Effects Of Trilobatin On Anti-T2DM In KK-Ay Mice And Its Underlying Mechanism

Objective:This study was designed to investigate the hypoglycemic effect of trilobatin（TLB）in type 2 diabetic（T2DM）KK-Ay mice and explore its possible mechanisms.Methods:8-week-old male KK-Ay mice were randomly divided into four groups:model group（KK-Ay）,model+TLB 10 mg/kg group（KK-Ay+TLB-10）,model+TLB 20 mg/kg group（KK-Ay+TLB-20）,model+Met 150 mg/kg group（KK-Ay+Met）;while,7-week-old male wild type C57BL/6J mice were randomly divided into blank group（WT）and blank+TLB 20 mg/kg group（WT+TLB-20）.KK-Ay group and WT group were intragastrically administered with volume-matched normal saline（NS）and the drug treatment groups were received with corresponding doses of TLB or Met by gavage for 28weeks.During the experiment,KK-Ay mice were fed with high-fat-food,and C57 mice were fed with ordinary food.Fasting blood glucose（FBG）was tested once a week,oral glucose tolerance test（OGTT）and insulin tolerance test（ITT）were performed in the 20th week and twenty-first week,respectively.Thereafter,the blood samples,pancreas,and liver tissues of mice were collected.The lipid metabolism indicators including triglyceride（TG）,high density lipoprotein-cholesterol（HDL-C）,low density lipoprotein-cholesterol（LDL-C）,free fatty acid（FFA）and oxidative stress indicators including reactive oxygen species（ROS）,glutathion peroxidase（GSH-Px）,catalase（CAT）,superoxide dismutase（SOD）and fasting insulin（FINS）were detected by enzyme-linked immunosorbent assay（ELISA）.Moreover,homeostasis model assessment-insulin resistence（HOMA-IR）was calculated based on FINS and FBG.The morphological changes of pancreas was observed by hematoxylin-eosin（H&E）staining.INS expression of mice was detected by immunohistochemistry（IHC）.The protein expressions of keap-1,Nrf-2,HO-1,NQO-1,IR,IRS-1,GSK-3β,Akt,and the levels of p-IR（Tyr 1185）,p-IRS-1（Ser 307）,p-Akt（Tyr 312+Tyr 315+Tyr 316）,p-GSK-3β（Ser9,Tyr 216+Tyr 279）,GLUT-2 of pancreas and GLUT-2 of liver were dected by Western blot.Results:The results showed that the FBG of mice in KK-Ay group was significantly higher than that of WT group,while,FBG of mice in KK-Ay+TLB-20 group was significantly decreased than that of KK-Ay group;The blood glucose of mice in KK-Ay group was significantly higher than that of WT group after given glucose 0,30,60,90,120 min.Compared with KK-Ay group,the blood glucose at 5 time points and area under curve of OGTT(AUC_OGTT)of mice in KK-Ay+TLB-20 group was significantly decreased;The blood glucose of mice in KK-Ay group was significantly higher than that of WT group after given insulin 0,30,60,90,120 min.Compared with KK-Ay group,the blood glucose at 0,60,90,120 min and AUC_ITTof mice in KK-Ay+TLB-20 group was significantly decreased;The HOMA-IR of mice in KKAy group was significantly higher than that of WT group.Compared with KK-Ay group,the HOMA-IR of mice in KK-Ay+TLB-10 and KK-Ay+TLB-20 groups were significantly decreased;The islets of mice in WT group appeared full and complete,round or oval,and the number of cells in the islets was also abundant and dense.However,the islets of mice in KK-Ay group were destroyed such as the volume was significantly reduced,the morphology appeared uneven,and the number of cells in the islets was significantly decreased;while,TLB markedly improved the pathological change of pancreatic islets in KK-Ay mice;The IOD value of INS of mice in KK-Ay group was dramatically lower than that of WT group.Compared with KK-Ay group,the IOD value of INS of mice in KK-Ay+TLB-10 and KK-Ay+TLB-20 groups were increased significantly.Moreover,the activities of CAT,GSH-Px,SOD and the level of HDL-C of mice in KK-Ay group were significantly lower than those of WT group,while,the levels of ROS,TG,LDL-C,FFA were significantly enhanced than those of WT group;However,TLB obviously increased the activities of CAT,GSH-Px,SOD,and the level of HDL-C in KK-Ay mice;Furthermore,the protein expressions of keap-1,nuclear Nrf-2,the phosphorylation levels of p-GSK-3β（Tyr 216+Tyr 279）and p-IRS-1（Ser 307）in pancreas of mice in KK-Ay group were significantly increased than those of WT group;While,the protein expressions of cytoplasm Nrf-2,HO-1,NQO-1,GLUT-2,the levels of p-GSK-3β（Ser 9）,p-Akt（Tyr312+Tyr 315+Tyr 316）,p-IR（Tyr 1185）in pancreas and GLUT-2 in liver of mice were decreased in KK-Ay group than those of WT group.However,compared with KK-Ay group,these above-mentioned protein expressions or phosphorylation levels were significantly reversed in KK-Ay+TLB-20 group except p-IR（Tyr 1185）.Conclusion:TLB not only reduces the high glucose level of KK-Ay mice,but also attenuates insulin resistance and lipid metabolism disorders.Its underlying mechanism may,at least partly,be related to the regulation of Nrf-2/ARE signaling pathway and IRS-1/GLUT-2 signaling pathway.