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Published on: July 30, 2018
Endostatin attenuates PDGF-BB- or TGF-β1-induced HSCs activation via suppressing RhoA/ROCK1 signal pathways
Haitao Ren1, Yuan Li2, Yan Chen3
1Department of Burns and Wound Care Center, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310009, P.R. China, rht@zju.edu.cn.
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.
Insights
Endostatin demonstrates antifibrotic effects by inhibiting hepatic stellate cell proliferation and collagen production. It modulates the RhoA/ROCK1 pathway, offering potential therapeutic strategies for liver fibrosis.
Area of Science:
- Cell Biology
- Biochemistry
- Pharmacology
Background:
- Liver fibrosis is a significant health concern characterized by excessive extracellular matrix deposition.
- Hepatic stellate cells (HSCs) are key players in liver fibrogenesis.
- Transforming growth factor β1 (TGF-β1) and platelet-derived growth factor-BB (PDGF-BB) are potent stimulators of HSC activation and fibrosis.
Purpose of the Study:
- To investigate the antifibrotic potential of endostatin in hepatic stellate cells (HSCs).
- To elucidate the role of endostatin in modulating the RhoA/ROCK1 signaling pathway in HSCs.
- To determine if endostatin inhibits TGF-β1- or PDGF-BB-induced fibrotic responses.
Main Methods:
- Primary HSCs were cultured and treated with TGF-β1, PDGF-BB, or endostatin.
- Cell proliferation was assessed.
- Collagen I and α-smooth muscle actin expression were quantified using ELISA and Western blot.
- RhoA and ROCK1 mRNA and protein levels were analyzed via real-time PCR and Western blot.
- Immunofluorescent staining was used to detect F-actin.
Main Results:
- TGF-β1 and PDGF-BB significantly promoted HSC proliferation and collagen I, F-actin, and α-smooth muscle actin expression.
- Endostatin significantly inhibited the proliferative and fibrotic effects induced by TGF-β1 and PDGF-BB.
- Endostatin treatment led to a significant downregulation of RhoA and ROCK1 expression in HSCs.
Conclusions:
- Endostatin exhibits significant antifibrotic properties in hepatic stellate cells.
- Endostatin exerts its antifibrotic effects by modulating the RhoA/ROCK1 signaling pathway.
- These findings suggest endostatin as a potential therapeutic agent for liver fibrosis.
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