Study On Smads Expression In Activated Rat Hepatic Stellate Cell Exposed To TGF-β₁

PrefaceIt has been generally agreed that liver fibrosis is characterized by excessive deposition of extracellular matrix ( ECM ). Transforming growth factor - β ( TGF- β) plays an important role in the pathogenesis of liver fibrosis. Hepatic stellat-e cell is the major cell that can synthesize a variety of ECM proteins in response to stimuli such as TGF - β.It has been shown that Smads proteins are the single TGF - β receptor ( T (R) substrate and intracellular mediators in TGF - β signaling. But the mechanism , such as how TGF - β regulates the expression of Smads, how TGF - β regulates the activation of Smads , is still unclear especially in the fields of liver fibrosis.The purpose of this study is to figure out the change of phospho - Smad2 or- Smad3 ( p - Smad2 or - Smad3 ) and Smad7 expression in activated rat HSC exposed to TGF - β, in vitro by the Western blotting, recognize the molecular mechanism of liver fibrosis and provide some experimental data in order to seek the new target of liver fibrosis treatment.Materials and methodsIn this study, p - Smad2 or - Smad3 and Smad7 protein expression were analysized in activated rat HSC exposed to TGF - β₁ in a time - dependent manner. We stimulated activated rat HSC with TGF - β₁ (5ng/ml) in vitro. The cells were collected at time point 0 hour,0. 5 hour, 1 hour, 4 hour, 8 hour and 24 hour perspectively: the cells were lysated with 100 μl lysate buffer and then add-ed 4μl PMSF, 1μl Aprotinin followed by collecting the cells on ice and centri-fuging at 10000 × g for 15 minutes at 4℃ The supernatants were stored at -130℃ for further study. Then the samples were analysized by Western blotting. The sample were mixed with sodium dodeyl sulfate - polyacrylamide gel electro-phoresis(SDS -PAGE) sample buffer,boiled for 5 minutes ,electrophoresed on a 10% SDS - PAGE and electroblotted on PVDF membranes. After the membranes stained,the blot was developed using the ECL detection kit to produce a chemiluminescence signal, which was captured on X - ray film followed by gel semi - quantitative analysis with GIS gel picture processor system. The data were expressed as the mean ± SD. Statistical analyses were performed using SPSS Software. Differences in p - Smad2 or Smad3 or Smad7 were assessed by one - way ANOVA.ResultsThe p - Smad2 or - Smad3 and Smad7 expression in activated rat HSC exposed to TGF - β₁ in vitroWestern blotting analyses show that: p - Smad2 or - Smad3/β - Actin levels in activated rat HSC after cultured perspectively at time point 0, 0.5 hour, 1 hour ,4 hour, 8 hour and 24 hour stimulated with TGF -β₁ were 0. 84 ±0.11,0. 97±0.09,1.02±0.16,1. 12 ±0.19,1.27 ±0.26,1.17 ±0.26. These results elucidated that p - Smad2 or - Smad3 expressions in activated rat HSC increased shortly and reached in the peak at 8 hour time point. The p - Smad2 or - Smad3 level in the group at 8 hour was much higher than those at 0 hour ( a-nalysis of variance, p =0.014and 0.048). And there was no difference among the other groups. The total F is 4. 667 and the value of P is 0.034. So the difference is of statistically value. At the same time, Smad7 levels were 0.71 ±0.22, 0.85 ±0.31,0.91 ±0.32,1.07 ±0.36,1.38 ±0.48 and 1.29 ±0.59 perspectively. The results showed that Smad7 expression in activated rat HSC increased at 4 hour and reached in the peak at 8 hour later. Smad7 levels in activated rat HSC at 4 hour and 8 hour was higher than those in the other groups ( analysis of variance, p =0.049 and 0.002). And there was no difference among the other...