Effect And Mechanism Of Electroacupuncture Combined With Diet Restriction On Microbiota-Gut-Brain-Axis Of Obese Rats With Insulin Resistance

ObjectivesObesity is closely related to disorders of appetite regulation.Hypothalamus,as the feeding center,is the key part of the body to control food intake and regulate energy metabolism.Insulin resistance is an important early pathogenesis of type 2 diabetes mellitus and obesity.Therefore,based on the previous studies,this study took obese rats with insulin resistance induced by high-fat diet as the research object.Starting from gut microflora regulating the content of brain-gut peptide which leads to central appetite regulation and energy metabolism disorders,which are closely related to the occurrence and development of insulin resistance,we observed the effects of electroacupuncture and diet restriction on gut microflora structure and function in obese rats with insulin resistance.The relationship between acupuncture and insulin sensitivity and body mass further elucidates the mechanism of electroacupuncture and diet restriction improving obesity in insulin resistance by regulating gut microflora-gut-brain axis.Methods100 SPF Wistar male rats were 8 weeks old and were all healthy.15 rats were choosed as the normal group at random and fed with 3.8kcal/g of common diet.The remaining 85 rats were fed with 5.4 kcal/g of high-fat diet to establish the model.After 8 weeks of feeding,52 obese rats were randomly divided into model group,electro-acupuncture group(EA group),diet restriction group(DR group)and electro-acupuncture combined diet restriction group(ED group),with 13 rats in each group.Three rats were randomly selected from each group for hyperinsulinemia-euglycemic clamp to determine the insulin sensitivity of rats in order to determine the success of insulin resistance model.Subsequently,each group was given corresponding intervention methods.(1)Normal group(n = 10): just only fed with regular diet;(2)Model group(n = 10): just only fed with high-fat diet;(3)EA group(n = 10): fed with high-fat diet continuely,Zhongwan(CV12),Guanyuan(CV4),Zusanli(ST36)and Fenglong(ST40)for acupuncture.Two electrodes of the same output of the HANS LH202 H EA therapeutic apparatus are connected with ST36 and ST40 on the ipsilateral side,and the other two electrodes are connected with CV12 and CV4.Continuous wave,frequency at 2 Hz and intensity at1 m A is selected.10 minutes a day,3 times a week,the total course of the treatment is 8 weeks.(4)DR group(n = 10): 30% calorie restriction was given every day for dietary control,feeding alone for 8 weeks;(5)ED group(n = 10): 30% calorie restriction was given every day and EA was given at the same time(the treatment was the same as that of EA group)for 8 weeks.The food intake,body weight and the nasal length of rats were measured before intervention and at the 2nd,4th,6th and 8th weeks of intervention respectively,and Lee’s index was calculated according to the formula.At the 6th week of intervention,the intraperitoneal glucose tolerance and intraperitoneal insulin tolerance were tested.At the end of all interventions,three rats in each group were choosed at random for hyperinsulin-normal glucose clamp to detect insulin sensitivity.Subsequently,the rats were perfused or fresh feces,blood,gut tract,nodular ganglion and brain tissue were taken for index detection.(1)Macrogenomics detection technology: Macrogenomics was used to detect the distribution of gut microflora,specific microflora and metabolic function of the fresh stool before the rats were executed.(2)Enzyme-linked immunosorbent assay(ELISA): Before the rats were executed,blood collected from rat heart was taken to detect the levels of serum insulin levels,leptin and CCK.(3)Western blotting(WB): The expression of leptin,leptin R,CCK,CCKR in the colon tissues and p-STAT 3 in nodal ganglion and arcuate nucleus of rats were detected by WB.(4)Real-time quantitative PCR(RT-PCR): RT-PCR was used to detect the levels of gene expression of leptin,leptin R,CCK and CCKR in colon tissues.(5)Immunohistochemistry method: Immunohistochemistry was used to detect the protein expression of phosphorylated STAT3 in efferent vagal neurons,and C-Fos expression in area postrema(AP)and nucleus tractus solitarius(NTS).Results1.EA and DR can reduce the food intake,body mass and blood sugar of obese rats with insulin resistance,improve insulin sensitivity.(1)The results of food intake showed that compared with the normal group,the food intake of rats in model group increased significantly(P<0.01).After 4 weeks of intervention,compared with the model group,the food intake of rats in the EA group decreased significantly(P<0.05).The difference became very significant until the 8th week of intervention(P < 0.01),suggesting that EA could inhibit the food intake of obese rats.However,due to the average daily food intake was 70% of the model group,so from the second week,the amount of food intake in the DR group and the ED group was significantly less than that in the model group and the EA group,the difference was very significant(P<0.01),but this didn’t mean that the active amount of food intake in the two groups decreased,but to the eighth week,the food intake of the two groups was still further reduced,indicating that DR combined with EA could inhibit appetite.At each time point,compared with the DR group,the intake of ED group was further reduced,indicating that electroacupuncture and diet restriction had synergistic effect on appetite inhibition.(2)The results of body weight showed that the weight of rats in each group was significantly higher than that in the normal group(P<0.01)at the 0th week of intervention,and all of them exceeded20% of the average body weight of rats in the normal group.In the first four weeks of intervention,the weight of rats in each group showed an upward trend.From the 4th week,compared with the model group,the weight of rats in other groups began to decrease,and the weight of ED group decreased significantly(P<0.05).In the6 th week of intervention,the weight of EA group,DR group and ED group decreased significantly(P < 0.01).The trend remained unchanged until the 8th week.Compared with the EA group,the ED group significantly decreased the weight(P<0.05).(3)The results of Lee’s index showed that the Lee’s index of each group was significantly higher than that of the normal group at week0(P<0.01).In the first four weeks of intervention,Lee’s index of rats in each group showed an upward trend.From the 4th week,compared with the model group,the Lee’s index of rats in other groups began to decrease,and the Lee’s index of ED group decreased significantly(P<0.05).In the 6th week of intervention,the Lee’s index of EA group,DR group and ED group decreased significantly(P < 0.01).The trend remained unchanged until the 8th week.Compared with the EA group,the ED group significantly decreased the Lee’s index(P<0.05).(4)The results of blood sugar showed that there was no statistical difference in the fasting blood sugar of rats in each set time during the whole experiment.At the 0th week of intervention,the postprandial blood sugar of rats in each group was significantly higher than that in the normal group(P < 0.01).After the intervention,compared with the normal group,the postprandial blood glucose in the model group were markedly up-regulated(P<0.01);Contrast to the model group,the postprandial blood glucose in the EA group,the DR group and the ED group decreased significantly(P < 0.05);Compared with the EA group,the postprandial blood glucose in the ED group decreased significantly(P<0.05).(5)The results of serum insulin showed that the level of serum insulin in the model group was significantly higher than that in the normal group(P<0.01),suggesting that the level of plasma insulin in obese rats with insulin resistance induced by high fat diet was higher.After intervention,the serum insulin levels in EA group,DR group and ED group were significantly lower than those in model group(P < 0.01),indicating that both EA and diet restriction can regulate the state of hyperinsulinemia.Compared with the EA group,the serum insulin in the ED group decreased significantly(P<0.05).(6)IPGTT test showed that there was no significant difference in the fasting state of blood glucose among the groups.The blood glucose increased 30 minutes after intraperitoneal injection of glucose and reached its peak value,then began to decline.Compared with the normal group,blood sugar in the model group increased significantly from 30 minutes to 120 minutes(P<0.05).Compared with the model group,the blood sugar of rats in EA group,DR group and ED group decreased significantly from 30 minutes to 120 minutes(P < 0.01).IPITT test showed that there was no significant difference in fasting blood glucose among groups.30 minutes after intraperitoneal insulin injection,the decrease of blood glucose in normal group,EA group,DR group and ED group was prominently shorter than which in model group(P <0.05).After 60 minutes,the blood sugar of rats in each group increased gradually.After 120 minutes,the blood sugar in model group was significantly higher than that in normal group(P<0.01).Compared with the model group,the blood sugar of rats in EA group,DR group and ED group decreased significantly(P<0.01).The GIR results showed that the GIR values of rats in the successful groups were significantly lower than those in the normal group at week 0(P<0.01),and there were no obvious differences among the groups.After 8 weeks of intervention,compared with model group,GIR in EA group,DR group and ED group increased significantly(P<0.01).2.EA and DR can regulate the content of serum brain-gut peptide and the structure and function of gut microflora in obese rats with insulin resistance.(1)The results of serum brain-gut peptide showed that the content of CCK in model group was obviously lesser than that in normal group(P<0.01).After intervention,the serum CCK level in EA group,DR group and ED group was significantly higher than that in model group(P<0.01).Compared with the EA group,the serum CCK in the ED group increased significantly(P<0.05).Compared with the normal group,the serum leptin level in the model group was significantly higher(P<0.01),suggesting that obese rats with insulin resistance induced by high fat diet existed leptin resistance.After intervention,the serum leptin level in EA group,DR group and ED group was significantly lower than that in model group(P<0.01),indicating that EA and DR can improve leptin resistance.Compared with EA group,serum leptin in ED group was significantly lower(P<0.05).(2)Effect of EA and DR on gut microflora structure in obese rats with insulin resistance: Compared with the normal group,the abundance of Bacteroides and Proteobacteria in model group was significantly reduced and the abundance of Firmicutes was significantly increased;Compared with model group,the abundance of Bacteroides and Proteobacteria in EA group,DR group and ED group all tended to increase.The abundance of Firmicutes tended to decrease.In the model group,the abundance of Bacteroides,Lactobacillus,Akkermansia and Clostridium decreased significantly,and the abundance of the above-mentioned flora increased significantly after electroacupuncture and diet restriction intervention.Genus level Heatmap showed that the structure and abundance of gut microflora were similar in EA group,DR group and ED group,and there were also some similarities between the three intervention groups and the normal group,but there were significant differences between model group and other groups.The results of beta diversity analysis showed that the species in the model group changed a lot,and both the electroacupuncture and diet restriction intervention could adjust the changed species to make them close to the normal species,and the species in the ED group was the closest to the normal group.(3)Effect of EA and DR on COG clustering of gut microflora in obese rats with insulin resistance: Compared with the normal group,the function of COG clustering of gut microflora were all decreased in model group in energy production and conversion,amino acid transport and metabolism,carbohydrate transport and metabolism,lipid transport and metabolism and inorganic ion transport and metabolism.While compared with the model group,the functional clustering of COG protein in EA group,DR group and ED group showed an upward trend.COG clustering Heatmap showed that EA group,DR group and ED group had similarities,and there were also some similarities between the three intervention groups and the normal group,while there was markable difference between the model group and other groups.(4)Effect of EA and DR on KEGG of gut microflora in obese rats with insulin resistance: Compared with the normal group,the function of metabolism of gut microflora in model group was obviously down-regulated.While contrast to the model group,the function of metabolism in EA group,DR group and ED group all increased significantly.In the model group,KEGG clusters of gut microbiotais were reduced in ko00785(Lipoic acid metabolism),ko00640(Propanoate metabolism),ko00650(Butanoate metabolism),ko00230(Purine metabolism),ko00240(Pyrimidine metabolism),ko00030(Pentose phosphate pathway),ko00600(Sphingolipid metabolism),ko00400(Phenylalanine,tyrosine and tryptophan biosynthesis)and ko00970(Aminoacyl-t RNA biosynthesis).After electroacupuncture and diet restriction,the above KEGG clusters of gut microbiotais were up-regulated.3.EA and DR can regulate the expression of brain-gut peptide and its receptor in colon tissue of obese rats with insulin resistance(1)The results of expression of CCK and CCKR showed that the expression of CCK and CCKR in colon tissue of model group was significantly lower than that of normal group(P<0.05,P<0.01).After 8 weeks of intervention,the expression of CCK and CCKR protein and m RNA in EA group,DR group and ED group were significantly increased compared with model group(P < 0.05).Compared with EA group,the expression of CCK and CCKR protein and m RNA in colon tissue of ED group were significantly increased(P<0.05).(2)The results of expression of leptin and leptin R showed that the expression of leptin in colon tissue of model group was significantly higher than that of normal group(P<0.05,P<0.01).After 8 weeks of intervention,compared with model group,the expression of leptin protein and m RNA in EA group,DR group and ED group were significantly decreased(P<0.05).Compared with EA group,the expression of leptin protein and m RNA in colon tissue of ED group decreased significantly(P<0.05).While the expression of leptin R in colon tissue of model group was significantly lower than that of normal group(P<0.05,P<0.01).After 8 weeks of intervention,compared with model group,the expression of leptin R protein and m RNA in EA group,DR group and ED group was notably raised(P<0.05).Contrast to EA group,the content of leptin R protein and m RNA in colon tissue of ED group increased significantly(P<0.05).4.EA and DR can improve the sensitivity of brain tissue to brain-gut peptide in obese rats with insulin resistance(1)The results of expression of p-STAT3 in the nodular ganglion showed that the expression of p-STAT3 in the model group was obviously down-regulated than which in the normal group(P<0.01).After 8 weeks of intervention,compared with model group,the expression of p-STAT3 protein in EA group,DR group and ED group was significantly increased(P<0.01).Compared with EA group,the expression of p-STAT3 in nodular ganglion of ED group increased significantly(P<0.05).(2)The results of expression of p-STAT3 in arcuate nucleus showed that the expression of p-STAT3 in model group was significantly lower than that in normal group(P < 0.01).After 8 weeks of intervention,compared with model group,the expression of p-STAT3 protein in arcuate nucleus was significantly increased in EA group,DR group and ED group(P<0.01).Compared with EA group,the protein expression of p-STAT3 in arcuate nucleus increased significantly in ED group(P<0.05).(3)The results of expression of p-STAT3 in efferent neurons of vagus nerve showed that the expression of p-STAT3 in dorsal vagal motor nucleus and nucleus ambiguus of model group was significantly lower than that of normal group(P < 0.01).After 8 weeks of intervention,compared with the model group,the expression of p-STAT3 in vagal dorsal motor nucleus and nucleus ambiguus was significantly increased in EA group,DR group and ED group(P<0.01).Compared with EA group,the expression of p-STAT3 protein in dorsal vagal motor nucleus and nucleus ambiguus increased significantly in ED group(P<0.05).(4)The results of expression of C-Fos in the AP of the model group was remarkably lesser than which in the normal group(P<0.01).After 8 weeks of intervention,compared with the model group,the expression of C-Fos in AP of EA group,DR group and ED group was significantly increased(P<0.01).Compared with EA group,the expression of C-Fos in the AP of ED group was significantly increased(P<0.05).(5)The results of expression of C-Fos in the NTS showed that the expression of C-Fos in the NTS of the model group was significantly lower than that of the normal group(P<0.01).After 8 weeks of intervention,compared with model group,the expression of C-Fos in NTS of EA group,DR group and ED group was significantly increased(P<0.01).Compared with EA group,the expression of C-Fos in NTS in ED group increased significantly(P<0.05).Conclusions1.EA and DR can reduce the body weight and Lee’s index of obese rats with IR,reduce the food intake,significantly reduce the content of serum insulin,and enhance the insulin sensitivity of obese rats with IR.2.EA and DR can improve the structure and function of gut microflora in obese rats with IR,regulate the content of serum brain gut peptide in obese rats with IR,reduce the content of serum leptin and increase the content of serum CCK.3.EA and DR can significantly increase the expression of CCK,CCKR and leptin R in the colon tissue of obese rats with IR,and decrease the expression of leptin.4.EA and DR can improve the sensitivity to brain gut peptide CCK and leptin of central(hypothalamus and brain stem)in obese rats with IR.5.By improving the structure and function of gut microflora in obese rats with IR,EA and DR can regulate the content of brain gut peptide both in serum and intestinal in IR obese rats,and further improve the sensitivity to CCK and leptin of central(hypothalamus and brain stem),so as to control the appetite,weight and improve insulin sensitivity of obese rats with IR.There was a synergistic effect between EA and DR.To sum up,EA and DR can improve insulin sensitivity and energy metabolism in obese rats with insulin resistance by regulating the structure and function of gut microflora,and central sensitivity to brain-gut peptide,that is,by regulating gut microflora-gut-brain axis by electroacupuncture and diet restriction.It has synergistic effect,which provides experimental basis for clinical application of acupuncture with diet restriction in the treatment of obesity and related diseases.