细胞内钙离子和活性氧在氟化钠致人神经母细胞瘤SH-SY5Y细胞损伤中的关系

目的 探讨细胞内钙离子水平([Ca2+]1)和活性氧(ROS)在氟化钠(NaF)致人神经母细胞瘤SH-SY5Y细胞损伤中的关系.方法 用40 mg/L NaF对SH-SY5Y细胞进行染毒,检测在染毒不同时间(0、3、6、12、18、24 h)的[Ca2+]1和ROS水平的变化,选择最佳染毒时间;观察最佳染毒时间(12 h)40 mg/L NaF与38.23 mg/L BAPTA-AM或380.40 mg/L乙二醇双(2-氨基乙醚)四乙酸(EGTA)或16.32 mg/L N-乙酰半胱氨酸(NAC)共同和单独作用下,[Ca2+]1、细胞内ROS和培养液乳酸脱氢酶(LDH)水平变化.结果 染毒3、6、12、18、24 h,[Ca2+]1水平(5620.0±226.3、4775.5±85.6、3312.3±87.5、3047.0±75.0、2717.0±66.5),与0 h(2115.0±24.0)比较,均明显升高(P<0.05);ROS水平(4449.53±324.61、7463.07±117.43、20 227.33±178.04、8817.56±200.13、7975.61±92.90),与0 h(4098.01±21.22)比较,除3 h外也明显升高(P<0.05);[Ca2+]1、ROS达到峰值的时间分别为3、12 h.[Ca2+]1、LDH水平,NaF组[3279.5±94.0,(1057.50±64.35)U/L]、NaF+NAC组[3583.0±350.7,(561.02±85.50)U/L]、NaF+EGTA组[3701.5±157.7,(1074.50±86.97)U/L]、NaF+BAPTA-AM组[2766.5±38.9,(521.43±40.80)U/L],与对照组[2022.5±118.1,(186.97±8.73)U/L]比较,均有明显升高(P<0.05);ROS水平,NaF组(19 003.04±332.34)、NaF+EGTA组(19 170.12±95.46)明显高于对照组(4060.98±145.66,P<0.05).在财ROS和LDH的影响中,NaF与NAC之间存在拮抗作用(F值分别为976.11、43.54,P<0.05).在对[Ca2+]1、ROS和LDH水平的影响中,NaF与BAPTA-AM之间存在拮抗作用(F值分别为15.65、1515.53、115.00.P<0.05).结论 NaF诱导SH-SY5Y细胞损伤的机制可能是通过胞内钙库的释放而提高[Ca2+]1水平,升高的[Ca2+]1促使ROS的产生,二者共同对细胞产生毒性,导致细胞培养液中LDH水平升高.

Relationship between intracellular calcium and reactive oxygen species in sodium fluoride-induced injury in human neuroblastoma SH-SY5Y cells

Objective To explore the relationship between intracellular calcium levels ([Ca2+]1) and reactive oxygen species (ROS) in sodium fluoride (NaF)-induced injury in human neuroblastoma SH-SY5Y cells. Methods The levels of [Ca2+]1 and ROS were measured in different exposed times(0,3,6,12,18,24 h) respectively after SH-SY5Y cells were exposed to 40 mg/L NaF in vitro, and the optimal expose time was selected. Furthermore, the changes of [Ca2+]1, ROS and LDH levels in 40 mg/L NaF-treated groups incubated with 38.23 mg/L BAPTA-AM or 380.40 mg/L ethylene glycol-bis-(beta-aminoethyl ether)-N, N, N', N'-tetraacetic acid (EGTA) or 16.32 mg/L N-acetyl-L-cysteine(NAC) also observed at the optimal expose time(12 h), respectively. Results At 3,6,12,18 and 24 h, [Ca2+]1 level(5620.0±226.3,4775.5±85.6,3312.3±87.5, 3047.0±75.0,2717.0±66.5) was significantly increased, and so was the ROS level(4449.53±324.61,7463.07±117.43,20 227.33±178.04,8817.56±200.13, 7975.61±92.90) except at 3 h, compared with 0 h(2115.0±24.0,4098.01±21.22, all P<0.05). The levels of [Ca2+]1 and ROS reached the peak at 3 h and 12 h, respectively. [Ca2+]1 and LDH levels in NaF-treated group [3279.5±94.0, (1057.50±64.35)U/L], NaF+NAC treated group[ 3583.0±350.7, (561.02±85.50)U/L], NaF+EGTA treated groups[3701.5±157.7, (1074.50±86.97)U/L], and BAPTA-AM treated group[2766.5±38.9, (521.43±40.80)U/L] had increased, compared with the control[2022.5±118.1, (186.97±8.73)U/L], the difference being statistically significant (P<0.05). ROS levels in NaF-treated group (19 003.04±332.34), and NaF+EGTA treated group(19 170.12±95.46) was higher than that in the controls(4060.98±145.66), the difference being statistically significant (P<0.05). NaF and NAC had antagonistic effect on ROS and LDH levels (F=976.11,43.54,P<0.05). And NaF and BAPTA-AM had antagonistic effect on [Ca2+]1, ROS and LDH levels (F=15.65,1515.53,115.00, P<0.05). Conclusions NaF-related calcium is released from the site of intracellular calcium storage, which induces ROS production, both of them caused cytotoxicity and the increase of LDH level in human neuroblastoma SH-SY5Y cells.