Research On Oxidative Damage And Apoptotic Pathway Of NRK Cells Induced By Melamine

Objective We aim to investigate the toxicity and mechanism of NRK cells induced by melamine so as to complement the mechanism of renal toxiry of melamine.Methods Melamine was dissolved in DMEM preheated in37℃,which was diluted to3mg/ml,2.5mg/ml,2.0mg/ml,1.5mg/ml,1.0mg/ml,0.5mg/ml,0mg/ml,amounting to7concentration groups.The impact on the proliferation of NRK cells induced by different concentration of melamine was detected by MTT assay.According to the influence of the proliferation of NRK cells induced by different concentration of melamine,we chose Omg/ml,1mg/ml,2mg/ml,3mg/ml as the concentrations of melamine that was utilized in the subsequent assays. Hoechst33342staining was used to observe the morphology of apoptotic NRK cells.The extent of oxidative damage of NRK cells was done by mesuring the content of SOD,MDA and GSH-Px. The apoptotic rate of NRK cells induced by different concentration of melamine and the action of Ac-LEHD-FMK were utililized by a quantitative flow cytometry assay. The measurement of mitochondrial transmembrane potential was stained by Rhodamine123.The expression levels of proteins of caspase3and caspase9were determined by Western Blot.Results Compared with the control group, the viability of NRK cells induced by the melamine concentration of0.5mg/ml,1.0mg/ml,1.5mg/ml,2.0mg/ml,2.5mg/ml,3mg/ml was descreased. The apoptotic NRK cells stained by Hoechst33342was showed by the colour of bright blue,nevertheless,the normal NRK cells was displayed by the colour of faint blue. Through the detection of oxidative damage indicators,we found the content of SOD and GSH-Px were declined,whose content was dropped with the increasing concentration of melamine,comparing with the control group.However,the content of MDA has its opposite effort.The content of SOD,MDA and GSH-Px changed in the dependent manner of concentration.After measuring the apoptotic rate of NRK cells induced by melamine, we found the apoptotic rate of NRK cells induced by the melamine concentration of lmg/ml,2mg/ml and3mg/ml, comparing with the control group.However,compared with the group of1mg/ml melamine,the apoptotic rate of the concentration of1mg/ml melamine pretreated by Ac-LEHD-FMK was descended and its number of normal NRK cells approximated to the control group,which indicates that Ac-LEHD-FMK can inhibite the apoptotic rate of NRK cells. Compared with the control group,the mitochondrial transmembrane potential of NRK cells induced by melamine was decreased,showing in the dependent manner of concentration. The expression levels of proteins of procaspase9and procaspase3of NRK cells induced by the melamine concentration of1mg/ml,2mg/ml and3mg/ml were falling,meanwhile,the expression levels of proteins of active-caspase9and active-caspase3of NRK cells induced by the melamine concentration of1mg/ml,2mg/ml and 3mg/ml were elevated.Moreover,the changes was increasing by the rising of concentration of melamine,showing the dependent manner of concentration.Conclusion Melamine inhibites the proliferation of NRK cells,causes the oxidative damage of NRK cells,and induces the apoptosis of NRK cells by the pathway of mitochondria.