| Objective:Electroacupuncture(EA)is effective in the treatment of simple obesity,without side effects,and can reduce the risk of multiple complications.Previous studies have shown that the effect of EA on weight loss is closely related to its promotion of adipocyte metabolism,but its specific biological mechanism has not been fully clarified.This study intends to use EA therapy to carry out studies from clinical trials and animal experiments respectively,which will clarify the clinical efficacy of EA on obese patients and observe the influence of EA on obesity phenotype and fat metabolism of experimental mice induced by high fat diet.Furthermore,the relationship between EA intervention and mitochondrial autophagy of mouse white adipocytes was further explored.The regulatory effect of EA intervention was discussed by targeting relevant molecular signaling pathways.Finally,part of the biological mechanism of EA treatment of simple obesity was illustrated from the perspective of mitochondrial autophagy of white adipocytes.Methods:Clinical study:A total of 110 cases were collected according to the inclusion criteria.They were randomly divided into obesity control group(Control,55 cases)and electroacupuncture group(EA,55 cases).The EA group was given 3 courses of electroacupuncture treatment,30 minutes each time,3 times a week(once every other day)for 3 consecutive months.Weight,waist circumference,hip circumference,sebum thickness and other obesity-related indexes were measured before and after treatment.Body mass index(BMI)and body fat percentage(F%)were also calculated.The clinical efficacy and safety of EA in treating simple obesity were evaluated as well.At the same time,the peripheral blood of two groups were extracted before and after treatment,and the levels of autophagy indexes such as LC3 Ⅱ and p62 in serum were detected by ELISA.Besides,observed the regulatory effect of electroacupuncture on autophagy levels.Experimental study part(Experiment 1):50 male C57BL/6N mice were randomly divided into blank control group(Control,10 mice)and obesity model group(HFD,40 mice).In the obesity model group,a total of 26 mice were successfully modeled by high-fat diet induction,and then randomly divided into obesity model group(HFD,13 mice)and obesity+EA group(HFD+EA,13 mice).The EA group was given bilateral "Tianshu" electroacupuncture intervention for 15 minutes,once a day,5 times/week,4 weeks in total.The body weight,liver tissue,white adipose tissue(WAT),epididymal adipose tissue,subcutaneous adipose tissue,retroperitoneal adipose tissue and mesenteric adipose tissue weight of mice in each group were measured before and after EA intervention.Fat mass/body weight ratio,respiratory gas exchange rate(RER)and obesity parameters(Lee’s index)were calculated.Blood biochemistry was used to detect blood glucose level,triglyceride(TG),total cholesterol(TC),aspartate aminotransferase(AST)and alanine aminotransferase(ALT)levels in plasma.The contents of thiobarbituric acid reactants(TBARS)were detected by the kit.The adipose tissue morphology of mice in each group was observed by H&E staining and oil red O staining.The mRNA expression levels of UCP1,Cidea,PRDM16,Dio2,PGC-1α,p53,Sirt3 and autophagy related molecules LC3,Beclin1,Parkin,PINK1 and p62 in WAT were detected by fluorescence quantitative PCR.Western Blot was used to detect the protein expression levels of UCP1,PGC-1α,p53,Sirt3 and autophagy related molecules LC3,Beclin1,Parkin,PINK1 and p62 in WAT.The expression of p53 in WAT was detected by immunohistochemistry.The expression and localization of LC3B in WAT were detected by immunofluorescence as well.Experimental research part(Experiment 2):40 C57BL/6N male mice successfully constructed with obesity model were randomly divided into 4 groups:obesity model control group(HFD),obesity+Sirt3 overpression group(HFD+Sirt3),obesity+EA group(HFD+EA+Sirt3),obesity+EA+Sirt3 overpression group(HFD+EA+SIRT3),with 10 mice in each group.Using the same modeling and electroacupuncture intervention method as Experiment 1,the Sirt3 overexpression group was treated with tail vein injection of Sirt3 overexpression virus.The body weight,liver tissue,epididymal adipose tissue,subcutaneous adipose tissue,retroperitoneal adipose tissue and mesenteric adipose tissue of mice in each group were measured before and after electroacupuncture intervention.Fat mass/body weight ratio,RER and Lee’s index were calculated.Blood biochemistry was used to detect blood glucose levels,TG,TC,AST and ALT levels,and TBARS content was detected with the kit.The adipose tissue morphology of mice in each group was observed by H&E staining and oil red O staining.The morphological changes of adipose tissue were detected by H&E staining and oil red O staining.The mRNA expression levels of UCP1,PGC-1α,p53,Sirt3 and autophagy related molecules LC3,Beclinl,Parkin,PINK1 and p62 in WAT were detected by fluorescence quantitative PCR.Western Blot was used to detect the protein expression levels of UCP1,PGC-1α,p53,Sirt3 and autophagy related molecules LC3,Beclin1,Parkin,PINK1 and p62 in WAT.The expression and localization of LC3B in WAT were detected by immunofluorescence.Results:Clinical studies showed that there was no statistical difference in baseline data between EA group and Control group before treatment.After 3 courses of EA treatment,compared with the Control group,the EA group had better weight loss effect,the total effective rate was 87.27%.At the same time,autophagy related indexes LC3 Ⅱ and p62 in peripheral blood of patients in EA group were significantly decreased after EA treatment(P<0.05).It suggested that electroacupuncture can regulate patients’ autophagy levels while achieving good weight loss effect.In the experimental research part(Experiment 1),HFD induced mouse obesity model was successfully established firstly.Compared with HFD group,EA intervention significantly reduced the body weight and the weight of liver tissue,epididymal adipose tissue,subcutaneous adipose tissue,retroperitoneal adipose tissue and mesenteric adipose tissue.Biochemical results showed EA significantly reduced the levels of Glu,TC,TG,AST,ALT and TBARS in HFD mice.H&E staining and oil red O fat staining showed that EA significantly reduced WAT adipocyte volume and lipid deposition in liver of HFD mice.PCR and Western Blot results pointed out EA intervention significantly down-regulate the mRNA and protein expression levels of p53 and autophagy related molecules LC3,Beclinl,Parkin and PINK1 in WAT of HFD mice,while up-regulate the mRNA and protein expression levels of UCP1,Cidea,PGC-1α,Sirt3 and autophagy related molecule p62.Immunofluorescence detection confirmed that EA intervention significantly decreased the expression level of LC3B in WAT of HFD mice,the differences were statistically significant(P<0.05).In the experimental research part(Experiment 2):Compared with HFD group,body weight and the weight of liver tissue,epididymal adipose tissue,subcutaneous adipose tissue,retroperitoneal adipose tissue and mesenteric adipose tissue significantly reduced by EA intervention or Sirt3 overexpression virus.Biochemical results showed whether EA intervention or Sirt3 overexpression virus significantly reduced the levels of Glu,TC,TG,AST,ALT and TBARS in HFD mice.H&E staining and oil red O fat staining showed that whether EA intervention or Sirt3 overexpression virus significantly reduced WAT adipocyte volume and lipid deposition in liver of HFD mice.PCR and Western Blot results pointed out whether EA intervention or Sirt3 overexpression virus intervention significantly down-regulate the mRNA and protein expression levels of p53 and autophagy related molecules LC3,Beclin1,Parkin and PINK1 in WAT of HFD mice,while up-regulate the mRNA and protein expression levels of UCP1,PGC-1α,Sirt3 and autophagy related molecule p62.Immunofluorescence detection confirmed that whether EA intervention or Sirt3 overexpression intervention significantly decreased the expression level of LC3B in WAT of HFD mice.Morover,the results showed that electroacupuncture EA intervention combined with overexpression of Sirt3 virus further promoted the above trend of changes,the differences were statistically significant(P<0.05).Conclusion:EA therapy can improve various obesity-related indexes and regulate autophagy levels in obese patients with significant clinical efficacy,can be used as a safe and effective way to lose weight.Electroacupuncture intervention and Sirt3 overexpression have a clear weight loss effect on HFD mice,improving their lipid metabolism and regulate the autophagy level of adipose mitochondria in white fat,moreover.Sirt3 overexpression significantly promotes the effect we metioned above.The mechanism of EA regulating obesity and fat metabolism may be related to up-regulation of Sirt3 expression,regulation of mitochondrial autophagy mediated by downstream Sirt3-p53/PGC-1α signaling pathway,and further promotion of browning of white adipocytes.It is expected to provide scientific basis for the widespread promotion and application of EA as a green weight loss therapy. |
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