The Impacts Of Locostatin On HSC Collagen Metabolism

HSC has been considered as the primary source of ECM.Under normallycircumstance, HSC is in the quiescent state. HSC often manifests as quiescentlipid droplet which is rich in VitaminA. The basic functions are:(1)metabolizing and keeping VitaminA.(2)storing fat (3) compounding andsecreting matrix components such as collagen, gluco protein and proteinpolysaccharide.(4) compounding MMPs(matrix metalloproteinase) andTIMPs(tissue inhibitor of metallo proteinases).(5) showing the expression ofcell factor and receptor.(6) participating in the regulation of hepatic sinusoidalblood flow. HSC would be activated if the liver is damaged by inflammationand mechanical stimulation. The phenotype has been transformed fromquiescence to activation. On the one hand, HSC takes part into the formationof liver fibrosis and reconstruction of inner liver structure by hyperplasia andsecreting ECM, on the other hand, HSC could make cell contraction topressurize the inner space of hepatic sinusoid. After being stimulated byphysical or chemical factors, activated HSC would be transformed to MFC,and HSC would also be considered as the target cell in the process offibrillation under the effects of different kinds of factors.The continuous activation of HSC has been considered as the key link inthe development of liver fibrosis. As mentioned above, HSC takes part intothe formation of liver fibrosis and reconstruction of inner liver structure byhyperplasia and secreting ECM, at the same time, HSC could make cellcontraction to pressurize the inner space of hepatic sinusoid. The changes haslaid pathogenetic foundations of hepatic fibrosis and portal hypertension inpathological way. Initialization phase and retention phase are major phase inthe complex activation process of HSC. Retention phase is been considered asthe maintenance process of HSC activated phenotype and it results theformation of liver hepatic fibrosis.Collagen, as the major ingredient of ECM, increases in number for5-7 times than normal times in the process of liver fibrosis. Collagen I andcollagen III increase primarily. The number of collagen I increase moresignificantly than collagen III. The collagen I content is60%-70%. Theactivated HSC has the fibrocystic features. The amount of VA contained incytoplasm reduces and even run out. After cell contraction, the cell has beenturned into collagenous fiber. The relevant genetic information of endonuclearcollagen would be transformed into a peptide chain after transcription,translation and modification. Procollagen, which is constructed by3spiralstructural peptide chains, has been secreted to extracellular space afterintra-cellular composition. Procollagen has been transformed into insolublecollagen. The degradation of collagen is made by collagenase. Underphysiological PH and temperature conditions, collagenase could hydrolyze the3D helical structure of natural collagen without harming other kinds of proteinand tissue.RKIP (raf kinase inhibitor protein, RKIP) belongs to PEBPs(phosphatidylethanolalmine-binding proteins, PEBPs) family and existsextensively in different species. This kind of protein takes part into theregulation of many kinds of signal transduction pathway. Also, the proteincould act as factors of negative accommodation in signal pathwayRaf-1/MEK/ERK1,2. However, the over expression of RKIP could enhancethe transfer ability of HSC. Locostation, an ephedroxane derivative withoutantimicrobial effects, is the caffeic acid phenethylester of RKIP. The migrationof HSC would be inhibited remarkably. Hepatic stellate cell LX-2would bechosen as an object in the research on the influence on Locostatin to thesecretion of HSC collagen metablic enzyme MMPs and TIMPs. In this way,the feasibility of application of Locostatin would be evaluated in liver fibrotictherapy.Objective: Do research and exploration on the influence ofTGF-β1-activated Locostatin to the secretion of MMPs and TIMPs forcollagen metabolism enzyme of HSC, and make sure whether Locostatincould accelerate the reversion of hepatic fibrosis. Methodology: PCR method would be used to analyze some index andfeatures such as Type I collagen, Type III collagen, Type IV collagen, MMP-1,MMP-2, TIMP-1, TIMP-2, MMP-7and the expression of GAPDH DNA. Elisawould be used to analyze Type I collagen, MMP-1and TIMP-1. Western blotwould be used for analyzing Type I collagen, MMP-1, MMP-2, TIMP-1,TIMP-2, MMP-7, MMP-9and the expression of GAPDH protein.Result：(1) According to the expression of type I collagen protein inwestern blot, RT-PCR, ELISA, we can give the conclusion. Compared withcontrol group the expression of type I collagen in locostatin group decredse(1.377±0.032vs1.252±0.007)(1.000±0.015vs0.083±0.034)(0.976±0.034vs1.094±0.002), P<0.05.Compared with control group, the result of groupTGF-β1and group TGF-β1+Locostatin have indifference relation.(2)Inwestern blot、RT-PCR、ELISA,the expression of MMP-1and TIMP-1increasein the TGF-β1, locostatin and TGF-β1+locostatin group compared withcontrol group. There was no difference of MMP-1/TIMP-1in the groups.(3)In western blot, RT-PCR, ELISA, the expression of MMP-2increased in theTGF-β1group and no difference in other groups. The expression of TIMP-2increased in the TGF-β1, TGF-β1+locostatin, locostatin group compared withcontrol group. The expression of MMP-2/TIMP-2decreased in theTGF-β1+locostatin group and no difference in other groups.(4) In westernblot, the expression of MMP-7decreased in the TGF-β1, TGF-β1+locostatingroups compared with control group.and the expression of MMP-9decreasedin the TGF-β1, TGF-β1+locostatin and locostatin groups compared withcontrol group.Conclusion: Locostatin intervention fibrosis of liver cell,make to reducethe type I collagen type III collagen type IV collagen,may with the increase ofMMP-1.