AT1 Receptor Regulates Apoptosis Induced By STZ In Islet Beta Cells And It’s Role In Irbesartan Protection

Objective: SiRNA interference silencing angiotensin II type 1 receptor To explore it’s regulation effect on apoptosis induced by streptozotocin （STZ）in insulinoma cells NIT-1 and then study the mechanism of irbesartan protection from that perspective.Methods: Part one, three pairs of siRNA targeting AT₁R were designed and synthesized. It was transfected into NIT-1 by liposome transfection. Using Fluorescence microscope to examine the transfection efficiency of different siRNA concentration and detecting AT₁R mRNA expression with Real-time PCR to decide which is the best interference sequence and time. Part two, to investigate the protective effects of irbesartan against the apoptosis damage of the NIT-1 cells induced by streptozotocin. experimental groups: Mock, STZ group, irbesartan+STZ group, si-AT₁R-2 group, si-AT₁R-2+STZ group and si-AT₁R-2+irbesartan+STZ group. Mock and si-AT₁R-2 group didn’t make any intervention, other groups treated with 5mM streptozotocin and/or with 0.01mM irbesartan as the experimental group. All experiments were totally repeated for 3-6 times. Microscope was used to observe Hoechst33342 staining, flow cytometer were used to detect apoptosis rate labbled AnnexinV-FITC/PI.Real-time PCR detected Caspase-3 mRNA, NADPH mRNA expression.Result: Part one, The maximum silence efficiency of AT₁R gene was at 24h with 40nM si-AT₁R-2. The expression of AT₁R mRNA was markedly down regulated （92.20± 1.61）% compared with that in normal（P <0.05）. Part two, The apoptosis rate and the expression of Caspase-3 mRNA, NADPH mRNA: in STZ group all of the indexs increased compared with mock group （P < 0.05）,and all of them were decreased in irbesartan+STZ group compared with STZ group（P < 0.05）, there is the same trend after silencing AT₁R gene. The difference of all the indexs between mock group and si-AT₁R-2 group has no statistical significance（P>0.05）. in si-AT₁R-2+STZ group the apoptosis rate decreased compared with STZ group （P < 0.05）. in si-AT₁R-2+ irbesartan +STZ group the apoptosis rate decreased compared with si-AT₁R-2+ STZ group （P <0.05）,and the expression of Caspase-3 mRNA,NADPH mRNA showed a decreasing trend. Silencing AT₁R gene can weaken irbesartan’s ability to antagonise apoptosis damage indused by STZ.Conclusion: This study successfully silence AT₁R gene of NIT-1 and obtain the best efficiency of silence with 40nM si-AT₁R-2 at 24h. a significant up-regulation of AT₁R expression mediate the process of apoptosis induced by STZ in NIT-1 cells. Irbesartan antagonise apoptosis induced by STZ via the way similar to the reverse agonism effect of AT₁R. May be there are other pathways irbesartan against the apoptosis damage of islet beta cells induced by streptozotocin.