Tanshinone ? Protects Ischemia/reperfusion Induced Renal Injury Via Activation Of Nrf2-ARE Pathway

Objective:To investigate the protective effect and mechanism of tanshinone I(T-I)on renal ischemia-reperfusion injury.Methods:Experiments in vitro:1.Cytotoxicity assay:the human renal tubular epithelial cells(HK-2)were treated with different concentrations of Tanshinone I(T-I),and the cell viabilities were detected by CCK-8 kit.2.Different groups of cells were treated with different concentrations of H2O2 for 2 hours,the cell viabilities were detected by CCK-8 kit to select the optimal H2O2 concentration for the oxidative stress model.3.Efficacy experiments:HK-2 cells were divided into four groups:control group 1(control)and model group 1(H2O2)were pretreated with solvent while control group 2(T-I)and model group 2(H2O2+T-I)were pretreated with T-1 for 24h,H2O2 group and H2O2+T-I group were treated by H2O2 for another 2h,the cell viabilities and percentages of apoptotic cells were tested to assess the cytoprotective effects of T-I.4.To study the treatment effect of T-I on oxidative stress injury,DCFH-DA was used to detecte the generation of reactive oxygen species in each group.5.RT-qPCR and Western Blot were used to measure the expressions of Nrf2 and its downstream genes in each group.6.The cellular levels of the mRNA specific for the siRNA transfections were verified by RT-qPCR,and then in vitro Efficacy experiments were repeated.Experiments in vivo:1.Male ICR Mice were randomly divided into four groups:The sham group and IR group received corn oil via intraperitoneal injection for 14 days before surgeried;the sham + T-I group and IR+T-I group received T-I dissolved in corn oil before surgeries.2.Mouse model of renal IR injury:Left renal pedicle was exposed and clamped with vascular clamp for 50 min to induce ischemia,and right kidney were exposed and removed afterwards.Sham-operated control animals underwent right kidney resection without occlusion of left renal artery.The blood sample and kidney tissue were collected 48 hours after surgeries.3.Serum creatinine(Cr)and blood urea nitrogen(BUN)were detected to evaluate the renal function.4.The scores of histopathology and apoptosis were used to evaluate the renal injury.5.Malondialdehyde(MDA)and superoxide dismutase(SOD)were measured to evaluate oxidative stress in the kidney.6.Immunohistochemical was used to evaluate the level of inflammation.7.TUNEL staining of kidney sections were used to detect the apoptosis cells.8.RT-qPCR and Immunoblotting were utilized to detect the activation of Nrf2 signaling pathways.Results:Experiments in vitro:Cytotoxicity experiments showed that 3.5?M and lower concentrations of T-I have no toxic effects on HK-2 cells,and the hydrogen peroxide toxicity test showed that 2 mM was the best concentration for oxidative stress models.Efficacy experiments show that T-I attenuated hydrogen peroxide-induced viability decrease and apoptosis of HK-2 cells.In addition,T-I reduced the generation of ROS induced by hydrogen peroxide in HK-2 cells.T-I was able to increase the expression of mRNA and proteins of Nrf2 downstream genes in HK-2 cell,also can increase the expression of Nrf2 proteins in nucleus and cytoplasm.And T-I can't attenuated hydrogen peroxide-induced viability decrease post Nrf2 knockdown.Experiment in vivo:Serum Cr and BUN were significantly decreased by the treatment of T-I in the RIRI,and T-I dramatically prevented ischemia reperfusion-induced histological damage,inflammation and apoptosis;Pretreatment with T-I significantly increased the SOD activity and decreased MDA content after IR in kidney tissues.T-I was able to increase the expression of Nrf2 protein in nucleus and cytoplasm,inaddition,T-I upregulate expression levels of mRNA and protein of Nrf2 downsteam genes,incuding GCLC,HMOX1 and GPX.Conclusion:The tanshinone I plays a protective role in renal ischemia-reperfusion injury via promoting Nrf2 signaling,This may supply a new strategy for renal ischemia/reperfusion injury.