摘要
目的探讨神经元型一氧化氮合成酶(nNOS)抑制剂7-硝基吲唑(7-nitroindazole,7-NI)对氟致体外培养细胞生长、凋亡保护作用的机制。方法体外培养SH-SY5Y细胞,在培养液中加入不同浓度Na F(0、0.02、0.2、2.0、4.0 mmol/L),分别培养24、48、72 h,同时在各组细胞培养液中联合应用7-NI(10^(-4)、10^(-3)、10^(-2) mmol/L);于倒置显微镜下观察细胞生长,以CCK-8试剂盒方法检测细胞存活率,以比色法和硝酸还原酶法测定细胞培养液中NO含量及NOS活力,以流式细胞仪检测细胞凋亡情况。结果与对照组比较,氟浓度为0.2 mmol/L(低氟组)和2.0 mmol/L(高氟组)时,24、48、72 h后细胞存活率分别下降至(83.76±1.04)%,(70.27±1.20)%,(54.40±0.98)%和(52.17±1.07)%,(40.60±1.11)%,(29.50±1.40)%。10^(-4)mmol/L 7-NI可刺激细胞增殖,24 h细胞存活率为(105.96±3.44)%;10^(-3) mmol/L 7-NI处理细胞24 h后的细胞存活率为(100±2.30)%;10^(-2) mmol/L 7-NI可使细胞存活率下降,24 h细胞存活率为(88.64±4.75)%。10^(-4)、10^(-3)、10^(-2) mmol/L 7-NI处理细胞24 h后细胞培养液中NO含量、NOS活力均降低,分别为(1.156±0.356)、(0.958±0.074)、(0.672±0.188)μmol/L和(0.403±0.089)、(0.398±0.010)、(0.103±0.076)U/ml。高剂量(10^(-2) mmol/L)7-NI组的SH-SY5Y细胞凋亡指数升高至(8.7±2.3)%,高于对照组(P<0.05);低剂量(10^(-4)、10^(-3) mmol/L)7-NI组的凋亡指数分别为(0.6±0.4)%和(1.0±0.2)%,与对照组差异无统计学意义(P>0.05);低剂量7-NI组凋亡指数低于高剂量组,差异有统计学意义(P<0.05)。染氟24、48、72 h后细胞培养液中NO含量及NOS活力升高,低氟组的NO含量、NOS活力分别为(3.074±0.160)、(5.604±1.374)、(10.173±1.307)μmol/L和(0.743±0.122)、(0.959±0.054)、(1.446±0.12)U/ml,高氟组分别为(8.974±0.919)、(14.729±1.241)、(20.976±0.712)μmol/L和(1.086±0.024)、(1.728±0.130)、(2.583±0.192)U/ml,高于对照组[(1.320±0.280)、(2.420±0.658)、(2.867±0.662)μmol/L,(0.452±0.012)、(0.517±0.072)、(0.607±0.013)U/ml],差异有统计学意义(P<0.05)。与氟单独处理组相比,NaF联合应用7-NI后,各组NO含量及NOS活力均下降,差异有统计学意义(P<0.05)。与对照组相比,氟可使SH-SY5Y细胞凋亡指数升高,差异有统计学意义(P<0.01),并随着氟染毒剂量及时间的增加,细胞凋亡指数随之增加;联合应用7-NI后,各组细胞凋亡指数下降,差异有统计学意义(P<0.01)。氟可使SH-SY5Y细胞数量减少,形态变为圆形、不规则形,细胞存活率降低;低剂量(10^(-4)、10^(-3) mmol/L)7-NI组在相同视野下细胞数增加,高剂量(10^(-2) mmol/L)7-NI组细胞数明显减少,大部分细胞呈圆形或不规则形。结论氟中毒所致细胞凋亡可能与NOS活力和NO含量升高有关,7-NI可降低细胞凋亡指数。
Objective To explore the mechanism of action of 7-nitroindazole（7-NI）,a specific nNOS-inhibitor, on fluorideinduced cell growth, apoptosis and NOS content in SH-SY5Y cells. Methods The SH-SY5Y cells were treated with sodium fluoride（Na F, 0, 0.02, 0.2, 2.0, 4.0 mmol/L） and 7-nitroindazole（7-NI, 10^-4、10^-3、10^-2mmol/L） at different concentrations for24 h, 48 h and 72 h respectively. After Na F-treatment for 24 h,48 h and 72 h or 7-NI treatment for 24 h, the cell survival rate was measured by the method of CCK-8. Nitric oxide（NO）in the culture media were determined with nitrate reduction method. The nitric oxide synthase（NOS） was determined with colorimetric analysis.The levels of the apoptosis among SH-SY5Y cells treated with NaF（0.2, 2.0 mmol/L） combined with 10^-3 mmol/L 7-NI for 24 h, 48 h and 72 h were determined with flow cytometer respectively. Results Compared with the control group, the cell survival rate of cells in 0.2、2.0 mmol/L NaF groups treated for 24 h, 48 h, and 72 h [（83.76±1.04）%,（70.27±1.20）% and（54.40±0.98）% for 0.2 mmol/L NaF,（52.17±1.07）%,（40.60±1.11）% and（29.50±1.40）% for 2.0 mmol/L NaF）] significantly decreased with statistically significant differences（P0.01）, the survival rate of cells in 10^-4mmol/L 7-NI group [（105.96 ±3.44）% ] treated for 24 h increased with statistically significant differences（P0.05）, in 10^-3mmol/L 7-NI group [（100±2.30）%] treated for 24 h showed no statistically significant differences（P 0.05）, in 10-2mmol/L 7-NI group [（88.64 ±4.75）% ] treated for 24 h decreased with statistically significant differences（P0.05）. The content of NO and the activity of NOS in the 7-NI groups [（1.156±0.356） μmol/L and（0.403±0.089）U/ml for 10-4mmol/L 7-NI,（0.958±0.074） μmol/L and（0.398±0.010） U/ml for 10^-3mmol/L 7-NI,（0.672 ±0.188） μmol/L and（0.103±0.076） U/ml for 10^-2mol/L 7-NI] treated for 24 h decreased, compared with the control group [（1.320 ±0.280） μmol/L and（0.452 ±0.012） U/ml] with statistically significant differences（P 0.05）. Compared with the control group, the level of apoptosis in SH-SY5Y cells exposed to high-dose 7-NI（10-2mmol/L） statistically increased [（8.7 ±2.3）% ],no statistically significant differences was found in the level of apoptosis among SH-SY5Y cells exposed to 10^-4and 10^-3mmol/L 7-NI[（0.6±0.4）% and（1.0 ±0.2）% ]（P 〈0.05）. Compared with the control group,the content of NO and the activity of NOS in fluoride groups increased [（3.074±0.160）,（5.604±1.374）,（10.173±1.307） μmol/L and（0.743±0.122）,（0.959±0.054）,（1.446±0.12）U/ml for 0.2 mmol/L NaF,（8.974±0.919）,（14.729±1.241）,（20.976±0.712）μmol/L and（1.086±0.024）,（1.728±0.130）,（2.583±0.192）U/ml for 2 mmol/L NaF for 24 h, 48 h, 72 h], compared with the control group [（1.320 ±0.280）,（2.420±0.658）,（2.867±0.662）] μmol/L and（0.452±0.012）,（0.517±0.072）,（0.607±0.013） U/ml],with statistically significant differences（P0.05）. After NaF treatment combined with 7-NI, the content of NO and the activity of NOS in SH-SY5Y cells decreased compared with the NaF treatment group（P0.05）. Compared with the control group[（0.5±0.2）%,（1.8±1.0）%,（3.7±1.3）% for 24 h, 48 h and 72 h],the level of apoptosis in SH-SY5Y cells exposed to fluoride increased [（8.5 ±0.4）%,（16.3±1.5）%,（24.2±2.8）% for 0.2 mmol/L Na F group treated for 24 h,48 h and 72 h,（25.6±2.0）%,（41.2±1.5）%,（58.9±2.8）% for 2 mmol/L NaF group treated for 24 h,48 h and 72 h], with statistically significant differences（P 〈0.01）. After Na F treatment combined with 7-NI, the level of apoptosis in SH-SY5Y cells decreased（ P〈 0. 01）. Under the inverted microscope it was found that the morphologic of the SH-SY5Ycells becoming circular, irregular shape etc, cell density decreased, the cell survival rate decreased. Conclusion The apoptosis of SH-SY5Y cells induced by excessive fluoride might be involved in the increases of the activity of NOS and the content of NO. 7-NI might decrease the level of apoptosis, to protect the SH-SY5Y cells.
出处
《环境与健康杂志》
CAS
北大核心
2017年第6期471-476,F0003,共7页
Journal of Environment and Health
基金
国家自然科学基金(81460482)
教育部博士学科点专项科研基金(20115215120003)
