MeHg-induced Mitochondrial-mediated Apoptosis In Human Proximal Tubular HK-2 Cells And Its Mechanism

Research Background Methylmercury(Me Hg)is an environmentally harmful pollutant that can enter the body through a variety of pathways and has a slow metabolism in the body.Me Hg entering the body can accumulate in the kidneys and cause kidney damage.Me Hg in the kidney is mainly deposited in the proximal tubules,and renal tubular cells are rich in mitochondria.Therefore,in this study,Me Hg was used as a processing factor,and human renal proximal tubule epithelial cell HK-2 was used as the research object to systematically explore the mitochondrial-mediated apoptosis and its mechanism induced by Me Hg in HK-2 cells.The study of the effect provides theoretical basis,and provides theoretical basis for Me Hg toxic action mechanism and effective protection.Research Aim To study the toxic effects of Me Hg on HK-2 cells,and to explore the mechanism and mechanism of Me Hg-induced mitochondrial-mediated Caspase 3dependent pathway and Caspase 3 non-pathway apoptosis in HK-2 cells.Research Methods MTT method was used to detect cell survival after HK-2 cells were administered with Me Hg;Rhodamine 123 flow cytometry was used to detect cell mitochondrial membrane potential;Annexin V/PI flow cytometry was used to detect apoptosis;Western blot test was used to detect mitochondrial cell apoptosis pathway related Protein expression;Cells were given PMN-1 inhibitor BMN-673 ts and / or Caspase 3 inhibitor Ac-DEVD CHO to explore related mechanisms.Research Results1.Effect of Me Hg on the survival rate of HK-2 cells Me Hg at different concentrations can reduce and reduce the survival rate of HK-2 cells,with a tendency to decrease with increasing concentration(P <0.01),there is a dose dependence(P < 0.01).Treatment of HK-2 cells at a certain concentration of Me Hg at different times,with the increasing treatment time,the survival rate of HK-2 cells showed a downward trend(P<0.01),and there was a time dependence(P<0.01).2.Effect of Me Hg on mitochondrial membrane potential of HK-2 cells The administration of different concentrations of Me Hg can cause the mitochondrial membrane potential of HK-2 cells to decrease.It can be seen in the flow overlay that the fluorescence intensity peak of the drug administration group shifted to the right.With the increase of Me Hg concentration,the percentage of cells in gate M1 decreased.Compared with the control group,the difference was statistically significant(P<0.01).3.Effect of Me Hg on apoptosis of HK-2 cells Different concentrations of Me Hg can cause apoptosis in HK-2 cells at different times.Compared with the control group,apoptosis,necrosis of 0.5,1.0,1.5,2.0,2.5?g / m L Me Hg group was statistically significant(P <0.01);1.5 ?g / m L Me Hg treated HK-2 cells 1,3,6,9,12,and 24 hours,Compared with the control group at,the difference was statistically significant(P<0.01).4.Effect of Me Hg on HK-2 Mitochondrial Mediated Apoptosis Pathway Related Proteins A certain dose of Me Hg affects HK-2 at different times1)Effect on apoptosis-related proteins of Caspase-dependent pathway.With the increase of Me Hg time,the expression of Cyt C protein increased,and the difference was statistically significant compared with the control group(P <0.01);Caspase 3 protein began to be expressed at 12 h,and Pro-Caspase 3 and Caspase at 24 h Compared with the control group,the expression of 3 protein was statistically significant(P<0.01);the level of Hsp 70 protein decreased,which was not statistically significant compared with the control group.Cytoplasmic protein showed that the expression of Hsp 70 protein increased at 1 h,and then decreased rapidly at 3 h,the difference was statistically significant(P<0.01).2)Effects on apoptosis-related proteins in Caspase-independent pathways Among the total proteins,compared with the control group,the expression levels of 116 and 89 k Da fragments of PARP-1 protein increased first and then decreased,and the differences between the 1,3,6,9,and 12 h groups were statistically significant compared with the control group(P < 0.01);PAR polymer showed an upward trend at 1,3,6,9 hours(P<0.01),and a downward trend at 12,24 hours.The 67 k Da fragment's expression of AIF protein decreased(P<0.01),and the 57 k Da fragment's expression was up-regulated.The difference between the 24 h group and the control group was statistically significant(P<0.01).In nuclear proteins,with the increasing treatment time of Me Hg,the expression levels of 116 and 89 k Da fragments of PARP-1 protein increased first and then decreased(P < 0.05);the expression of PAR polymer increased first and then decreased(P <0.05);AIF protein(57 k Da)expression gradually increased(P <0.01).5.Discussion on related mechanisms HK-2 was added with PARP-1 and / or Caspase 3 inhibitors,and the mechanism of apoptosis induced by Me Hg was explored by MTT and Annexin / PI.1)Add different concentrations of PARP-1 inhibitor BMN-673ts1,10,100 n M,1 ?M BMN-673 ts alone acting on HK-2 had no effect on cell survival rate and apoptosis.With the addition of 1.5 ?g / m L Me Hg,the cell survival rate was higher than that in the Me Hg group(P<0.01).The apoptosis was lower than that in Me Hg group(P<0.01).2)Add different concentrations of Caspase 3 inhibitor Ac-DEVD CHO1.10,100 ?M Ac-DEVD CHO alone affected HK-2 cells,and had no effect on cell survival rate and apoptosis.With the addition of 1.5 ?g / m L Me Hg,the cell survival rate was greater than the Me Hg group(P <0.01),and the apoptosis was lower than the Me Hg group(P<0.01).3)Join BMN-673 ts and Ac-DEVD CHO HK-2 10 n M BMN-673 ts and 100 ?M Ac-DEVD CHO had no effect on apoptosis.With the addition of 1.5 ?g / m L Me Hg,the apoptosis was lower than that in the Me Hg group(P<0.01).It was also lower than the 10 n M BMN-673 ts +1.5 ?g/ m L Me Hg group,but it was no different from the 100 ?M Ac-DEVD CHO + 1.5 ?g/ m L Me Hg group.Conclusions 1.Me Hg can induce apoptosis of HK-2 cells through mitochondrial-mediated PARP-1/AIF and Cyt C/Caspase 3.2.Inhibition of PARP-1/AIF and Cyt C/Caspase 3 can reduce HK-2 cell apoptosis.3.Me Hg caused apoptosis of HK-2 cells with mitochondrial pathway accompanied by cell necrosis.