| BackgroundLiver fibrosis is an excessive accumulation of extracellular matrix (ECM) proteins including collagen, and could occur in most types of chronic liver diseases. Currently, liver fibrosis is considered as a physiological wound-healing response to chronic liver injury, concomitantly with the processes of repair and regeneration.Hepatic stellate cells (HSCs) activation is a critical event in fibrosis. HSCs are activated by fibrogenic cytokines such as PDGF, IL-6, TGF-β and TNF-α. Meanwhile, activated HSCs can secrete a series of cytokines and growth factors, such as TGF-β, PDGF, osteopontin (OPN), leptin, FGF, HGF and angiotensin Ⅱ, which are beneficial for sustaining the active status. Among those cytokines, TGF-β is considered the most potent fibrogenic cytokine in the liver, which stimulates the synthesis of ECM proteins.Full length OPN (OPN-FL) is a matricellular soluble protein and a matrix-bound phosphoglycoprotein. At the center of the molecule, OPN-FL contains a classical cell-binding motif, an arginine-glycine-aspartic acid (RGD) domain that is recognized by cell surface including αvβ3, αvβ5, α4β1 and α8β1 integrins. These RGD-recognizing integrins are expressed by variety of cells including fibroblasts, smooth muscle cells, endothelial cells, epithelial cells and immune cells. Next to RGD domain, OPN-FL can be cleaved by thrombin. It has been shown that OPN-FL cleaved by thrombin (Thr-OPN) exposes an additional integrin-binding motif, SVVYGLR (SLAYGLR in mice) sequence, and this motif is recognized by integrins, such as α4β1 and α9β1 integrins.Liver fibrosis could occur in most types of chronic liver diseases such as chronic HBV infection, chronic HCV infection, nonalcoholic steatohepatitis (NASH), alcoholic liver disease (ALD) and primary biliary cirrhosis (PBC). Recent reports have indicated that the elevated plasma OPN-FL level is predictive of liver cirrhosis, via inducing the activation of HSCs. In short, OPN is involved in liver fibrosis of most types of chronic liver diseases. However, the mechanisms of OPN regulate the progress of liver fibrosis remain unclear.Thr-OPN exposes an additional integrin-binding motif, SWYGLR (SLAYGLR in mice) sequence, promoting the adherence of cells expressing a4 and a9 integrins. Thr-OPN has been implicated in the pathogenesis of alcoholic hepatitis and ConA-induced hepatitis in mice. Moreover, recent study reported that thrombin-cleaved OPN enhanced the synthesis of collagen type III in myocardial fibrosis.However, little evidence can be required to explain the role of thrombin-cleaved form of OPN in human disease, and less is known about the function of thrombin-cleaved OPN in liver cirrhosis. Therefore, in this study, we explored to determine the expression of Thr-OPN in liver cirrhosis and whether the thrombin-cleaved form of OPN critically contributed to liver cirrhosis.Part 1:The expression of Thr-OPN in liver fibrosis and its influence on the activation and function of HSCsAims:To determine the expression of Thr-OPN in liver fibrosis and to investigate its influence on the activation and function of HSCs.Methods:Samples were collected from health volunteers, chronic hepatitis B patients and liver cirrhosis patients, and then Thr-OPN, OPN-FL and TGF-β expression were detected by ELISA. Meanwhile, the correlations between plasma Thr-OPN and non-invasive markers of fibrosis (HA, PCIIINP and CFV) as well as Thr-OPN and TGF-β levels were performed. Moreover, the influence of Thr-OPN on HSCs activation, proliferation, migration and collagen synthesis were determined by western blot, PCR, MTS and Cell ASIC Microfluidic Chemotaxis Assay.Result:Plasma Thr-OPN levels in liver cirrhosis patients were obviously higher than those in health volunteers and chronic hepatitis B patients. The expression of Thr-OPN was positively correlated with the levels of non-invasive markers of fibrosis (HA, PCⅢNP and CⅣ) as well as TGF-β. Moreover, compared with OPN-FL, Thr-OPN presented a more powerful ability in promoting HSCs activation, proliferation and migration as well as the ability producing collagen.Part 2:CC14 induced liver fibrosis model was established to investigate the role of Thr-OPN in liver fibrosis progressionAims:To determine the role of Thr-OPN in the progression of liver fibrosis induced by CC14 injection in mice models.Methods:CC14-induced liver fibrosis in mice model was established. The expressions of Thr-OPN and OPN-FL in liver were detected by ELISA; the degree of liver fibrosis was evaluated by Masson trichromatic staining; HSCs activations in liver tissue were determined by western blot, PCR and immunohistochemistry. Moreover, integrins α4 and α9 exprssions in liver tissue of human and mice were detection by western blot, PCR, immunohistochemistry and immunofluorescence. And integrin α4 and α9 expressions of LX2 cells after stimulation of Thr-OPN and OPN-FL respectively, were determined by western blot.Results:Thr-OPN levels in liver tissue were gradually elevated in liver fibrosis progression model. The expression of Thr-OPN was positively correlated to the degree of liver fibrosis, HSCs activation and collagen expression, respectively. Moreover, integrins α4 and α9 exprssions in liver tissue were increased in liver cirrhosis patients and gradually elevated in liver fibrosis progression model. Also, the activated HSCs after Thr-OPN stimulation could express high levels of integrin α4 and α9 compared with OPN-FL stimulation, which is beneficial for Thr-OPN involved in promting the progress of liver fibrosis.Part 3:OPN-deficient mice and the effective peptide from thr-OPN were used to further investigate the role of Thr-OPN in liver fibrosis progressionAims:To further investigate the role of Thr-OPN in liver fibrosis progression, OPN-deficient mice and the effective peptide from thr-OPN were used in CC14-induced liver fibrosis in mice model.Methods:OPN-deficient mice were applied to induce liver fibrosis mice model by CCL4 injection. The effective peptide from thr-OPN (M5 peptide) was administrated to evaluate the degree of fibrosis by Masson trichromatic staining.Result:OPN deficiency could alleviate liver fibrosis. When the effective peptide from thr-OPN (M5 peptide) was administrated in OPN-deficient mice, the liver fibrosis became aggrevated in vivo.Conclusion:In our study, plasma Thr-OPN levels in liver cirrhosis patients were obviously higher than those in health volunteers and chronic hepatitis B patients. The expression of Thr-OPN was positively correlated with the levels of TGF-β. In vivo experiments indicated that Thr-OPN expression is associated with liver fibrosis degree, HSCs activation and collagen formation. OPN deficiency could alleviate liver fibrosis. When the effective peptide from thr-OPN (M5 peptide) was administrated in OPN-deficient mice, the liver fibrosis became aggrevated in vivo. Moreover, compared with OPN-FL, Thr-OPN presented a more powerful ability in promoting HSCs activation, proliferation and migration as well as the ability producing collagen. In addition, Thr-OPN could promote the high expression of a 4 and a 9 integrins in HSCs. Thus, we speculated that Thr-OPN was critically involved, via a9 and a4 integrins, in the pathogenesis of liver fibrosis and played an important role in promoting liver fibrosis progression. |
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