| Objective:Based on the mechanism of regulation of TLRs/NF-κB signaling pathway by intestinal flora changes,we investigated the regulation of Wumei Wan on blood glucose,insulin and inflammatory factors in type 2 diabetic rats.Methods:Sixty healthy male SD rats were used as experimental subjects.Ten were selected as the normal group on a random basis.Fifty were given high-sugar and high-fat emulsion for eight weeks,and low-dose intraperitoneal injection of STZ(35 mg/kg)to establish T2 DM model.The blood glucose was greater than 11.10 mmol/L,which was successful in modeling.Rats with successful modeling were randomly divided into five groups,namely model group,metformin group(ig200 mg/kg),Wumei Wan high dose group(ig20 g/kg),Wumei Wan middle dose group(ig10 g/kg),Wumei Wan in low dose group(ig5 g/kg),ten in each group.The rats were sacrificed after four weeks of treatment,and the femoral artery was centrifuged for use,and feces and colon tissues were taken for examination.The body weight and blood glucose of each group were monitored regularly.The fecal DNA of rats was extracted with 16S-rDNA to analyze the microbial diversity of the intestines.The expression of TLR-4 and NF-κB p65 protein was detected by Western blotting using rat immunoblotting.The expressions of serum insulin,LPS,TNF-α and IL-10 were detected by enzyme-linked immunosorbent assay(ELISA).The morphological changes of the rat pancreas were observed by HE staining.Results:1.Rat general observation and weight change:In the normal group,the rats were free to move,everything was normal,and the body weight increased steadily.The weight gain of the high-sugar and high-fat emulsion was significantly higher than that in the normal group(P<0.05).After STZ injection,there was obvious polydipsia and more food.Symptoms of urinary weight loss.After four weeks of treatment,the symptoms of the Wumei Wan group and the metformin group were gradually relieved,and the body weight was significantly higher than that of the model group.2.Blood glucose and insulin test results:The fasting blood glucose of the rats before the model establishment was in the normal range,and the blood glucose of the rats was significantly increased after the model establishment(P<0.01).The blood glucose of the metformin group and the Wumei Wan group were significantly lower than that of the model group(P<0.05 or P<0.01).The results of serum insulin detection around the drug showed that compared with the normal group,the INS level of the model group was significantly decreased.Compared with the model group,the INS levels of the Wumei Wan group and the metformin group were increased,which was statistically significant(P<0.01).3.Changes in intestinal flora structure:3.1 Species analysis results:The number of unique OTUs in the normal group is greater than the model group.The similarity of the OTU between the middle and low dose groups of Wumei Wan and the normal group was higher than that of the model group and the normal group.The number of bacteroidetes in the model group was higher than that in the normal group.Compared with the model group,the number of bacteroidetes in the high,medium and low dose groups of Wumei Wan decreased.Compared with the normal group,the number of firmicutes decreased in the model group.The number of firmicutes in the high and medium dose groups of Wumei Wan was higher than that in the model group.In the model group,the proteobacteria abundance decreased,and compared with the model group,the proteobacteria abundance increased in the Wumei Wanl high and low dose groups.The amount of proteobacteria in metformin group and Wumei Wan medium-dose group was less than that in model group.TM7 bacteria were not detected in the samples of the model group and the Wumei Wan high-dose group,while TM7 bacteria were detected in the normal group,metformin group,and Wumei Wan medium-dose and low-dose groups.3.2 Alpha diversity test results:The sobs of the normal group,the low-dose group of Wumei Wans and the metformin group were significantly higher than that of the model group(P<0.05).The sobs of the Wumei Wan group had an increasing trend,but the high-dose group of Wumei Wan was significantly reduced.The model group had a reduced chao index compared to the normal group.Compared with the model group,the chao index of the metformin group and the Wumei Wan group was significantly increased(P<0.01).The ace index of the model group was significantly lower than that of the normal group(P<0.05).The ace index of the middle and low dose groups of Wumei Wan increased.There was no significant difference between the groups of shannon index.4.The results of Western-bloting of NF-κB p65 and TLR-4 in colon tissue:Compared with the normal group,the expression levels of TLR-4 and NF-κB p65 protein in the model group were significantly increased(P<0.01).Compared with the model group,the high-,medium-,and low-grade groups of Wumei Wan and the Metformin group were TLR-4,NF-κB p65 protein expression decreased significantly,statistically significant(P <0.01 or P <0.05).5.The results of ELISA in the expression of rat LPS,TNF-α and IL-10 in serum:Compared with the normal group,the serum LPS and TNF-α levels in the model group increased significantly(P<0.01);compared with the model group The levels of serum LPS and TNF-α in the high,middle and low dose groups of Wumei Wan decreased significantly(P<0.01).Compared with the normal group,the serum IL-10 level in the model group was significantly decreased(P<0.01).Compared with the model group,the serum IL-10 level in the Wumei Wan middle dose group increased significantly(P<0.01).IL-10 levels were increased in the low-dose group and the metformin group(P<0.05).6.Morphological changes of pancreas:Pancreatic HE staining results in normal rats showed that the number of islets was abundant.The shape was regular.And the boundaries of islet cells were clear.In the type 2 diabetic rats,the number of islets was reduced,the shape was irregular,and the edges were not neat and other pathological changes.The Wumei Wan high,middle dose group and the metformin group improved significantly.Conclusions:Wumei Wan can regulate the behavior and weight of type 2 diabetic rats,reduce fasting blood glucose and increase insulin levels.It can also improve the pathological damage of the pancreas.Wumei Wan may reduce the LPS level,improve intestinal mucosal permeability and reduce NF-κB p65 and TLR-4 protein expression by regulating the diversity of the intestinal flora and species abundance.Thereby Wumei Wan can reduce the level of LPS in model rats,inhibit TNF-α,promote the production of IL-10,and control the occurrence of chronic low-grade inflammation,so as to achieve the purpose of prevention and treatment of type 2 diabetes. |
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