Effect And Mechanism Of Electroacupuncture On Glucose Metabolism In Type 2 Diabetic Mice By Regulating Gut Microbiota

Objective Although electroacupuncture(EA)has been reported to reduce blood glucose levels and change intestinal flora in diabetic mice,there have been few related studies and the relationship between the three has not been clearly clarified.In this study,a model of type 2 diabetes mellitus(T2DM)was stimulated by EA,and the effect of EA on blood glucose levels and intestinal flora of diabetic mice was observed.The effect of EA-altered flora on blood glucose of diabetic mice was used to prove whether EA can regulate blood glucose levels by changing the intestinal flora.Methods 6 to 8 weeks of C57BL/6 mice(male)induced by high-fat diet and STZ into type 2 diabetes,randomly divided into five groups: normal group,high-fat diet group(HFD),type 2 diabetes group(DM group),DM plus pseudo EA stimulation group(DM+SEA group),DM plus EA stimulation group(DM+EA group).Monitoring daily weight and random blood glucose levels(RBG).After 8 weeks,the RBG and fasting blood glucose levels(FBG)of the mice in each group were evaluated,the kit was used to detect glycosylated hemoglobin,the glucose tolerance and insulin resistance were evaluated by OGTT and ITT,and the 16 s r DNA amplicon sequencing technology was used to detect the diversity and composition of intestinal flora;Establishment a model of T2 DM sterile mouse faecal transplantation,randomly divided into 5 groups,namely,recipient mice plus PBS transplantation group(ABX+PBS),recipient mice plus normal control mice fecal transplantation group(ABX+Control),recipient mice plus diabetic mice fecal transplantation group(ABX+DM),recipient mice plus sham EA stimulated mice fecal transplantation group(ABX+SEA),recipient mice plus EA stimulated mice fecal transplantation group(ABX+EA).The blood glucose level was monitored every week.4 weeks after the FMT,the level of insulin and Hb A1 c in the serum,OGTT and ITT were performed to assess the insulin resistance.Results(1)Compared with the normal group,the blood glucose level of diabetic mice was significantly increased.After EA treatment,compared with the DM group,the blood glucose and serum insulin levels of the mice in EA group were reduced.The total areas under the OGTT and ITT curve were distinctly reduced,and there was no significant change in the level of glycated hemoglobin.There was no significant change in blood glucose of mice in SEA group.(2)The Chao 1 index was not significantly different in each group,while the Shannon index was significantly reduced in the DM group and significantly increased in the EA group;At the Phylum level,the abundance of Firmicutes increased in the DM group,and significantly decreased in the EA group;In the genus level,Lachnoclostridium,Lachnospiraceae_UCG-006,Odoribacter and Oscillibacter increased in the DM group,but decreased after EA stimulation;The content of caproic acid in the EA group was increased,but there was no significant changes in the total and other SCFAs content.(3)After FMT,compared with ABX+DM group,the blood glucose level and serum insulin level of mice in the ABX+EA group decreased significantly,and the area under the curves of OGTT and ITT decreased.Conclusion EA changed the diversity and composition of intestinal flora,thereby effectively improving the insulin resistance and ultimately reducing blood glucose levels in T2 DM mice.Objective Although in the previous part of the experiment we have confirmed that EA can reduce the blood glucose level of T2 DM mice by regulating the intestinal flora,but the specific mechanism has not yet been explained.This section we will further explore the specific mechanism of effect of EA on glucose metabolism by after changing the intestinal flora.Methods The serum,feces,intestinal tissue,liver tissue,and muscle tissue from mice model in the first part.The following tests were performed: Ultra-high-performance gas chromatography-mass spectrometry(GC-MS/MS)was used to detect seven kinds of SCFAs(acetic acid,propionic acid,isobutyric acid,butyric acid,isovaleric acid,valeric acid and hexanoic acid)in the feces.CBA technology was used to detect the levels of inflammatory factors(IL6,IL10,IL12P70,IFN-?,MCP-1 and TNF-?)of serum;Western blot was used to detect the colonic tight junction protein level(ZO-1,occludin,claudin-1),colonic inflammatory factors(TLR4,IL1-?,IL6,IL10,TNF-?),and p-IKK?,p-JNK,IRS-1,p-IRS-1,AKT and p-AKT in liver and muscle tissue;q RT-PCR was used to detect the tight junction protein m RNA level(ZO-1,occludin,claudin-1),and inflammatory factors(TLR4,IL1-?,IL6,IL10,TNF-?)in colon tissue of mice.Results(1)The contents of SCFAs in feces of diabetic mice were significantly lower than normal control group.EA could increase the content of caproic acid in the feces of diabetic mice,but had no significant effect on the contents of total and other SCFAs.(2)Compared with Control group,the expression level of intestinal tight junction proteins ZO-1,occludin,claudin-1 was significantly reduced in DM group mice,and EA promoted the m RNA and protein expression of these molecules.(3)Diabetic mice showed the increased expression of pro-inflammatory factors and decreased anti-inflammatory factors in colon tissue,and EA can inhibit the expression of pro-inflammatory factors IL1-?,IL6 and TNF-? in colon tissue,and promote the expression of anti-inflammatory factor IL10.(4)EA reduced the expression of proinflammatory factors IL-6,MCP-1 and TNF-? in serum,but had no significant effect on IL10,IL12P70 and IFN-?.(5)EA inhibits the protein phosphorylation levels of IKK?,JNK and IRS-1 in liver and muscle tissues,and promotes the protein phosphorylation of AKT.Conclusion EA may promote the expression of intestinal tight junction proteins and maintain the integrity of the intestinal barrier by regulating the intestinal flora,thereby reducing the inflammation and promoting the normal conduction of insulin signaling through IKK?/ JNK-IRS-1-AKT pathway in liver and muscle tissues,ultimately lower glucose levels and increase insulin sensitivity of T2 DM mice.Objective The first two parts have confirmed that EA can improve the glucose metabolism of diabetic mice by regulating the intestinal flora,but how EA can regulate the intestinal flora needs further confirmation.Our previous research has confirmed that EA can repair the intestinal ICC of type 1 diabetic mice and promote gastrointestinal motility.We speculate whether EA also promotes intestinal motility by repairing intestinal ICC of type 2 diabetic mice,thereby regulating intestinal bacteria group.Methods The model of type 2 diabetic mice with EA treatment and the Kit W/Wv gene mice with EA treatment were established,and the intestinal motility function of the mice was evaluated by measuring the intestinal transit time,defecation frequency and fecal water content;The glucose levels of mice in each group were detected at the end of EA treatment.The Immunofluorescence technique(c-Kit and Ano1 co-staining)was used to detect the expression level and distribution range of ICC in the intestinal muscularis of mice.Western blot was used to detect the content of c-Kit and m SCF in the intestinal muscle of mice,and RT-PCR was used to detect the content of m SCF and c-Kit in the intestine of mice to evaluate the expression level of intestinal ICC;16s r DNA amplicon sequencing technique was used to detect the diversity and composition in the intestinal flora of the mice after EA treatment?Results(1)Compared with the DM group,the intestinal transit time of the mice in the DM + EA group was reduced,and the fecal water content was significantly increased.(2)After EA treatment,immunofluorescence results showed that c-Kit and Ano1 co-staining coincidence rate of intestinal muscularis in diabetic mice increased significantly,and the network structure was more complete.(3)The expression levels of c-Kit and m SCF proteins and genes in the colon tissue of mice in the DM + EA group were significantly higher than those in the DM group.(4)In the model of Kit W/Wv gene mice with EA treatment,the blood glucose level of the mice after EA treatment was not significantly improved compared with that in the DM group.(5)In the model of Kit W/Wv gene mice with EA treatment,the intestinal transit time,fecal water content,and defecation frequency of mice in each group showed no significant changes.(6)In the model of Kit W/Wv gene mice with EA treatment,results about the diversity and composition of intestinal flora in mice after EA treatment had not been reported.Conclusion EA increases the number of ICC and repairs the network structure of ICC in diabetic mice,which promotes intestinal motility and improved the glycometabolism,but whether EA affects the intestinal flora by promoting the intestinal motility,thereby reducing the glucose level has not been concluded.