Effect Of Irbesartan On Hyperglycemia-Induced Apoptosis In Isolated Islet Cells

Background and Objectives:Hyperglycemia is a very important syndrome of diabetes and high glucose can induce apoptosis of islet cells (glucose toxicity).Besides hyperglycemia,renin-angiotensin system disturbance, hyperlipemia,and oxidative stress can be involved in apoptosis in process.We aimed:(1)to create high glucose-induced apoptosis in isolated islet cells.(2)to explore the effects of irbesartan on hyperglycemia-induced islet cells apoptosis.Methods:Two steps in our study.In order to investigate the mechanism of apoptosis,we first induced apoptosis in isolated islet cells after prolonged exposure to high glucose levels.The detailed procedure included:①islet isolation:SD rat islets free of exocrine tissue were obtained by repeated hand-picking under a stereomicroscope and cultured in DMEM medium.6 samples(20 islets/sample)were collected in one group.②group designing: NG group 1(glucose 5.6mmol/L,normal glucose,24 hours-incubation),NG group 2(glucose 5.6mmol/L,normal glucose,72 hours-incubation),NG group 3 (glucose 5.6mmol/L,normal glucose,120 hours-incubation);HG group 1 (glucose 25.6 mmol/L,high glucose,24 hours-incubation),HG group 2(glucose 25.6 mmol/L,high glucose,72 hours-incubation),HG group 3(glucose 25.6 mmol/L,high glucose,120 hours-incubation).Then we explored the blockade of irbesartan,an antagonist of renin-angiotensin-receptor blocker,on hyperglycemia-induced apoptosis in islet cells.The influencing factors included glucose concentration and irbesartan(serum test),Four groups(24 hours-incubation):NG group(glucose 5.6mmol/L,normal glucose),HG group(glucose 25.6 mmol/L,high glucose); NG+A group(glucose 5.6mmol/L+1ml irbesartan serum),HG group (glucose 25.6 mmol/L+1ml irbesartan serum).the parameters and measurements were as follows:①Apoptotic cells of islet were detected by TUNEL assay.②The expression of gene Caspase-3,Bax,AT1R,AngⅡmRNA were measured through reverse transcription-fluorescence quantitative-polymerase chain reaction(RT-FQ-PCR).These parameters indicate the mechanisms of apoptosis and RAS.③p22phox,gp91phox mRNA obtained by RT-PCR.TAOC,SOD,GSH,MDA were detected by colorimetry.④Insulin concentration in culture medium was detected by ELISA the changes of glucose-stimulated insulin secretion was calculated as the stimulation ratio of stimulated release(15 mmol/L glucose)to basal release(3.3 mmol/L glucose).Results:Our results of partⅠindicated:①The apoptotic rates of islet cells in the HG groups were significantly higher than those of islet cells in NG groups (P＜0.05).gene Caspase-3 expression showed a remark increase in HG groups (P＜0.05).From the 1^stday to 3^rdday after incubation,gene Caspase-3 expression increased gradually both in high glucose levels and normal glucose levels (P＜0.05).The results of partⅡwere as follows:①both apoptotic rate of islet cells and gene expression of Caspase-3 and Bax in HG groups were significantly higher than those in NG groups(P＜0.05);HG+A group were significantly less than HG group(P＜0.05).②AT1R and AngⅡgene expression:the expression of AT1R,AngⅡgene and p22phox,gp91phox mRNA in HG groups were significantly higher than NG group(P＜0.0005);HG+A group were signify-cantly less than HG group,respectively(P＜0.0005).③ROS:TAOC,SOD,GSH in HG groups were significantly less than NG group(P＜0.05);but they were high level in HG+A group.MDA was higher in HG groups than NG group,but it was lower in HG+A group(P＜0.05).④Glucose-stimulated insulin secretion:the stimulation ratio was markedly higher in HG groups than NG group(P＜0.0005).There were no significant difference with NG,NG+A and HG,HG+A groups(p＞0.05).Conclusion:①High glucose can induce islet cell apoptosis in vitro,the important mechanism was associated with enhanced ROS by NADPH oxidase which was mediated by AngⅡ,and increase expression of the apoptosis-related genes Bax,Caspase-3.②Irbesartan can markedly reduce the apoptosis of islet cell in high glucose,the mechanism have relate to inhibiting of AT1R,AngⅡmRNA expression,blocking of RAS and anti-oxidation,down-regulating gene expression of Bax and Caspase-3.③Irbesartan do not significantly affect insulin secretion of isolated islet cells in high glucose for 24 hours.