| Objective : Danzhi Jiangtang Capsule(DJC) is a experienced prescription in First Affiliated Hospital of Anhui Medical University. Based on the preliminary experiments, in this study we observed some main pharmacodynamic indexes of DJC in treatment of diabetes with vascular lesionsthe through the establishment of type 2 diabetic rat model with vascular lesions. Meanwhile in order to study the material basis and mechanism of DJC in treatment of diabetes with vascular lesions, a metabolomics method based on UPLC/QTOF-MS was employed to study the possible metabolism and metabolic profile changes in urine and serum caused by DJC after 4 weeks’ treatment. We got pattern recognition and parsing of the obtained corresponding metabolic profile and identified the possible potential biomarkers. This approach have inspiration on the research of traditional Chinese medicine.Methods:1. Male SD rats were fed on high-fat diet for 4 weeks and intraperitoneal injection of 35mg/kg streptozotocin(dissolution in 0.1mmo L/L citrate buffer,PH=4.2), 3 days after the injection, we chose type 2 diabetic rats with vascular lesions model whose random blood sugar were higher than 16.7mmo L/L. After the modeling, continuous administration with DJC for 4 weeks, detected the blood glucose through tail vein weekly. After administered two weeks,selected six rats in each group randomly, intraperitoneal injection of 3.5%chloral hydrate anesthesia, abdominal aortic blood 5.0m L in heparinized tubes.After 4 weeks, sacrificed all the rats, narcotic abdominal aortic blood.Dynamic Observed changes in indicators of substance administered 2 weeks and 4 weeks of content. The resulting collection of blood, standing 2h after centrifugation plasma. Determination of plasma NO, ET-1, PAI, t-PA, TXB2and 6-keto-PGF1α. Take the thoracic aorta, HE staining of vascular endothelial cells.2.Established an urine metabolomics technology platform based on UPLC/QTOF-MS, applied this technique for metabolomics study on high-fat diet combined with a small dose of STZ-induced rat model of vascular disease in type 2 diabetes mellitus, study the possible metabolism and metabolic profile changes in urine, combined the principal component analysis(Principal Components Analysis, PCA) with the partial least squares(Partial Least Squares Diseriminant analysis, PLS-DA) compared the differences of endogenous metabolites in each group. Based on VIP values and P values, we determined the related biomarkers of DJC prevention of type 2 diabetes with vascular lesions, based on the Human Metabolome Database and Kegg Compound Database database, analyzed possible pathways of metabolic biomarker.3.Established a serum metabolomics technology platform based on UPLC/QTOF-MS, applied this technique for metabolomics study on high-fat diet combined with a small dose of STZ-induced rat model of vascular disease in type 2 diabetes mellitus, study the possible metabolism and metabolic profile changes in serum, combined the principal component analysis(Principal Components Analysis, PCA) with the partial least squares(Partial Least Squares Diseriminant analysis, PLS-DA) compared the differences of endogenous metabolites in each group. Based on VIP values and P values, we determined the related biomarkers of DJC prevention of type 2 diabetes with vascular lesions, based on the Human Metabolome Database and Kegg Compound Database database, analyzed possible pathways of metabolic biomarker.Results:1.Blood sugar in type 2 diabetic rats was significantly higher(P<0.01)compared with normal rats; 2 weeks after administration, blood glucose compared with the model group for each treatment group has declined, which metformin group(P<0.01) Shenqijiangtang group(P<0.05) was statistically significant; Compared to three weeks after the administration with the model group, Danzhi high dose group was significantly lower blood glucose(P<0.05), positive control group declined more significantly(P<0.01); Were significantly decreased glucose each treatment group compared with the model group after 4 weeks(P<0.01).And normal rats compared with the model group rat plasma NO, t-PA and6-keto-PGF1αlevels were significantly lower(P<0.01), plasma ET-1, PAI and TXB2 levels were significantly increased(P<0.01). Administered two weeks, and the model group compared to the group administered rat plasma NO, t-PA and 6-keto-PGF1αlevels increased, decreased plasma ET-1, PAI and TXB2 levels. After 4 weeks, and compared with the model group, DJC group may reduce plasma NO, t-PA and 6-keto-PGF1αlevels(P<0.01 or P<0.05), increased plasma ET-1, PAI and TXB2(P<0.01 or P<0.05).HE staining showed normal rat aorta smooth, no local defects;endothelium intact; the layers of smooth muscle cells arranged in neat rows,no proliferation phenomenon. Aortic intima type 2 diabetic rats had significant damage, continuity is broken; part of endothelial cell loss, connect the gap between cell volume and cell increases; medial smooth muscle disorder, endothelial cell damage is obvious. Each treatment group after 4weeks of treatment, compared with the model group, aortic disease status were all reduced.2.Experiments in the positive ion mode identified seven with DJC intervention in type 2 diabetic Vascular lesions biomarkers: creatine,creatinine, indole-3-carboxylic acid, kynurenic acid, uric acid, 5-hydroxy-6-methoxy-indole-glucuronic acid and 3-indole carboxylic acid ofglucuronic acid; negative mode identified six associated biomarker: thio bran,N- acetyl-L- tyrosine sulfate estrone sulfate, ascorbic acid-2, 2-phenylethanol glucuronic acid and sulfuric acid catechol. Through the analysis of metabolic pathways, we found DJC type 2 diabetes by improving Vascular lesions in rats glucose metabolism and amino acid metabolism, regulate the secretion of estrogen, reducing oxidative stress in rats, repair damaged the prevention and treatment of liver and kidney function in type 2 diabetic patients with vascular disease.3.Experiments found in the positive ion mode selected 11 markers difference: monoglycerides, eicosanoids, L- phenylalanine, chenodeoxycholic acid succinate, three kinds of LPC substances, a the LPE substances, two kinds of PC and isoleucine substances. Negative mode selected four difference markers: eicosanoids, monoglycerides, chenodeoxycholic acid and succinic acid. Analysis of the various differences in the metabolic pathway metabolites, we found DJC adjustable type 2 diabetic rats metabolize amino acids, bile acid and lipid metabolism and oxidative stress. DJC prevention and treatment of type 2 diabetes possible be an early Vascular lesions through the above channels.Conclusion:1.Pharmacodynamic studies of Danzhi Jiangtang Capsule in treatment of type 2 diabetic rats with vascular lesions showed that DJC have a protective effect on vascular endothelial cells in type 2 diabetic rats and have a clear treatment effect on type 2 diabetes-induced vascular lesions in rats. The possible mechanism cloud be repair the damaged vascular endothelial cells,improve vascular smooth muscle function, adjust anticoagulant/procoagulant and fibrinolytic/pro-fibrinolytic balance.2.The urine metabolomics studies based on UPLC / QTOF-MS technique about Danzhi Jiangtang Capsule in treatment of type 2 diabetic rats withascular lesions indicate the mechanism of DJC in treatment of type 2 diabetes with vascular lesions exist in the regulation of energy metabolism, amino acid metabolism and oxidative stress.3.The serum metabolomics studies based on UPLC / QTOF-MS technique about Danzhi Jiangtang Capsule in treatment of type 2 diabetic rats with ascular lesions indicate the mechanism of DJC in treatment of type 2 diabetes with vascular lesions exist in the regulation of amino acid metabolism, bile acid and lipid metabolism and oxidative stress.In summary, the DJC have a preventive and therapeutic effect on early type 2 diabetic rats with vascular lesions indued by high-fat diet and STZ. The possible mechanism could be the regulation of energy metabolism, amino acid metabolism, bile acid and lipid metabolism and oxidative stress status. |
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