Protective Effect Of Hepatic Knockout Foxo1 Gene On Hepatic Fibrosis Induced By Insulin Resistance

Objectives To investigate the role of Foxo1 in liver fibrosis induced by insulin resistance.Methods Twenty 2-month-old specific liver Foxo1 gene knock-out(L-F1KO)mice and twenty normal control mice were randomly divided into four groups: CNTR,L-F1 KO,CNTR+HFD and L-F1KO+HFD.CNTR+HFD and L-F1KO+HFD were given 60% high fat diet(HFD)for 4M to induce insulin resistance(IR),and CNTR and L-F1 KO groups were given maintain normal diet for the same time.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,respectively.The protein and/or gene expressions of ECMs,fibrosis-related genes,changes of both TGF-β1→Smad2/3 signaling pathway and IRS1/2→Akt→Foxo1 insulin signaling pathway in the livers were detected through Western-blot and/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;(3)Fasting and random blood glucose levels obviously went up;(4)HE and Masson staining showed that the hepatocytes were swollen with loose intercellular connections,fat granules were obvious,and fibrosis was appeared in the liver;(5)According to the results of Westernblot and Real-Time PCR,firstly,the expressions of extracellular matrix proteins,such as Collagen1 increased significantly,and Collagen3 showed an non-statistic increasing trend.The expressions of MMP2 increased,while MMP9 decreased,whose functions were promoting ECM degradation.Secondly,insulin signaling pathway IRS1/2→Akt→Foxo1in mouse liver was down-regulated,demonstrated by decreased IRS2 expression,and the reduced ratio of p-Akt/Akt and p-Foxo1/Foxo1.Thirdly,the TGF-β1→Smad2/3 signaling pathway was activated,exhibited by the increased expression of TGF-β1 and its downstream signaling molecules p-Smad2/3 and Smad2/3.2 After 4M of normal diet treatment,compared with CNTR mice,L-F1 KO mice showed,first,the expressions of Foxo1 and TGF-β1 genes at protein and m RNA levels dramatically went down;Second,the concentrations of both ALT and AST in serums were markedly reduced.3 After 4M of HFD stimulation,compared with CNTR+HFD mice,L-F1 KO +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 blood glucose and random blood glucose levels were non-statistically reduced.(4)HE and Masson staining revealed: the swelling degree of hepatocytes in L-F1KO+HFD mice was obviously reduced,the loose of intercellular connections were improved,the amount of fat granules was significantly decreased,and the accumulation of fibrosis was rescued;(5)The results of Western-blot and Real-Time PCR discovered that,first,the expression of extracellular matrix protein,such as Collagen1,was significantly decreased.The proteins,whose functions were promoting ECM degradation,such as MMP2,was decreased,while MMP9 was increased.Second,the signaling pathway TGF-β1→Smad2/3 related to fibrosis was weakened,demonstrated by TGF-β1 expression decreased at both m RNA and protein levels,and the ratio of p-Smad2/3 to Smad2/3 was also reduced.Conclusions 1 After stimulation of HFD,the liver insulin signaling pathway is impaired,dephosphorylation of Foxo1 is increased and activated.Thus insulin resistance is induced.At the same time,the liver function is impaired,and liver fibrosis is developed.Moreover,the fibrosis-related signaling pathway TGF-β1→Smad2/3 is activated.2 The expression of TGF-β1 gene is decreased when Foxo1 gene is specific knock-out in mouse liver.3 Liver specific knock-out of Foxo1 gene can improve impaired liver function and liver fibrosis induced by insulin resistance,which mechanism is related to inhibition of the fibrosisrelated signaling pathway TGF-β1→Smad2/3.Figure 25;Table 7;Reference 125...