Effects Of Hypoglycemic Xiaoke Granule Combined With Metformin On Glucose And Lipid Metabolism And GLP - 1 In Diabetic Mice

Objectives:The study is conducted to investigate the combined therapy that Jiangtang Xiaoke granule (JTXKG) combined with metformin exerts effects on the blood glucose and lipids in type 2 diabetic mice. At the second stage of the study, the pharmacological action of the combined therapy, based on the glucagon-like peptide-1 (GLP-1), is explored. With the study above conducted, the appropriate dose of JTXKG in the combined therapy is explored.Methods:1. To induce the model of type 2 diabetic mice, ICR mice aged 8 weeks are randomly divided into normal group and high-fat-diet group after 7-day acclimation. The mice fed by high-fat diet for 4 weeks are given intraperitoneal injection of streptozocin (STZ) in 100mg·kg-1. Randomly, the model mice are divided into DM group (untreated), metformin group (0.19 g·kg-1MET only) and combined therapy groups (JTXKG in 1.75g·kg-1,3.5 g·kg-1 and 7 g·kg-1, combined with 0.19 g·kg-1MET). During the 4-week intervention, the normal group and the DM group are administrated with the same volume of water.2. During the intervention, the general condition of mice is observed. The intake, the body weight and the fasting blood glucose (FBG) are recorded weekly. The oral glucose tolerance test (OGTT) is conducted after 4-week treatment.3. At the end of intervention, the blood is collected from the orbit to measure fasting serum insulin (INS), glucagon, triglyceride (TG), serum total cholesterol (TC), high density lipoprotein (HDL), low density lipoprotein (LDL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine (Cr). The liver is removed and weighed. The insulin sensitivity index (ISI) and the hepatic index are calculated.4. The pancreas which is removed and formalin-fixed is embedded in paraffin and made into paraffin sections, using HE stain for the pathological observation of pancreas.5. The intestine homogenate is centrifuged for measurement of active GLP-1 by the enzyme-linked immunosorbent assay (ELISA).6. The intestine and the pancreas which are removed and formalin-fixed are made into paraffin-embedded sections. The expression of GLP-1, GLP-1R and PC1/3 in intestine and the expression of GLP-1 R in pancreas are measured by immuneohistochemistry method.Results:1. General conditions:Compared with DM group, both the food intake and the weight in combined therapy groups decrease. The JTXKG 3.5 g·kg-1 combined with metformin is more effective (p<0.01).2. Glucose metabolism:The combined therapy shows the positive effect on reducing FBG (p<0.01), INS level (p<0.05), Glucagon level (p<0.05), and improving ISI (p<0.05) and glucose tolerance (p<0.05) in type 2 diabetic mice. The JTXKG 3.5 g·kg-1 combined with metformin improves glucose tolerance more effectively (p<0.01). Compared with the MET group, the JTXKG 3.5 g·kg-1 combined with metformin is more effective on reducing FBG (p<0.05).3. Lipid metabolism:The combined therapy shows the effect on decreasing TG level (p<0.01), hepatic weight (p<0.05), hepatic index (p<0.01), and increasing HDL level (p<0.01). The level of TC and LDL between DM group and combined therapy groups does not show significant statistical difference (p>0.05).4. Pathomorphology:The combined therapy shows the effect on protecting pancreatic islet.5. Liver and kidney function:The combined therapy shows the effect on decreasing serum ALT level (p<0.01), while there is no significant statistical difference in AST, BUN and Cr between the DM group and combined therapy groups (p>0.05).6. ELISA results:The combined therapy shows the effect on promoting GLP-1 secretion in intestine tissue (p<0.01). Compared with the MET group, the JTXKG 3.5 g·kg-1 combined with metformin is more effective on GLP-1 secretion (p<0.01).7. Immuneohistochemistry results:The combined therapy shows the effect on enhancing the expression of GLP-1 (p<0.01), GLP-1R (p<0.01), PC1/3 (p<0.05) in intestine and the expression of GLP-1R in pancreas (p<0.05). Compared with the MET group, the JTXKG 3.5 g·kg-1 combined with metformin is more effective on GLP-1 (p<0.01) and GLP-1R (p<0.05) secretion.Conclusion:1. The combined therapy can decrease food intake, body weight, FBG and improve glucose tolerance and ISI, implying the efficacy of protecting pancreatic islet and regulating glucose metabolism. The JTXKG 3.5 g·kg-1 combined with metformin shows superior efficacy on reducing food intake, body weight, FBG and improve glucose tolerance. When compared the efficacy on reducing FBG with metformin alone, the JTXKG 3.5 g·kg-1 combined with it is superior.2. The combined therapy can decrease hepatic weight and hepatic index, and regulate blood lipid level.3. The combined therapy can promote the expression of GLP-1, GLP-1R, and PC1/3 in intestine, as well as the expression of GLP-1R in pancreas. The effect of JTXKG 3.5 g·kg-1 combined with metformin on promoting the products above is more significant and on enhancing GLP-1 in intestine is superior to administrating metformin alone, which suggests that the combined therapy on glucose metabolism may exerts influence through the mechanism of GLP-1.4. The combined therapy dose not aggravate the injury of liver and kidney in T2DM mice, while it does not show the improvement in the hepatic and renal function, which suggests that a further toxicity research on the combined therapy is needed.5. The potential role of the combined therapy on the spleen and stomach of traditional Chinese medicine (TCM) is illuminated from the GLP-1-related mechanism, and the crucial function of the spleen and stomach of TCM in the onset and progression of DM is explored, which are a benefical attempt from Chinese medical theory to explore the treatment pattern of DM using Chinese medicine and western medicine together.