The effects of co-culture of L-iminoethyl ornithine and excessive fluorine on the expression of nitric oxide/endothelial nitric oxide synthase in human neuroblastoma SH-SYSY cells / 中华地方病学杂志

ABSTRACT

Objective To investigate the effects of endothelial nitric oxide synthase (eNOS) specific inhibitor L-iminoethyl ornithine hydrochloride (L-NIO) for all the fluorine in vitro cultivation of SH-SY5Y cells apoptosis,eNOS mRNA and protein expression,and effect of nitric oxide (NO) content and nitric oxide synthase (NOS) activity change.Methods The SH-SY5Y cells cultured in vitro were divided into control group,low fluoride group,high fluoride group,L-NIO group,low fluoride with L-NIO group,high fluoride with L-NIO group,n =3.The control group added equal volume culture liquid with the experimental group,the concentration of sodium fluoride (NaF) in the low fluoride group and the high fluoride group were 0.2 and 2.0 mmol/L,respectively.The L-NIO group added 3 μmol/L L-NIO,the low fluoride with L-NIO group and the high fluoride with L-NIO group were added to 0.2 mmol/L NaF and 3 μmol/L L-NIO,2.0 mmol/L NaF and 3 μmol/L L-NIO,respectively.The incubation time was 48 h.The expression level of eNOS protein in cells was detected by Western blotting.The expression level of eNOS mRNA in cells was detected by Real-time fluorescence quantitative PCR method.The apoptosis of cells was detected by flow cytometry,NO content and NOS activity in cell culture liquid were detected by nitrate reductase and colorimetric assay.Results Compared with the control group (1.000 ± 0.026),the expression of eNOS protein in the low and high fluoride groups (1.108 ± 0.071,1.349 ± 0.057) increased (P ＜ 0.05),and the L-NIO group (0.755 ± 0.148) decreased (P ＜ 0.05);compared with the low fluoride group,the high fluoride group increased and the low fluoride with L-NIO group (0.802 ± 0.115) decreased (P ＜ 0.05);compared with the high fluoride group,high fluoride with L-NIO group (0.988 ± 0.135) decreased (P ＜ 0.05).Compared with the control group (1.000 ±0.018),the expression of eNOS mRNA in the low and high fluoride groups (1.809 ± 0.099,2.416 ± 0.295) increased (P ＜ 0.05),the L-NIO group (0.609-± 0.077) decreased (P ＜ 0.05);compared with the low fluoride group,the high fluoride group was elevated(P ＜ 0.05),the low fluoride with L-NIO group (1.040 ± 0.034) decreased (P ＜ 0.05);compared with the high fluoride group,high fluoride with L-NIO group (1.233 ± 0.152) decreased (P ＜ 0.05).Compared with the control group [(1.66 ± 0.07)％],the cell apoptosis rate in the low and high fluoride groups [(8.81 ± 0.27)％,(17.60 ± 0.20)％] increased,L-NIO group [(1.03 ± 0.04)％] decreased (P ＜ 0.05);compared with the low fluoride group,the high fluoride group increased and the low fluoride with L-NIO group [(6.03 ± 0.10)％] decreased (P ＜ 0.05);compared with the high fluoride group,the high fluoride with L-NIO [(12.12 ± 0.08)％] decreased (P ＜ 0.05).Compared with the control group [(2.773 ± 0.145)μmol/L],the content of NO in the cell culture medium in the low and high fluoride groups [(8.251 ± 1.047),(14.287 ± 1.062) μmol/L] increased (P＜ 0.05),and the L-NIO group [(1.648 ± 0.155) μmol/L] decreased (P ＜ 0.05);compared with the low fluoride group,the high fluoride group was elevated (P ＜ 0.05),the low fluoride with L-NIO group [(4.622 ± 0.252) μmol/L] decreased (P ＜ 0.05);compared with the the high fluoride group,high fluoride with L-NIO group [(7.899 ± 0.385) μmol/L] decreased (P ＜ 0.05).Compared with the control group [(0.507 ± 0.041) U/ml],the activity of NOS in the cell culture medium in the low and high fluoride groups [(0.772 ± 0.032),(2.258 ± 0.062) U/ml] increased (P ＜ 0.05),and the L-NIO group [(0.346 ±0.015) U/ml] decreased (P ＜ 0.05);compared with the low fluoride group,the high fluoride group was elevated (P ＜0.05),the low fluoride with L-NIO group [(0.637 ± 0.026) U/ml] decreased (P ＜ 0.05);compared with the the high fluoride group,high fluoride with L-NIO group [(1.161 ± 0.071) U/ml] decreased (P ＜ 0.05).Conclusions Excessive fluoride can lead to overexpression of eNOS protein and mRNA in SH-SY5Y cells,increase of apoptosis rate,increase the content of NO in cell culture and enhance the activity of NOS.After co-culture of L-NIO and fluorine,it can antagonize the damage of fluorine to SH-SY5Y cells and play a certain neuroprotective effect.