| BackgroundType 2 diabetes mellitus(T2DM)is a progressive metabolic disease characterized by pancreatic conduction β-cell dysfunction and peripheral insulin resistance,which results in defective glucose metabolism and chronic low-grade inflammation.Its prevalence continues to increase with severe macrovascular and microvascular complications,and it not only causes physical and psychological suffering to patients,but also leads to a significant healthcare economic burden.Therefore,it is important to explore new pathogenesis and treatment methods for T2DM.In recent years,many studies have confirmed that the intestinal microbiota is closely related to the development of T2DM.The impaired intestinal barrier of T2DM patients causes intestinal leakage,and the massive release of its intestinal toxins such as Lipopolysaccharides(LPS)for circulation triggers the activation of inflammatory pathways such as TLR4/NF-κB triggering insulin resistance.Chinese medicine has achieved remarkable efficacy in the treatment of T2DM with its multi-target and low side effects.Prof.Liu Weijing,the supervisor of the study,took the "incubative pathogen" as the starting point,combined with the pathogenesis of T2DM as "spleen deficiency and deficiency of fluids,phlegm and heat," and established Shenling Jiangtang Formula for lowering glucose,which benefits the stomach and generates fluids,clears heat and phlegm,and reconciles and penetrates.This study aims to investigate its clinical efficacy and further elucidate its potential mechanism.Objective1.With the help of clinical efficacy indexes,T2DM patients were divided into two groups before and after treatment,and biochemical indexes as well as changes in intestinal microbiota before and after treatment were analyzed.2.To observe the regulation of LPS/TLR4/NF-κB inflammatory signaling pathway by Shenling Jiangtang Formula using T2DM mouse model,and to explore the regulatory mechanism of Shenling Jiangtang Formula on intestinal microbiota in T2DM mice.Method1.Basic clinical information was collected from T2DM patients who met the inclusion criteria and were treated with Shenling Jiangtang Formula for 12 weeks,and serum and stool were collected from patients before and after treatment to analyze the differences in glucose,glycated hemoglobin,glucose tolerance,insulin resistance,blood lipids,liver function,and changes in intestinal flora before and after treatment.2.C57BL/6Jmice were used as the study subjects in this experiment,and the T2DM mouse model was constructed using the high-fat diet feeding modeling method.The experimental mice were randomly divided into control group,model group,Shenling Jiangtang Formula group and metformin group.The mice were tested for body weight,blood glucose,glycated hemoglobin,insulin resistance,triglycerides,total cholesterol and serum LPS levels after 12 weeks of drug administration.Colon tissues were collected to observe the pathological changes by HE staining,and the ZO-1 protein level was detected by western blot(WB);pancreatic tissues were collected to detect TLR4 and NF-κB p65 protein levels by WB;feces were also collected for intestinal flora analysis.ResultsPart Ⅰ Clinical studies:1.After T2DM patients took Shenling Jiangtang Formula,the levels of blood glucose related indexes:HbAlc,FPG,OGTT 2h decreased significantly(P<0.001)compared with those before treatment;insulin resistance related indexes:insulin resistance index(P<0.01)and area under the insulin curve(P<0.01)decreased significantly,insulin secretion index(P<0.001),early phase insulin secretion index(P<0.01),2-h C-peptide variability index(P<0.01)and 3-h C-peptide variability index(P<0.05)were significantly higher;lipid-related indexes:TG(P<0.05),TC(P<0.001)and LDL(P<0.001)levels were significantly lower;liver function-related indexes:ALT(P<0.01)and AST(P<0.01)levels were significantly decreased.2.The results of the analysis of intestinal flora showed differences between the intestinal flora after treatment and before treatment.The relative abundance of the Firmicutes phylum was significantly decreased after treatment with Shenling Jiangtang Formula(P<0.001),the relative abundance of the Bacteroidetes phylum and Proteobacteria phylum was significantly increased after treatment(P<0.001),and the ratio of the relative abundance of the Firmicutes phylum to the Bacteroidetes phylum was significantly down-regulated after treatment with Shenling Jiangtang Formula(P<0.001).Part Ⅱ Animal experiments:1.Compared with the control group,the mice in the model group had significantly higher body weight and blood glucose level(P<0.01),and the intervention with Shenling Jiangtang Formula could significantly reduce the body weight and blood glucose of T2DM mice(P<0.01),and the efficacy was similar to that of metformin.Compared with the control group,glycosylated hemoglobin and insulin resistance index were increased in the model group(P<0.01),and the hemoglobin and insulin resistance index were significantly decreased after the intervention with Shenling Jiangtang Formula(P<0.01),and the efficacy was similar to that of metformin.In addition,triglyceride and total cholesterol levels were significantly increased in the model group mice compared with the control group(P<0.01),and Shenling Jiangtang Formula could regulate lipid metabolism(P<0.01).2.In terms of colon pathology,compared with the control group,mice in the model group showed significant colon histopathological damage and decreased ZO-1 protein level(P<0.01),while the colon pathological damage was reduced,and ZO-1 protein level was increased in the Shenling Jiangtang Formula and metformin groups(P<0.05).Compared with the control group,LPS level and TLR4/NF-κB p65 inflammatory protein expression were significantly increased in the model group mice(P<0.05),and Shenling Jiangtang Formula group could significantly reduce LPS level(P<0.05)and TLR4/NF-κB p65 inflammatory protein level(P<0.05).3.In terms of intestinal flora alteration,the relative abundance of Firmicutes phylum was significantly higher in the model group compared with the control group(P<0.05),and its relative abundance decreased significantly after the use of Shenling Jiangtang Formula compared with the model group(P<0.05);the relative abundance of Bacteroidetes phylum was significantly lower in the model group compared with the control group(P<0.05),and it tended to increase after the use of Shenling Jiangtang Formula compared with the model group,but there was no statistic difference;compared with the control group,the ratio of Firmicutes phylum to Bacteroidetes phylum was significantly higher in the model group(P<0.05),and after using Shenling Jiangtang Formula,its ratio decreased significantly compared with the model group(P<0.05).Conclusion1.Shenling Jiangtang Formula can significantly reduce blood glucose level,improve insulin resistance,regulate lipid level,and affect the species abundance and diversity of intestinal flora in both clinical and animal experiments.2.Shenling Jiangtang Formula can increase intestinal ZO-1 protein expression,reduce intestinal injury,protect intestinal barrier,and reduce serum LPS level in T2DM mice.3.Shenling Jiangtang Formula can reduce the expression of TLR4 and NF-κB p65 protein in pancreas of T2DM mice,reduce inflammatory response and improve insulin resistance,which may be related to regulating intestinal flora and improving intestinal barrier function. |
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