| Liver fibrosis is essentially a wound healing response in chronic liver damage caused by a variety of etiology and it is mainly characterized by the excessive deposition of intrahepatic extracellular matrix in Disse space. As a result of liver function damage, liver fibrosis is a essential phase for the development of chronic liver disease to cirrhosis of the liver. A large number of experimental studies have confirmed that collagen is the main ECM producing in the process of liver fibrosis. Therefore, inhibiting collagen deposition may slow the development of liver fibrosis.TGF-β1 is recognized as an control factor in the process of liver fibrosis, its main role in the process of liver fibrosis is prompting the ECM production, inhibitting of collagenase and matrix metalloproteinases, reducing collagen degradation, and aggravate hepatic fibrosis gradually. With structurally similarity but functionally diversity, Smad2 and Smad3 interact with each other to mediate transforming growth factor-β(TGF-β)-triggered signaling transduction. However, in the hepatic fibrosis, the detailed roles of R-Smads, and interaction between Smad2 and Smad3 are still undefined. In this setting, we established a rat model of CCl4-induced hepatic fibrosis in vivo and TGF-β1-treated hepatic stellate cell model in vitro. Laser Scanning Confocal Microscopy, molecular biology and Gelatin zymography assay were used to detect whether Smad2 and Smad3 play distinct roles in mediating liver fibrogenesis. Results indicated :(1) Both phosphorylation of Smad2 and Smad3 were detected in the hepatic stellate cells of liver fibrotic tissues and cells.CCl4-induced hepatic injury model is a well-established model for the study of liver fibrogenesis. In the liver of CCl4-treated group, severe morphology changes including fat degeneration, ballooning, necrosis and infiltration of inflammatory cells were observed compared with normal control. The collagen deposition were also detected by Masson trichrome staining, which showed total collagen was significantly increased in the model of CCl4-induced hepatic fibrosis, expression of Col.I elevated in rat liver. Immunofluorescence double dye of α-SMA and p-Smad2, α-SMA and p-Smad3 indicated that p-Smad2 and p-Smad3 were expressed in the hepatic stellate cell. Results of Western blot showed that p-Smad2 and p-Smad3 were increased during rat liver fibrosis. This result provided the basis of selects the LX-2 cell model for the next research(2) In vitro data demonstrated that knockdown of Smad2 in human hepatic stellate cells increased expression of collagen I(Col.I), tissue inhibitor of metalloproteinase-1(TIMP-1) whereas decreasing expression of the matrix metalloproteinases-2(MMP-2) in presence of TGF-β1 compared with control group. The balance of TIMP-1 and MMP-2 was broken. The Matrix production increased but degradation was reduced. In contrast, knockdown of Smad3 significantly reduced TGF-β1-induced Col.I production.(3) overexpression of Smad2 attenuated the expression of Col.I and TIMP-1, but enhanced MMP-2 whereas overexpression of Smad3 showed the opposite effect.(4) Smad2 suppressed the phosphorylation and nuclear translocation of Smad3, which may protect against Smad3-mediated fibrotic response. knockdown of Smad2 with RNAi largely enhanced Smad3 signaling as identified by a marked phosphorylated Smad3(phospho-Smad3) and nuclear translocation. Western blot analysis confirmed that knockdown of Smad2 in LX-2 cells significantly enhanced TGF-β1–induced phosphorylation of Smad3, overexpressing Smad2 attenuated TGF-β1-induced Smad3 phosphorylation significantly.Collectively, Smad2 may be a potential therapeutic target for the treatment of hepatic fibrosis. |
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