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

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

目的探讨细胞内钙离子水平（[Ca^2＋]i）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40mg／LNaF对SH—SY5Y细胞进行染毒，检测在染毒不同时间（0、3、6、12、18、24h）的[Ca^2＋]，和ROS水平的变化，选择最佳染毒时间；观察最佳染毒时间（12h）40mg／LNaF与38．23mg／LBAPTA—AM或380．40mg/L乙二醇双（2-氨基乙醚）四乙酸（EGTA）或16．32mg／LN-乙酰半胱氨酸（NAC）共同和单独作用下，[Ca^2＋]i、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、24h，[Ca^2＋]i水平（5620．0±226．3、4775．5±85．6、3312．3±87．5、3047．0±75．0、2717．0±66．5），与0h（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、20227．33±178．04、8817．56±200．13、7975．61±92．90），与0h（4098．01±21．22）比较，除3h外也明显升高（P〈0．05）；[Ca^2＋]i、ROS达到峰值的时间分别为3、12h。[Ca^2＋]i、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（19003．04±332．34）、NaF＋EGTA组（19170．12±95．46）明显高于对照组（4060．98±145．66，P〈0．05）。在对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[Ca^2＋]i、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[Ca^2＋]i水平，升高的[Ca^2＋]i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。

Objective To explore the relationship between intracellular calcium levels （ [ Ca^2＋ ]i ） and reactive oxygen species （ROS） in sodium fluoride （NaF）-induced injury in human neuroblastoma SH-SY5Y cells. Methods The levels of [ Ca^2＋ ]i 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 [ Ca^2＋ ]i, ROS and LDH levels in 40 mg/L NaF-treated groups ineubated 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- aeetyl-L-cysteine（NAC） also observed at the optimal expose time（12 h）, respectively. Results At 3,6,12,18 and 24 h, [ Ca^2＋ ]i 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 [ Ca^2＋ ]i and ROS reached the peak at 3 h and 12 h, respectively. [ Ca^2＋ ]i 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 [ Ca^2＋ ]i, 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 eytotoxicity and the increase of LDH level in human neuroblastoma SH-SY5Y cells.