| ObjectivesHepatic fibrosis is a common histological change of chronic liver disease by a variety of etiological factors.Researchers have indicated that the key point and center link of hepatic fibrosis is the activation and proliferation of HSC.Transforming growth factor-β1(TGF-β1) plays a crucial role in regulating the hepatic fibrosis development. Smads protein are the most important cytokine in cytoplasm during signal transduction of TGF-β1.They can conduct the signal of TGF-β1 from cellular membrane into cell nucleus;The main causes of hepatic fibrosis are the overexpression of Smad3 and the inhibition of Smad7.The recent studys have showed that IFN-αhas effects on anti-hepatic fibrosis.It is uncertain if IFN-αinfluents the expression of Smads.We provide more theoretreal basis for the clinical hepatic fibrosis drug treatment by investigating the effect of IFN-αon the expressions of Smad3 and Smad7 in Hepatic Stellate Cell excited by TGF-β1 in vitro and probe the possible antifibrotic mechanism of IFN-α.Materials and methods1.Materials(1) Rat hepatic stellate cell strain(r-HSC99),provided by Dr.Leng xisheng of Peking University People's Hospital,conserved by laboratory of Infectious Diseases, Affiliated Shengjing Hospital of China Medical University.R-HSC99 are cultured and passaged for a long time,it is in line with natural characters of HSC,and it is the ideal model of liver fibrosis.(2) main reagents:TGF-β1;Recombinant Human Interferon Alpha 2b(IFN-α-2b);β-actin primer,Smad3 primer,Smad7 primer,RT-PCR kit,Goat-anti-rat Smad2/3 polyclonal antibody;Goat-anti-sat Smad7 polyclonal antibody,rabbit-anti goat IgG. Antibody. 2.MethodsHepatic stellate cells(rHSC-99) were cultured in vitro.Two groups were divided: group of TGF-β1 and group of TGF-β1+IFN-α.we detect the levels of Smad3 mRNA and Smad7 mRNA by semi-quantitative reverse-transcription polymerase chain reaction(RT-PCR),and the levels of Smad3 protein and Smad7 protein by the semi-quantitative Western blotting at the 0,2,4,8,16,24 hour.3.Statistical analysisWe analyzed the variance by SPSS V13.0 software.Results1.The expressions of Smad3,Smad7 mRNA by HSCThe ratio of Smad3 mRNA/β-actine at the 0 hour is 0.524±0.022,The ratios of Smad3 mRNA/β-actine of group TGF-β1 at the 2,4,8,16,24 hours are respectively 0.591±0.039,0.761±0.023,0.896±0.033,0.779±0.020,0.648±0.037;The ratios of Smad3 mRNA/β-actine of group IFN-α+ TGF-β1 at the 2,4,8,16,24 hours are respectively 0.562±0.038,0.593±0.033,0.705±0.031,0.647±0.020,0.579±0.024.The expression of Smad3 mRNA increased with time and peaked at 8 hour with the effect of TGF-β1.Since then,the expressions began to decrease.The variances of mRNA between the 4,8,16,24 hour and 0 hour are statistically significant(P<0.05);The mRNA level of IFN-α+ TGF-β1 group was lower than that of TGF-β1 group at the same time point,the variances at the 4,8,16 hour are statistically significant(P＜0.05).The ratio of Smad7 mRNA/β-actine at the 0 hour is 0.513±0.135,The ratios of Smad7 mRNA/β-actine of group TGF-β1 at the 2,4,8,16,24 hours are respectively 0.604±0.034,0.764±0.022,0.855±0.027,0.775±0.032,0.668±0.033;The ratios of Smad7 mRNA/β-actine of group IFN-α+ TGF-β1 at the 2,4,8,16,24 hours are respectively 0.672±0.029,0.949±0.038,1.126±0.026,0.933±0.015,0.631±0.088.The expression of Smad7 mRNA increased with time and peaked at 8 hour with the effect of TGF-β1.Since then,the expressions began to decrease.The variances of mRNA between the 4,8,16 hour and 0 hour are statistically significant(P＜0.05);The mRNA level of IFN-α+ TGF-β1 group was higher than that of TGF-β1 group at the same time point,the variances at the 4,8,16 hour are statistically significant(P＜0.05). 2.The expression of Smad3,Smad7 protein by HSCThe ratio of Smad3 protein/β-actine at the 0 hour is 0.291±0.031,The ratios of Smad3 protein/β-actine of group TGF-β1 at the 2,4,8,16,24 hours are respectively 0.321±0.055,0.393±0.034,0.569±0.035,0.556±0.034,0.395±0.022;The ratios of Smad3 protein/β-actine of group IFN-α+ TGF-β1 at the 2,4,8,16,24 hours are respectively 0.307±0.072,0.390±0.029,0.451±0.027,0.433±0.022,0.330±0.026, The expression of Smad3 protein increased with time and peaked at 8 hour with the effect of TGF-β1.Since then,the expressions began to decrease.The variances of protein between the 4,8,16,24 hour and 0 hour are statistically significant(P＜0.05); The protein level of IFN-α+ TGF-β1 group was lower than that of TGF-β1 group at the same time point,the variances at the 4,8,16 hour are statistically significant (P＜0.05).The ratio of Smad7 protein/β-actine at the 0 hour is 0.18±0.010,The ratios of Smad7 protein/β-actine of group TGF-β1 at the 2,4,8,16,24 hours are respectively 0.201±0.014,0.270±0.043,0.422±0.035,0.330±0.042,0.259±0.027;The ratios of Smad7 protein/β-actine of group IFN-α+ TGF-β1 at 2,4,8,16,24 hours are respectively 0.182±0.011,0.303±0.031,0.528±0.049,0.437±0.037,0.304±0.030.The expression of Smad7 protein increased with time and peaked at 8 hour with the effect of TGF-β1.Since then,the expressions began to decrease.The variances of protein between the 4,8,16,24 hour and 0 hour are statistically significant(P＜0.05);The protein level of IFN-α+ TGF-β1 group was higher than that of TGF-β1 group at the same time point,the variances at the 4,8,16 hour are statistically significant(P＜0.05).Conclusion1.TGF-β1 upregulated the expression of Smad3 mRNA and protein by HSC, also,the effects were time dependence.2.TGF-β1 upregulated the expression of Smad7 mRNA and protein by HSC, also,the effects were time dependence.3.IFN-αinhibitted the expression of Smad3 and upregulated the expression of Smad7 by HSC at mRNA and protein level excited by TGF-β1 so that it could inhibit the progression of hepatic fibrosis.
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