Effects Of IFN-α On The Expressions Of ECM And TGF-β₁ In Hepatic Stellate Cell Excited By PDGF-BB

ObjectivesHepatic fibrosis is a common pathological change of chronic liver disease by a variety of etiological factors. The dominant feature of hepatic fibrosis is increasing of ECM and the change of ingredient. At present, researches had indicated that the key point and central link of hepatic fibrosis is the activation and proliferation of HSC.HSC has the ability to secrete ECM and cytokines, and can contract. It is known that HSC plays main role in the development of the cirrhosis and portal hypertension. Previous studies show that cytokine plays significant role in the formation of hepatic fibrosis. Transforming growth factor-(β₁ (TGF-β₁) plays a crucial role in hepatic fibrosis development.Platelet-derived growth factor (PDGF) as HSC's drastic mitogen can promote disintegration and proliferation of HSC, and transform HSC to fibroblast which can synthesizes massive ECM. PDGF plays main role in the development of hepatic fibrosis.ECM contain collagen,in-collagen glycoprotein and proteoglycans, mainly by collagen. In normal conditions, the secretary collagen by HSC is mainly collagen III,IV, only synthesizes the few collagen I. When liver is damaged, HSC is activated to synthesize profuse ECM which is mainly collagen I,III.Collagen I can promote the activation and proliferation of HSC, inhibit the proliferation of hepatic cell and antro-endothelial cell, promoting the development of hepatic fibrosis. At present, IFN-αas antiviral medicine have been used in clinical treatment. But the exact mechanism of anti-hepatic fibrosis is still uncertain. We will provide new theory for the clinical anti-hepatic fibrosis treatment by investigating the effect of IFN-αon the expressions of Collagen I and TGF-β₁ in hepatic stellate cell stimulated by PDGF-BB and it maybe the possible antifibrogenic mechanism. Material and methods1. material(1) rat hepatic stellate cell strain(r-HSC99):laboratory of Infectious Diseases, Affiliated Shengjing Hospital of China Medical University. (2) main reagent: Recombinant Human Interferon Alpha 2b(IFN-a 2b Human): (Prospec company); Platelet-derived growth factor-BB(PDGF-BB): (Peprotech Asia company); MTT: (Boster biotechnology wuhan); DMEM high glucose culture fluid,fetal bovine serum:(Gibco company American); agarose:(Sigma company); Trizol RNA extract kit:(Invitrogin company);RT-PCR kit,β-actin primer,Col-I primer,TGF-β₁ primer: (TaKaRa Biotechnology Dalian); DNA marker (MBI company).2. methodsHepatic stellate cells (rHSC-99) cultured in vitro were exposed to various concentrations of IFN-αand/or PDGF-BB, HSC proliferation was measured by MTT. The levels of Col-I mRNA and TGF-β₁ mRNA were measured by the quantitative reverse-transcription polymerase chain reaction (RT-PCR).3. statistical analysisanalysis of variance by SPSS V13.0 software.Results1. The results of MTTHepatic stellate cell (HSC) was exposed to various concentrations of IFN-α(0.4,0.2,0.1,0.05,0.025,0.0125ng/ml) and /or 20ng/ml PDGF-BB. We set up control group. The absorbances are detected by MTT. The difference of 20ng/ml PDGF-BB and these four groups of (0.4,0.2,0.1,0.05 ng/ml)IFN-α+PDGF-BB were significant compared with the control group (p<0.05). The absorbance of the group of PDGF-BB was higher than that of the control group. Absorbances of these four groups of (0.4,0.2,0.1,0.05 ng/ml)IFN-α+PDGF-BB were lower than that of the control group. It indicate that PDGF-BB promote the proliferation of HSC. The proliferation of HSC had no remarkable change when HSC was exposed only to IFN-α., but the proliferation of HSC was remarkably inhibited when HSC was exposed to both PDGF-BB and different concentrations of IFN-α. Furthermore, the inhibition of HSC's proliferation was dose-dependent in the range of 0.025ng/ml to 0.1ng/ml.2. The results of RT-PCRHepatic stellate cell (HSC) was exposed to various concentrations of IFN-αand/or PDGF-BB 12hr, 24hr, 48hr and 72hr. Then the levels of Col-1mRNA and TGF-β₁mRNA were measured by the quantitative reverse-transcription polymerase chain reaction (RT-PCR). The levels of Col-I mRNA and TGF-β₁mRNA of the group of PDGF-BB(20ng/ml) were higher than that of corresponding control group at 12hr, 24hr, 48hr and 72hr.These differences were statistically significant (p<0.05). The levels of Col-I mRNA and TGF-β₁mRNA of these groups of(0.2,0.1,0.05ng/ml)IFN-α+ PDGF-BB were lower than that of corresponding control group at 12hr, 24hr,48hr and 72hr. These differences were statistically significant (p<0.05). Furthermore, with the increasing of IFN-αconcentrations and time prolonging, the differences were more significant. While the difference between the levels of the other groups and the control group were not statistically significant. PDGF-BB increased the expression of Col-I mRNA and TGF-β₁ mRNA. The expression of Col-I mRNA and TGF-β₁mRNA had no remarkable change when HSC was only exposed to IFN-α., but the expression of Col-I mRNA and TGF-PimRNA were remarkably inhibited when HSC was exposed to both PDGF-BB and IFN-α. Furthermore, the effects were dose- dependent and time-dependent.Conclusions1. The proliferation of HSC and the expression of Col-I mRNA and TGF-β₁ mRNA had remarkably increased when exposed to PDGF-BB;2. The proliferation of HSC and the expression of Col-I mRNA and TGF-β₁ mRNA had no remarkable change when HSC was exposed only to IFN-α;3. The proliferation of HSC and the expression of Col-I mRNA and TGF-β₁ mRNA were remarkably inhibited when HSC was exposed to both PDGF-BB and IFN-α. Furthermore, the effects were dose dependent and time dependent.