| Objective: As the obese population continues to expand worldwide,the prevalence of metabolic associated fatty liver disease(MAFLD)is increasing each year.MAFLD is a chronic liver disease caused by multiple factors and is a manifestation of metabolic syndrome in the liver.Disturbances in cholesterol metabolism in the liver are an important mechanism in the development and progression of MAFLD.Bariatric surgery is effective in reducing body weight and improving the pathological changes of MAFLD in obese patients,but the mechanisms involved are still not fully elucidated.Irisin is a recently discovered adipokine(myokine)produced by the cleavage of fibronectin type III domain containing protein 5(FNDC5).Irisin has been shown to play an important role in the alleviation of many metabolic diseases and in the regulation of disorders of lipid and cholesterol metabolism.In this study,the changes in irisin levels following bariatric metabolic surgery and its therapeutic effects on MAFLD were further demonstrated by examining liver and serum samples from patients undergoing bariatric surgery as well as from mouse bariatric surgery models.The role and mechanism of irisin in the regulation of cholesterol metabolism was also investigated by in vitro cellular experiments.Our study provides a new target and basis for the mechanism of bariatric surgery for treating MAFLD.Methods: In the first part of this study,a total of 70 obese patients undergoing laparoscopic sleeve gastrectomy(LSG)in the Department of General Surgery of the Fourth Affiliated Hospital of China Medical University were collected according to the inclusion and exclusion criteria established beforehand,and 6 patients were excluded according to the exclusion criteria during the study,and 64 patients were actually enrolled.All patients underwent LSG after completing preoperative assessment and abdominal ultrasound.Liver biopsy were obtained during the operation.After HE staining and by pathological diagnosis,the enrolled patients were divided into the obese without hepatic steatosis group(OB CON)and the obese combined MAFLD group(OB MAF).The preoperative serum biochemical indicators and irisin levels were analyzed and the differences were compared between groups.Protein and RNA were then extracted from liver biopsy samples to compare FNDC5 expression differences.All enrolled patients underwent postoperative diet and lifestyle guidance and returned to the hospital for re-examination on time 6 months after operation.Body weight and serum biochemical indicators such as irisin levels were collected,and abdominal ultrasound was performed again,and compared with the corresponding indicators before surgery.In the second part of this study,6-8 week-old male C57BL/6J mice were used and fed a high-fat-diet for about 12 weeks to establish high-fat-diet induced obesity mouse model.Afterwards,sleeve gastrectomy(SG)and gastric bypass(GB)were performed on the obese mice,and high-fat-diet was continued after surgery.Two weight-paired-sham groups were set corresponding to the two surgical groups.Mice in the two sham groups were fed the same high-fat-diet after surgery,ensuring that there was no significant difference in weight from the surgical groups.In addition,there was a chow diet(CD)group,which was always fed with standard chow diet,and a high-fat-diet(HFD)group,which was always fed with high-fat-diet and no surgical intervention.The body weight and food intake of each group of mice were measured weekly after surgery.All mice were killed 6 weeks after surgery,and liver tissue and serum samples were collected.The liver tissues of each group of mice were stained with HE and oil red O to observe the hepatic steatosis.The serum and liver total cholesterol(TC),triglyceride(TG)and irisin levels of each group of mice were measured and analyzed.The protein and m RNA expressions of FNDC5,sterol-regulatory element binding proteins 2(SREBP2)and the key enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMG-Co A reductase,HMGCR)were measured in the liver tissues of each group of mice.The effect of bariatric surgery on the expression of FNDC5/irisin in mice and the regulation of hepatic cholesterol metabolism were analyzed based on the results.In the third part of this study,Hep G2 cell lines were used for the study.The MAFLD model of Hep G2 was established after intervention with oleic acid and palmitic acid.The Hep G2 cells were treated with recombinant irisin(r-irisin)at different concentrations.TC and TG contents and the expression of SREBP2 and HMGCR proteins in Hep G2 cells were detected.The differential expression sites of SREBP2 in Hep G2 were detected by immunofluorescence to further verify the regulatory effect of irisin on SREBP2.Finally,the Hep G2 cells were treated with receptor antagonist of irisin and r-irisin simultaneously,and the TC and TG contents and the expression of SREBP2 and HMGCR protein and m RNA in the cells were detected.To verify the effect of irisin on the downstream cholesterol synthesis pathway after its inhibition,to clarify the role of irisin in the regulation of hepatocyte cholesterol metabolism and to demonstrate its possible mechanism.Results: The results of the clinical study showed that patients in the OB MAF group had elevated levels of TC and TG in the liver and showed more pronounced liver damage and lipid accumulation compared to patients in the OB CON group.However,hepatic FNDC5/irisin expression was lower in the OB MAF group than in the OB CON group.The detection of the preoperative serum indicators in each group revealed that the levels of serum lipid metabolism indicators TC and TG and serum liver injury indicators alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in the OB MAF group were significantly higher than that of OB CON group,however,the serum irisin levels was slightly lower than that in the OB CON group,and was statistically significant(P=0.031).Six months after LSG in the enrolled patients,a significant decrease in weight and BMI was observed in all groups.48 patients with fatty liver detected by abdominal ultrasound before surgery showed remission of the degree of hepatic steatosis after surgery,and 37(77%)of them had complete disappearance of hepatic steatosis.Patients in the OB MAF group showed a significant decrease in serum TC,TG,ALT,and AST levels after surgery,but patients in the OB CON group showed only a slight decrease in serum TC and TG levels.Postoperative serum irisin levels increased in both groups,but were not statistically significant in the OB CON group(P=0.07),whereas they were statistically significant in the OB MAF group(P<0.001),indicating that bariatric surgery may have a regulating effect on serum irisin levels.Correlation analysis of serum TC,TG,ALT,AST levels and irisin levels in patients in the preoperative and postoperative OB MAF groups revealed that all four serum indicators showed negative correlation with serum irisin levels.This indicates that irisin has an important regulatory role in the development of lipid metabolism,cholesterol metabolism and MAFLD.The results of animal experiments showed that the livers of obese mice induced by high-fat-diet showed significant lipid deposition,and both SG and GB bariatric surgery significantly reduced body weight,improved liver steatosis and reduced intrahepatic TC and TG contents of obese mice.The expression of FNDC5/irisin in the liver of mice in the HFD and sham groups was low,while the expression appeared to be significantly higher after SG and GB operations,and the same results were obtained after the serum irisin content of mice in each group was examined.This indicates that bariatric surgery can upregulate the FNDC5/irisin content in mice.Subsequently,SREBP2 and HMGCR proteins and m RNA were examined in mouse liver.The results showed that SREBP2-HMGCR was activated and showed high expression in the liver of mice in the HFD group.Both SG and GB groups resulted in the inhibition of SREBP2-HMGCR expression.Based on the reported role of irisin in regulating cholesterol metabolism,it is speculated that bariatric surgery may inhibit hepatic cholesterol synthesis by upregulating the expression of irisin,which may reduce hepatic cholesterol deposition and alleviate the severity of MAFLD.The results of in vitro cellular experiments showed that a 1.0 m M concentration of hepatocyte steatosis inducer could successfully induce steatosis in Hep G2 cells and establish a hepatocyte MAFLD model.Detection of SREBP2-HMGCR protein expression and TC and TG contents in cells showed that both SREBP2 and HMGCR protein expression were significantly up-regulated after hepatocyte steatosis,and cellular TC and TG contents were also significantly increased.After administration of r-irisin at five different concentrations of 1n M,5n M,10 n M,20 n M,and 40 n M to the MAFLD cell model,the protein expression of SREBP2 and HMGCR gradually decreased with increasing intervention concentrations in a concentration-dependent manner.Intracellular TC and TG contents also appeared to decrease at higher intervention concentrations.Subsequently,using immunofluorescence to detect the localization of SREBP2 protein expression in Hep G2 cells,it could be found that irisin could inhibit the nuclear translocalization of SREBP2 and suppress its activation to the active form.Finally,after the application of irisin receptor inhibitor to the cells,the inhibitory effect of irisin on SREBP2-HMGCR pathway appeared to be attenuated,while the intracellular TC and TG contents also appeared to be increased to some extent.Conclusions: In individuals with MAFLD,a disturbance in cholesterol metabolism occurs,causing an increase in hepatic lipid and cholesterol accumulation.At the same time FNDC5/irisin expression is inhibited and fails to play a role in regulating cholesterol metabolism.Bariatric surgery upregulates the levels of FNDC5/irisin in the liver and serum,thereby inhibiting the activation of hepatic SREBP2.It also inhibits the expression of the downstream target HMGCR.This process reduces the hepatic cholesterol de novo synthesis,alleviates cholesterol accumulation and hypercholesterolemia,improves the pathological manifestations and acts as a treatment for MAFLD. |
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