Effect Of Sitagliptin Combined With HMS5552 On Glucose Metabolism In Type 2 Diabetic Rats And Mechanism Discussion

OBJECTIVE:?1?To study the effect of sitagliptin combined with a novel glucokinase activator HMS5552 on glucose metabolism in type 2 diabetic SD rats;?2?To explore the mechanism of action of HMS5552 by drug combination.METHODS:?1?100 healthy male SD rats of one month old were fed with normal feed for 7 days,and 8 rats were randomly selected as normal control group?NC?,fed with normal feed,and the rest rats were fed with high-fat and high-sugar diet.Two months later,streptozotocin?STZ,40 mg/kg?was intraperitoneally injected one-time to induce type 2 diabetes,then fasting blood glucose was measured twice and the concentration were all?8 mmol/L,the modeling was successful.All models were fasted for 12 hours,and blood was collected for the measuring of fasting blood glucose?FBG?,triglyceride?TG?,total cholesterol?TC?,fasting insulin?FIns?,fasting glucagon?FGC?,dipeptidyl peptidase-IV?DPP-IV?and glucagon-like polypeptide-1?GLP-1?.Models with type 2 diabetes were randomly divided into 7 groups,6 rats in each group:diabetes model group?DM?,HMS5552 low dose treatment group?HMS-L,10 mg/Kg?.,HMS5552 high-dose treatment group?HMS-H,30mg/Kg?,sitagliptin low-dose treatment group?Sitagliptin-L,SIT-L,10mg/Kg?,sitagliptin high-dose treatment group?SIT-H,30mg/Kg?,HMS5552 low dose and sitagliptin low dose treatment group?HMS-L10mg/Kg and SIT-L10mg/Kg?,HMS5552 low dose and sitagliptin high dose treatment group?HMS-L10mg/Kg and SIT-H30mg/Kg?,after grouping,the blood was first taken from the tail vein for oral glucose tolerance test?OGTT?,and then the rats were intragastrically treated according to the grouping for a month,during the treatment period,the blood glucose was measured regularly.After treatment,fasting for 12 hours,fasting blood glucose was measured in the tail vein,followed by OGTT test;the next day,the anesthetized rats were sacrificed and blood was taken for testing FBG,TC,TG,FINS,FGC,DPP-IV and GLP-1;taking liver tissue,pancreatic tissue and small intestine tissue;detection of liver GK protein and small intestinal GLP by Western blotting;oil red O staining method to observe the presence of liver fat;the tissue structure of liver,pancreas and small intestine was analyzed by HE staining.;using immunohistochemical methods for liver GK,pancreas INS and GC and small intestine GLP-1,the distribution was analyzed;the expression of GK mRNA in liver tissue and GLP-1 mRNA in small intestine were measured by semi-quantitative RT-PCR.?2?Select NC,DM,HMS-L,SIT-H and HMS-L and SIT-H from the drug treatment group to discuss the discussion of mechanism,SIT-H group as a drug control group,the mechanism of HMS5552 was explored;the metabolism of glycolipids was investigated by oil red O staining;caspase-3,insulin-like growth factor-1?IGF-1?and pancreatic tissue were detected by immunohistochemical staining,the distribution of GLP-1 and PANDER was analyzed;western blotting was used to detect phosphatidylinositol 3-kinase?PI3K?,phosphorylated-PI3K?p-PI3K?,in the tissue of liver and pancreas,the protein expression of caspase-3,cleaved caspase-3,cytochrome c?Cyt-C?of cytoplasmic and mitochondrial in liver and pancreas tissue was analyzed.RESULTS:1.Results of drug combination1)Modeling of type 2 diabetes was successful,and the fasting blood glucose values of each group were?8mmol/L;2)OGTT test results:the blood glucose peak of the normal group was about 15minutes,and gradually returned to normal after 2 hours.The glucose tolerance of the model group decreased,the blood glucose peak shifted to the right after eating,and the recovery time of blood glucose exceeded 2 hours,and even 3 hours did not fully recover to the normal level;the blood glucose peak of each group was shifted to the left compared with the DM group,and the blood glucose recovery time was advanced.Compared with the area under the curve,?AUC_0-240-240 compared with the DM group,the changes of each treatment group were a statistical difference?P<0.05 or P<0.01?,and the drug combination group was more effective than the single drug group?P<0.05 or P<0.01?.3)Results of serological indicators:?1?Blood lipids:Compared with DM group,blood lipid levels decreased in different degrees after treatment?P<0.05 or P<0.01?,Compared with HMS-L or SIT-H,TC of drug combination groups were respectively reduced,the changes were more obvious?P<0.01?,the changes of TG are were smaller;?2?GC:GC in the blood increased in the DM group,after treatment,GC expression was significantly reduced?P<0.01?,compared with HMS-L or SIT-H,the decrease of GC was statistically significant?P<0.05?;?3?Blood glucose,INS andInsulin resistance index?HOMA-IR?:blood glucose of each treatment group after treatment were significantly lower than that of the DM group?P<0.05 or P<0.01?,the drug combination group was significantly lower than the HMS-L or SIT-H monotherapy groups,and the change of blood glucosewas statistically significant?P<0.05?,while the expression of INS in the DM group was significantly increased,and the expression of each group decreased significantly after treatment?P<0.01?,compared with the drug alone group The changes of HMS-L+SIT-H were statistically different?P<0.05or P<0.01?;the reduction level of HOMA-IR was consistent with the level of insulin change;?4?DPP-IV and GLP-1:The blood content of DPP-IV in DM group was significantly increased,and the treatment group had different degrees of decline?P<0.05 or P<0.01?.the effect of sitagliptin was better than HMS5552 group,the expression of DPP-IV in the drug combination group was better than that in the monotherapy group?P<0.05?;The expression of GLP-1 was significantly decreased in serum of DM group,after treatment,the expression level of GLP-1 in each group was higher than that in DM group?P<0.05 or P<0.01?,The expression of GLP-1 in HMS-L and SIT-H combination group was significantly higher than that of HMS-L or SIT-H group?P<0.05?.4)GK activity of liver homogenate:The activity of GK enzyme in DM group was significantly decreased,and the activity of GK enzyme in each treatment group was improved?P<0.05 or P<0.01?,Compared with HMS-L or SIT-H,the change of the drug combination group was in respectively,the increase of GK enzyme activity was statistically significant?P<0.05 or P<0.01?;5)Liver morphology:The liver of the normal group is soft and moist,with bright color and smooth surface,the liver surface of the DM group is rough,tarnished,light and yellow,and brittle,after treatment in each group,the liver has a certain appearance.improvement,the liver of the drug dual-use group improved significantly;6)RT-PCR results:The expression levels of mRNA in liver GK and small intestine GLP-1 were significantly decreased in DM group,and the expression of GK mRNA in each treatment group was significantly increased?P<0.05 or P<0.01?,while the expression of GLP-1mRNA was significantly increased?P<0.01?,and the expression of the two molecules was significantly higher in the HMS-L and SIT-H combination group than in the HMS-L or SIT-H single drug group?P<0.05 or P<0.01?,it was found that HMS5552 was superior to sitagliptin in GK mRNA expression,and sitagliptin was superior to HMS5552 in terms of GLP-1 mRNA expression;7)HE staining:The liver,pancreas and small intestine of NC group have clear structure,uniform cell distribution,typical cell structure characteristics,intact cell membrane and complete structure.The tissue structure of DM group is disordered,and most of the intact structure was destroyed,and most of the membranes were damaged,and the intercellular boundaries were unclear.After treatment,the tissue structure of each group was restored,and the combined treatment group was better.8)Oil red O staining results:The fat distribution in the liver tissue of DM group was significantly increased,the fat distribution of each group was significantly reduced by drug treatment?P<0.01?,and the fat distribution of HMS-L and SIT-H drug combination group was respectively compared with HMS-L or SIT-H single drug group,it was significantly reduced?P<0.01?;9)Immunohistochemical staining results:?1?The expression of GK in liver tissue was significantly decreased in DM group,after treatment,the expression of GK in each group was significantly increased?P<0.01?,and the drug combination group had higher GK than the single drug group,and the change had significantly statistically significant?P<0.01?,the results found that the HMS group was superior to the sitagliptin drug group in the expression of GK;?2?The expression of INS and GC in DM group were significantly inhibited,the expression decreased,after drug treatment the expression of INS in each group was significantly increased?P<0.01?,compared with HMS-L or SIT-H,the improvement effect of drug combination treatment group had better improvement?P<0.05 or P<0.01?;the expression of GC was increased in different extents?P<0.05 or P<0.01?,and the was more effective than the monotherapy group?P<0.01?;?3?The expression of GLP-1 in the small intestine tissue was significantly decreased in DM group,and improved after drug treatment?P<0.05 or P<0.01?,the effect of the drug combination group was significantly enhanced compared with the single drug?P<0.01?.10)Western blot results:The expression of GK protein in treatment groups were significantly higher than that in DM group?P<0.01?.the effect of the HMS-L and SIT-H combination treatment group was better than that of the HMS-L or SIT-H?P<0.05 or P<0.01?,and the HMS group was slightly better than the SIT in the expression of GK.In the expression of GLP-1 protein,the expression of GLP-1 protein was higher in the drug-treated group than the DM group?P<0.05 or P<0.01?,and the combination group was more effective than the single-agent group?P<0.01?,and sitagliptin is superior to HMS.2.Results of drug mechanism1)Immunohistochemistry results:?1?The distribution of hepatic insulin-like growth factor-1?IGF-1?in the DM group was significantly reduced,after treatment,the IGF-1of each group was significantly increased?P<0.01?,compared with the single-drug group,the effect of the combination group was stronger?P<0.01?;the distribution of caspase-3 in the liver was significantly increased in DM group,and the distribution of caspase-3 was significantly decreased after treatment in each drug treatment group?P<0.01?,in the same way,the effect of the drug combination group was better?P<0.01?;?2?the expression of GLP-1 in the pancreatic tissue of the DM group was significantly reduced,and the expression of GLP-1 in each group was significantly increased after drug treatment?P<0.01?,GLP-1 in combination group was increased in different degrees in the drug-treated group compared with HMS or SIT group?P<0.05 or P<0.01?;the expression of pancreatic-derived factor?PANDER?was significantly increased in pancreatic tissue of DM group,after treatment,the expression of PANDER was significantly decreased in each group?P<0.01?,the drug combination group was significantly more significant than the single drug group?P<0.01?;2)WB results:?1?The expression of p-PI3K and PI3K in liver tissue were significantly lower than those in DM group,after treatment,this ratio was significantly increased?P<0.01?,compared with monotherapy,the ratio of the the drug combination group was increased,and the change was statistically significant?P<0.01?.?2?The gray ratios of cleaved caspase-3 and caspase-3 in liver and pancreas tissues of DM group were significantly increased,after treatment,the ratio of cleaved caspase-3/caspase-3 in each group was significantly decreased?P<0.01?,the drug combination group was more effective than the monotherapy group?P<0.01?;?3?Cytochrome C?Cyt-c?Western blot results:The expression of Cyt-c in the cytoplasm of hepatocytes and pancreatic cells in the DM group was significantly increased,while Cyt-c in the mitochondria was significantly decreased,after drug treatment,the expression of Cyt-c in cytoplasm was significantly decreased?P<0.01?,and the expression of Cyt-c in mitochondria was significantly increased?P<0.01?,the drug combination group was compared with the cytoplasm of single drug group,the expression of Cyt-c was significantly decreased?P<0.01?,while the expression in mitochondria was significantly higher than that in the single drug group?P<0.01?.CONCLUSION:1.The effect of sitagliptin combined with HMS5552 on the regulation of glucose metabolism is better than that of the single drug group,and the effect on lowering blood sugar level is better than that of single drug;2.In terms of GK regulation,HMS5552 is superior to sitagliptin.In terms of GLP-1regulation,sitagliptin is superior to HMS5552,and the effect of combination is more significant;3.HMS5552 may protect the islet?cells and inhibit its apoptosis through the PI3K pathway.