| Objective To explore the protective effect of YAP gene knockout on liver fibrosis induced by insulin resistance and its mechanism.Methods Twenty 2-month-old specific liver YAP gene knockout(L-YKO)mice and twenty normal control(CNTR)mice were randomly divided into four groups: CNTR,L-YKO,CNTR+HFD,L-YKO+HFD: Among them,CNTR + HFD and L-YKO + HFD were given 60% high-fat diet(HFD)for 4 months to induce insulin resistance(IR),CNTR and L-YKO groups were given regular maintenance feed for 4 months.Then,after measurement of body weight,fasting blood glucose and random feed blood glucose concentration,all the mice were sacrificed under anesthesia.The liver tissues were weighed and the liver tissue samples were taken and stored.At the same time,serums were prepared and the concentrations of ALT and AST in the serums were detected respectively.The hepatic morphological changes were observed by HE staining,and the distribution of liver fibrosis evaluated using Masson staining.The protein and gene expressions of ECMs,fibrosis-related genes,YAP signaling pathway and IRS1/2→Akt→FoxO1 insulin signaling pathway in the livers were detected through Western-blot or Real-Time PCR respectively.Results 1 After 4M of HFD stimulation,compared with CNTR mice,CNTR+HFD mice exhibited:(1)the body weight and liver weight were significantly increased;(2)Both the concentrations of ALT and AST in the serums were significantly increased,and liver function was impaired;(3)HE and Masson staining showed that the hepatocytes were swollen with loose intercellular connections,fat drop were obvious,and fibrosis was appeared in the liver;(4)Fasting and random blood glucose levels obviously went up,and both glucose tolerance and insulin tolerance declined;(5)Liver glycogen synthesis gene,such as G6 pase,is significantly increased,and the expression of Glut2 gene,which is involving in glucose transpotation in the liver,is increased.All this suggested that hepatic glucose metabolism was abnormal;(6)The expression of extracellular matrix protein Collagen1 in the liver increased significantly.Although the expression of MMP9 that promotes the degradation of ECM was increased,while MMP2 was significantly reduced;(7)α-SMA,the marker gene for hepatic stellate cell activation,significantly increased at protein and gene levels,which suggested that hepatic stellate cells have been activated.In addition,TGF-β1,as a key factor involved in fibrosis,was also significantly increased at protein and gene levels;(8)Insulin signaling pathway IRS1/2→Akt→FoxO1 in mouse liver was down-regulated,demonstrated by decreased IRS2 expression,and the ratio of p-FoxO1/FoxO1 decreased significantly which indicated that FoxO1 dephosphorylation was increased and activated;(9)The expression of Ankrd1,the target gene of YAP signaling pathway was significantly increased.All these results indicated that the stimulation of a high-fat diet led to IR,abnormal glucose metabolism,liver fibrosis,impaired liver function,impaired insulin signaling,FoxO1 was activated,and the YAP target gene Ankrd1 was up-regulated.2 After YAP gene being specifica knockout in the liver,compared with that of CNTR mice,the protein levels of Foxo1 and TGF-β1 in L-YKO mouse liver were significantly reduced which implys that: YAP gene knockout in the liver can inhibit the expression of TGF-β1 and FoxO1.3 After 4M of HFD stimulation,compared with CNTR+HFD mice,L-YKO +HFD mice exhibited:(1)the body weight and liver weight were obviously reduced;(2)The concentrations of both ALT and AST in serums were statistically decreased;(3)The fasting and random blood glucose of mice were significantly reduced,and the glucose tolerance and insulin tolerance was improved;(4)HE and Masson staining revealed that: the swelling degree of hepatocytes in L-YKO+HFD mice was obviously reduced,the loose of intercellular connections were improved,the amount of fat drop was significantly decreased,and the accumulation of fibrosis was rescued;(5)Liver glycogen synthesis genes,such as Pepck and G6 pase,were significantly reduced,and the expression of Glut4 gene was increased,which suggested that the liver’s glucose metabolism ability has been improved;(6)The expression of ECM such as Collagen1 in the liver was significantly reduced,the expression of MMP2 and MMP9 that promoted the degradation of ECM were increased;(7)With the inhibion of YAP signaling pathway,the expressions of TGF-β1 and α-SMA at the protein level were reduced;(8)Ankrd1,the target gene of YAP,was further inhibited;(9)FoxO1 in the liver was inhibited.All these data indicated that the knockout of YAP gene in the liver can improve IR-induced liver fibrosis and liver function,and inhibit the activation of hepatic stellate cells,which the underlying mechanism are related to the inhibition of TGF-β1 and FoxO1.Conclusion Liver specific knockout of YAP gene has a protective effect on hepatic fibrosis and liver injury induced by insulin resistance,which underlying mechanisms are related to the inhibition of the activities of TGF-β1 and FoxO1.Figure 36;Table 6;Reference 122... |
PDF Full Text Request