Attenuation Of Renal Ischemia/reperfusion Injury By Oleanolic Acid Preconditioning Via Its Antioxidant,anti-inammatory,and Anti-apoptotic Activities

Background: The advancement in vascular anastomosis and organ preservation,plus the development and application of immunosuppressants like mycophenolate mofetill and irolimus,has all contributed to the constant improvement of organ transplant technologies.Among them,renal transplant,which was conducted earliest,has developed rapidly and recorded the greatest number of clinical surgical procedures,thus becoming the primary option for terminal renal diseases.Due to limited donors,great medical resources involved and patient’s expenses,higher expectation is placed on successful renal transplant.There are many reasons for transplant renal function injuries and early renal transplant failures,one of which is ischemia/repeffusion injury(IRI),a leading factor for infection,cardiocerebrovascular events,and immunologic rejection.Therefore,the study of the action and molecular mechanism of Ischemia/Reperfusion injury,and the probe into effective drugs and the molecular therapeutic target to attenuate and avoid Ischemia/Reperfusion injury,have great significance in clinical prevention and theorectical research.Objects: Aimed to examine the effects of Ischemia/Reperfusion(I/R)on renal functions;to investigate the influences of Oleanolic acid(OA)preconditioning on renal Ischemia/Reperfusion injury on renal function loss,oxidative stress,and inflammatory and apoptotic activities;to reveal the pharmacological action and molecular mechanism of leanolic acid preconditioning on renal Ischemia/Reperfusion injury.Methods: The present study consisted of two-parts.Part I:1.Constructed a renal I/R model.Prior to bilateral renal I/R induction,rats were administered OA(12.5,25 and 50 mg/kg)by peritoneal injection.Set up the control group.2.Venous blood samples were collected.With biochemical techniques,the concentrations of blood urea nitrogen(BUN)and creatinine(Cr)were determine.3.Serum samples and kidneys were then collected.With Enzyme-linked immunosorbent assays(ELISA),the expressions of LDH,Kim-1,SOD,MDA,CAT,GPx,GSH,INF-γ,IL-6,IL-10,and MPO were determined,and apoptosis rate was detected.The influences of Oleanolic acid(OA)preconditioning on renal Ischemia/ Reperfusion injury on renal function loss,oxidative stress,and inflammatory and apoptotic activities were observed.Part II:1.Constructed a renal I/R model.Prior to bilateral renal I/R induction,rats were administered OA(50 mg/kg)or OA(50 mg/kg)+ BSO by peritoneal injection.Set up the control group.2.Venous blood samples were collected.With biochemical techniques,the concentrations of blood urea nitrogen(BUN)and creatinine(Cr)were determine.3.Serum samples and kidneys were then collected.With Enzyme-linked immunosorbent assays(ELISA),the expressions of Kim-1,LDH,MDA,and INF-γ were determined,and apoptosis rate was detected.Whether the protection from attenuating renal ischemia/reperfusion injury by oleanolic acid,as well as anti oxidative stress,inflammatory and apoptotic activities,was dependent on the maintenance of GSH function was observed.4.Total RNA was extracted with Trizol.The m RNA expression levels of nuclear factor erythroid 2-related factor 2(Nrf2)and γ-glutamylcysteine ligase(GCLc)were detected by RT-q PCR.Their roles in OA preconditioning on renal I/R were evaluated.Results:Part I :1.The renal I/R model of rats was successfully constructed by blocking renal blood perfusion in the procedure of surgical clipping.The intraperitoneal injection of OA or BSO on rats 15 days prior to the experiment proceeded well2.In the case of renal Ischemia/Reperfusion injury,BUN,Cr,LDH,and KIM-1 levels were significantly increased.Conversely,OA preconditioning dosedependently prevented the increased levels of BUN,Cr,LDH,and KIM-1 in the I/R rats.3.In the case of renal Ischemia/Reperfusion injury,the expression of MDA levels was significantly increased whereas SOD,CTA,GPx and GSH activities were markedly decreased.OA preconditioning resulted in the decreased expression of MDA and the increased activities of SOD,CTA,GPx and GSH.This effect was observed to be dose-dependent.4.In the case of renal Ischemia/Reperfusion injury,the expression of IFN-γ,IL-6 and MPO was elevated whereas IL-10 was decreased.OA preconditioning markedly prevented the increased levels of IFN-γ,IL-6 and MPO as well as I/R-induced reductions in IL-10 levels in I/R rats.5.In the case of renal Ischemia/Reperfusion injury,the number of apoptotic cells in the kidneys and the expression of Caspase 3 content were significantly increased.OA preconditioning notably and dose-dependently reduced the number of apoptotic cells and nd the expression of Caspase 3 content.Part II:1.The expresssion level of BUN,Cr,KIM-1,LDH,MDA and IFN-γ and the apoptosis rate in the OA+BSO+I/R rats was evidently higher than those in the OA+I/R rats.2.Nrf2 m RNA in I/R rat group was 0.217?0.032,definitely lower than that in the control group(1.024?0.085).Nrf2 m RNA in the OA+I/R rats was manifestly higher than that in I/R rat group.The relative expression of Nrf2 m RNA in the OA+I/R rats was dose-dependent.3.GCLc m RNA in I/R rat group was 0.158?0.014,definitely lower than that in the control group(1.089?0.052).GCLc m RNA in the OA+I/R rats was manifestly higher than that in I/R rat group.The relative expression of GCLc m RNA in the OA+I/R rats was dose-dependent.Conclusions:OA preconditioning can attenuate I/R-induced renal injury,oxidative stress,and inflammatory and apoptotic activities;the OA protective mechnism in I/R-induced renal injury was dependent on GSH,and Nrf2/GCLs might act as its downstream signaling pathways.