The Effect Of MMPs And Type Ⅰ Collagen During The Activation Of HSC And Its Intervation By IFN-α

objectLiver fibrosis is the excessive accumulation of extracellular matrix(ECM),its center is the activation of hepatic stellate cells(HSC),which is the main source of ECM. HSC can also excrete tissue inhibitor of metalloproteinase(TIMPs),which can inhibit the degradation of ECM,this would cause the imbalance between the synthesis and degradation of ECM,eventually facilitate the formation of hepatic fibrosis(HF).On the other hand ECM can also influence HSC trough other mechanism.During the process of HF the normal ECM like basement membrane was substituted by fibrotic matrix rich of typeⅠand typeⅢcollagens(Col-Ⅰand Col-Ⅲ),and this process can cause the change of liver constitution,more importantly it can activate HSC and maintain the state of its activation.Inflammatory factors play an important role in the fibrotic diseases.Evidence has shown that tumor necrosis factor(TNF-α) can induce the activation of pro-MMP-2 in dermal fibroblasts embedded in Col-Ⅰ,but Col-Ⅰand TNF-αalone had little influence on the activation of pro-MMP-2.Interleukin-1(IL-1) promoted the activation of HSC embedded in Col-Ⅰrequired the participation of matrix metalloprotease(MMPs),but Col-Ⅰand IL-1 alone can't cause the production of MMPs and the activation of HSC. TNF-α,one of the most important inflammatory factors,whether need the participation of other factors in the action of mediating HSC's activation is not clear.And also Interferon-α-2b(IFN-α-2b),a specific anti-fibrosis drug,has been generally applied in patients of hepatitis B and hepatitis C,better curative effect was obtained in patients of inflammatary stage.Whether the anti-fibrosis effect of interferon-αwas achieved by anti-flammatary factors is unknown.To provide more theoretical bases for the use of anti-fibrosis drugs,we determine Col-ⅠmRNA,MMP-2 mRNA,MMP-14 mRNA and protein MMP-2 secreted by HSCs in vitro by exposing to different combines of TNF-α,Col-Ⅰ,IFN-αat different time to expatiate some mechanisms of the activation of HSC and anti-fibrosis of IFN-α. Materials and methods1.(1)materials:rat hepatic stellate cell strain(r-HSC99),(2) main reagent: Interferon Alpha 2b(IFN-a 2b),tumor necrosis factor Alpha(TNF-a),Col-Ⅰprimer, MMP-2 primer,MMP-14 primer,Rabbit antimouse MMP-2 antibody,Rabbit antimouseβ-actin antibody and goat antirabbit antibody.2.methods:Hepatic stellate cell(rHSC-99) cultured in vitro was exposed to TNF-α(group TNF-α);Col-Ⅰ(group Col-Ⅰ);TNF-αand Col-Ⅰ(group TNF-αand Col-Ⅰ); TNF-α,Col-Ⅰ,IFN-α-2b(group TNF-α,Col-Ⅰand IFN-α-2b) and control group at 4h, 12h,24h,36h;The level of Col-ⅠmRNA,MMP-2 mRNA and MMP-14 mRNA was measured by the half-quantitative reverse-transcription polymerase chain reaction (RT-PCR).The level of protein MMP-2 was measured by western blotting.3.Statistical treatment:analysis of variance by SPSS software.Results1.The expression of Col-ⅠmRNA of HSC at different time:the level of Col-ⅠmRNA of the group Col-Ⅰwere higher than corresponding control group,this difference had statistical significance by analysis of variance(P＜0.05),the level of Col-ⅠmRNA of the group TNF-αwas similar to corresponding control group,this difference had no statistical significance(P＞0.05),the level of Col-ⅠmRNA of the group Col-Ⅰand TNF-αwas higher than the group Col-Ⅰ,this difference had statistical significance (P＜0.05),the level of Col-ⅠmRNA of the group Col-Ⅰ,TNF-αand IFN-αwas lower than the group Col-Ⅰand TNF-α,this difference had statistical significance(P＜0.05).2.The expression of MMP-2 mRNA of HSC at different time:the level of MMP-2 mRNA of group Col-Ⅰand group TNF-αwas similar to corresponding control group,this difference had no statistical significance by analysis of variance(P＞0.05), the level of MMP-2 mRNA of group Col-Ⅰand TNF-αwas higher than corresponding control group,this difference had statistical significance(P＜0.05),the level of MMP-2 mRNA of group Col-Ⅰ,TNF-αand IFN-αwas lower than the Col-Ⅰand TNF-α,this difference had statistical significance(P＜0.05).3.The expression of MMP-14 mRNA of HSC at different time:the level of MMP-14 mRNA of the group Col-Ⅰand group TNF-αwas similar to corresponding control group,this difference had no statistical significance by analysis of variance(P＞0.05),the level of MMP-14 mRNA of the group Col-Ⅰand TNF-αwas higher than corresponding control group,this difference was statistical significance (P＜0.05),the level of MMP-14 mRNA of the group Col-Ⅰ,TNF-αand IFN-αwas lower than the group Col-Ⅰand TNF-α,this difference had statistical significance (P＜0.05).4.The level of pro-MMP-2 protein of HSC in different groups was detected at 24h, active MMP-2 protein can be detected in the group Col-Ⅰand group Col-Ⅰand TNF-α, when added IFN-αit can not be detected.Conclusions1.Col-Ⅰalone can increase the expression of Col-ⅠmRNA of HSC,the expression of Col-ⅠmRNA had no remarkable change when HSC was only exposed to TNF-α,but the expression of Col-ⅠmRNA had remarkably increased when HSC was exposed to both Col-Ⅰand TNF-α.2.The expression of MMP-2 mRNA had no remarkable change when HSC was only exposed to TNF-αor Col-Ⅰ,the expression of MMP-2 mRNA had remarkably increased when HSC was exposed to both Col-Ⅰand TNF-α,Col-Ⅰalone can stimulated the expression of active MMP-2 protein,TNF-αdo not have the effect.3.The expression of MMP-14 mRNA had no remarkable change when HSC was only exposed to TNF-αor Col-Ⅰ,the expression of MMP-14 mRNA had remarkably increased when HSC was exposed to both Col-Ⅰand TNF-α.4.IFN-αcan remarkably inhibit the expression of Col-ⅠmRNA,MMP-2 mRNA,MMP-14 mRNA and active MMP-2 protein by HSC induced by Col-Ⅰand TNF-α.This may be one way IFN-αeduce the effect of anti-hepatic fibrosis.