| In recent years,the non-alcoholic fatty liver disease(NAFLD)to human health hazards continue to increase,is a research hotspot and challenge in the field of contemporary medicine.This study was designed to investigate the regulatory mechanisms of Yap and TGF-β/Smad signaling pathway and their interactions on the development of NAFLD,which play an important role in the discovery of therapeutic targets of NAFLD.Monkeys high-fat feed free to eat for two years,liver puncture 4% paraformaldehyde were fixed and liquid nitrogen frozen,and fasting blood to do blood biochemical tests.HE,Masson and Oil Red O staining were used to screen for simple steatosis,non-alcoholic steatohepatitis(NASH)and liver fibrosis in monkeys.Immunohistochemical SABC blue staining was used to detect the expression of protein and its expression level.Real-time PCR The relative expression of each target gene;rat hepatocytes cultured in 5% CO2,10% FBS medium,MTT assay Verteporfin cell toxicity test;Real-time fluorescence quantitative PCR detection of the relative expression of the target gene of each cell;Western blot detection the amount of protein expressed by the cells.1.The pathological results of macaque liver biopsy showed that there were 23 normal Monkeys,45 degenerated hepatic steatosis,inflammatory cell infiltration,45 macaques which did not reach NASH in total ballooning,9 macaque reached NASH,and 0 19 macaques in the-1 phase and 5 macaques in the 2-3 phases.Blood biochemical statistics of monkeys after screening showed that FPG and TG were significantly increased(P <0.01)in macaques with NASH compared with monkeys with normal and simple fatty liver,no abnormalities were found in other groups.2.NASH monkey liver study results showed that the expression of YAP,TGF-β,Smad3,CTGF and α-SMA in livers of NASH monkeys were significantly higher than those in simple steatosis monkeys(P < 0.01).The expression of YAP,TGF-β,Smad3,CTGF and α-SMA in livers of simple steatosis monkeys were significantly higher than those in normal monkeys(P <0.01).The expression of Smad7 in the livers of NASH monkeys was significantly lower than that in simple fatty degeneration monkeys(P <0.01).The simple fatty degeneration monkeys were significantly lower than normal monkeys(P <0.01).the mRNA expression of YAP,TGF-β,Smad3,and Smad7 in the liver of NASH monkeys was significantly higher than that in simple fatty degeneration monkeys(P < 0.01).However,there was no significant difference in the expression of hepatic cells in the simple steatotic monkeys and normal monkeys(P> 0.05).The expression of CTGF mRNA increased with the severity of the disease,and the difference was extremely significant(P < 0.01).Compared with normal monkeys and simple hepatic steatosis monkeys,the expression of α-SMA mRNA in livers of NASH monkeys increased significantly(P < 0.01).3.Liver fibrosis study results showed that The expression of YAP,TGF-β,Smad3,CTGF and α-SMA in liver of hepatic fibrosis monkeys increased with the degree of hepatic fibrosis.The expression of YAP,TGF-β and α-SMA,CTGF were significantly different among the groups(P <0.05 or P <0.01).There was no significant difference in the expression of Smad3 between F1 a and F1c(P>0.05).Compared with the control group,the expression of Smad7 in hepatic fibrosis was significantly decreased(P < 0.05).The expression of Smad7 in F1 c was significantly higher than that in F2-3(P < 0.01).There was no significant difference in the expression of Smad7 between F1 a and F2-3(P>0.05).The mRNA expression of YAP,α-SMA,TGF-β and CTGF increased with the degree of fibrosis,and there was a significant difference among the groups(P <0.05 or P <0.01).There was no significant difference in the expression of CTGF mRNA between normal monkey group and F1a(P > 0.05).The expression of Smad3 mRNA in F1 c was significantly lower than F1 a,2-3 and normal monkeys(P <0.05 or P <0.01),but there was no significant difference in other groups(P > 0.05).The expression of Smad7 mRNA in F1 a was significantly higher than that in the other three groups(P < 0.01).There was no difference in the expression level of normal monkeys group and F2-3(P> 0.05).The expression of F1 c was significantly lower than that in normal monkeys group and F2-3(P < 0.01).4.Cell test results showed the result of MTT showed that when the Verteporfin concentration was 250 nmol/L,the survival rate of rat hepatocytes was the highest,and significantly higher than that of other concentrations.The results of Western blotting showed that protein content of Smad7 was significantly increased(P <0.01)compared with that without Verteporfin treatment.After adding Verteporfin,the protein content of Smad3,YAP decreased significantly(P <0.01).The result of real-time PCR showed that the expression of YAP mRNA in Verteporfin group was significantly lower than that in the non-Verteporfin group(P <0.05)at 0h and 8h,at 1h and 2h,the expression of YAP mRNA in rat hepatocytes without Verteporfin significantly decreased(P <0.01),at 0.5h and 4h,there was no significant difference in the expression of YAP mRNA between Verteporfin group and Verteporfin group(P> 0.05).The expression of Smad3 RNA in Verteporfin group was significantly higher than that in Verteporfin group at 0h,1h,2h,8h(P <0.01),and the expression of Smad3 RNA in Verteporfin group was significantly decreased at 4h(P <0.05)The difference was not significant(P> 0.05);the expression of Smad7 RNA in Verteporfin group at 0.5h,2h,4h,8h was significantly higher than that without Verteporfin group(P <0.01),and at 0h,1h,Verteporfin group was significantly higher than that without Verteporfin group(P <0.05).In monkeys NAFLD,key factors of YAP and TGF-β signaling pathway are involved in the important regulation of its development. |
