7-Nitroindazole Against Fluorosis That Induces The Increased Expression Of Nitric Oxide And Nitric Oxide Synthetase And The Toxic Effect Of Cells In Cultured SH-SY5Y Cells

Objective: To discuss the pathogenesis of nerve injury by observe the expression of neuronal nitric oxide synthase(nNOS)and nitric oxide(NO)of SH-SY5 Y cells with fluorosis.To disscus the effect of the nNOS inhibitor7-nitroindazole(7-NI)by observe the change of nNOS and NO in SH-SY5 Y cells cultured with fluorine and 7-NI.Then to explore the relationship between nNOS/NO and nerve injury with fluorosis,the protection of 7-NI in nerve injury with fluorosis,and the experimental basis for the research of target treatment of nerve fluorosis was provided.Methods:(1)SH-SY5 Y cells were cultured with high-glucose DMEM containing 10% fetal bovine serum,the condition of culture was37℃,5% CO2.(2)To screen the best appropriate sodium fluoride solution and 7-NI concentration by the cell counting kit 8(CCK 8).(3)The experiment was divided into control groups(0mmol/L sodium fluoride solution,0 μ mol/L 7-NI),fluorine groups(0.2mmol/L and 2.0mmol/L sodium fluoride solution)and fluorine and 7-NI groups(1.0μmol/L 7-NI,0.2mmol/L sodium fluoride solution +1.0μmol/L 7-NI,and2.0mmol/L sodium fluoride solution+1.0μmol/L 7-NI),for 48 h.(4)The morphology of SH-SY5 Y cells were observed by inverted microscope,the mitochondria,endoplasmic reticulum,ribosomes,golgiapparatus,such as ultrastructure were observed by transmission electron microscope(TEM).The content of nitric oxide(NO)was detected by nitrate reductive enzymatic,and the activation of nitric oxide synthase(NOS)was detected by colorimetry.The mRNA and protein expression of nNOS were detected by Real-time PCR and Western Blot.Apoptosis SH-SY5 Y cells was detected by flow cytometry.All index difference wer compared among the control groups,fluorine groups,fluorine and 7-NI groups,and the differences in the contentamong each group were analyzed statistically.Results:(1)The cell proliferation rate of the SH-SY5 Y cells with fluorine-contaminated groups was lower than the control group,higher fluorine groups was lower than the lower groups,the difference was statistically significant(P<0.05).Compared with fluorine-contaminated groups,the cell proliferation rate of the fluorine and 7-NI groups was increased significantly,the difference was statistically significant(P<0.05).Compared with the control groups,the cell proliferation rate of 7-NI groups was no difference(P>0.05).(2)Under the inverted microscope,the cell density of the fluorine-contaminated groups was decreased gradually,some cells in the lower fluorine groups became to shriveled,circular,there has grain appearance changes in the cytoplasm,most cells in the higher fluorine groups became to shriveled,circular,karyopyknosis,cells gap increased,net like arranged,the desquamation of small number of cells.The cell density of fluorine and 7-NI groups was increased gradually,most of the cells was spindle,only a few cells were circular and irregular.(3)Under the transmission electron microscope(TEM),the cells of the fluorine-contaminated groups were observed shrinkage,nuclear chromatin pyknosis,condensation and edge accumulation.The number of organelles in the cytoplasm seriously reduced,disordered arrangement of mitochondria,mitochondrial cristae broken,deletion,and appeared different degrees of vacuolization,endoplasmic reticulum expansion.Compared with the fluorinecontaminated groups,the number of organelles in the cytoplasm seriously increased in the fluorine and 7-NI groups,and the vacuolization reduced significantly.(4)The content of nitric oxide(NO),the activation of nitric oxide synthase(NOS),and the mRNA and protein expression of nNOS of the fluorine-contaminated groups,were higher than the control group(P<0.05),higher fluorine groups was higher than the lower groups(P<0.05).Compared with the fluorine-contaminated groups,the all index above-mentioned were seriously reduced(P<0.05).(5)The cells apoptosis rate of the fluorine-contaminated groups was higher than the control group,and higher fluorine groups was higher than the lower groups,the difference was statistically significant(P<0.05).Compared with the fluorine-contaminated groups,the cellsapoptosis rate of the fluorine and 7-NI groups seriously reduced(P<0.05).Compared with the control groups,the cells apoptosis rate of 7-NI groups was no difference(P>0.05).Conclusion:(1)Excessive fluoride can restrain the cells proliferation and promote apoptosis,and promote the injury of cellular morphology,which may play an important role in the pathogenesis of nerve damaged by fluorosis.The nNOS may play an important role in the mechanism of nerve injury by fluorosis.(2)7-Nitroindazole can against the up-regulation of nNOS that induced by fluorosis.Which may provid a experimental basis for the research of target treatment of nerve injury induced by fluorosis.