| Purpose:In this experiment,we established an animal model of type 2 diabetes to explore whether Tang-Ping-San decoction(raw astragalus 15 g,pueraria 15 g,coptis 10 g,carambola 20 g,fried radish seed 15 g,polygonatum 10 g,ground bark 10 g,eucommia 10 g,fried anemarrhena 15 g)can regulate type 2 diabetes by improving the intestinal flora and repairing the intestinal mucosal barrier,improving the content of short chain fatty acids,activating autophagy,and interfering with the mRNA expression level of autophagy related genes,the mechanism of Tang-Ping-San Decoction in regulating T2 DM was discussed.Methods:After feeding with HFD for 6 weeks and fasting overnight for 12 hours,STZ 100mg/kg was injected intraperitoneally(the Control group was injected intraperitoneally with the same volume of citric acid-sodium citrate buffer solution).After 72 hours,fasting for 12 hours and taking blood from the tail to measure blood glucose,the blood glucose value ≥11.1 mmol/L was considered as successful modeling.The T2 DM mouse model was successfully replicated.After the model was successfully established,the decoction of Tang-Ping-San decoction,short chain fatty acids and chloroquine were administered by gavage for 4 weeks respectively.Analysis of active ingredients in Tang-Ping-San decoction by UHPLC-Q Active non-target metabonomics.Fasting blood glucose(FBG)was measured by glucose meter.The contents of fasting insulin(FINS)and lipopolysaccharide(LPS)in serum were determined by ELISA.Serum triglyceride(TG),total cholesterol(TC),high density lipoprotein(HDL-C)and low density lipoprotein(LDL-C)levels were measured by the kit..The injury of pancreatic islet cells was observed by making HE staining pathological sections;HE staining and oil red O staining were used to observe the inflammatory cell infiltration and lipid deposition in liver tissue.HE stained pathological sections were made to observe the infiltration of inflammatory cells in intestinal tissues and the damage degree of intestinal mucosal barrier.16 S r RNA was used to detect the abundance and diversity of intestinal flora in mice.The changes of short-chain fatty acids in mouse intestinal contents were detected by GC-MS highthroughput target metabolomics.Real-time quantitative fluorescence PCR(Q-PCR)was used to detect the relative mRNA expression of related genes.Western blot(WB)was used to detect the expression of Beclin 1,LC3 B and p62 protein in liver tissue,and the expression of ZO-1,Occludin and Claudin 1 protein in intestinal tissue.Results:Compared with the model group,the FBG of mice in each dose group of Tang-Ping-San decoction decreased significantly,the levels of FINS,TC,TG,LDL-C in serum decreased,and the level of HDL-C increased.Compared with the control group,the other groups of mice showed obvious pancreatic lesions,including hyalinosis of islets,atrophy of islets,vacuolar degeneration of acinar cells,atrophy of pancreas,etc.The number of lesions in the model group was the largest,the range was the largest,the forms were diverse and serious,and the pancreatic lesions could be reduced after treatment with Tang-Ping-San decoction.Compared with the model group,the degree of vacuolar changes and lipid deposition in liver tissue of mice in each dose of Tang-Ping-San decoction intervention group were alleviated.Compared with the model group,the abundance and diversity of intestinal flora in the model group were decreased,and the abundance and diversity of intestinal flora in the Tang-Ping-San decoction intervention groups were increased.TangPing-San decoction intervention can significantly reduce the infiltration of inflammatory cells in the intestinal tissue of model mice,increase the expression of ZO-1,Occludin and Claudin 1,repair the intestinal mucosal barrier,and reduce LPS into the blood.The content of short-chain fatty acids in the intestines of mice in the model group decreased,especially acetic acid,propionic acid and butyric acid.Short-chain fatty acids can significantly improve T2 DM in mice.Inhibition of autophagy in model mice aggravated the symptoms of type 2 diabetes.Tang-Ping-San decoction and SCFAS can activate liver autophagy in T2 DM mice.Compared with the model group,the expressions of autophagyrelated genes in the liver were changed after the intervention of Tang-Ping-San decoction and SCFAs.Conclusion:1.Tang-Ping-San decoction can improve glucose homeostasis and dyslipidemia in T2 DM mice,reduce pancreatic and liver lesions,restore liver function,and reduce liver lipid deposition.2.Tang-Ping-San decoction improve the intestinal flora of T2 DM mice,increase the abundance and diversity of the flora,repair the intestinal mucosal barrier of the model mice,reduce the inflow of endotoxin into the blood,and alleviate type 2 diabetes.3.SCFAs can improve glucose and lipid metabolism disorders,repair intestinal mucosal barrier,and promote autophagy to alleviate type 2 diabetes in T2 DM mice.4.Alb,Apoa1,Tat and Scd1 as the target genes of autophagy were identified to participate in the intervention process of Tang-Ping-San decoction on T2 DM through SCFAs. |
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