| Objective: Sinomenine(SIN),an alkaloid from Sinomenium acutum,has been used in the treatment of various inflammatory diseases including rheumatism and arthritis.However,the effect and mechanisms of sinomenine on kidneys from ischemia-reperfusion(I/R)injury have not been well understood.This study aimed to test whether sinomenine attenuate renal I/R injury and reveal its underlying molecular mechanism.Methods: Kidney ischemia-reperfusion mice model was established on 1 h before reperfusion mice celiac injection of SIN(200 mg/kg),Sham group and I/R group intraperitoneal injection capacity such as saline solution.The serum Cr and BUN concentration of mice were detected by automatic biochemical analyzer.The content of MDA in kidney tissues of mice was determined by using xanthine oxidase assay to determine the activity of SOD activity and myeloperoxidase in mouse kidney tissues.The renal tissue cells apoptosis was detected by Terminal deoxynucleoitidyl Transferase Mediated Nick End Labeling(TUNEL).A cellular model of HK-2 cell hypoxia/reoxygenation(H/R)was established by mineral oil covering cell method.MTT assay was used to detect the activity of HK-2 cells after H/R.Flow cytometry was used to detect the apoptosis of HK-2 cell H/R in different concentrations.According to the literature,mi R-21,mi R-320,mi R-124,mi R-92 a,mi R-29 and mi R-378 were identified as candidate mi RNAs,and the expression of these mi RNAs was detected by real-time quantitative PCR for HK-2 cell H/R.Using Target Scan to analyze the mi R-124 target gene,the combination of mi R-124 and target gene caspase-9 was detected by the double-luciferase reporter gene detection method.After detection of mi R-124 mimic and inhibitor transfection of HK-2 cells,Westblot detected the expression of caspase-9 after H/R of HK-2 cell.Results:1.Compared with Sham group,the serum Cr and BUN concentrations were both increased at 8h and 24 h after renal ischemia reperfusion in I/R group and SIN group(P<0.01).Compared with the I/R group,the serum Cr and BUN concentrations of the SIN+I/R group were reduced at 8h and 24 hours after renal ischemia reperfusion,which was statistical significance(P<0.01).2.Compared with the Sham group,the I/R group and SIN group in the renal ischemia-reperfusion 8 h and 24,kidney tissue cell apoptosis rate,MDA concentration and MPO activity were increased,while SOD activity decreased,which was statistical significance(P<0.01);Compared with the I/R group,the apoptosis rate,MDA concentration and MPO activity of the renal tissue cells were decreased in the SIN+I/R group at 8h and 24 h after ischemia reperfusion,while the activity of SOD increased,which was statistically significant(P<0.05).3.Compared with the control group H/R,24 h HK-2 cells,the activity of HK-2 cells in the SIN+H/R group of each concentration increased,and the apoptosis rate decreased of each concentration group,which was statistically significant(P<0.05).4.The expression of mi R-21,mi R-29 and mi R-378 of HK-2 cells after H/R 24 h was up-regulated,and the expression of mi R-320,mi R-124 and mi R-92 a were down-regulated,which was statistically significant(P<0.05).Compared with the control group,mi R-378 expression was significantly up-regulated,while the expression of mi R-124 was significantly down-regulated,with statistical significance(P<0.05).Compared with the control group,H/R caused the expression of mi R-124 expression in HK-2 cells to decrease with the increase of H/R time,which was statistically significant(P<0.05).5.The expression of mi R-124 in H/R HK-2 cells was increased with the concentration of sinomenine.Compared with the control group H/R,mi R-124 expression of HK-2 cells in the SIN+H/R group after 24 h was up-regulated in the other concentration,except for the concentration of 0.1?M,which statistical significance(P<0.05).6.When mi R-124 mimic transfected HK-2 cells,compared with NC,mi R-124 was significantly increased;while inhibitor transfect HK-2 cells,and compared with NC,mi R-124 expression decreased significantly(P<0.01).7.Compared with control group,the expression of caspase-9 of p GL 3'-WT Caspase 9 3'-UTR + mi R-124 mimic group(P<0.01),while the expression of caspase-9 of p GL 3-WT Caspase 9 3'-UTR + mi R-124 inhibitor group increased significantly(P<0.01).Compared with control group,the expression of caspase-9 of Mut caspasase 9 3'-UTR no obvious change,which was no statistical significance(P?0.05).8.Compared with the control group,when the HK-2 cells were transfected by mi R-124 mimic,the expression of caspase-9 was significantly reduced(P<0.01),while the expression of caspase-9 was significantly increased when the HK-2 cells were transfected with mi R-124inhibitor(P<0.01).9.Sinomenine(50?M)can improve the HK-2 H/R cell activity and decrease the rate of cell apoptosis,but when the mi R-124 inhibitor inhibiting HK-2cells expression of mi R-124,SIN + mi R-124 hibitor group HK-2 H/R cell activity and reduce significantly the effect of cell apoptosis rate decreased significantly.Compared with Sin group,the cell activity of HK-2 cells decreased significantly,but the cell apoptosis rate increased significantly after H/R 24 h of the SIN+ mi R-124 inhibitor group,which was statistical significance(P<0.01).Conclusion: Sinomenine can alleviate the tissue damage of renal ischemia reperfusion in mice,and its mechanism is related to the inhibition of oxidative damage and decreased renal cell apoptosis rate.The expression of HK-2 cells after H/R was significantly down-regulated,indicating that mi R-124 was associated with HK-2 cell H/R.Mi R-124 was combined with caspasase 9 3'-UTR to down-regulate the expression of caspasase 9 and inhibit the process of apoptosis.Sinomenine can reduce the damage of HK-2 cells after hypoxia/reoxygenation,and its mechanism is related to the up-regulation of mi R-124 induced by sinomenine and the inhibition of cell apoptosis. |
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