| ObjectivesObesity has become a public health problem affecting the health of millions of people in developing and developed countries.Chronic low-grade inflammation of the whole body is closely related to obesity and is an important cause of islet resistance in obese people.Insulin resistance is a key initiating factor of obesity harmful to health,which can lead to glucose intolerance,dyslipidemia,type 2 diabetes,atherosclerosis and hypertension.At present,the sources of inflammation and pro-inflammatory cell factors in obese people or animal models all focus on adipose tissue,but the latest findings point out that obese diet may lead to the imbalance of intestinal microflora homeostasis,resulting in the activation of intestinal inflammation and changes in gastrointestinal motility,which is closely related to the development of insulin resistance.Therefore,taking relevant intervention measures on obesity and insulin resistance as soon as possible is of great significance to prevent or limit the adverse effects of obesity on health.In this experiment,the model was induced by high-fat diet.The successful obese Wistar rats with insulin resistance were intervened by electroacupuncture.From the point of view of intestinal inflammation,the effects of this method on silencing information regulatory factors(SIRT1),Toll-like receptor 4(TLR4),nuclear factor-kappa B(NF-?B)and inflammatory state of small intestinal mucosa in obese rats with insulin resistance were observed.To analyze the correlation between electroacupuncture enhancing insulin sensitivity and improving small intestinal inflammation,and to explore the mechanism of electroacupuncture regulating small intestinal inflammation through SIRT1/TLR4 to improve insulin resistance and obesity,in order to provide experimental basis for clinical application of acupuncture in the prevention and treatment of obesity and related metabolic diseases.MethodsA total of 90 8-week-old SPF male Wistar rats were selected.After one week of adaptive feeding,the rats were randomly divided into two groups: normal group(n=15)and high-fat diet group(n=75).After 8 weeks of normal feeding,5 rats were randomly selected from the normal group,20 rats were randomly selected from the model group,and then high insulin-euglycemic clamp was performed to evaluate the success of insulin resistance model.In addition,the body weight and anal length of each rat need to be measured to calculate the Lee's index,so as to judge whether the obesity model is successful or not,and the model that meets the above two evaluation criteria can be considered as successful.The rats fed with high-fat diet were randomly divided into 5 groups: model group,electroacupuncture(EA)group,electroacupuncture(EA)+ inhibitor group,inhibitor group and resveratrol(RSV)group,with 10 rats in each group.10 rats fed with general diet were randomly selected as normal group.After the end of the group,the normal group continued to be fed with ordinary diet without any intervention measures;the other five groups were fed with high-fat diet,and the model group did not take any intervention measures.The electroacupuncture(EA)group was treated with electroacupuncture,selecting Zusanli acupoint and Fenglong acupoint on both sides of lower extremities,Guanyuan acupoint and Zhongwan acupoint on abdomen,the acupuncture depth was 3-5 mm,and using Korean electroacupuncture instrument,the frequency of electroacupuncture instrument was set to 2Hz,the intensity of electroacupuncture instrument was set to 1mA,continuous wave was used,the intervention time lasted 10 minutes,3 times a week,and the whole course of treatment lasted 8 weeks.The electroacupuncture(EA)plus+ inhibitor group was given the same electroacupuncture treatment as the electroacupuncture(EA)group,based on the body weight of each rat,according to the unit proportion of 1mg/kg,the injection volume of Sirtinol inhibitor in each rat was calculated by tail vein injection,3 times a week,the intervention lasted 8 weeks,and the inhibitor group was only given Sirtinol inhibitor via tail vein injection for 8 weeks.In the resveratrol(RSV)group,according to the body weight of each rat and the unit proportion of 200mg/kg,the intragastric administration of resveratrol in each rat was calculated,3 times a week,and the intervention lasted for 8 weeks.During the intervention period,the body weight of rats in each group was measured and recorded at the 0th,2nd,4th,6th and 8th week of the intervention,in addition,after the completion of the intervention treatment at the 6th week,the abdominal glucose tolerance(IPGTT)and intraperitoneal insulin tolerance(IPITT)of all rats were detected and recorded.After eight weeks of intervention,3 rats from each group were randomly selected and treated with hyperinsulinemic euglycemic clamp,during which the glucose infusion rate(GIR)was measured at the same time.After the completion of the intervention,blood was taken from the apex of the heart,and the rats in each group were killed and samples were taken.The levels of serum endotoxin(LPS),serum inflammatory factors CRP,TNF-?,IL-6 and serum insulin were detected by ELISA method,and the levels of TC,TG and FFA were detected by biochemical method.In addition,the morphological changes of small intestinal mucosa were observed by hematoxylin-eosin staining(HE),the protein expressions of SIRT1,TLR4 and NF-?B in small intestinal tissues of each group were detected by Western blot(Western blotting),and the gene expression levels of SIRT1,TLR4 and NF-?B in intestinal tissues of each group were detected by real-time fluorescence quantitative PCR(RT-PCR).Results 1.Effects of high-fat diet on the weight,Lee's index and GIR of rats after eight weeks of feedingAfter eight weeks of feeding,the body weight of the model group fed with high fat diet was significantly higher than that of the normal group fed with general diet(P <0.01),and the Lee's index of the model group fed with high fat diet was higher than that of the normal group(P <0.05).The GIR index of the model group fed with high fat diet was significantly lower than that of the normal group(P <0.01).2.Effects of EA on body weight,lipid metabolism,serum Insulin,insulin sensitivity and GIR in obese rats with insulin resistanceAfter eight weeks of electroacupuncture treatment,compared with the model group,the changes of body weight in the electroacupuncture group and agonist group were significantly decreased(P < 0.01),and those in the inhibitor group were significantly higher than those in the electroacupuncture group(P < 0.05).Compared with the electroacupuncture group,there were significant differences between the inhibitor group and the RSV group(P < 0.01).In terms of lipid metabolism,compared with the model group,the contents of TC,TG and FFA in the EA group and RSV group decreased significantly,the contents of TC,TG and FFA in the EA + inhibition group decreased significantly(P <0.05),and the contents of TC,TG and FFA in the inhibitor group increased significantly(P < 0.01),indicating that electroacupuncture can improve the disorder of lipid metabolism in IR obese rats.Compared with the EA group,the contents of TC,TG and FFA in the EA + inhibition group and inhibitor group increased significantly(P <0.01),while the contents of TC,TG and FFA in the inhibitor group and RSV group were significantly different from those in the EA + inhibition group(P <0.01).In terms of serum insulin(INS),compared with the model group,the serum insulin(INS)level in the EA group,the EA + inhibition group and the agonist group decreased significantly(P <0.01),while the serum insulin(INS)level in the inhibitor group increased significantly(P <0.01),and the serum insulin(INS)level in the EA + inhibition group and inhibitor group was significantly higher than that in the EA group(P <0.01).Compared with the EA + inhibition group,the serum insulin(INS)level in the inhibitor group increased significantly(P <0.01),and the serum insulin(INS)level decreased significantly in the RSV group(P <0.01).In terms of IPGTT,the blood glucose level in the EA group and RSV group was significantly lower than that in the model group(P <0.01),the blood glucose level in the EA + inhibition group was lower than that in the model group(P <0.05),and the blood glucose level in the inhibitor group was significantly higher than that in the model group(P <0.01),and the blood glucose level in the EA + inhibition group was significantly higher than that in the EA group(P <0.05).The level of blood glucose in the EA + inhibition group was significantly lower than that in the inhibitor group(P <0.01)and higher than that in the RSV group(P <0.01).In terms of IPITT,the blood glucose level in the EA group and RSV group was significantly better than that in the model group(P <0.01),and the blood glucose level in the inhibitor group was significantly higher than that in the model group(P <0.01),and the blood glucose level in the EA group was significantly lower than that in the EA + inhibitor group(P <0.01)and the inhibitor group(P <0.01).The level of blood glucose in the EA + inhibition group was significantly lower than that in the inhibitor group(P <0.01)and higher than that in the agonist group(P <0.01).In terms of GIR,compared with the model group at the same period after treatment,the GIR value in the EA group and the RSV group increased significantly(P <0.01),the GIR value in the EA + inhibition group increased significantly(P <0.05),and the GIR value in the inhibitor group decreased significantly compared with the EA group in the same period after treatment(P <0.05).Compared with the EA group in the same period after treatment,the GIR value in the inhibitor group also decreased significantly(P <0.01).Compared with the EA + inhibition group at the same period after treatment,the value of GIR in the inhibitor group decreased significantly(P <0.01),and the value of GIR in the RSV group increased significantly(P <0.01).3.Effects of EA on serum endotoxin(LPS),serum inflammatory factors CRP,TNF-?,IL-6 and small intestinal mucosal morphology in obese rats with insulin resistanceThe level of serum LPS: compared with the model group,the content of serum LPS in EA group and RSV group decreased significantly(P <0.01),and the level of serum LPS in EA + inhibition group also decreased(P <0.05),while the content of serum LPS in inhibitor group increased significantly(P < 0.01).Compared with the EA group,the serum LPS content in the EA + inhibition group was significantly increased(P <0.05),and the serum LPS content in the inhibitor group was also significantly increased(P <0.01),while the serum LPS content in the inhibitor group was significantly higher than that in the EA + inhibition group(P <0.01),and the serum LPS content in the agonist group was significantly lower than that in the RSV group(P <0.05).Compared with the model group,the contents of serum CRP,TNF-? and IL-6 in the EA group and RSV group were significantly lower than those in the model group(P <0.01),and the contents of CRP,TNF-? and IL-6 in the EA + inhibition group were also decreased(P <0.05),while the Serum CRP and TNF-? in the inhibitor group increased significantly(P <0.01),and serum IL-6 levels also increased(P <0.05).Compared with the EA group,the contents of serum CRP,TNF-? and IL-6 in the EA + inhibition group were significantly higher than those in the EA group(P <0.05),and the contents of CRP,TNF-? and IL-6 in the inhibitor group were significantly higher than those in the EA group(P <0.01).Compared with EA + inhibition group,serum CRP,TNF-? and IL-6 levels in inhibitor group increased significantly(P <0.01),while the serum CRP and TNF-? in the RSV group were significantly decreased(P <0.05),and serum IL-6 levels were also significantly reduced(P <0.01).The morphological changes of intestinal mucosa and Chiu's score: in the normal group,the structure of each layer of intestinal mucous membrane was intact,the arrangement of epithelial cells was relatively neat,the arrangement of villi was regular and there was no rupture,compared with the normal group,the intestinal mucosal cells of model group and inhibitor group had the phenomena of atrophy,erosion,inflammatory infiltration and so on.Compared with the model group,the tissue structure of small intestinal mucosa in electroacupuncture group and agonist group was clearer,and the phenomena of atrophy,erosion and inflammatory infiltration were alleviated.Compared with the model group,the Chiu's score of the EA group,the EA + inhibition group and the agonist group decreased significantly(P < 0.01),while the Chiu's score of the inhibitor group increased significantly(P < 0.01).Compared with the EA group,the Chiu's score of the EA + inhibition group and the inhibitor group increased significantly(P < 0.01).Compared with the EA + inhibition group,the Chiu's score in the inhibitor group was significantly higher than that in the agonist group(P < 0.01),and the Chiu's score in the RSV group was significantly lower than that in the EA + inhibition group(P < 0.01).4.Effect of EA on the expression of SIRT1,TLR4,NF-?B protein and mRNA in small intestine of obese rats with insulin resistanceIn terms of protein expression,compared with the model group,the expression of SIRT1 protein in the EA group and RSV group was significantly increased(P < 0.01),while the expression of TLR4 and NF-?B protein decreased significantly(P < 0.01),and the expression of SIRT1 protein in the EA + inhibition group was higher than that in the model group(P < 0.05).The expression of NF-?B protein in the EA + inhibition group was significantly lower than that in the model group(P < 0.05).The expression of SIRT1 protein in the inhibitor group was significantly lower than that in the model group(P < 0.01),while the expression of TLR4 and NF-?B protein was significantly increased in the inhibitor group(P < 0.01).Compared with EA group,SIRT1 protein expression decreased significantly in EA + inhibition group and inhibitor group(P < 0.01),while TLR4 and NF-?B protein expression increased significantly in EA + inhibition group and inhibitor group(P < 0.01).Compared with the EA + inhibition group,the expression of SIRT1 protein in the inhibitor group was significantly decreased(P < 0.01),while the expression of TLR4 and NF-?B protein was significantly increased in the inhibitor group(P < 0.01),and the expression of SIRT1 protein in the RSV group was significantly increased(P < 0.01),while the expression of TLR4 and NF-?B protein was significantly decreased in the RSV group(P < 0.01).In terms of mRNA gene expression: compared with the model group,the expression of SIRT1 mRNA in EA group and RSV group was significantly increased(P < 0.01),while the expression of TLR4 and NF-?B mRNA was significantly decreased in EA group and RSV group(P < 0.01).SIRT1 mRNA expression in EA + inhibition group was significantly increased(P < 0.05),TLR4 and NF-?B mRNA expression was also decreased(P < 0.05).The expression of SIRT1 mRNA in the inhibitor group was significantly lower than that in the model group(P < 0.05),while the expression of TLR4 and NF-?B mRNA in the inhibitor group was significantly higher than that in the model group(P < 0.01).Compared with EA group,the expression of SIRT1 mRNA in EA + inhibition group and inhibitor group decreased significantly(P < 0.01),while the expression of TLR4 and NF-?B mRNA in EA + inhibition group increased significantly(P < 0.05),and the expression of TLR4 and NF-?B mRNA in inhibitor group increased significantly(P < 0.01).Compared with the EA + inhibition group,the expression of SIRT1 mRNA in the inhibitor group was significantly decreased(P < 0.01),while the expression of TLR4 and NF-?B mRNA was significantly increased in the inhibitor group(P < 0.01),and the expression of SIRT1 mRNA in the RSV group was significantly increased(P < 0.01),while the expression of TLR4 and NF-?B mRNA was significantly decreased in the RSV group(P < 0.05).Conclusions 1.Both electroacupuncture and SIRT1 agonists can reduce the body weight and growth rate of IR obese rats,reduce the contents of blood lipids TC,TG and FFA,and enhance the islet sensitivity of IR obese rats,thus improving the state of IR.It shows that electroacupuncture can improve obesity and IR status by specifically up-regulating SIRT1.Compared with the EA group,the body weight growth rate of the EA + inhibition group treated with SIRT1 inhibitor increased significantly,the levels of blood lipids TC,TG and FFA increased significantly,and the insulin sensitivity decreased,indicating that SIRT1 inhibitor can partially reverse the effect of electroacupuncture.2.Both electroacupuncture and SIRT1 agonists can reduce the contents of serum inflammatory factors TNF-? and IL-6 in IR obese rats,reduce the level of serum LPS in IR rats,and reduce the IR state.At the same time,electroacupuncture can also reduce the atrophy,erosion and inflammatory infiltration of small intestinal mucosa.It shows that electroacupuncture can improve the inflammatory state of the body and reduce the inflammatory reaction of the small intestine by specifically up-regulating SIRT1.In the EA + inhibition group treated with SIRT1 inhibitor,the levels of serum LPS and inflammatory factors were higher than those in the EA group,and the atrophy,erosion and inflammatory infiltration of small intestinal mucosa were aggravated,indicating that SIRT1 inhibitor could partially block the effect of electroacupuncture on improving the inflammatory state of the body.3.Both electroacupuncture and SIRT1 agonists can up-regulate the expression of SIRT1 protein and gene in the small intestine of IR obese rats,thus down-regulate the expression of TLR4 protein and gene,and reduce the synthesis of NF-?B protein and gene.Compared with the EA group,the expression of SIRT1 protein and gene in the EA + inhibition group treated with SIRT1 inhibitor decreased significantly,while the protein and gene expression levels of TLR4 and NF-?B increased,indicating that SIRT1 inhibitor could partially antagonize the effect of electroacupuncture.4.SIRT1 inhibitor can partially reverse the effect of electroacupuncture,indicating that electroacupuncture can down-regulate the expression of toll-like receptor 4(TLR4)by promoting the expression of SIRT1,improve the disorder of intestinal flora,inhibit the expression of NF-?B,exert anti-inflammatory effect,then control diet,regulate body weight,and improve peripheral insulin sensitivity,thus improving IR,which is a new mechanism of electroacupuncture in improving insulin resistance. |
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