| 1.Effect of hepatic stellate cells and hepatocyte FOXO1 specific knockout on CCl4-induced hepatic fibrosisBackground:Hepatic fibrosis is a pathological process of chronic liver injury.Most chronic liver diseases,including non-alcoholic fatty liver disease(NAFLD)and non-alcoholic steatohepatitis(NASH),have liver fibrosis,which is a serious threat to human health.The activation of hepatic stellate cells(HSC)is an important initiating link in the development of liver fibrosis.Forkhead box O1(FOXO1)not only plays an important role in the regulation of hepatic glycolipid metabolism homeostasis,but also participates in the regulation of HSC activation during liver fibrosis.Carbon tetrachloride(CCl4)is the most commonly used drug to construct animal liver fibrosis models.Therefore,we speculate that:HSC-specific and liver-specific expression of FOXO1 may have a significant contribution to CCl4-induced liver fibrosis.However,the effect of HSC and hepatic FOXO1 on the metabolic phenotype and liver fibrosis of CCl4-induced liver fibrosis has not been reported.Therefore,to test our hypothesis,the present study targeted knockout of hepatic stellate cell and hepatocyte FOXO1 expression by genetic engineering techniques,respectively,aiming to investigate the metabolic phenotypic effects and possible mechanisms of HSC-FOXO1-KO and LFOXO1-KO on CCl4-induced hepatic fibrosis.Methods:In this experiment,the Cre/LoxP recombinase system was used to construct the HSC-FOXO1-KO and L-FOXO1-KO mouse model as experimental groups,while age-and sex-matched wild type littermates(WT littermates)were screened as control groups.All strains were derived from C57BL/6J mice.To investigate the effects of HSC-FOXO1-KO and L-FOXO1-KO on glycolipid metabolism in CCl4-induced liver fibrosis mouse model,we divided the mice into four groups and all groups of mice injected CCl4 for 4 weeks:(i)male HSC-FOXO1-KO mice(n=13,6 weeks old)and male WT littermate mice(n=7,6 weeks old);(ii)female HSC-FOXO1-KO mice(n=11,6 weeks old)and female WT littermate mice(n=8,6 weeks old);(iii)male L-FOXO1-KO mice(n=13,6 weeks old)and male WT littermates(n=8,6 weeks old);(iv)female L-FOXO1-KO mice(n=12,6 weeks old)and female WT littermates(n=9,6 weeks old).The metabolic phenotypic changes including body weight(BW),blood glucose(BG),glucose tolerance and area under the curve of glucose(AUC),insulin tolerance,serum lipid levels[total cholesterol(TC),triglyceride(TG),non-esterified fatty acids(NEFA)],liver lipid profile(TC,TG),whole body mass distribution(fat mass,lean mass)and liver histopathology were used to investigate the metabolic phenotypic effects of HSCFOXO1-KO and L-FOXO1-KO on CCl4-induced liver fibrosis mice and the possible mechanisms.Results:(1)Effects of HSC-FOXO1-KO on body weight changes and blood glucose in CCl4-induced hepatic fibrosis miceDuring CCl4 injection,the body weight of male HSC-FOXO1-KO was significantly higher than that of WT mice from the 4 weeks of injection,while the body weight changes of female HSC-FOXOl-KO and WT mice were not significantly different.After 4 weeks of CCl4 injection,HSC-FOXO1-KO male mice body weight significantly higher than WT mice under non-fasting conditions(P<0.05),while there was no significant difference in body weight during fasting condition.The blood glucose was significantly lower than WT mice under both fasting and non-fasting conditions(P<0.05).There was no significant difference in body weight and blood glucose between KO and WT groups of female mice under fasting and non-fasting conditions.(2)Effect of HSC-FOXO1-KO on glucose tolerance in CCl4-induced liver fibrosis miceAfter 4 weeks of CCl4 injection,the glucose tolerance of HSC-FOXO1-KO male mice was significantly lower(P<0.05)than that of WT mice in terms of blood glucose levels at 90 and 120 min as well as AUC.HSC-FOXO1-KO female mice had significantly lower blood glucose levels at 120 min than WT mice,while no significant differences were found in blood glucose levels at other time points.Female KO group mice had lower AUCs than WT mice,but no significant differences were found.The results indicate that HSC-FOXO1-KO improved glucose tolerance in CCl4-induced hepatic fibrosis mice and had a more significant effect on male mice.(3)Effect of HSC-FOXO1-KO on insulin sensitivity in CCl4-induced liver fibrosis miceAfter 4 weeks of CCl4 injection,HSC-FOXO1-KO male mice had significantly lower blood glucose at 0 min and higher blood glucose at 120 min than WT mice(all P<0.01).When the degree of blood glucose decrease was presented as the percentage level of blood glucose at each time points versus 0 min,male HSC-FOXO1-KO mice had significantly lower blood glucose decreases at 90 min,120 min than WT mice(all P<0.01).The blood glucose level of female HSC-FOXO1-KO mice at 30 min was significantly higher than that of WT mice(P<0.05).When the degree of blood glucose decrease was presented as the percentage level of blood glucose at each time points versus 0 min,the degree of blood glucose decrease of female HSC-FOXO1-KO mice at 30 min was significantly higher than that of WT mice(P<0.05).This indicates that the effect of HSC-FOXO1-KO on insulin sensitivity in CCl4-induced liver fibrosis mice has gender differences,with male mice having significantly lower insulin sensitivity and female mice having slightly improved insulin sensitivity.(4)Effect of HSC-FOXO1-KO on body mass distribution in mice with CCl4induced liver fibrosisAfter 4 weeks of CCl4 injection,the absolute value of lean mass in male HSCFOXO1-KO mice was significantly higher than that in WT mice(P<0.05),and there were no significant differences in the absolute value of fat mass,percentage of fat mass and lean mass relative to body weight(%BW)and fat/lean ratio.There were no significant differences in absolute fat mass,%BW of fat mass and lean mass and fat/lean ratio in female HSC-FOXO1-KO mice compared with WT mice.This indicates that the effect of HSC-FOXO1-KO on the distribution of body fat in CCl4induced liver fibrosis mice was small,and only the lean mass in the KO group of male mice was higher than that in the WT group.(5)Effect of HSC-FOXO1-KO on serum lipid levels in CCl4-induced liver fibrosis miceOAfter 4 weeks of CCl4 injection,serum TC was significantly higher in both male and female HSC-FOXO1-KO mice than in WT mice,while serum TG was significantly lower,and more significant in males(P<0.05 for females and P<0.01 for males).Serum NEFA was significantly lower in male HSC-FOXO1-KO mice than in WT mice(P<0.05),while there was no significant difference in serum NEFA in female mice compared with the two groups.This indicates that HSC-FOXO1-KO had a significant effect on lipid levels in CCl4-induced liver fibrosis mice,and the effect was more significant in male mice.(6)Effect of HSC-FOXO1-KO on liver weight and liver lipid deposition in CCl4induced liver fibrosis miceAfter 4 weeks of CCl4 injection,the liver appearance of HSC-FOXO1-KO mice has no difference compared with WT mice.Body weight,absolute liver weight,and percent liver relative to body weight(%BW)were significantly higher in male HSCFOXO1-KO group mice than in WT group(all P<0.05).There were no significant differences in body weight,absolute liver weight,and percentage of liver relative to body weight(%BW)in female mice compared to the two groups.This indicates that there was a sex difference in the effect of HSC-FOXO1-KO on liver weight in mice with CCl4-induced liver fibrosis,with a significant increase in body weight and liver weight in male mice.The results of liver H&E staining showed that the two groups of mice showed insignificant hepatic steatosis.The liver TC content of mice in both male and female HSC-FOXO1-KO groups was significantly lower than that in WT group(P<0.01 for males and P<0.05 for females),while the liver TG content of mice in female HSC-FOXO1-KO group has no difference compared with WT group.This indicates that HSC-FOXO1-KO ameliorated hepatic lipid deposition in CCl4-induced hepatic fibrosis mice and had a more significant effect on male mice.(7)Effect of HSC-FOXO1-KO on the degree of liver fibrosis in CCl4-induced liver fibrosis miceAfter 4 weeks of CCl4 injection,Sirius Red staining and Masson Trichrome staining of liver slides showed that both groups of mice showed obvious liver fibrosis.Male HSC-FOXO1-KO mice showed less obvious liver fibrosis and the liver collagen level was significantly lower than that in the WT group(both P<0.05).In contrast,there were no significant differences in liver collagen levels,liver Sirius Red staining and Masson Trichrome staining in female HSC-FOXO1-KO group mice compared with WT group.It indicates that the effect of HSC-FOXO1-KO on the degree of liver fibrosis in CCl4-induced fibrosis mice was gender-specific,liver fibrosis was significantly improved in male mice and knockout of FOXO1 in male HSC had a protective effect on preventing liver fibrosis.(8)Effect of L-FOXO1-KO on body weight change and blood glucose in CCl4induced liver fibrosis miceAfter 4 weeks of CCl4 injection,the fasting body weight of L-FOXO1-KO male mice was significantly higher than that of WT group mice,and the fasting blood glucose was significantly lower than that of WT group mice(P<0.05),while KO group body weight changes during injection,body weight and blood glucose levels under nonfasting conditions after 4 weeks of CCl4 injection have no differences compared with WT group.The differences in body weight during injection,body weight and blood glucose levels under fasting and non-fasting conditions 4 weeks after CCl4 injection in L-FOXO1-KO female mice were not statistically significant compared to the WT group.(9)Effect of L-FOXO1-KO on glucose tolerance in mice with CCl4-induced liver fibrosisAfter 4 weeks of CCl4 injection,the glucose tolerance of male L-FOXO1-KO mice was significantly lower than that of WT mice at all points of blood glucose level and AUC(all P<0.05),and that of female L-FOXO1-KO mice was significantly lower than that of WT mice at 30 min,60 min,and 90 min(all P<0.001).This indicates that L-FOXO1-KO had significant effects on glucose tolerance in male and female mice with CCl4-induced liver fibrosis,and glucose tolerance was significantly improved in both KO groups.(10)Effect of L-FOXO1-KO on insulin sensitivity in mice with CCl4-induced liver fibrosisAfter 4 weeks of CCl4 injection,the blood glucose level at 30 min was significantly lower in male L-FOXO1-KO mice than in WT mice,and there was no significant difference between the two groups in female mice.When the degree of blood glucose decrease was presented as the percentage level of blood glucose at each time points versus 0 min,male L-FOXO1-KO mice showed significantly lower blood glucose decrease at 30 min,60 min,and 90 min than WT mice(all P<0.05),and there was no significant difference between the two groups in female mice.This indicates that the effect of L-FOXO1-KO on insulin sensitivity in female and male mice with CCl4induced liver fibrosis was gender-specific,with significant improvement in insulin sensitivity in male mice and no effect on female mice.(11)Effect of L-FOXO1-KO on body fat composition in CCl4-induced liver fibrosis miceAfter 4 weeks of CCl4 injection,there were no significant differences in the absolute value of fat mass,absolute value of lean mass,percentage of fat mass and lean mass relative to body weight(%BW)and fat/lean ratio in both male and female L-FOXO1KO mice compared with the WT group.It indicates that L-FOXO1-KO has no effect on body fat distribution in CCl4-injected mice.(12)Effect of L-FOXO1-KO on serum lipid levels in mice with CCl4-induced liver fibrosisAfter 4 weeks of CCl4 injection,serum TC was significantly higher in both male and female L-FOXO1-KO mice than in WT mice(all P<0.001),and serum TG was significantly lower in male L-FOXO1-KO mice than in WT mice(P<0.05).There was no significant difference in serum NEFA between male and female L-FOXO1KO mice compared with WT mice.This indicates that L-FOXO1-KO had a significant effect on lipid levels in CCl4-injected mice,with elevated serum TC and decreased TG in males,while only elevated serum TC in females.(13)Effect of L-FOXO1-KO on liver weight and liver lipid deposition in CCl4induced liver fibrosis miceAfter 4 weeks of CCl4 injection,the absolute liver weight(mg)as well as the percentage of liver relative to body weight(%BW)were significantly higher in the male L-FOXO1-KO group than in the WT group(all P<0.05),in the absence of significant differences in body weight(g)between the two groups of mice.There were no significant differences in body weight,absolute liver weight(mg),and percent liver relative to body weight(%BW)between the two groups of mice compared to the female group.It indicates that L-FOXO1-KO has gender differences on liver weight in female and male mice,and liver weight was significantly increased in male KO group mice,while liver weight was not affected in female mice.Liver H&E staining results showed that both groups of mice showed insignificant hepatic steatosis.Liver TG content was significantly higher in both male and female L-FOXO1-KO group mice than in WT group(P<0.01 for males and P<0.001 for females),and liver TC content was significantly lower in male L-FOXO1-KO group mice than in WT group(P<0.05).This indicates that L-FOXO1-KO had a significant effect on liver lipid profile in CCl4-induced liver fibrosis mice,and liver TG content was significantly increased in both male and female mice,and liver TC content was significantly decreased in male mice.(14)Effect of L-FOXO1-KO on liver fibrosis in CCl4-induced liver fibrosis mice After 4 weeks of CCl4 injection,liver Sirius Red staining and Masson Trichrome staining showed significant manifestations of liver fibrosis in both groups of mice,and the liver collagen levels,Sirius Red staining and Masson Trichrome staining of fibrotic areas in male and female L-FOXO1-KO group mice compared with WT group were not significantly different from each other.This indicates that L-FOXO1KO did not have a significant ameliorating effect on liver fibrosis in mice.Conclusion:(1)HSC-FOXO1-KO had significant effects on glucolipid metabolism in CCl4induced hepatic fibrosis mice and the effects were more pronounced in males:Compared with WT mice,the male HSC-FOXO1-KO mice had metabolic phenotypic changes such as increased body weight,improved glucose tolerance,decreased insulin sensitivity,and improved lipid metabolism.Compared with WT mice,the female HSC-FOXO1-KO mice had metabolic phenotypic changes such as increased insulin sensitivity and improved lipid metabolism.HSC-FOXO1-KO mice showed changes in metabolic phenotypes such as increased insulin sensitivity and improved lipid metabolism compared to WT mice.(2)L-FOXO1-KO had significant effects on glucolipid metabolism in CCl4-induced liver fibrosis mice and the effects were more pronounced in males:compared with WT mice,male L-FOXO1-KO mice showed changes in metabolic phenotypes such as improved glucose tolerance,increased insulin sensitivity,and improved lipid metabolism.Compared with WT mice,female L-FOXO1-KO mice had changes in metabolic phenotypes such as improved glucose tolerance and improved lipid metabolism.(3)HSC-specific knockout of FOXO1 attenuated the extent of hepatic lipid deposition in CCl4-induced hepatic fibrosis mice,whereas hepatocyte-specific knockout of FOXO1 significantly increased the extent of hepatic lipid deposition,and FOXO1 has different metabolic regulatory mechanisms for hepatic steatosis and lipid deposition in HSC and hepatocytes of hepatic fibrosis mice.(4)Hepatic fibrosis was only improved in male mice in the HSC-FOXO1-KO group,while no significant changes were observed in both male and female mice in the LFOXO1-KO group,indicating that FOXO1 in HSC of male mice promoted the development of hepatic fibrosis and played different regulatory roles from FOXO1 in hepatocytes in HSC activation and hepatic fibrosis,a finding reported for the first time in this study.FOXO1 in HSC may be a potential target for the treatment of liver fibrosis,but its specific mechanism needs to be further investigated and explored.2.Effect of hepatic stellate cell FOXO1 specific knockout on NASHBackground:With socio-economic development and lifestyle changes,the prevalence of NAFLD is rapidly increasing and often accompanied by a variety of metabolic diseases,such as obesity,type 2 diabetes and metabolic syndrome,etc.NASH is a developmental situation of NAFLD,accompanied by inflammation and steatosis with hepatocyte damage,which can lead to advanced liver fibrosis,cirrhosis,liver failure and liver tumor production,and is a serious threat to human health.Activation of HSC is an important initiating link in the process of NASH liver fibrosis.It was found that FOXO1 is not only a key regulator of glycolipid metabolism,but also involved in the regulation of hepatic lipid deposition and liver fibrosis.Therefore,we speculated:HSC-specific expression of FOXO1 may have a significant role in promoting hepatic lipid deposition and liver fibrosis in NASH.However,the effect of hepatic FOXO1 on hepatic lipid deposition and liver fibrosis in NASH has not been reported.Therefore,to test our hypothesis,the present study targeted knockout of hepatic stellate cell FOXO1 expression by genetic engineering technology,aiming to investigate the effect of HSC-FOXO1-KO on high fat high cholesterol(HFHC)dietinduced hepatic lipid deposition and hepatic fibrosis in NASH mice and the possible mechanism.Methods:In this experiment,the HSC-FOXO1-KO mouse model was constructed using the Cre/LoxP recombinase system as the experimental group,and age-and sex-matched wild type littermates(WT littermates)were screened as the control group.All strains were derived from C57BL/6J mice.To investigate the effect of HSC-FOXO1-KO on HFHC diet-induced glucolipid metabolism in NASH mice,we divided the mice into two groups,both of the group were fed HFHC diet for 16 weeks:(i)male HSC-FOXO1-KO mice(n=14,6 weeks old)and female HSCFOXO1-KO mice(n=13,6 weeks old);(ii)male WT littermates mice(n=9,6 weeks old)and female WT littermates mice(n=9,6 weeks old).The effect of HSCFOXO1-KO on glycolipid metabolism was investigated by assessing glycolipid metabolic indexes in both groups of mice.The metabolic phenotypic changes including body weight(BW),blood glucose(BG),glucose tolerance and area under the curve of glucose(AUC),insulin tolerance,serum lipid levels[total cholesterol(TC),triglyceride(TG),non-esterified fatty acids(NEFA)],24-h food intake,hepatic lipid profile(TC,TG),body mass distribution(fat mass,lean mass),liver histopathology and other metabolic phenotypic changes were used to investigate the effects of HSC-FOXO1-KO on liver lipid deposition and liver fibrosis in HFHCinduced NASH mice.Results:(1)Effects of HSC-FOXOl-KO on body weight changes and blood glucose in HFHC-induced NASH miceDuring HFHC feeding,there were no significant differences in body weight changes between HSC-FOXO1-KO and WT groups in both male and female mice.At the 16th week of HFHC feeding,female HSC-FOXO1-KO mice had significantly lower blood glucose levels than WT mice under non-fasting condition,while there were no significant differences in blood glucose under fasting condition and body weight under fasting and non-fasting conditions.There was no difference between male HSCFOXO1-KO mice and WT mice in body weight and blood glucose under fasting and non-fasting conditions.This indicates that the effect of HSC-FOXO1-KO on nonfasting blood glucose in HFHC-induced NASH mice is gender-specific and only female mice were significantly improved.(2)Effect of HSC-FOXO1-KO on glucose tolerance in HFHC-induced NASH miceAt the 16th week of HFHC feeding,male HSC-FOXO1-KO had significantly lower blood glucose levels than WT mice at 30 min,while there were no significant differences in blood glucose and AUC at other time points;there were no significant differences in glucose tolerance and AUC between female HSC-FOXO1-KO and WT mice.It indicates that HSC-FOXO1-KO slightly improved glucose tolerance in HFHC-induced NASH male mice,while it had no significant effect on female mice.(3)Effect of HSC-FOXO1-KO on insulin sensitivity in HFHC-induced NASH miceAt the 16th week of HFHC feeding,the blood glucose level of male HSC-FOXO1-KO mice was significantly lower than that of WT mice at 30 min,while the blood glucose level at 120 min was significantly higher than that of WT mice;when the degree of blood glucose decrease was presented as the percentage level of blood glucose at each time point versus 0 min,the degree of blood glucose decrease at 120 min in male HSC-FOXO1-KO mice was significantly lower than that in WT mice(all P<0.05).In female HSC-FOXO1-KO mice,the blood glucose level at 0 min was significantly lower than that of WT mice,while that at 60,90,and 120 min was significantly higher than that of WT mice(all P<0.05).The degree of decrease in blood glucose was significantly lower in female HSC-FOXO1-KO mice than in WT mice at 60,90 and 120 min(all P<0.05).It indicates that HSC-FOXO1-KO reduced insulin sensitivity significantly in HFHC-induced NASH mice and the reduction was more pronounced in female mice.(4)Effect of HSC-FOXO1-KO on body mass distribution in HFHC-induced NASH miceAt the 16th week of HFHC feeding,the absolute value of lean mass in male HSCFOXO1-KO mice was significantly higher than that in WT mice(all P<0.05),and the absolute value of fat mass,percentage of fat mass and lean mass relative to body weight(%BW)and fat/lean ratio in both groups of male HSC-FOXO1-KO and WT mice had no differences.The fat mass(%BW)and fat/lean ratio of female HSCFOXO1-KO mice were significantly higher than those of WT mice while the lean mass(%BW)was significantly lower than that of WT mice(P<0.05),and the absolute values of fat mass and lean mass had no differences.This indicates that HSCFOXO1-KO had a significant effect on body mass distribution in HFHC-fed female mice and the percentage of adipose tissue increased,while it had less effect on body mass distribution in male mice.(5)Effect of HSC-FOXOl-KO on serum lipid levels in HFHC-induced NASH miceAt the 16th week of HFHC feeding,serum TC,TG and NEFA were significantly lower in male and female HSC-FOXO1-KO mice than in WT mice(TC:P<0.001 in both female and male mice,TG:P<0.05 in male mice and P<0.001 in female mice,NEFA:P<0.05 in both female and male mice).It indicates that HSC-FOXOl-KO significantly improved the lipid levels in HFHC-fed mice and serum TC,TG and NEFA were significantly decreased.(6)Effect of HSC-FOXO1-KO on 24-hour food intake in HFHC-induced NASH miceAt the 16th week of HFHC feeding,the 24-h food intake(g)of female HSC-FOXO1KO mice was significantly higher than that of WT mice(P<0.05),but there was no significant difference in body weight and 24-h food intake relative to body weight(Kcal/gBW).Male HSC-FOXO1-KO mice also had higher 24-h food intake(g)than WT mice,but there was no significant difference in body weight and 24-h food intake relative to body weight(Kcal/gBW).This indicates that the difference in metabolic phenotypes between HSC-FOXOl-KO mice and WT group mice under HFHC feeding may not be related to food intake.(7)Effect of HSC-FOXO1-KO on the liver weight and liver lipid deposition of HFHC-induced NASH miceAt the 16th week of HFHC feeding,the absolute liver weight(mg)and the percentage of liver relative to body weight(%BW)were significantly higher in male and female HSC-FOXO1-KO mice than in WT group(all P<0.05),in the absence of significant differences in body weight(g)between KO and WT groups.It indicates that HSCFOXO1-KO can affect the increase of liver weight in HFHC-induced male and female NASH mice.The results of liver H&E staining and oil red O staining showed that both groups of mice showed significant hepatic steatosis,which was more pronounced in the WT group.The liver TC and TG contents of male and female HSC-FOXO1-KO group mice were significantly lower than those of WT group(all P<0.05).It indicates that HSC-FOXO1-KO can improve hepatic lipid deposition in HFHCinduced NASH mice.(8)Effect of HSC-FOXO1-KO on liver fibrosis in HFHC-induced NASH miceAt the 16th week of HFHC feeding,liver Masson Trichrome staining and Sirius Red staining showed that both groups of mice showed liver fibrosis but no significant difference.The liver collagen content of both male and female HSC-FOXO1-KO group mice was not significantly different from that of WT group.It indicates that HSC-FOXO1-KO may have no effect on liver fibrosis in HFHC-induced NASH mice.Conclusion:HSC-FOXO1-KO had significant effects on glucolipid metabolism in HFHC-induced NASH mice.Female HSC-FOXO1-KO mice showed changes in metabolic phenotypes such as decreased insulin sensitivity,altered body mass distribution,and improved lipid metabolism compared to WT mice.Male HSC-FOXOl-KO mice showed decreased insulin sensitivity and improved lipid metabolism.HSC-FOXO1KO significantly improved lipid metabolism and reduced hepatic lipid deposition in HFHC-induced NASH mice,but had no significant effect on the improvement of liver fibrosis.FOXO1 in HSC may be a new target for NASH treatment and its specific mechanism needs further study and exploration. |
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