatin-alone groups (P<0.01). Therefore, we selected 72 hours as the working time in the subsequent experiments.
Figure 1 Endostatin inhibits HSC-T6 cells proliferation.
Abbreviations: CCK-8, Cell Counting Kit-8; HSCs, hepatic stellate cells; PDGF, platelet-derived growth factor; TGF, transforming growth factor.
Effect of endostatin on the expression of collagen I
As shown in , PDGF-BB and TGF-β1 induced a significant increase in collagen I protein level. The upregulated expression of collagen I induced by PDGF-BB and TGF-β1 was significantly inhibited by endostatin (P<0.01, ).
Figure 2 ELISA of collagen I.
Abbreviations: PDGF, platelet-derived growth factor; TGF, transforming growth factor.
Endostatin inhibited the expression of F-actin
The expression of F-actin in TGF-β1- or PDGF-BB-induced HSC-T6 cells was significantly upregulated than that in the control group. Endostatin reduced the TGF-β1- or PDGF-BB-induced expression of F-actin significantly (, P<0.01).
Figure 3 Effects of endostatin on the expression of F-actin in HSCs induced by PDGF-BB or TGF-β1.
Abbreviations: HSCs, hepatic stellate cells; PDGF, platelet-derived growth factor; TGF, transforming growth factor.
Endostatin inhibited the expressions of α-SMA, RhoA, and ROCK1 at the mRNA level
The expression of α-SMA, RhoA, and ROCK1 at mRNA level in the PDGF-BB- or TGF-β1-induced groups was significantly higher than that in the control group. Endostatin significantly inhibited the expression of α-SMA, RhoA, and ROCK1 at the mRNA level (P<0.01, ).
Figure 4 Endostatin inhibits the expression of α-SMA, RhoA, and ROCK1 at mRNA level.
Abbreviations: α-SMA, α-smooth muscle actin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HSC, hepatic stellate cell; RhoA, ras homolog gene family, member A; ROCK1, Rho-associated protein kinase 1; RT-PCR, real-time PCR.
Endostatin inhibited the expressions of α-SMA, RhoA, and ROCK1 at the protein level
The expressions of α-SMA, RhoA, and ROCK1 at protein level were significantly upregulated in the PDGF-BB- and TGF-β1-induced groups. Endostatin significantly inhibited the expressions of α-SMA, RhoA, and ROCK1 at the protein level (P<0.01, ).
Figure 5 Endostatin inhibits the expressions of α-SMA, RhoA, and ROCK1 at protein level.
Abbreviations: α-SMA, α-smooth muscle actin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HSC, hepatic stellate cell; PDGF, platelet-derived growth factor; RhoA, ras homolog gene family, member A; ROCK1, Rho-associated protein kinase 1; TGF, transforming growth factor.
Discussion
Hepatic fibrosis is actually the result of a wound-healing process. HSCs are located in the Disse space between hepatocytes and sinusoidal endothelial cells. HSCs have been recognized as liver-specific pericytes.Citation9 HSCs could be activated by stimulations and differentiated into myofibroblasts.Citation10–Citation12 In our study, an in vitro TGF-β1- or PDGF-BB-induced HSCs fibrosis model was used. TGF-β1 and PDGF-BB promote the expressions of collagen I, α-SMA, and F-actin significantly.
Endostatin is the C-terminal of collagen type XVIII present in the liver sinusoidal and basement membrane, and discovered to ameliorate liver fibrosis.Citation13 Our previous studies found that endostatin ameliorates scar formation in the rabbit ear modelCitation14 and inhibits fibrosis in human fibroblasts.Citation8,Citation15 In the present study, endostatin significantly inhibited the upregulated expressions of collagen I, α-SMA, and F-actin induced by PDGF-BB and TGF-β1 in HSCs.
Hepatic fibrosis is a complex pathological process that involves a variety of signaling pathways. RhoA/ROCK1 pathways participated in liver fibrosis.Citation16 The transformation from fibroblasts into myofibroblasts has a close relationship with the RhoA/ROCK1 signal pathways.Citation17 RhoA regulates the cell cytoskeleton and activates HSCs.Citation18 The actin filament production is directed by the RhoA/ROCK1 signal pathways.Citation19 RhoA/ROCK1 signaling pathways control cell morphology, proliferation, and adhesion. In addition, ROCK1 plays important roles in PDGF-BB-induced cell proliferation in human aortic vascular smooth muscle cells.Citation20
We have uncovered here a novel mechanism of endostatin to inhibit HSC activation by selectively modulating the RhoA/ROCK pathways. For the limitations, we investigated only the effect of endostatin on RhoA and ROCK1 expressions in mRNA and protein levels. Further research is needed to clarify the molecular mechanisms behind these effects.
Conclusion
Endostatin attenuates PDGF-BB- or TGF-β1-induced HSC activation via suppressing RhoA/ROCK1 signal pathways.
Acknowledgments
This research was supported by Zhejiang Provincial Natural Science Foundation of China under Grant Nos LY15H150004 and LY17H160035, Foundation of Teaching Department of Zhejiang Province (No Y201330073), and Foundation of Health Department of Zhejiang Province (No 2013KYB132).
Disclosure
The authors report no conflicts of interest in this work.
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