氟、砷对原代培养大鼠海马神经细胞氧化应激及凋亡影响的实验观察

Effects of fluoride and arsenic on oxidative stress and apoptosis in primary hippocampal neurons of rats in culture

目的本研究通过体外细胞实验,观察氟、砷单独及联合染毒对大鼠海马神经细胞氧化应激及细胞凋亡的影响。方法采用2因素3水平的析因设计,将体外培养的海马神经细胞分为9组,分别为对照(细胞培养液)组、低剂量砷(2μmol/L亚砷酸钠)染毒组、高剂量砷(10μmol/L亚砷酸钠)染毒组、低剂量氟(10μmol/L氟化钠)染毒组、高剂量氟(50μmol/L氟化钠)染毒组、低剂量砷(2μmol/L亚砷酸钠)+低剂量氟(10μmol/L氟化钠)联合染毒组、低剂量砷(2μmol/L亚砷酸钠)+高剂量氟(50μmol/L氟化钠)联合染毒组、高剂量砷(10μmol/L亚砷酸钠)+低剂量氟(10μmol/L氟化钠)联合染毒组及高剂量砷(10μmol/L亚砷酸钠)+高剂量氟(50μmol/L氟化钠)联合染毒组。采用MTT还原法检测细胞存活情况;采用微量酶标法测定细胞中谷胱苷肽(GSH)含量;采用硫代巴比妥法(TBA)测定谷胱苷肽过氧化物酶(GSH-Px)含量;采用WST-I法测定超氧化物歧化酶(SOD)活力;采用化学比色法测定丙二醛(MDA)含量及过氧化氢酶(CAT)活力;采用流式细胞仪检测细胞早期凋亡率。结果与对照组比较,高剂量砷、高剂量氟、低剂量砷+高剂量氟、高剂量砷+低剂量氟以及高剂量砷+高剂量氟联合染毒组海马神经细胞MTT吸光度值明显降低(P<0.05);高剂量砷+低剂量氟联合组及高剂量砷+高剂量氟联合组细胞CAT活力明显降低(P<0.05);高剂量砷染毒组、高剂量氟染毒组、低剂量砷+高剂量氟联合组、高剂量砷+低剂量氟联合组及高剂量砷+高剂量氟联合组细胞GSH含量明显下降(P<0.05);高剂量氟染毒组、高剂量砷+低剂量氟联合组及高剂量砷+高剂量氟联合组细胞GSH-Px活力明显下降(P<0.05);高剂量砷染毒组、高剂量氟染毒组、低剂量砷+高剂量氟联合组、高剂量砷+低剂量氟联合组及高剂量砷+高剂量氟联合组细胞MDA含量明显升高(P<0.05);高剂量砷染毒组、高剂量氟染毒组、低剂量砷+高剂量氟联合组、高剂量砷+低剂量氟联合组及高剂量砷+高剂量氟联合组细胞凋亡率明显升高(P<0.05)。结论氟、砷可导致大鼠海马神经细胞氧化应激并引起细胞凋亡率升高,这可能是氟、砷对动物学习记忆能力产生影响的重要原因。

Objective To observe the effects of fluoride and arsenic on oxidative stress and apoptosis in primary hippocampal neurons of rats.Methods Primary hippocampal neurons of rats in culture were randomly divided into 9groups,control group（cell culture）,low dose NaAsO_2（2μmol/L）group,high dose NaAsO_2（10μmol/L）group,low dose NaF（10μmol/L）group,high dose NaF（50μmol/L）group,low dose NaAsO_2（2μmol/L）and low dose NaF（10μmol/L）combined group,low dose NaAsO_2（2μmol/L）and high dose NaF（50μmol/L）combined group,high dose NaAsO_2（10μmol/L）and low dose NaF（10μmol/L）combined group,high dose NaAsO_2（10μmol/L）and high dose NaF（50μmol/L）combined group.MTT test was used to detect survival rates of primary hippocampal neuron cells;contents of amounts of reduced glutathione（GSH）were detected with micro-ELISA;activity of catalase（CAT）was detected with visible light;activity of superoxide dismutase（SOD）was detected with WST-1;contents of malondialdehyde（MDA）,activity of glutathione peroxidase（GSH-Px）were detected with colorimetry;the cell apoptosis rate was determined by flow cytometer.Results Compared with the control group,MTT absorbance value of primary hippocampal neuron cells treated with high-dose NaAsO_2 group,highdose NaF group,low-dose NaAsO_2 and high-dose NaF combined group,high-dose NaAsO_2 and low-dose NaF combined group,high-dose NaAsO_2 and high-dose NaF combined group were significantly decreased（P〈0.05）;CAT contents of hippocampal neuron cells treated with high-dose NaAsO_2 and low-dose NaF combined group,high-dose NaAsO_2 and high-dose NaF combined group were significantly decreased（P〈0.05）;GSH contents of hippocampal neuron cells treated with high-dose NaAsO_2 group,high-dose NaF group,low-dose NaAsO_2 and high-dose NaF combined group,high-dose NaAsO_2 and low-dose NaF combined group,high-dose NaAsO_2 and high-dose NaF combined group were significantly decreased（P〈0.05）;GSH-Px contents of hippocampal neuron cells treated with high-dose NaF group,high-dose NaAsO_2 and low-dose NaF combined group,high-dose NaAsO_2 and high-dose NaF combined group were significantly decreased（P〈0.05）;MDA contents of hippocampal neuron cells treated with high-dose NaAsO_2 group,high-dose NaF group,low-dose NaAsO_2 and high-dose NaF combined group,high-dose NaAsO_2 and low-dose NaF combined group,high-dose NaAsO_2 and high-dose NaF combined group were significantly increased（P〈0.05）;the cell apoptosis rate in high-dose NaAsO_2 group,high-dose NaF group,low-dose NaAsO_2 and high-dose NaF combined group,high-dose NaAsO_2 and low-dose NaF combined group,high-dose NaAsO_2 and high-dose NaF combined group were significantly increased（P〈0.05）.Conclusions Fluoride and arsenic exposure might cause oxidative stress and cell apoptosis rate increase in hippocampal neuron cells.These effcts might be related to damaged learning and memory of rats.