H₂O₂ Induced Apoptosis And The Metabolic Changes Of Extracelluar Matrix After Apoptosis In Hepatic Stellate Cells

Objective Liver fibrosis is a scarring process that is associated with an increased and altered deposition of extracelluar matrix in liver. Lots of aberram cytokins excreted from the activated Hepatic stellate cells ,such as transforming growth factor-β(TGF-β), insulin like growth factor-1(IF-1), which are capable of paracrine activation of HSC and of autocrine stimulation. Activation of HSC is characterized by contractility,proliferation,the increasing of extracellular matrix (ECM) ( including collagen, laminin ,proteoglycans)and endogenous tissue inhibitors of MMPs (TIMPs),the decreasing of matrix metalloproteinase(MMPs) ,So HSC become the center of liver fibrosis. Current evidence indicated that liver fibrosis could be reversed in the earlier period,so how to reverse liver fibrosis became the urgently problem for us. More and more investigation indicated that: to remove activated HSC by apoptosis were the key to reverse liver fibrosis. There were many ways to induce apoptosis, such as MMP-9 and COL I-degradation had the capability of inducing apoptosis. So we would had a presumption: If MMPs were increased, and collagenⅠwas degradated, those would not only induce apoptosis but also degradate the redundant ECM. So to investigate the change of ECM after the apoptosis of HSC was the precondition of our presumption.Methods HSC-T6 were cultured in DMEM containing 10% fetal bovine serum (FBS), then cultured in DMEM containing no FBS after passaged. HSC were allocated into normal groups, and model groups. Normal groups: N1: HSC were cultured in DMEM, and collected at 8h; N2: HSC were cultured in DMEM, and collected at 24h; N3 HSC were cultured in DMEM at 8h, then changed into fresh DMEM, collected at 24h. Model groups: M1: HSC were cultured in DMEM containing H2O2 (100nmol/L), collected at 8h; M2:HSC were cultured in DMEM containing H2O2 (100nmol/L), collected at 24h; M3: HSC were cultured in DMEM containing H2O2 (100nmol/L) at 8h, then changed into fresh DMEM, collected at 24h.The apoptosis of HSC-T6 was evaluated by flow cytometry.To measure the changes of TIMPs,MMPs and COLⅠⅢduring the different apoptosis levels,we choose 3 different levels of apoptosis groups(5% ,58% ,71% named group A,B,C).RT- PCR was carried out to determinate the gene expression levels of TIMP-1,TIMP-2,MMP-2,MMP-9,COLⅠ,COLⅢ;The Proteinum expression levels of TIMP-1 was analysed by Western blotting; MMP-2 and MMP-9 were shown by gelatin zymography.Results The apoptosis rates of three normal groups were 5.56%,13.75%,12.58%, respectively, and the apoptosis rates of model groups were 35.78%,71.98%,57.55% respectively.The changes of TIMPs,MMPs and COLⅠ,Ⅲin different apoptosis levels: TIMP-1 B>A>C; TIMP-2 A≈B>C;MMP-2 B≈C>A;MMP-9 B≈C>A;COLⅠA>B>C;COLⅢA>B>C; and the changes of COLⅠwas much obvious than COLⅢ;Conclusion During the natural apoptosis process of HSC-T6 , the difference apoptotic rates between group N1 and N2, group N1 and N3 were statistical significance ,But no statistical significance between N2 and N3 .So we could conclude that: during the process of culturing of HSC-T6, apoptosis increased over time,but no influence had been found from the replacement of fresh medium (reduced effects of ECM,TIMPs and MMPs on HSC) .After adding H2O2 to stimulate the apoptosis of HSC-T6 , the apoptosis rates between each two of M1,M2,M3 was statistical significance . With this phenomenon, we can get 3 possible inference: first: the capability of MMPs inducing the apoptosis of HSC was larger than that of TIMPs refraining the apoptosis; second:the absolute number of MMPs was larger than those of TIMPs; third: the infulence of other factors could promoting apoptosis,e.g the degradation of COLⅠ. so we could get the conclusion: the change of ECM,TIMPs,MMPs had effects on H2O2 induced the apoptosis of HSC.The metabolic changes of ECM in differents levels of apoptosis: at the earlier stage of apoptosis: the expression of TIMP-1 increased obviously, COLⅠCOLⅢgene expression were at high levels, but MMP-2,MMP-9 gene expression were at low levels, So the ability of anti-apoptosis was stronger than the ability of inducing apoptosis.The increased apoptosis also induced more TIMP-1 which inhibited MMPs excretion and COLⅠ,COLⅢdegradation. At the later stage of apoptosis, the expression of TIMP-1, COLⅠ,COLⅢdecreased sharply, but the expression of MMP-2,MMP-9 increased ,which inhanced the capability of apoptosis;Gene expression of TIMP-2 on low levels in the whole process of apoptosis.Therefore, we conclude that COLⅠ,COLⅢ, MMPs,TIMP-1 played an important role in the whole process of Apoptosis in HSC.