The Role And Mechanism Of Osteopontin In The Rat Non-alcoholic Fatty Liver Fibrosis

Non-alcoholic fatty liver disease (NAFLD), including simple fatty liver (NAFL), nonalcoholic steatohepatitis(NASH), liver fibrosis and cirrhosis ,is a group of clinicopathologic syndrome. The prevalence of NAFLD is increasing, which does serious harm to the health of people, but the pathogenesis of NAFLD is not clear. It is the traditional concept that NAFL is benign lesions, but it is recently reported that the NAFL fat healthy liver cells are not static in that it can be the existence of hepatic stellate cell activation and stimulation of apoptosis. Meanwhile, NASH can progress to liver fibrosis, cirrhosis, and some studies have shown that NASH patients with severe obesity were found significantly associated with liver fibrosis as high as 30%～42%. Therefore, a study of non-alcoholic fatty liver fibrosis is of great significance.Osteopontin (OPN) is the secreted phosphorylated glycoprotein containing RGD (Arg - Gly - Asp) integrin-binding domain ,which can be synthesized and secreted by various cells and involved in many physiological and pathological process. Previous studies have shown OPN is related to fibrosis. However, OPN involved in non-alcoholic liver fibrosis and the incidence of specific molecular mechanism has been rarely reported. The study showed that OPN combined with the integrinβ3 can activate downstream signaling molecules which activated cells phosphorylation cascade, controlling cell biological effects. FAK is an important downstream signal molecules with the OPN signaling transduction. FAK, a non-receptor tyrosine kinase, is a number of signaling pathway convergence points in vivo, which play an important role in the development of cell adhesion, migration , proliferation, apoptosis and cytoskeleton reorganization. We use high-fat diet-induced NAFLD pathological process in rats to observe the expression of OPN, integrinβ3 and FAK in non-alcoholic fatty liver fibrosis progression and the relationships in particular with the HSC activation and liver fibrosis. In this paper, the effect and possible mechanism of OPN in rat nonalcoholic fatty liver fibrosis is explored ,try to find a new target for the effective prevention and treatment of clinical disease.Method:(1)To establish a rat fatty liver fibrosis model by a high fat feeding : A total of 56 Wistar rats (4 weeks old, male)were randomely divided into the basic diet-control group (nomal control group,Group C),the high-fat diet group(model group,Group F)and the high-fat diet group rats were subdivided into 6 subgroups (4,8,12,16,20 and 24 weeks). Each subgroup had 8 rats.(2) The following parameters were observed dynamically in each group:①changes of liver pathology by HE and Masson staining with microscopy;②expression of collagen typeⅠ,Ⅲby picric acid Sirius red staining③changes of the serum triglyceride (TG), free fatty acid (FFA) , alamine-aminotrasferase (ALT), and aspartate- aminotrasferase (AST);④hepatic expression ofα-SMA were observed by immunohistochemistry ;⑤The expression of OPN and Integrinβ3 and FAK was detected by RT-PCR and Western blot。Results:1,HE and Masson staining: for group C, no abnormality was found in rat liver; after 4 weeks, rat liver in Group F displayed fatty degeneration,up to F(0-1); after 8 weeks, mild to moderate fatty degeneration appeared, up to F(1-3); In addition to mild to moderate steatosis, in the 12th week, some rats had been progress in non-alcoholic steatohepatitis, up to NASH-F (1-4)G(1-2)S0; in the 16th -20th weeks, the rats showed moderate - severe fatty liver, part of rat liver tissue showed sinus Week / cell-week fibrosis, up to NASH-F(2-4)G(1-3) S(0-2); in the 24th week, the rats with severe fatty liver, inflammation in rats compared with the 16th week was reduced, sinus weeks / cell-week fibrosis in all rats, yet can be seen bridging fibrosis-like, up to NASH-F(2-4)G(1-2)S(1-3).2,Picric acid Sirius red staining: liver tissue in Group C rat contained a small amount of collagen fibers, existing in the central vein, the portal area, small blood vessel wall.The expression of collagen in group F was increasing with feeding time, the expression of typeⅢcollagen in 16th-week rat was significantly increased, accompanied by a small amount of increase in the expression of typeⅠcollagen, mainly in the portal area, and some liver tissue sinus Chow shows the expression of collagen. In the 24th week the hyperplasia of typeⅠcollagen in liver tissue predominates, and some collagen fibers separated hepatic lobules forming false lobules.3,Serological indicators: Group F serum TG level in the 4th to 16th weeks was significantly higher (P <0.05 or P <0.01), and decreased in the 20th week and the 24 week, compared with the insignificant difference in the control group. For Group F, serum FFA levels have increased significantly since the 12th week till 24th week (P <0.05). For group F, serum AST levels increased in the fourth week, but with huge differences among individuals. Therefore, compared with the control group, there was no significant difference, from the 16th to the 24th week compared with the control group were significantly different (P <0.05 or P < 0.01). Still for Group F, serum ALT levels in the 4th week was significantly higher (P <0.01), from the 12th to the 24th week it is still higher than that in the normal control group (P <0.05 or P <0.01).4,Immunohistochemical staining:α-SMA in Group C and Group F, in the 4th and 8th week displayed only a small amount of expression in the vessel wall, showing Light brown yellow staining. In addition to positive expression of the vascular wall found, in Group F, in the 12th week, the lobule can be seen in the expression of a small number of positive cells, scattering brown Light brown stain. In the 16th week, a number of positive cells began to increase in fiber bundles in the colored part of the deep sediment, in the 20 week, a further increase was found in positive staining, in the 24th week the number of positive cells, staining deep , showed brown positive cells located in portal area, liver sinusoids and the fiber spacing.5,RT-PCR and Western blot results: In Group F ,rat liver tissue expression of OPN mRNA and protein was gradually increasing, compared with the control group in which significant difference appeared from the 12th to the 24th week (P <0.05 or P <0.01). Group F liver tissue Integrinβ3 mRNA and protein expression are increased, but did not change significantly in the 4th to 16th week, compared with the insignificant difference in Group C,in the 20the to the 24th week, Integrinβ3 mRNA and protein expression was significantly increased, and there was a significant difference compared with Group C(P <0.01).In group F,rat liver tissue FAKmRNA and protein expression in FAK-PY397 gradually increased with the feeding time , Protein expression of FAK-PY397 dropped in the 24th week compared with appearance of significant difference from the 16th to the 24th week in the control group (P <0.05 or P <0.01). Conclusion:1,Replicate the animal model of non-alcoholic fatty liver fibrosis similar to the human NASH and liver fibrosis.2,High-fat diet can induce OPN expression in liver tissue, and its high expression and non-alcoholic liver fibrosis are closely related, OPN plays an important role in the process from simple fatty liver to steatohepatitis and liver fibrosis.3,OPN involved in non-alcoholic fatty liver fibrosis may be Integrinβ3-FAK pathway.