L-亚氨基乙基鸟氨酸与过量氟共培养对人神经母细胞瘤SH-SY5Y细胞一氧化氮／内皮型一氧化氮合酶表达的影响

The effects of co-culture of L-iminoethyl ornithine and excessive fluorine on the expression of nitricoxide/endothelial nitric oxide synthase in human neuroblastoma SH-SY5Y cells

目的 探讨内皮型一氧化氮合酶（endothelial nitric oxide synthase，eNOS）特异性抑制剂L-亚氨基乙基鸟氨酸 （L-iminoethyl ornithine hydrochloride，L-NIO）对氟致体外培养人神经母细胞瘤SH-SY5Y细胞凋亡、eNOS mRNA和蛋白表达以 及一氧化氮（Nitric oxide，NO）含量和一氧化氮合酶（nitric oxide synthase，NOS）活性改变的影响。方法 将体外培养的 SH-SY5Y细胞分为对照组、低氟组、高氟组、L-NIO组、低氟 ＋ L-NIO组、高氟 ＋ L-NIO组，n = 3。对照组加入与实验组等体积的 培养液，低氟组和高氟组加入氟化钠（sodium fluoride，NaF）浓度分别为0.2、2.0 mmol／L，L-NIO组加入3 μmol／L L-NIO， 低氟 ＋ L-NIO组和高氟 ＋ L-NIO组分别加入0.2 mmol／L NaF和3 μmol／L L-NIO、2.0 mmol／L NaF和3 μmol／L L-NIO，培养时 间为48 h。采用蛋白免疫印迹（Western blot）法检测细胞中eNOS蛋白表达水平，实时荧光定量PCR（Real-time fluorescent quantitative PCR）法检测细胞中eNOS mRNA表达水平，流式细胞仪检测细胞的凋亡情况，硝酸还原酶法和比色法检测细胞培养液 中NO含量和NOS活性。结果 与对照组（1.000 ± 0.026）比较， 低、高氟组eNOS蛋白表达量（1.108 ± 0.071、1.349 ± 0.057 ）升高，L-NIO组（0.755 ± 0.148）下降（P均 〈 0.05）；与低氟组比较，高氟组升高，低氟 ＋ L-NIO组（0.802 ± 0.115）下 降（P均 〈 0.05）；与高氟组比较，高氟 ＋ L-NIO组（0.988 ± 0.135）下降（P 〈 0.05）。与对照组（1.000 ± 0.018）比较， 低、高氟组eNOS mRNA表达量（1.809 ± 0.099、2.416 ± 0.295）升高，L-NIO组（0.609 ± 0.077）下降（P均 〈 0.05）；与低 氟组比较，高氟组升高，低氟 ＋ L-NIO组（1.040 ± 0.034）下降（P均 〈 0.05）；与高氟组比较，高氟 ＋ L-NIO组（1.233 ± 0.152）下降（P 〈 0.05）。与对照组［（1.66 ± 0.07）％］比较，低、高氟组细胞凋亡率［（8.81 ± 0.27）%、（17.60 ± 0.20）%］升高，L-NIO组［（1.03 ± 0.04）%］下降（P均 〈 0.05）；与低氟组比较，高氟组升高，低氟 ＋ L-NIO组［（6.03 ± 0.10）%］下降（P均 〈 0.05）；与高氟组比较，高氟 ＋ L-NIO组［（12.12 ± 0.08）％］下降（P 〈 0.05）。与对照组 ［（2.773 ± 0.145）μmol／L］比较，低、高氟组细胞培养液中NO含量［（8.251 ± 1.047）、（14.287 ± 1.062）μmol／L ］升高，L-NIO组［（1.648 ± 0.155）μmol／L］下降（P均 〈 0.05）；与低氟组比较，高氟组升高，低氟 ＋ L-NIO组［（4.622 ± 0.252）μmol／L］下降（P均 〈 0.05）；与高氟组比较，高氟 ＋ L-NIO组［（7.899 ± 0.385）μmol／L］下降（P 〈 0.05） 。与对照组［（0.507 ± 0.041）Ｕ／ｍｌ］比较，低、高氟组细胞培养液中NOS活性［（0.772 ± 0.032）、（2.258 ± 0.062 ）Ｕ／ｍｌ］升高，L-NIO组［（0.346 ± 0.015）Ｕ／ｍｌ］下降（P均 〈 0.05）；与低氟组比较，高氟组升高，低氟 ＋ L-NIO 组［（0.637 ± 0.026）Ｕ／ｍｌ］下降（P均 〈 0.05）；与高氟组比较，高氟 ＋ L-NIO组［（1.161 ± 0.071）Ｕ／ｍｌ］下降 （P 〈 0.05）。结论 过量氟可导致SH-SY5Y细胞eNOS蛋白和mRNA过度表达以及细胞凋亡率上升，细胞培养液中NO含量增多以及NOS 活性增强，加入L-NIO与氟共培养后可拮抗氟对SH-SY5Y细胞的损伤，起到一定的神经保护作用。

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.