JNK And Cyclophilin D In Mitochondrial Necroptosis Induced By Renal Ischemia Reperfusion

Objective: We establish the renal ischemia-reperfusion injury model in rats to observe its biochemical indexes,renal pathology expression of injury substances.At the same time,observe the correlation between JNK and cyclophilin D.To explore the mechanism of cyclophilin D initiating mitochondrial damage mediating necroptosis in renal tubular epithelial cells.Methods: 1.Nine healthy male adult SD rats weighing 220-250 g were randomly(random number)divided into four groups: Normal control group 1,model group 2,nec-1 group 3,three rats in each group.Necroptosis model of renal ischemia-reperfusion was established by clamping bilateral renal arteries for 45 minutes and then restoring perfusion for 24 hours in group 2 and 3.Nec-1 was given through injecting the peritoneal cavity one hour before reperfusion in group 3.In group 2,the ischemia-reperfusion model was made by the same method,and DMSO solution of equal volume was given one hour before reperfusion.In group 1,only bilateral renal pedicles were free,and no other treatment was done.Then,the expression of LC3-II were detected by Western blot.2.Twenty-four healthy male adult SD rats weighing 220-250 g were randomly(random number)divided into four groups: sham group A,model group B,JNK inhibitor group C,cyclophilin D inhibitor group D,six rats in each group.After 7 days of adaptive feeding,necroptosis model of renal ischemia-reperfusion was established by clamping bilateral renal arteries for 45 minutes and then restoring perfusion for 24 hours in group C and D.Drugs were given through injecting the peritoneal cavity one hour before reperfusion.In group B,the ischemia-reperfusion model was made by the same method,and DMSO solution of equal volume was given one hour before reperfusion.In group A,only bilateral renal pedicles were free,and no other treatment was done.After successful modeling,the blood sample of four groups were taken to measure serum creatinine and blood urea nitrogen,and renal tissue morphology dyed with HE and renal necrosis score were observed with microscope.The expression of LC3-II,c-Jun Nterminal kinase(JNK)and cyclophilin D(CYPD)were detected by Western blot.Results:1.Compared with group 1,the expressions of LC3-II in group 3 were higher than those in group 1(P < 0.05),but lower than those in group 2(P < 0.05)?2.Compared with sham group,plasma levels of Scr and BUN levels were significantly increased in model group,JNK inhibitory group and cyclophilin D inhibitory group up and cyclophilin D inhibitory group were lower than those in model group(P < 0.05),especially in model group.The levels of Scr and BUN in JNK inhibitory group.2.HE staining: the structure of tissues were normal,the cells tubular epithelial were complete in the sham group.Compared with the sham group,the cell exfoliation and necrosis,swelling,turbidity,tubular formation and inflammatory cell infiltration were significantly increased in model group?JNK inhibitory group and cyclophilin D inhibitory group(P<0.05).Compared with model group,the renal necrosis score was decreased in JNK inhibitory group and cyclophilin D inhibitory group(P<0.05).3.WB staining: 1)The expression of LC3-II,cyclophilin D and JNK were significantly increased in model group compared with the sham group(P<0.05)?The expressions of LC3-II,cyclophilin D and JNK in JNK inhibition group and cyclophilin D inhibition group were higher than those in sham operation group(P < 0.05),but lower than those in model group(P < 0.05)?2)In JNK inhibition group,the expression of JNK and cyclophilin D decreased,but there was no significant difference between them(P > 0.05).In cyclophilin D inhibition group,the expression of JNK was significantly higher than that of cyclophilin D,and the difference was statistically significant(P< 0.05).Conclusion: 1.JNK inhibitor and cyclophilin D inhibitor can alleviate renal ischemia-reperfusion injury and have a protective effect on renal ischemiareperfusion injury.2.JNK inhibitors improve renal ischemia-reperfusion injury in rats may be related to down-regulation of cyclophilin D expression.