The Effects Of Epigallocatechin Gallate (EGCG) On Insulin Signaling Pathway And TLR4 Signaling Pathway In Skeletal Muscle Tissue Of Obese Rats

Introduction: The prevalence of obesity has become a popular issue in the past few decades.Overweight and obesity are considered to be the major causes of metabolic diseases such as type 2 diabetes,dyslipidemia and atherosclerosis.In recent years,it has been found that obesity is a chronic low inflammation,which is closely related to the occurrence of insulin resistance.Nowadays studies have shown that toll-like receptor 4(TLR4)is the key factor affecting insulin resistance that is caused by obesity.Obesity inhibits insulin signaling pathway by activating TLR4 signaling pathway and associated inflammatory factors.EGCG,which has the function of free radicals clearance,anti-tumor,anti-inflammation,anti-virus and anti-mutation,is the main element of green tea's biological effect.According to the current epidemiological study of EGCG and obesity,EGCG can reduce weight and BMI of obese and diabetic patients.Based on the previous experiments of our researh team,the results have shown that EGCG can reduce inflammation and improve insulin resistance through negative regulation of TLR4 signaling pathway,after intervening with the adipose and liver tissues of obese rats.However,it is still unknown whether EGCG has the improvement function on insulin resistance in skeletal muscle-the main target organ for insulin.Skeletal muscle is the main place where insulin stimulates glucose absorption and utilization.80% of glucose in human body is consumed by skeletal muscle.Skeletal muscle,as the main target organ for insulin,plays an important role in the occurrence of fat-induced insulin resistance and type 2 diabetes mellitus.The experiment chose the skeletal muscle of obese rat's hind legs as the research object,aiming to find out EGCG's effect on TLR4 signaling pathway and insulin signaling pathway and its correlation in between,so as to improve the insulin resistance and provide the theoretical basis for the prevention of type 2 diabetes.Methods: 30 male SD rats of 4 weeks old were sampled to be fed in a standard cage of sterile conditions for 1 week.Then they are randomly divided into 2 groups: normal contrasting group(NC group,n=10)and high-fat diet group(n=20).The normal control group fed on normal diet(containing 10% fat);the other group fed on high fat diet(containing 60% fat).After 16 weeks feeding,the weight of the two groups rats appearedto be distinctly different(p < 0.05).The high-fat diet group was then divided into another two groups at random,i.e.High fat diet group(HFD group,n=10)and EGCG intervention group(EGCG group,n=10).Then the HFD group rats continued to feed on a high-fat diet,while the EGCG group were fed on high fat diet with EGCG added i.e.3.2g EGCG per kg of high-fat feeding.The normal contrasting group(NC group,n=10),the high-fat diet group(HFD group,n=10)and the EGCG intervention group(EGCG group,n=10)were all maintained their own feeding ways for a further 16 weeks time,following by intraperitoneal injection anesthetization with 10% chloral hydrate,before the rats' skeletal muscle were separated and removed from the hind legs,and put into the container full of liquid nitrogen,which was then refrigerated at-80?.In this experiment,the expression levels of GLUT4,TLR4,TRAF-6,IKK?,TNF-a m RNA were detected through Real time PCR in the skeletal muscle of the three groups of rats.Western methods were used to detect the expression levels of GLUT4,p-IRS-1ser307,TLR4,TRAF-6,p-IKK?,p-NF-?B,TNF-a protein.The software of SPSS 20.0 was used for the statistical analysis.Results: 1.The effect of EGCG on the expression of insulin signaling pathway in skeletal muscle tissue of obese rats.After 32 weeks of high fat feeding,all the experiment results were different statistically.The the GLUT4 m RNA and protein expression level of the high fat diet group(HFG group)decreased in,comparing with the normal contrasting group(NC group).The p-IRS-1 ser307 protein level of the HFG group were higher than that in NC group(p < 0.05).The GLUT4 m RNA and protein expression levels of the EGCG intervention group(EGCG group)were higher than those of the HFG group(p <0.05),and the p-IRS-1ser307 protein level of the EGCG group(p < 0.05)was lower than that of the HFG group(p < 0.05).These results have indicated that EGCG can improve insulin signaling pathway in skeletal muscle tissue of obese rats.2.The effect of EGCG on the expression of TLR4 signaling pathway in skeletal muscle tissue of obese rats.After 32 weeks of high fat feeding,all the experiment results were different statistically.The TLR4,TRAF-6,IKK?,TNF-a m RNA levels of the HFD group went higher that those of the NC group(p < 0.05).And also comparing with the NC group(p < 0.05).the levels of TLR4,TRAF-6,p-IKK?,p-NF-?B,TNF-a m RNA were higher.Furthermore,with EGCG intervening for 16 weeks,TLR4,TRAF-6,IKK?,TNF-a m RNA levels of theEGCG group went lower than those of the HFG group(p < 0.05),and TLR4,TRAF-6,p-IKK?,p-NF-?B,and TNF-a protein levels were lower than those of the HFG group(p< 0.05).The above results have shown that EGCG can reduce TLR4 signaling pathway in skeletal muscle tissue of obese rats.3.The Effects of EGCG on general metabolic indexes of SD rats.Based on the previeous experiments of our research team,after 32 weeks of high fat feeding,the level of weight,FINS,FFA and HOMA-IR of the HFD group were higher than those of the NC group(p < 0.01).Also with the EGCG intervening for 16 weeks,the FINS,FFA and HOMA-IR of the EGCG group were lower than those of the HFD group(p < 0.01).Although there was declining indication in the rats weight of the EGCG group,in comparison with that in HFD group(p >0.05),but there was no difference statistically.There was also no difference statistically in FBG level among the three groups(p>0.05).The above results have shown that the levels of free fatty acids and insulin resistance in HFD group were all higher than those of the NC group,and EGCG intervention could reduce the level of free fatty acids,improving insulin resistance in obese rats.Conclusion:EGCG can expression of IRS-1 serine phosphorylation in skeletal muscle tissue of obese rats induced by high-fat diet and up-regulated expression of GLUT4,performing negative regulation of TLR4 signaling pathway and improving the state of inflammation.