Effects Of Epigallocatechin-3-gallate On Insulin Resistance And Intestinal Microbiota In Type 2 Diabetic Rats

Objective To observe the effects of epigallocatechin-3-gallate(EGCG)on insulin resistance and intestinal microbiota in type 2 diabetic rats and to explore the mechanism of improving type 2 diabetes.Methods1.Animal grouping: SD rats were randomly divided into normal control group(n = 10)and model group(n = 80)after adaptive feeding for one week.The model group was fed with high-fat and high-sugar diet combined with intraperitoneal injection of 35 mg/kg streptozotocin to establish the model of type 2 diabetic rats.After excluding the unmodeled and dead rats,50 successfully established rats were randomly divided into model group(distilled water),metformin group(300 mg/kg),EGCG low dose group(25 mg/kg),middle dose group(50 mg/kg)and high dose group(100 mg/kg),with 10 rats in each group.After intragastric intervention for 6 weeks,the rats were anesthetized with 3% pentobarbital sodium,the blood was collected from the abdominal aorta and the serum was retained for biochemical index detection;the liver and skeletal muscle tissue were reserved for protein detection;and the ileum tissue and its contents were collected for subsequent intestinal microbiota sequencing and tight junction protein detection.2.During the experiment,the health and daily behavior of each group of rats were observed daily,and the body weight were measured weekly.3.Fasting blood glucose(BG)and serum insulin(INS)were measured.Insulin sensitivity index(ISI)and insulin resistance index(HOMA-IR)were calculated.4.The mRNA and protein expression levels of phosphoenolpyruvate carboxykinase(PEPCK)in liver and glucose transporter 4(GLUT4)in skeletal muscle were detected by real-time fluorescence quantitative PCR and Western blotting(WB).5.16 s r DNA high-throughput sequencing was used to analyze the structure and molecular ecology of intestinal microbiota in ileal contents.6.The ultrastructural changes of ileum were observed under transmission electron microscope.7.The expression of tight junction protein ZO-1,Occludin and Claudin-1 in ileum of rats was detected by WB.Results1.During the intervention period,compared with the control group,the body weight of rats in other groups decreased significantly(P<0.05).There was no significant difference in body weight among other groups.2.Compared with the control group,the serum BG,HOMA-IR levels in other groups were significantly increased(P<0.05),and the ISI levels were significantly decreased(P<0.05).Compared with the model group,the serum BG,INS and HOMA-IR levels of the metformin group were significantly decreased,while the ISI levels were significantly increased(P<0.05).The serum INS levels of EGCG medium dose group were significantly decreased(P<0.05).The levels of INS and HOMA-IR in the serum of the high-dose EGCG group decreased significantly(P<0.05),and the levels of ISI increased significantly(P<0.05).3.Compared with the model group,the mRNA levels of GLUT4 in metformin group were significantly increased(P<0.05).The m RNA and protein expression levels of PEPCK in liver were significantly decreased and the protein expression levels of GLUT4 in skeletal muscle were significantly increased in the EGCG middle dose group(P<0.05).The m RNA and protein expression levels of PEPCK in liver were significantly decreased in EGCG high dose group,while the m RNA and protein expression levels of GLUT4 in skeletal muscle were significantly increased(P<0.05).4.The Shannon index and the relevant abundance of Verrucomicrobia and Akkermansia in the EGCG high dose group were significantly higher than those in the model control group(P<0.05).The results of Lefse analysis showed that Akkermansia was a species with significant difference in the high dose group of EGCG.5.Transmission electron microscopy showed that the ileum microvilli of the control group were abundant and orderly,and the tightly connected structure of the cells was complete and clear.Compared with the control group,the close connections of the ileum epithelial cells in the model group were blurred.After the intervention of metformin and high-dose EGCG,the tightly connected structure of the ileum epithelial cells were improved to varying degrees.6.Compared with the control group,the ZO-1,Occludin and Claudin-1 protein expression levels of the model group and the metformin group were significantly reduced(P<0.05).The rats in the high-dose EGCG group showed a significant reduction in the protein expression levels of Occludin and Claudin-1(P<0.05).Compared with the model group,the protein expression levels of Occludin in the metformin group were significantly increased(P<0.05),and the expression levels of ZO-1,Occludin and Claudin-1 in the high-dose EGCG group were significantly increased(P<0.05).Conclusion1.EGCG may directly improve insulin resistance by enhancing insulin sensitivity and inhibiting hepatic gluconeogenesis and increasing glucose uptake in skeletal muscle tissues by activating the PI3K/Akt insulin signaling pathway.2.High dose of EGCG intervention significantly increased the abundance of Akkermansia in the intestinal tract of diabetic rats and regulated the intestinal barrier function,thereby indirectly improving insulin resistance.