| Objectives Obesity is the result between calorie intake and calorie consumption of energy imbalance.When the body is in a state of obesity for a long time,its adipose tissue will secrete a large amount of non-esterified fatty acids,pro-inflammatory factors,adipokines and others to induce the occurrence of insulin resistance(IR).IR is the key pathological basis for obesity to develop into type 2 diabetes.Therefore,timely improvement of IR status can not only reduce the burden of disease caused by obesity,but also improve people’s health and quality of life.The latest research has found that the decreased proliferation ability of intestinal epithelial cells and the inability to repair the barrier function changes caused by intestinal mucosal damage are important causes of metabolic endotoxemia in mice induced by high-fat diet,and are closely related to the development of insulin resistance.Proliferation and apoptosis of intestinal epithelial cells of the gut is the basic way of self-renewal and restoration,but also to maintain the integrity and function of the intestinal basis,is essential for intestinal homeostasis.It is worth noting that Wnt/β-catenin signaling plays a key role in driving the proliferation of epithelial cells.Therefore,in this experiment,a high-fat diet was used to induce an obese insulin resistance rat model,electroacupuncture was used as an intervention method,and the intestinal mucosal injury was used as an entry point to observe the influence of SIRT1 on the Wnt/β-catenin pathway in the small intestine tissue of obese insulin-resistant rats,combining with the changes of intestinal mucosal injury,apoptosis of intestinal epithelial cells and barrier function,to explore the mechanism of electroacupuncture(EA)through SIRT1-mediated Wnt/ β-catenin repairing the intestinal mucosal injury and barrier function of obese insulin-resistant rats,which is a clinical application Electroacupuncture provides experimental evidence for the treatment of obesity and insulin resistance related diseases.Methods120 5-week-old SPF-grade Wistar male rats were used to mark one by one with ear tags.15 rats of them were selected as the general food group by random number table method and fed with ordinary food.The remaining 105 rats were used as the modeling group to give high diet food(HFD)for 10 weeks.After the model was completed,12 rats in the general food group were randomly selected as the normal group(N group)and continued to be fed with ordinary food.60 rats were randomly selected as the model group(M group),the electroacupuncture group(EA group),the sham electroacupuncture group(SEA group),the electroacupuncture combined SIRT1 inhibitor group(EAI group)and SIRT1 agonist group(A group),after the modelwas successfully established,each with 12 rats,continued to be fed with HFD.(1)N group: no other intervention for 8 weeks;(2)M group: model rats,no other intervention for 8 weeks;(3)EA group: Selected acupoints Zhongwan(CV12),Guanyuan(CV4),Zusanli(ST36)and Fenglong(ST40).Connect the Han’s EA instrument,connect a pair of EA clips to CV12 and CV4,and connect another pair of EA clips to ST36 and ST40 on the same side,frequency 2Hz,intensity 1m A,continuous wave,15min/time,3 times/week,for 8weeks;(4)SEA group: After shallowly puncturing the subcutaneous side of the acupuncture point of the rat with a filiform needle,the electrode is clamped,and no electricity is applied.The rest is the same as the above-mentioned EA method for 8 weeks;(5)EAI group:On the basis of EA intervention,SIRT1 specific inhibitor Sirtinol(1mg/kg,administered by tail vein injection)was simultaneously administered 3 times a week for 8 weeks;(6)A group:Give SIRT1 activator resveratrol as a positive control(200mg/kg,intragastric administration),3 times a week for 8weeks.During the modeling period,the body weight of each group of rats was measured on the last day of the 10 th week of the HFD modeling,and then 3 rats were selected from the general diet group,and 15 rats were selected from modeling group for the hyperinsulinemic euglycemic clamp experiment to detect the glucose infusion rate(GIR).The body weight and GIR were used to judge whether the obese insulin resistance rats were successful in modeling.During the intervention,the body mass and Lee’s index weremeasured before intervention(0 week),and at the 2th,4th,6th and8 th week of intervention,and the introperitoneal insulin tolerance test(IPITT)and introperitoneal glucose tolerance test(IPGTT)were measured at the 6th week.After 8 weeks of intervention,3 rats in each group were randomly selected to measure the glucose infusion rate(GIR).Before the rats in each group were killed,the blood was taken from the apex of heart,and the contents of insulin(INS)and lipopolysaccharide(LPS)in serum were detected by ELISA;The total cholesterol(TC),triglyceride(TG)and free fatty acid(FFA)in serum were detected by automatic biochemical analyzer,and then the white fat in abdomen and peritestis of rats were cut and weighed.After the rats were sacrificed,fresh small intestine tissues were taken,and Western blotting(WB)was used to detect the protein expression levels of ZO-1,Occludin,SIRT1,β-catenin,p-β-catenin,Cyclin D1,and caspase3 in the small intestine tissues of rats in each group.The immunofluorescence double-labeling method was used to detect the co-expression of SIRT1 and β-catenin in rat small intestine tissues,and RT-PCR was used to detect the m RNA expression levels of ZO-1,Occludin,SIRT1,β-catenin,Cyclin D1,and caspase3 in rat small intestine tissues.After perfusion and fixation,the small intestine was cut to make paraffin section and frozen section,the morphological changes of small intestine were observed by HE staining,and the apoptosis of small intestine was detected by TUNEL.Results1.The effect of high-fat diet for 10 weeks on the body mass and GIR of rats(1)Body mass: After 10 weeks of intervention,compared with thegeneral food group,the body mass of 78 rats in the modeling group was significantly increased(P<0.01).(2)GIR: After 10 weeks of intervention,the GIR index of the modeling group was significantly lower than that of the general food group(P<0.01).2.The effect of electroacupuncture on body mass,white fat weight,lipid metabolism,insulin sensitivity,GIR and serum insulin in obese rats with insulin resistance(1)Body mass: In the 0th week of intervention,compared with the N group of rats,the weight of the other five groups of rats fed with HFD increased significantly(P<0.01).The body mass of the M group was significantly higher than that of the N group in the following 2,4,6,and 8 weeks(P<0.01).Starting from the 4th week,the body mass of the rats in the A group was lower than that of the M group(P<0.05),and showed a significant downward trend in the6 th and 8th weeks(P<0.01).Compared with the M group,the body mass of the EA group decreased after 6 weeks of intervention(P<0.05),and the difference was more obvious at the 8th week(P<0.01).However,the body mass of the SEA group was not statistically different from the M group at each time period(P>0.05),and was significantly higher than that of the EA group at the 8th week(P<0.05).The body mass of the EAI group was less than that of the M group after 8 weeks of intervention(P<0.05).(2)Lee’s index: In the 0th week of intervention,compared with the N group,the Lee’s index of rats in the other 5 groups increased significantly(P<0.01).At the 6th week,only the A group’s Lee’s index was lower than that of the M group(P<0.05);at the 8th week,both the EA group and the A group could reduce the model rats’ Lee’sindex(P<0.05,P<0.05,P<0.01).However,the Lee’s index of the SEA group and the EAI group was not significantly different from the M group at each time period(P>0.05).(3)The weight of White adipose tissue(WAT): Compared with the N group,the weight of WAT in the M group increased significantly(P<0.01).Compared with the M group,the WAT weight of the EA group,the EAI group,and the A group were all reduced(P<0.01,P<0.05,P<0.01).However,there was no significant difference in the weight of WAT between the SEA group and the M group(P>0.05),and it was higher than that of the EA group(P<0.05).(4)Serum TC,TG,FFA content: Compared with the N group,the serum TC,TG,FFA content of the M group increased significantly(P<0.01).Compared with the M group,the EA group and the A group can significantly down-regulate the serum TC,TG,and FFA levels(P<0.01);the serum TC and TG levels of the SEA group are also lower than the M group(P<0.01),but still higher than the EA group(P<0.01,P<0.05);and the EAI group can also reduce the serum TC,TG,FFA content when compare with the M group(P<0.05,P<0.01,P<0.05),but the effect of adjusting TC is not as good as that of EA group(P<0.05).(5)IPITT results and IPGTT results: IPITT results showed that compared with the N group,the blood glucose level of the M group was significantly higher from the 30 th min(P<0.01).Compared with the M group,the blood glucose level of the EA group decreased at the 60 th and 120 th min(P<0.05),and the blood glucose level of the A group was lower than that of the M group from the 30 th min(P<0.05),and at the 60 th min,the degree of decline was the most significant(P<0.01).In different periods,the blood glucoselevels of the SEA group and the EAI group were not significantly different from the M group(P>0.05).The IPGTT results showed that compared with the N group,the blood glucose level of the M group increased from the 30 th min(P<0.05),and the difference was considered significant at the 30 th min(P<0.01).Compared with the M group,the blood glucose level of the EA group gradually decreased from the 60 th min(P<0.05),and was significantly lower than the M group at the 120 th min(P<0.01).The blood glucose level of the A group was lower than that of the M group from the 30 th min(P<0.05),and the difference was expanded to be more significant at the 120 th min(P<0.01).The blood glucose levels of the SEA group and the EAI group were not statistically different from the M group at each time period(P>0.05),and were higher than the EA group at the 120 th min(P<0.05).(6)The GIR level: Before the intervention,compared with the N group,the GIR levels of the other five groups were significantly lower(P<0.01).After intervention,the GIR level of rats in the M group was still significantly lower than that in the N group(P<0.01).Compared with the M group,the EA group,EAI group,and A group can all increase the GIR level of rats(P<0.01,P<0.05,P<0.01).The GIR level of the SAE group was not only indistinguishable from the model group(P>0.05),but also significantly lower than that of the EA group(P<0.01).(7)The Serum INS content: Compared with the N group,the serum INS level of the M group was significantly increased(P<0.01).Compared with the M group,the serum INS levels in the EA group and A group were significantly decreased(P<0.01),but the INS levels of SEA group and EAI group were not significantly different fromthose of M group(P>0.05),and the INS levels in the two groups were still higher than those in the EA group(P<0.01,P<0.05).3.Effect of EA on LPS and intestinal barrier function in obese rats with insulin resistance(1)The Serum LPS content: Compared with the N group,the serum LPS of the M group increased significantly(P<0.01).Compared with the M group,the EA group,EAI group,and A group can all down-regulate the serum LPS content of model rats(P<0.01,P<0.05,P<0.01).The serum LPS content of the SEA group was not significantly different from that of the M group(P>0.05),and was significantly higher than that of the EA group(P<0.01).(2)The protein expression of ZO-1 and Occludin in the small intestine tissue: Compared with the N group,the protein expression of ZO-1 and Occludin in the small intestine tissue of the M group was significantly decreased(P<0.01).Compared with the M group,the EA group,EAI group,and A group can up-regulate the protein expression of ZO-1 and Occludin(P<0.01,P<0.05,P<0.01),and the protein expression of Occludin in the SEA group was also higher than that in the M group(P<0.05).Compared with the EA group,the protein expressions of ZO-1 and Occludin in the small intestine tissue of the SEA group and the EAI group were significantly reduced(P<0.05).(3)The gene expression of ZO-1 and occludin in small intestine:Compared with M group,m RNA expression of ZO-1 and occludin in small intestine of M group was significantly decreased(P<0.01).Compared with the M group,the A group could significantly up regulate the m RNA expression of ZO-1 and occludin(P<0.01),and EA intervention could also increase the m RNA expression of ZO-1 andoccludin(P<0.01,P<0.05).However,the m RNA expressions of ZO-1and occludin in the SEA group were not significantly different from those in the M group(P>0.05),and they were lower than those in the EA group(P<0.05).The EAI group could only increase the expression of ZO-1 m RNA(P<0.05),but the m RNA expression levels of ZO-1 and occludin were still significantly lower than EA group(P<0.05).4.The effect of EA on the intestinal mucosa morphology,Chiu’ score and apoptosis of intestinal epithelial cells in obese rats with insulin resistance(1)Morphological changes of the small intestine and Chiu’s score:Compared with the N group,the small intestinal mucosa of the M group was severely damaged,with a large amount of villus shedding and necrosis,and the glandular structure was disordered,and the Chiu’s score was significantly higher than that of the N group(P<0.01).Compared with the M group,the villi in the EA group and the A group were arranged more neatly,the gland structure was more complete,and the Chiu’s score was significantly lower than that of the M group(P<0.01).The SEA group and the EAI group had obvious mucosal damage and disordered villi arrangement.There was no difference in Chiu’s score compared with the M group(P>0.05),and the Chiu’s score of the two groups increased compared with the EA group(P<0.01,P<0.05).(2)Apoptosis of intestinal epithelial cells: Compared with the N group,apoptosis rate of intestinal epithelial cells of the M group was significantly increased(P<0.01).Compared with the M group,the four intervention groups can effectively reduce the apoptosis rate of intestinal epithelial cells(P<0.01).Among them,theapoptosis rate of intestinal epithelial cells in the EA group decreased more than that of the SEA group and the EAI group(P<0.01,P<0.05).5.The effect of EA on the protein and m RNA expression of Wnt/β-catenin related genes mediated by SIRT1 in the small intestine tissue of obese insulin-resistant rats(1)The protein and m RNA expression of SIRT1 in the small intestine tissue: Compared with the N group,the protein and m RNA expression of SIRT1 in the small intestine tissue of the M group was significantly reduced(P<0.01).Compared with the M group,the EA group and the A group can significantly up-regulate the protein and m RNA expression of SIRT1 in the small intestine tissue(P<0.01).The protein and m RNA expression of SIRT1 in the SEA group was also higher than that of the M group(P<0.05),but the protein content of SIRT1 was still lower than that of the EA group(P<0.05).The expression of SIRT1 protein and m RNA in the EAI group was not different from that of the M group(P>0.05),and was significantly lower than that of the EA group(P<0.01).(2)The co-expression of SIRT1 and β-catenin in the small intestine tissues of rats in each group: Compared with the N group,the positive cell rates of SIRT1 and β-catenin in the M group were significantly reduced(P<0.01).Compared with the M group,the positive cell rate of SIRT1 and β-catenin increased significantly in the EA group and the A group(P<0.01).The positive cell rate of SIRT1 and β-catenin in the EAI group was also higher than that in the M group(P<0.05),but the positive cell rate of β-catenin was lower than that in the EA group(P<0.05).Compared with the EA group,the positive cell rates of SIRT1 and β-catenin in theSEA group were lower(P<0.05,P<0.01),and only the positive cell rate of SIRT1 was higher than that of the M group(P<0.05).(3)The protein expression of related genes in the Wnt/β-catenin pathway in the small intestine: Compared with the N group,the protein expression of β-catenin and Cyclin D1 in the small intestine tissue of the M group was significantly reduced(P<0.01),the expression of p-β-catenin and caspase3 protein increased significantly(P<0.01).Compared with the M group,the A group can significantly up-regulate the protein expression of β-catenin and Cyclin D1(P<0.01),and down-regulate the protein content of p-β-catenin and caspase3(P<0.01).The protein expression of β-catenin and Cyclin D1 in the EA group was also higher than that in the M group(P<0.01,P<0.05),and the protein expression of p-β-catenin and caspase3 was significantly lower than that in the M group(P<0.01).Compared with the M group,the SEA group can only reduce the protein expression of p-β-catenin and caspase3(P<0.05).In addition,compared with the EA group,the SEA group has a certain difference in regulating the protein expression of β-catenin,p-β-catenin,Cyclin D1,and caspase3(P<0.05).The protein expression of β-catenin in the EAI group was higher than that in the M group(P<0.05),and the protein expression of p-β-catenin and caspase3 was lower than that in the M group(P<0.05,P<0.01),but the EAI group’s regulatory effect on the expression of p-β-catenin and Cyclin D1 protein was still different than that of the EA group(P<0.05).(4)The m RNA expression of related genes in the Wnt/β-catenin pathway in the small intestine tissue: Compared with the N group,the m RNA expression of β-catenin and Cyclin D1 in the smallintestine tissue of the M group was significantly reduced(P<0.01),and the expression of caspase3 m RNA was significantly increased(P<0.01).Compared with the M group,both the EA group and the A group could up-regulate the m RNA expression levels of β-catenin and Cyclin D1 in the small intestine tissues(P<0.01),and at the same time down-regulate the caspase3 m RNA expression levels(P<0.01).The m RNA expression of β-catenin and Cyclin D1 in the EAI group was also increased compared with the M group(P<0.05),and the expression of caspase3 m RNA was decreased compared with the M group(P<0.05).Among them,the EAI group’s regulatory effect on β-catenin m RNA is not as good as the electroacupuncture group(P<0.05).Compared with the M group,the SEA group can increase the m RNA expression levels of β-catenin and Cyclin D1 in the small intestine tissue(P<0.05),but the EA group has better regulatory effects on β-catenin,Cyclin D1,and caspase3 m RNA than SEA group(P<0.05).Conclusions1.EA can effectively reduce the body mass,Lee’s index,WAT weight,correct lipid metabolism disorders,enhance systemic insulin sensitivity,and significantly reduce serum insulin levels in obese insulin-resistant rats,thereby improving insulin resistance.2.EA can promote the expression of tight junction related proteins ZO-1 and Occludin in the small intestine tissue of obese insulin-resistant rats,improve intestinal barrier function,decrease intestinal permeability,and reduce serum LPS levels.3.EA can inhibit the apoptosis of small intestinal cells in obese insulin-resistant rats,repair intestinal mucosal damage,andmaintain the structural integrity of intestinal epithelium。4.EA can up-regulate SIRT1 protein and gene expression in the small intestine tissue of obese insulin-resistant rats,increase β-catenin transcription and translation,reduce p-β-catenin protein expression,activate Wnt/β-catenin pathway,and make the expression of caspase3 protein and gene decreased,and the expression of Cyclin D1 protein and genes increased.5.EA and SIRT1 agonists have similar regulatory effects on obese insulin-resistant rats,while SIRT1 inhibitors can partially block the effect of EA,indicating that EA promotes the expression of SIRT1,thereby up-regulating the expression of β-catenin,activating the Wnt/ β-catenin pathway,regulating the transcription and translation of downstream target genes,inhibiting the apoptosis of small intestinal cells,repairing intestinal mucosal damage,promoting intestinal barrier function,and reducing Intestinal permeability,reducing LPS leakage,to achieve the goal of improving obesity and insulin resistance.In summary,EA can enhance the proliferation of intestinal epithelial cells by regulating SIRT1 to mediate the Wnt/β-catenin signaling pathway,change the intestinal barrier function to reduce the level of LPS entering the circulation,and to improve the state of obesity and insulin resistance.This is a new mechanism for EA to improve insulin resistance,and may provide experimental evidence for electroacupuncture to prevent obesity and insulin resistance-related diseases. |
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