Study On The Mechanism Of Sleeve Gastrectomy Via Nesfatin1/AMPK/Nrf2 Pathway To Improve NAFLD

Objective:The prevalence of obesity is increasing rapidly.NAFLD as a disease with multiple pathogenesis,also developing rapidly.The primary treatment for NAFLD is sustained weight loss,other main therapeutic directions include improving cell metabolism,antioxidant stress and antiapoptotic,antagonizing inflammatory response and anti-fibrosis therapy.Bariatric surgery not only improves NAFLD by reducing body weight,but also plays an important role in reshaping oxidative stress balance and alleviating inflammatory processes.In this study,the effects of sleeve gastrectomy on body weight,hepatic steatosis and liver oxidative stress were investigated.To investigate the changes of gastrointestinal hormone Nesfatin-1 levels after sleeve gastrectomy and the mechanism of Nesfatin-1 improving oxidative stress imbalance by affecting AMPK/NRF2 pathway.To provide experimental basis for further discovery of liver anti-oxidative stress target drugs and in-depth understanding of the mechanism of weight loss surgery.Methods:Firstly,a total of 68 obese patients underwent laparoscopic sleeve gastrectomy in the General Surgery Department of the Fourth Affiliated Hospital of China Medical University were selected.Nonalcoholic fatty liver disease was diagnosed by preoperative ultrasonography.Exclusion criteria;Patients with diabetes and abnormal glucose tolerance;Patients with cardiopulmonary insufficiency,very high risk of surgery,serious chronic diseases(such as severe autoimmune diseases or cancer);Increased alcohol consumption(>30g/d for men and>20g/d for women);Hepatitis or other chronic liver disease;Patients with perioperative complications;Mental disorders that may affect clinical studies,including dementia,episodic psychosis,severe depression,or a history of suicide attempts;Patients who have received an implanted device or radioactive opaque substance that may affect the test results.Postoperative rehabilitation was performed according to dietary guidance,and the patient returned to the hospital for re-examination on time one year after the operation.Fasting blood samples were collected before and 1 year after surgery,and metabolism-related indexes such as Nesfatin-1,blood lipid,liver function and blood glucose were examined.In the animal experiment,male 6-week-old C57BL/6J mice were fed with high-fat diet for 14 weeks to establish the NAFLD mouse model.The animal experiments were divided into three groups:non-operative high-fat diet group,sham operation food intake matching group and sleeve gastrectomy group.After the model was successfully established,the corresponding intervention measures were given to each group.After successful modeling,the corresponding intervention measures were given to each group.After 4 weeks of high-fat feeding,the samples were collected and tested for triglyceride,cholesterol,HE staining,oil red O staining,SOD activity and MDA content.The levels of Nrf2,Nrf2 nucleoprotein,HO-1 protein,p-AMPKαand AMPKα in liver tissues were detected by Western Blot.In the cell experiment,AML 12 cell culture medium containing 1 mmol/L free fatty acid mixture(FAA)(oleic acid/palmitic acid=2:1)was used to induce AML 12 cells and establish the model of non-alcoholic fatty liver cells.Give concentration gradient Nesfatin-1(10-7M,10-9M,10-11M)to cells,detection of TC,TG,oil red O staining,ROS,MDA content in cell detection,SOD,CAT and GSH-px activity detection,cell mitochondrial membrane potential detection.Western Blot detection AMPK α,p-AMPK α,Nrf2 protein,Nrf2 nucleoprotein and HO-1 protein expression levels.To determine whether Nesfatin-1 regulates oxidative stress through AMPK/NRF2.SiRNA was used to knockdown AMPK-α,Western Blot and qRT-PCR were used to detect the transfection efficiency.Nesfatin-1 with a concentration of 10-7 M was given for intervention.Cell TC,TG,oil red O staining,ROS were detected.MDA content detection,SOD,CAT,GSH-Px activity detection,and mitochondrial membrane potential detection were performed.The expression levels of Nrf2 protein,Nrf2 nucleoprotein and HO-1 protein were detected by Western Blot.Results:Laparoscopic sleeve gastrectomy is effective in weight control.The body weight of 29 patients was(124.8±28.77)Kg before surgery and(83.65±16.34)Kg one year after SG.BMI was(42.63±8.91)Kg/m2 before surgery,and body weight was(28.54±5.63)Kg/m2 one year after SG.TWL%at 1 year after SG was(32.11±7.10)%.One year after laparoscopic sleeve gastrectomy,the liver function indexes ALT,AST and NAFLD were significantly improved.ALT and AST levels were significantly lower than those before operation(P<0.05).ALT was(124.8±28.77)U/L before surgery and(29.97±10.33)U/L one year after SG.The AST was(79.93±26.41)U/L before surgery and(26.69±9.58)U/L one year after SG.The patient’s HSI was(52.55±9.17)before surgery and(38.84±5.82)after 1 year(P<0.05).One year after laparoscopic sleeve gastrectomy,the preoperative Nesfatin-1 value of patients was(3.04±0.81)nglml,and the postoperative Nesfatin-1 value was(5.52±1.55)ng/ml(P<0.05).There was a positive correlation between the increase of Nesfatin-1 and the decrease of HSI after surgery(P<0.05;R=0.44).One year after laparoscopic sleeve gastrectomy,the patient’s blood glucose and triglyceride levels improved.Fasting blood glucose was(5.8±0.8)mmol/L before surgery and(4.9±0.3)mmol/L one year after SG.The triglycerides were(2.0±0.6)mmol/L before surgery and(1.3±0.4)mmol/L one year after SG.In the animal experiment,the weight of the non-surgical high-fat diet group increased continuously over time.The body weight of the sham operation food intake matching group before operation was(39.3±3.61)g,and the body weight slightly decreased(38.5±3.21)g one week after operation,then gradually stabilized without significant increase,showing slight fluctuation.In the gastric sleeve resection group,the body weight before surgery was(381.±1.34)g,the body weight decreased rapidly(32.7±1.25)g at 1 week after surgery,followed by a slight fluctuation,and showed a rebound trend at 4 weeks after surgery.HE staining and oil red O staining showed that the liver sinuses were narrowed,the hepatic cord was disorganized,the hepatocyte edema and the presence of a large number of lipid droplets,and the hepatic lobules were not clearly defined,suggesting the presence of fatty liver.In the sleeve gastrectomy group,the hepatic cords were arranged neatly,and the structures of hepatic lobules,sinuses and hepatocytes were nearly normal.The sleeve gastrectomy group had TC of(3.05±0.07)mmol/L,the non-operative high-fat diet group had TC.of(6.24±0.11)mmol/L,and the sham-operation food intake matching group had TC of(4.95±0.08)mmol/L.The TG of the sleeve gastrectomy group was(1.64±0.04)mmol/L,the TG of the non-operative high-fat diet group was(3.1±L0.05)mmol/L,and the food intake of the sham operation matched group was(2.3 8±0.05)mmol/L.The above results showed that SG significantly decreased the levels of TC and TG in serum of mice(P<0.05).The SOD activity detection in sleeve gastrectomy group(106.01±1.54)U/mgprot,the non-operative high-fat diet group(55.35±1.09)U/mgprot,and the sham operation food intake matching group(78.05±1.16)U/mgprot.MDA was(4.324±0.203)nmol/mgprot in sleeve gastrectomy group,(7.915±0.234)nmol/mgprot in the non-operative high-fat diet group,and(6.255±0.146)nmol/mgprot in the sham food intake matching group.The above results showed that SG reduced the content of MDA and enhanced the activity of SOD in liver tissue of mice.Western blot analysis showed that there was no significant difference in NRF2 protein in liver tissues among all groups.The expression levels of Nrf2 nucleoprotein,HO-1 protein and p-AMPKα protein in the gastric sleeve resection group were higher than those in the non-operative high-fat diet group and the sham-operation matched food intake group,with statistical significance(P<0.05).In cell experiment,liver cells showed obvious lipid droplet deposition after FFA induction-The cells were detected by TC,TG and oil red O staining.TC and TG in model group(6.33±0.32)mmol/L and(4.21±0.28)mmol/L were significantly higher than those in control group(1.62±0.49)mmol/L and(0.97±0.19)mmol/L.Compared with the model control group,when the concentration of Nesfatin-1 added into the cell culture medium was 10-7M,10-9M,10-11M,respectively,the reactive oxygen species level of AML12 cells induced by FFA decreased successively.When the concentration of Nesfatin-1 increased from 10-11M to 10-7M,the content of MDA decreased gradually.Compared with the model control group,the content of MDA decreased from(5.52±0.26)nmol/mgprot to(2.82±0.14)nmol/mgprot_The results of SOD,CAT and GSH-Px activity detection showed that,compared with the model control group,the activities of SOD,CAT and GSH-Px in AML 12 cells induced by FFA increased successively when the concentrations of Nesfatin-1 added in the cell culture medium were 10-11M,10-9M and 10-7M,respectively-SOD increased from(26.84±0.42)U/MgProt to(46.84±0.34)U/MgProt,CAT increased from(7.92±0.57)U/MgProt.GSH-Px increased from(17.23±0.62)U/MgProt to(27.66±1.09)U/Mgfrot.Nesfatin-1 can reduce mitochondrial membrane potential loss caused by HFD-induced cells.When the concentration of Nesfatin-1 is 10-7M,the LR ratio is(9.14±0.89)%;when the concentration of Nesfatin-1 is 10-9 M,the LR ratio is(10.95±0.85)%;when the concentration of Nesfatin-1 is 10-11 M,the LR ratio is(12.94±0.78)%.However,there was no significant difference in the effect of medium-dose and high-dose Nesfatin-1 on mitochondrial membrane potential loss.Western blot analysis showed that there was no significant difference in the expression of Nrf2 protein among all groups.By detecting p-AMPKα,AMPKα,Nrf2 nucleoprotein and HO-1,it was found that Nesfatin-1 activated AMPK in a concentration-dependent manner and up-regulated the levels of nuclear Nrf2 protein and downstream HO-1 protein,and the results were statistically significant compared with the control group(P<0.05).The intervention concentration of Nesfatin-1 was fixed at 10-7 M,and the ROS synthesis was detected by adding FAA-induced AML 12 cells,negative transfected cells and AMPKα1 siRNA transfected cells respectively.It was found that the inhibition of ROS generation by Nesfatin-1 was inhibited to a certain extent after AMPKα1 knockdown,suggesting that AMPK is one of the ways that Nesfatin-1 plays an anti-oxidative stress role.The changes of MDA content and the activity levels of SOD,CAT and GSH-Px were further detected after blocking AMPKα1.MDA content in negative transfected model group was the lowest(2.72±0.33)nmol/mgprot after Nesfatin-1 intervention,then it increased to(4.61±0.26)mnol/mgprot after AMPKα1 knockdown,which was still lower than that in AMPKα1 siRNA transfected group without Nesfatin-1(7.51±0.22)nmol/mgprot,P<0.05.These results suggested that AMPK was involved in the regulation of Nesfatin-1 on oxidative stress balance,but it was not the only mechanism.The activity tests of SOD,CAT and GSH-Px also reflected this result.When Nesfatin-1 interfered with negative transfection model group,SOD,C.AT and GSH-Px were(45.86±0.91)U/mgprot,(12.53±0.58)U/mgprot,and(27.39±0.83)U/mgprot.When AMPKα1 was knocked down,SOD and CAT decreased to(30.89±0.38)U/mgprot and(8.82±0.32)U/mgprot.GSH-Px decreased to(20.50±0.92)U/mgprot,with statistical significance P<0.05.The LR ratio of Nesfatin-1 intervention was(8.24±0.59)%in the negative transfection model group,and the LR ratio increased to(17.11±1.73)%after AMPKα1 knockdown.Nesfatin-1 with a concentration of 10-7 M was used to intervene the cells and AMPKα1 was knocked down.The levels of Nrf2,Nrf2 nucleoprotein and HO-1 protein were detected by Western blot.After AMPKα1 knockdown,the level of Nrf2 protein in the nucleus and the HO-1 protein were down-regulated(P<0.05).Conclusion:Sleeve gastrectomy can effectively alleviate NAFLD,reduce the weight of obese patients,improve blood glucose,lipid and other metabolism-related indicators.Nesfatin-1 changes were positively correlated with HSI changes after sleeve gastrectomy.Sleeve gastrectomy and food restriction reduced body weight in NAFLD mice.Sleeve gastrectomy can effectively reduce lipid levels,alleviate hepatic steatosis,activate AMPK/NRF2 pathway,and reduce liver oxidative stress levels in NAFLD mice.Nesfatin-1 can improve the oxidative stress state of non-alcoholic fatty liver cells in a concentration-dependent manner,and reduce the oxidative stress injury of hepatocytes induced by high fat by activating AMPK/NRF2 signaling pathway.