The Role Of RGD-Dependent Integrin Signaling Pathways In Regulating Collagen Metabolism During Fibrosis In Rats And In HSCs And Effect Of Salvia Miltiorrhiza Monomer IH764-3 On Them

Hepatic fibrosis is defined by increased synthesis and deposition of extracellular matrix (ECM) components in response to chronic liver injury of various causes and represents the common end point of the majority of chronic liver injuries. It gives rise to significant morbidity and mortality. The initiating events include viral hepatitis, alcohol abuse and bile duct obstruction. At the cellular level there is now a wealth of evidences indicating that the hepatic stellate cells (HSCs) represent the pivot of the fibrotic process. In normal liver, HSCs store vitamin A and show minimal proliferation and collagen synthesis. However, in injured liver, HSCs lose vitamin A and transform to myofibroblastic cells, termed as activated HSCs, which expressα-smooth muscle actin (α-SMA) and synthesize various extracellular matrix protein components.In addition to expressing matrix proteins, particularly type I collagen, activated HSCs also express matrix degrading metalloproteinases and the potent metalloproteinase inhibitors tissue inhibitor of metalloproteinases. Members of the collagenase subgroup of MMPs, i.e. MMP-1(in human)and MMP-13(in rat), are the principal neutral proteinases capable of degrading native fibrillar collagens in the extracellular space. And TIMP-1 is the main inhibitor of them. A lot of cytokines and growth factors may affect the activation of HSCs, however, the interaction between the ECM and HSCs plays a role as important as the factors. The integrins are widespread transmembrane glycoproteins on the surface of the cells and the main receptors of ECM, so they are responsive for the exchange of the information between intracellular and extracellular. Integrins may participate in the modulation of MMPs and TIMPs induced by ECM.A number of observations strongly suggest that ligand occupancy,integrin receptor clustering and their combination trigger the aggregation of focal adhesion kinase (FAK) in focal adhesion plaque(FAP) and autophosphorylation of FAK at Tyr397 in the N-terminal domain. The activation of FAK may initiate many of the signaling pathways. Among them, RAS-dependent mitogen-activated protein kinase (MAPK) pathway is the clearest. Extracellular signal-regulated kinase1/2 (ERK1/2) is a member of MAPK family. The RAS-RAF-MEK-ERK pathway is involved in many cellular behaviors. We set about our research from the interaction of HSCs and ECM. Through experiments in vivo and in vitro,the roles of integrin signal pathways in production of MMP-13 and TIMP-1 were studied in hepatic fibrosis and HSCs. The experiments contained four parts as below:Part 1: The Dynamic Expression of MMP-13/TIMP-1 during Hepatic Fibrogenesis in RatsObjective:To investigate the dynamic expression of MMP-13 and TIMP-1 and the relation with the deposition of ECM, especially collagenⅠin the hepatic fibrogenesisMethods:A rat model of common bile duct ligation (BDL)-induced hepatic fibrosis was used to assess the expression of collagenⅠ, collagen-degrading proteinase MMP-13 and its inhibitor-TIMP-1 during the progression of fibrosis. Distributions of collagenⅠin the livers were assessed immunohistochemistrically. Expressions of MMP-13 were evaluated by RT-PCR and TIMP-1 was analyzed by in-situ hybridization at mRNA level and by Western blot at protein level , respectively. While histopathological changes of the livers were evaluated both by hematoxylin and eosin staining and by Masson's trichrome method. The species were from liver tissue isolated at 2h,6h,2d,1wk,2wk,3wk and 4wk after BDL and sham operation. Results: ①Following BDL 1～4wk, the areas of collagen fibers increased from 10.41%±1.33% to 42.63%±1.68%, obviously larger than the sham operated group(7.35%±1.14%),P＜0.05; ②With the development of hepatic fibrosis, the deposition of collagen Ⅰ increased obviously. They mainly extended from portal tracts, and resided in the fiber septa and around the proliferated bile ducts. The areas around the central veins were stained positively. Typical pseudo-lo...