Mechanism Of Empagliflozin Improving Nonalcoholic Steatohepatitis Through AMPK/FOXA2/MCAD Pathway

Objective:Non-alcoholic fatty liver disease(NAFLD)is a clinical syndrome in which the main lesion is located in the hepatic lobule.It is characterized by hepatocyte steatosis and lipid deposition,but there is no history of excessive drinking.The disease spectrum includes simple steatosis,nonalcoholic steatosis hepatitis,cirrhosis and hepatocyte carcinoma.Studies have reported that the incidence of NAFLD in patients with type 2 diabetes(T2DM)is as high as 55.5%,about twice that of the general population,and the incidence of NASH is as high as 37.3%,more than twice that of the general population.However,due to the complicated pathogenesis of NASH,which has not been fully elucidated,there is currently no targeted and effective therapeutic drugs.Sodium-glucose cotransporter 2 inhibitor(SGLT2i)is a new type of anti-diabetic drug.Clinical and experimental studies have shown that SGLT2 i can reduce liver lipid content in addition to its hypoglycemic effect,but the specific mechanism is not yet clear.Therefore,in this study,T2 DM patients with NASH and T2 DM patients without fatty liver were selected as the research subjects.The i TRAQ protein profiling test was used to screen for proteins with significant differences and related to lipid metabolism and predict their related signaling pathways.Then we further validated and explored the effect of empagliflozin on AMPK/FOXA2/MCAD pathway and liver lipid metabolism in vitro and in vivo.Methods:Six T2 DM patients with NASH and six T2 DM patients without fatty liver were selected.During the surgical resection of benign lesions such as hepatic hemangioma or gallbladder polyp,liver tissue specimens were obtained for i TRAQ protein spectrum detection and subsequent immunohistochemical,western blot,and q RT-PCR detection.8-week-old male db/m mice were selected as the normal control group.8-week-old db/db mice were used as the experimental group,and were randomlydivided into two groups: placebo intervention group and empagliflozin treatment group.The mice in the control group had a normal diet for 8 weeks,the mice in the experimental group had a high-fat diet for 8 weeks,and drug intervention for 8 weeks.During the feeding period,the mice were regularly tested for body weight and blood glucose.At the end of the 8th week,mice were anesthetized with chloral hydrate,eyeball blood was used to detect plasma lipid levels,liver tissue was used to detect liver lipid levels and MCAD,FOXA2,AMPK,p-AMPK and IL in the liver-1β,TNFα and other protein expression levels.With normal hepatocytes as the control group and steatotic hepatocytes as the experimental group,the protein expression levels of p-AMPK,FOXA2 and MCAD were detected after the intervention of empagliflozin,AMPK activator,AMPK inhibitor and si RNA-FOXA2.Results:1.The results of i TRAQ protein spectrum,immunohistochemistry,western blot and q RT-PCR in human liver tissues showed that compared with the control group,the levels of phosphorylated AMPK,nuclear FOXA2 and MCAD proteins in the liver of the T2 DM NASH group were significantly reduced,and the levels of IL-1β and TNFα were significantly increased.2.The general data of mouse showed that: compared with the normal group,the body weight,blood glucose,blood lipid and liver lipid levels of the db/db group were significantly increased,while the body weight,blood glucose,blood lipid and liver lipid levels were significantly reduced after the empagliflozin intervention.The results of oil red staining showed that: compared with the normal group,the lipid deposition in the liver of the db/db group was significantly increased,while the lipid deposition in the liver was significantly reduced after the empagliflozin intervention.Immunofluorescence and Western blot results showed that compared with the normal group,p-AMPK,nuclear FOXA2 and MCAD protein levels in the liver of the db/db group were significantly reduced,and IL-1β and TNF α levels were significantly increased.However,after the empagliflozin intervention,p-AMPK,nuclear FOXA2 and MCAD levels in the liver of the db/db group were significantly increased,while IL-1β and TNF α levels were significantly reduced.3.The results of cell oil red showed that: compared with the control group,the lipid deposition in the high-glucose and high-lipid group was significantly increased,while the lipid deposition in the cells was significantly reduced after the empagliflozin intervention.Immunofluorescence and Western blot results showed that compared with the normal group,p-AMPK,nuclear FOXA2 and MCAD protein levels in the high-glucose and high-fat group were significantly reduced,while p-AMPK,nuclear FOXA2 and MCAD protein levels in the cells were significantly increased after the empagliflozin intervention.Western blot results showed no significant changes in p-AMPK,nuclear FOXA2,and MCAD protein levels in cells treated with AMPK activator,while significantly reduced p-AMPK,nuclear FOXA2,and MCAD protein levels in cells treated with AMPK inhibitor.Western blot results showed that compared with the empagliflozin group,the levels of p-AMPK,total FOXA2 and MCAD protein in the si RNA-FOXA2 intervention group were significantly reduced.However,after si RNA-FOXA2 and empagliflozin co-intervention,the levels of p-AMPK protein in the cells increased significantly,but the levels of total FOXA2 and MCAD proteins did not change significantly.Conclusion:1.The AMPK/FOXA2/MCAD pathway was significantly impaired in the liver of T2 DM patients with NASH.2.Empagliflozin improves NASH by activating the AMPK/FOXA2/MCAD pathway,promoting fatty acid oxidation and reducing lipid deposition and inflammatory response in the liver.