Protective Effect Of Salvianolic Acid B On Pancreatic Islet Cells In Diabetic Rats With Fluctuating Blood Glucose And The Potential Mechanisms Implicated

Objective: To observe the protective effect of Salvianolic acid B(Sal B)on pancreatic islet cells in diabetic rats with fluctuating blood glucose and the possible mechanisms involved.Methods: 1.Animal experiment: Diabetic rat model was established by keeping on high sugar high fat diet and intraperitoneally injection with a single does of STZ(40mg/kg).Rats with radom blood glucose(RBG)levels exceeding 16.7 mmol/L were considered diabetic rats and divided into intermittent high glucose group(IHG,distilled water 5 ml·kg-1·d-1),Sal B 160 group(160 mg·kg-1·d-1)and Sal B 80 group(80 mg·kg-1·d-1).Rats in Sal B groups were given Sal B for 6 weeks,while the rest groups were given the same volume of distilled water.Rat model of blood glucose fluctuation was established by subcutaneous injection with regular insulin or gavaging of glucose twice daily.Body weight and daily blood glucose levels of 5 different times were recorded every week and fasting blood glucose(FBG)was recorded every two weeks.After 6 weeks of treatment,rats were subjected to an oral glucose tolerance test(OGTT)and the values of area under curve(AUC)were calculated.Levels of total cholesterol(TC),glycosylated hemoglobin(GHb)and fasting insulin(FINS)were measuresd and insulin sensitivity index(ISI)were calculated to assess the extent of insulin resistance(IR).Pancreatic tissue levels of Caspase-9,Caspase-3 activity and the contents of total antioxidant capacity(T-AOC),superoxide dismutase(SOD)activity and malondialdehyde(MDA)in both serum and tissues were determined with commercially available kits.Histopathological changes and cell apoptosis in pancreatic islet were observed by HE and TUNEL staining,respectively.Levels of JNK and NF-?B activation and the protein expression of PDX-1,Bax,Bcl-2,and Bim in pancreatic tissue were determined by western blot.2.INS-1 cell experiment: INS-1 cells were preincubated with Sal B(1×10-5,10-6,10-7mol/L)for 24 h,followed by exposure to intermittent high glucose(IHG,11.1 mmol/L 12 h,33.3 mmol/L 12 h)for 72 h.Cell viability was assessed by CCK8 assay and insulin secretion capacity was measured by enzyme-linked immunosorbent(ELISA)and cell apoptosis was evaluated by flow cytometry analysis.Intracellular reactive oxygen species(ROS)and mitochondrial membrane potential(MMP)were evaluated by fluorescent probe DCFH-DA and JC-1,respectively.Levels of T-AOC,MDA,Caspase-9 and Caspase-3 activity were determined by colorimetric method according to the manufacture?s instructions.Protein levels of JNK,p-JNK,NF-?B,PARP,PDX-1,Bax,Bcl-2 and Bim were determined by Western-blot analysis.Results: 1.Animal experiment:(1)Treatment with Sal B for 6 weeks significantly improved the level of glucose tolerance(P <0.05 or P <0.01),with body weight increased and GHb decreased(P <0.05 or P <0.01)in diabetic rats.(2)Sal B significantly increased FINS secretion and decreased levels of FBG and TC(P <0.05 or P <0.01),accompanied by alleviation of insulin resistance in diabetic rats(P <0.05).(3)Pancreatic islets in diabetic rats became smaller and decreased in numbers and injury cells,with JNK and NF-?B activation increased notably(P <0.05 or P <0.01).(4)Treatment with Sal B significantly reduced pancreatic islet cell apoptosis,Caspase-9 and Caspase-3 activity(P <0.05 or P <0.01),with protein expression of Bax,Bim down-regulated and PDX-1,Bcl-2 up-regulated markedly(P <0.05 or P <0.01).(5)In addition,Sal B effectively enhanced T-AOC and SOD activity in both serm and pancreatic tissues,with MDA content reduced evidently(P <0.05 or P <0.01).2.INS-1 cell experiment:(1)Sal B notably improved INS-1 cell apoptosis and injury(P <0.05 or P <0.01)and increased cell viability,with the levels of Bax,Bim and PARP cleavage decreased and Bcl-2 protein expression increased evidently(P <0.05 or P <0.01).Preincubation with Sal B also markedly decreased the activity of Caspase-9 and Capase-3 in INS-1 cells(P <0.05 or P <0.01).(2)Preincubation with Sal B significantly increased insulin secretion capacity and mitochondrial membrane potential(P <0.05 or P <0.01),with protein expression of PDX-1 up-regulated(P <0.05 or P <0.01).(3)Levels of T-AOC were significantly increased(P <0.05 or P <0.01),while the protein activation of JNK and NF-?B,the levels of intracellular ROS and MDA content were decreased markedly in INS-1 cells(P <0.05 or P <0.01).Conclusion:(1)Sal B can significantly improve pancreatic islet dysfunction and suppresss pancreatic islet apoptosis in diabetic rats with fluctuating blood glucose.(2)Sal B is capable of suppressing pancreatic islet apoptosis and increasing its insulin secretion capacity,which might be attributed to enhancement of antioxidant capacity,suppression of JNK and NF-?B activation and up-regulation of PDX-1 protein expression.(3)Sal B is capable of suppressing IHG-induced injury and apoptosis in pancreatic islet cells,which might be derived from regulation of Bcl-2 family protein expression,attenuation of mitochondrial membrane potential and subsequent suppression of Caspase-9 and Caspase-3 activity.