The Role Of Resveratrol In The Oxidative Damage Of Renal Tubular Epithelial Cells And Its Molecular Mechanism

PART?Objective:To explore the role of resveratrol in the oxidative damage of renal tubular epithelial cells and its molecular mechanism.Methods:(1)HK-2 cell was challenged by hydrogen peroxide(100u M,250 u M,500 u M,750 u M),after 48 h the activity of cell was detected by CCK-8.(2)HK-2 cell was treated by 250 u M hydrogen peroxide with resveratrol(5u M,10 u M,20 u M),after 48 h the activity of cell was detected by CCK-8.After 10 u M Res was applied to HK-2 48 H,the apoptosis rate was detected by flow cytometry.Western Blot was used to detect Y-H2 ax and P-Akt expression of DNA fragmentation markers.Results:(1)The results of CCK-8 test showed that with the increase of hydrogen peroxide concentration,the activity of cells decreased,and the results of CCK-8 detection showed that with the increase of the concentration of resveratrol(5u M,10 u M,20 u M),the cell activity decreased gradually,P<0.05.(3)Western Blot results showed that the DNA rupture after the hydrogen peroxide alone was given to 250 u M.The expression of the marker Y-H2 ax increased significantly;at the same time,after Res treatment with hydrogen peroxide and 10 u M,the Y-H2 ax expression level of the DNA breakage marker was further increased,and the DNA fracture marker Y-H2 ax increased significantly after the resveratrol alone was given to 10 u M.(4)the flow pattern showed that the apoptosis rate of hydrogen peroxide alone was 56.7%,and the apoptosis rate of 250 u M was increased to 69.1% after 10 u M and 10 u M Res were given.5.The results of Western Blot showed that P-Akt was significantly up-regulated when hydrogen peroxide was given alone,while P-Akt decreased significantly when 250 u M hydrogen peroxide and 10 u MRes were added,and P-Akt was significantly down regulated when Res was given alone.We further studied that adding 10 u M MK-2206(Akt inhibitor)into H2O2 induced injury further aggravated the injury induced by H2O2.Conclusion: Res plays a protective role in hydrogen peroxide induced injury of renal tubular epithelial cells,but plays a further role in aggravating cell damage.The down-regulation of Akt induced by Res plays an important role in promoting the injury induced by hydrogen peroxide.PART?Objective: Further study on the damage and mechanism of Res induced HK-2 cells.Methods:(1)After 48 H of different concentrations of Res(5u M,10 u M,20 u M),CCK-8 was used to detect cell activity;apoptosis was detected by flow cytometry,and the expression of Western Blot detection protein was detected.(2)HK-2 cells with Res concentration of 10 u M were used to detect reactive oxygen species(Reactive Oxygen Species,ROS)and mitochondrial membrane potential,and Western Blot was used to detect the expression of related proteins.Apoptosis.(3)the rate of apoptosis was detected by flow cytometry with the simultaneous action of the Caspase inhibitor of 10 u M(a cell osmotic,irreversible pan caspase inhibitor,Z-VAD-FMK,FMK)with 48 H of 10 u M.(4)The diffusion of Cytc by immunofluorescenceResults:(1)CCK-8 results showed that the increase of cell count gradually decreased with the increase of Res concentration,and the flow pattern showed that the survival rate of cells decreased with the increase of Res,and the apoptosis rate of cells increased,P<0.05;Western Blot results showed that Y-H2 ax increased gradually with the increase of Res concentration..Flow cytometry showed that compared with the control group,the ROS level of Res group increased significantly,and the mitochondrial membrane potential decreased significantly,P<0.05.(3)flow cytometry showed that NAC significantly reduced Res induced reactive oxygen species(P<0.05),but had no significant effect on HK-2 cell apoptosis,P>0.05.The results showed that compared with group Res,the rate of apoptosis decreased significantly after FMK was given to P<0.05.The immunofluorescence staining of cytochrome c(Cytochrome C,Cytc)after incubation with 10 u M Res showed that the mitochondria of the Con group were more than the Res group,while the con group did not see the Cytc dispersion out of the mitochondria,while the Res group showed that the number of mitochondria decreased significantly.It was found that the increase of P53 and acetylation P53,the expression of Noxa,Bax and Bcl2 of the downstream protein of P53 did not increase,but the expression of the downstream protein Bcl-xl was reduced,and the expression of Puma was up regulated.Different concentrations of P53(10u M,5u M,1u M,0.5u M)inhibitor Pifithrin-alpha(PFT a)were used to detect cell activity after 48 H,and there was no difference in cell activity between 1u M PFT alpha and P>0.05,and the apoptosis rate was detected after 48 hours of Res group.It was found that there was no difference in cell apoptosis rate after P53 inhibition,P>0.05.Join Sp600125(SP600125 is a broad-spectrum JNK inhibitor,acting on JNK1,JNK2 and JNK3,Sp).It is found that Sp cell activity is not inhibited when 1u M is given.In group Res,apoptosis rate was detected by 1u M48 H,and apoptosis rate was significantly decreased,P<0.05.As we detected the expression of P-jnk protein,Res in group Con was significantly higher than that in group Con.Conclusion: when renal tubular epithelial cells(HK-2 cells)were given Res alone,they could induce renal tubular epithelial cell injury,and increased with the increasing concentration of Res.The damage degree of HK-2 cells was directly proportional to the concentration of Res.Res causes the increase of ROS and the decline of JC-1,which is not the cause of apoptosis and necrosis,but only the accompanying symptoms of cell damage.It was found that P53 and Ac-P53 were upregulated,and the P53 apoptosis protein Puma was upregulated.However,the apoptosis of PTF cells did not decrease significantly after giving PTF alpha.Therefore,Res induced HK-2 cell injury is not mediated by P53 signal pathway.Jnk plays an important role in Res induced HK-2 cell damage.