| Objective:Based on the changes of intestinal flora,the mechanism of Wumei Wan’s intervention in type 2 diabetes(T2DM)model rats was studied,and whether Wumei Wan could affect T2 DM rats through TLRs/NF-κB signaling pathway was investigated.Methods:Eighty male SD rats were selected as the research object.After one week of normal breeding,ten male rats were selected as the normal group.The remaining 70 rats were given a high fat and high sugar emulsion as a model for 8 weeks.After intragastric injection of 35 mg/kg streptozotocin(STZ),a model of T2 DM was induced.The fasting blood glucose value ≥11.1mmol / L was regarded as a successful model.The rats that failed the modeling were excluded,and the remaining rats were randomly divided into the model group,metformin group,Wumei Wan high-dose group,Wumei Wan middle-dose group,and Wumei Wan low-dose group,with 10 rats in each group.Rats in the normal group and the model group were orally administered with saline at 20 ml/kg,metformin group was administered with metformin at 200mg/kg,and Wumei Wan high,medium,and low dose groups were administered with Wumei Wan decoction at 20g/kg,10g/kg and 5g/kg were administered by gavage,and fasting blood glucose and body weight of rats were monitored regularly during the experiment.After four weeks of administration,rats were treated,and serum and feces,intestinal contents,pancreatic tissue,and colonic tissue were collected for examination.16S-r DNA was used to sequence the feces of rats to observe the changes of intestinal microbial diversity in each group of rats.Enzyme-linked immunosorbent assay(ELISA)was used to detect rat serum tumor necrosis factor α(TNF-α)and interleukin 1(IL-1),interleukin 6(IL-6),interleukin 22(IL-22)levels;gas chromatography to detect the content of short-chain fatty acids(SCFAs)in rat intestinal contents;Western blotting method was used to detect the expression levels of NF-κB p65,P-NF-κB p65,IκBα,P-IκBα,and My D88 in rat colon tissue;immunohistochemical methods were used to detect the expression of NF-κB p65 and TLR4 in rat pancreatic tissue;HE The morphological changes of pancreas in rats were observed by staining.Results:1.Observation of general condition and weight of rats: The rats in the normal group were in good condition,eating and drinking normally,their hair was neat and white,and their weight continued to rise steadily.The hair color of the model group and the medication group rats gradually became yellow after high-fat and high-sugar emulsion,and the weight gain was very significant and higher than that of the normal group(P﹤0.05).After modeling,the frequency of litter replacement increased,the odor increased,the diet increased,and the weight showed a significant downward trend(P﹤0.05).After four weeks of treatment in the treatment group,the weight loss trend of the rats in each treatment group became slower,and the diet and urine output improved.2.Monitoring of blood glucose in rats: The blood glucose of rats in the normal group was kept within the normal range.Before the model group and the medication group,the blood glucose of the rats also belonged to the normal range.After the model was established,the blood glucose of the rats increased significantly and was significantly higher than the normal group(P ﹤ 0.01).Decrease(P ﹤ 0.01 or P ﹤0.05).3.Intestinal flora diversity test results:3.1.Species analysis: Compared with the normal group,the model group has low species richness and uneven distribution,Firmicutes,Clostridia,Actin0 bacteria,Lactobacillus,Bacteroides reduced abundance,Bacteroidetes,Prevotella,Escherichia Escherichia abundance increased;compared with the model group,the metformin group and Wumei Wan low-dose group have higher species richness and a more uniform distribution.The metformin group and Wumei Wan high-,medium-,and low-dose groups increased the abundance of Firmicutes and Bacteroides,and decreased the prevotella abundance.Clostridia and Lactobacillus in the metformin group and Wumei pill in medium and low doses The abundance increased in the group,while the Escherichia abundance decreased;Actin0bacteria increased the abundance in the metformin group and Wumei Wan high and low dose groups,while the abundance in the Wumei Wan medium dose group decreased.3.2.Alpha diversity analysis: Compared with the normal group,the sobs,chao,and ace indexes in the model group were reduced,and the differences were significant(P﹤0.01);compared with the model group,the metformin group,the Wumei Wan medium dose group,and Wumei Wan were lower.The sobs,chao,and ace indexes increased in the dose group,and the differences were statistically significant(P ﹤0.01 or P﹤0.05).The sobs,chao,and ace indexes in the high-dose group of Wumei Wan decreased(P﹤0.05);the Shannon index was different among the groups.No significant difference.4.ELISA detection of rat serum TNF-α,IL-1,IL-6,IL-22 content: Compared with the normal group,the contents of TNF-α,IL-1,IL-6,and IL-22 in the model group increased significantly.(P ﹤ 0.01).Compared with the model group,the contents of TNF-α,IL-1,IL-6,and IL-22 in the serum of the metformin group and Wumei Wan high,medium and low dose groups were all higher.The difference was statistically significant(P﹤0.01 or P﹤0.05).5.Detection of SCFAs in rat intestinal contents by gas chromatography:Compared with the normal group,the content of acetic acid,propionic acid and n-butyric acid in the model group rats decreased significantly,and the difference was statistically significant(P﹤0.01 or P﹤0.05);Compared with the model group,the levels of acetic acid,propionic acid,and n-butyric acid in the metformin group and Wumei Wan high,medium,and low dose groups were all increased,and the differences were statistically significant(P﹤0.01 or P﹤0.05).6.Western blot detection of colonic tissue NF-κB p65,P-NF-κB p65,IκBα,P-IκBα,Myd88 protein expression levels: compared with the normal group,the model group of colon tissue NF-κB p65,P-NF-κB p65 the protein expression levels of P-IκBα and My D88 all increased,and the expression levels of IκBα protein decreased(P﹤0.05).Compared with the model group,the metformin group and Wumei Wan high,medium and low dose groups had the expression levels of NF-κB p65,P-NF-κB p65,P-IκBα,and My D88 all decreased,and the expression of IκBα protein increased,and the difference was statistically significant(P﹤0.01 or P﹤0.05).7.Immunohistochemical detection of the expression of NF-κB p65 and TLR4 in rat pancreatic tissue: Compared with the normal group,the positive expressions of NF-κB p65 and TLR4 in the pancreas tissue of the model group were significantlyincreased(P﹤0.01).Compared with the model group,the metformin group,Wumei Wan high-dose group,Wumei Wan middle-dose group,and Wumei Wan The expression of NF-κB p65 and TLR4 in pancreas tissue of rats in the low-dose group decreased to varying degrees,and the difference was statistically significant(P ﹤0.01).8.HE staining to observe the morphology of rat pancreas: in the normal group,the islets of the normal group were clumped,the islet cells were intact,arranged neatly,the nucleus was clearly visible,and the border was obvious;in the model group,the islets were irregular in shape,there were gaps between cells,and some cells The changes were vacuole-like and the borders were blurred.The pathological changes in pancreatic tissue of the metformin group and Wumei Wan high,medium and low dose groups were significantly improved.Conclusions:Wumei Wan can regulate the abundance and diversity of intestinal flora,can reduce blood glucose in T2 DM rats,slow down the trend of weight loss,increase the content of short chain fatty acids,and reduce inflammatory factors TNF-α,IL-1,IL-6.The release of IL-22 improves the inflammatory response;and can reduce the expression of NF-κB p65,P-NF-κB p65,P-IκBα,My D88 protein in colon tissues,and increase the expression of IκBα protein;In addition,Wumei Wan can also Decreased the expression of NF-κB p65 and TLR4 in T2 DM pancreatic tissue,and improved pathological changes in pancreatic tissue.In summary,Wumei Wan may intervene TLRs / NF-κB signaling pathway in T2 DM model rats by regulating the intestinal flora,thereby achieving the therapeutic effect on T2 DM. |
PDF Full Text Request