Experimental Study On The Toxic Effect Of Selenium On NRK-52E Cells Exposed To Fluoride And The Underlying Mechanism

Objective:In our research, we chose rat renal tubular epithelial cells(NRK-52E) to detect the toxic effect of selenium and the mechanism. NRK-52 E cells were cultured and respectively divided into control group, fluoride groups, selenium groups, fluoride and selenium joint action groups.Methods:NRK-52 E cells were cultured with fluoride in various concentrations, selenium in various concentration,and fluoride and selenium combination for 48 h and 72 h. The proliferation of cells were measured by MTT assay. The morphological changes of cells were observed by HE staining; The apoptosis rates and mitochondrial membrane potential(ΔΨm)were detected by flow cytometry; The activity of enzymes and the content of free radicals were examined by relevant reagent kit; The m RNA and protein expression level of Caspase-3,Caspase-9,Bax and Bcl-2 were examined by Real-Time PCR and Western-blot.Results:1. After exposed to different concentrations of fluoride for 48 h, 72 h and 96 h,fluoride with dose of 5 mg/L,10 mg/L,20 mg/L and 40 mg/L had inhibitive effects on cell proliferation. Low dose of selenium could promote cell proliferation, while high concentration of selenium had inhibition effect. Compared with the isodose sodium fluoride group, cell proliferation of selenium and fluoride in combinition group significantly increased.2.Observation of cell morphology under the microscope.In the control group, cell morphology was natural, and they were arranged orderly. The volume of cells in fluoride groups increased;cell gap became wide;and multicore phenomenon appeared;the deformation quantity of cell increased; Cell morphology in selenium groups were similar to the control group: compared with the isodose fluoride group, cell morphology inselenium and fluoride in combinition group returned to normal shape.3.Test results showed that oxidative stress in cell cultures of indicators, with the increase of fluoride concentrations, The activity of SOD, CAT, T-AOC and GSH-PX gradually reduced; NO and MDA increased gradually. Compared with the control group,the difference of the enzyme activity and the content of free radical in selenium group was not statistically significant. Compared with the isodose sodium fluoride group, the activity of SOD, CAT, T-AOC and GSH-PX in selenium and fluoride in combinition group increased, while NO and MDA were down-regulated.4.Cell apoptosis and mitochondrial membrane potential changed, with the increase of fluoride concentrations.The apoptosis rates and the loss of mitochondrial membrane potential were up-regulated. The apoptosis rate and mitochondrial membrane potential significantly decreased in selenium groups. Compared with the isodose sodium fluoride group,the apoptosis rate and mitochondrial membrane potential in selenium and fluoride in combinition group significantly decreased.5. Apoptosis related gene m RNA expression. With the increase of fluoride concentrations, the m RNA expression level of Caspase-3, Caspase-9 and Bax increased significantly. Compared with the isodose sodium fluoride group,the m RNA expression level of Bcl-2 increased in selenium and fluoride in combinition group,while the m RNA expression level of Caspase-3, Caspase-9 and Bax were significantly reduced.6.The determination results of protein expression apoptosis related factors.With the increase of fluoride concentrations, compared with the control group, the protein expression of Cleaved Caspase-3, Cleaved Caspase-9 and Bax were up-regulated, while the protein expression of Bcl-2 were down-regulated. The difference was not statistically significant between selenium groups and control group. Compared with the isodose sodium fluoride group, the protein expression of Cleaved Caspase-3、Cleaved Caspase-9 and Bax were down-regulated, while the protein expression of Bcl-2 were up-regulated.Conclusions:1.Selenium may antagonize the apoptosis of fluoride-induced NRK-52 E cells.2.Selenium can adjust the activity of antioxidant enzyme, improve the cell antioxidant capacity and promote the removal of the free radicals, so as to keep the cells from oxidative damage.3.Selenium can regulate the apoptosis related factors of Caspase-3,Caspase-9,Bax and Bcl-2, restrain the progress of apoptosis signal transduction, and thus antagonize the apoptosis.