7-硝基吲唑对氟致SH-SY5Y细胞凋亡及一氧化氮合成酶含量的影响

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(P0.05);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(P0.05);低剂量7-NI组凋亡指数低于高剂量组,差异有统计学意义(P0.05)。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(P0.05)。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(P0.05)。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(P0.01),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(P0.01)。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。

Protective effect of 7-nitroindazole on fluoride-induced cell growth,apoptosis and NOS content in SH-SY5Y cells

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 (NaF,0,0.02,0.2,2.0,4.0 mmol/L) and 7-nitroindazole (7-NI,10-4、10-3、10-2 mmol/L) at different concentrations for 24 h,48 h and 72 h respectively.After NaF-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 (P＜ 0.01),the survival rate of cells in 10-4 mmol/L 7-NI group (105.96±3.44)％] treated for 24 h increased with statistically significant differences (P＜0.05),in 10-3 mmol/L 7-NI group (100±2.30)％] treated for 24 h showed no statistically significant differences (P＞0.05),in 10-2 mmol/L 7-NI group (88.64±4.75)％] treated for 24 h decreased with statistically significant differences (P＜0.05).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 10-4 mmol/L 7-NI,(0.958±0.074) μmol/L and (0.398±0.010) U/ml for 10-3 mmol/L 7-NI,(0.672±0.188) μmol/L and (0.103±0.076) U/ml for 10-2 mol/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 (10-2 mmol/L) statistically increased (8.7±2.3)％],no statistically significant differences was found in the level of apoptosis among SH-SY5Y cells exposed to 10-4 and 10-3 mmol/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 fluoridegroups 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/rml],with statistically significant differences (P＜0.05).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(P＜0.05).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 NaF 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 NaF 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.