Effects Of Curcumin On Renal Tubular Epithelial Cells In High Glucose By Regulating PINK1/Parkin-mediated Mitophagy

ObjectiveTo observe the effect of Curcumin on the Renal Tubular Epithelial Cells in high glucose environment through the PINK1/Parkin pathway.MethodsCCK-8 assay was used to detect the cell viability of HK-2 cells treated with different concentrations of Curcumin(0,2.5,5,10,20,30 μM)for 48 h.According to CCK-8 results,HK-2 cells experimental groups were divided into blank control group(NC,5.5 m M glucose),High glucose group(HG,35 m M glucose)and curcumin group(HG+C,35 m M glucose with 10 μM curcumin).After 48 h of culture,the expression levels of PINK1/Parkin pathway protein,autophagy related proteins p62 and LC3-II,as well as apoptotic proteins caspase-3 were detected by western blotting.The level of intracellular reactive oxygen species(ROS)was detected by DCFH-DA using fluorescence microplate reader.Mito SOX was used to detect mitochondrial reactive oxygen species(mt ROS).Mitochondrial membrane potential(MMP)was detected by JC-1.Results1.Curcumin inhibited HK-2 cell proliferation in a dose-dependent manner.Curcumin had no significant effect on HK-2 cell viability when the concentration of curcumin was lower than 10 μM,and had obvious inhibitory effect when the concentration was 20 μM.2.High glucose inhibited PINK1/Parkin pathway in HK-2 cells,and the addition of 10 μM curcumin increased PINK1/Parkin pathway activation in HK-2cells.3.Western blot revealed that the expression level of p62 was increased and the expression level of LC3-II was decreased by high glucose treatment.However,10 μM curcumin treatment reversed this phenomenon,that is to say,the expression level of p62 was decreased and the expression level of LC3-II was increased.Cell immunofluorescence staining of LC3 also supported this result.4.48 hours of high glucose treatment increased the expression level of caspase-3 in HK-2 cells,and 10 μM curcumin treatment partially reversed the increase of caspase-3 expression level caused by high glucose.5.The results of reactive oxygen species(ROS)test showed that high sugar treatment could increase the intracellular ROS content,and the addition of 10 μM curcumin partially reversed this situation.The intracellular mt ROS content was detected by Mito SOX,and the results showed a similar trend.6.The mitochondrial membrane potential of cells was detected by JC-1.MMP of most cells in the control group was at normal level.Mitochondrial membrane potential decreased after high glucose treatment.The addition of 10μM curcumin partially reversed the depolarization of mitochondrial membrane potential.Conclusions1.When HK-2 cells were treated by high-glucose environment,the PINK1/Parkin pathway expression was down-regulated,mitophagy level was decreased,apoptosis was increased,intracellular ROS and mt ROS contents were increased,and mitochondrial membrane potential was depolarized.2.In a high-glucose environment,the PINK1/Parkin pathway expression of HK-2 cells was up-regulated by curcumin treatment,mitophagy was increased,apoptosis was decreased,intracellular ROS and mt ROS contents were decreased,and mitochondrial membrane potential depolarization was decreased.