摘要
目的 观察神经型尼古丁受体α3亚单位（α3nAChR）及丝裂原活化蛋白激酶（MAPK）信号通路中细胞外调节蛋白激酶（ERK1/2）、c-Jun氨基末端激酶（JNK）、p38激酶在氟暴露神经母细胞瘤细胞系SH-SY5Y中的表达,探讨过量氟暴露对细胞形成损害的信号传递机制.方法 以α3nAChR基因沉默的SH-SY5Y细胞作为α3nAChR沉默组,以正常SH-SY5Y细胞作为对照组,采用蛋白印迹法和实时荧光定量PCR法检测α3nAChR基因沉默效果.分别用0.000、0.005、0.050、0.500、1.000、2.500、5.000 mmol/L氟化钠（NaF）处理正常SH-SY5Y细胞,用四甲基偶氮唑盐（MTT）法检测细胞存活率,筛选染氟安全浓度.根据染氟安全浓度筛选结果,选择4.000 mmol/L NaF处理α3nAChR沉默组和对照组SH-SY5Y细胞0、4、8、12、24、36、48 h,用蛋白印迹法检测细胞中ERK1/2、JNK、p38蛋白表达.结果 基因沉默效果检测结果显示,在α3nAChR沉默组,α3nAChRmRNA（0.04±0.03）和蛋白（12.0±2.5）表达明显低于对照组（1.00±.11、100.0±11.3,t=24.58、28.80,P均＜ 0.05）.筛选染氟安全浓度结果显示,在染氟5.000 mmol/L组,SH-SY5Y细胞存活率（0.53±0.15）明显低于0.000 mmol/L组（1.05±0.05,P＜0.05）,而其他染氟剂量组未见明显改变（P均＞0.05）.随着染氟时间延长,α3nAChR沉默组和对照组磷酸化ERK1/2表达逐渐升高,其中24、36、48 h（188.33±7.33、200.00±10.01、213.33±11.55,125.33±5.69、136.00±4.52、155.33±6.51）与同组0 h（100.00±0.00、100.00±0.00）比较,差异有统计学意义（P均＜ 0.05）,且24、36、48 h α3nAChR沉默组明显高于对照组（t=9.26、7.63、5.72,P均＜0.05）.随着染氟时间延长,α3nAChR沉默组和对照组磷酸化JNK表达逐渐升高,其中12、24、36、48 h α3nAChR沉默组（154.00±6.25、149.00±5.57、156.00±6.08、141.67±2.52）和8、12、24、36、48 h对照组（133.33±10.69、173.00±4.00、175.00±11.79、200.67±11.93、200.33±18.58）与同组0 h（100.00±0.00、100.00±0.00）比较,差异有统计学意义（P均＜ 0.05）,且8、12、24、36、48 h α3nAChR沉默组明显低于对照组（t=-428、-5.02、-2.89、-8.33、-6.18,P均＜0.05）.24、36、48 h对照组磷酸化p38激酶表达（120.33±4.51、122.00±7.55、119.67±7.57）明显高于0h（100.00±0.00,P均＜0.05）,且高于同时间点α3nAChR沉默组（93.33±9.61、94.00±5.01、98.33±5.69,t=-4.01、-6.73、-5.59,P均＜0.05）.两组细胞中总ERK1/2、总JNK、总p38激酶表达随染氟时间延长未见明显改变（P均＞ 0.05）.结论 在过量氟暴露条件下,ERK1/2信号通路的激活不完全依赖于α3nAChR的表达;而JNK和p38两条通路的激活在一定程度上依赖于α3nAChR.
Objective To observe the expression of neural nicotinic acetylcholine receptor subunit α3 （α3nAChR） and extracellular regulated protein kinases （ERK1/2）,c-Jun N-terminal kinase （JNK）,p38 kinases of mitogen-activated protein kinase （MAPK） pathway in human neuroblastoma cell line SH-SY5Y overexposed to fluoride,and try to investigate the molecular mechanism of cell damage caused by overexposure of fluoride.Methods The SH-SY5Y cell with low expression of α3nAChR suppressed by silence interference RNA served as α3nAChR silence group;the normal SH-SY5Y cell served as control group,and the effect of silencing of αt3nAChR gene in SHSY5Y was detected by Western blotting and real-time PCR;SH-SY5Y cell was treated with different concentrations of fluoride （0.000,0.005,0.050,0.500,1.000,2.500,5.000 mmol/L）,the safe concentration of fluoride in SHSY5Y cell was detected by 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide （MTT） assay;the SH-SY5Y cell of control group and α3nAChR silence group were treated with 4.000 mmol/L fluoride for 0,4,8,12,24,36,48 h according to the results of MTT assay;the expression of ERK1/2,JNK,p38 kinases of MAPK pathway in SH-SY5Y at protein levels was measured by Western blotting.Results The expression of α3nAChR mRNA （0.04 ＆#177; 0.03） and protein （12.0 ＆#177; 2.5） in α3nAChR silence group was decreased significantly compared with those of control group （1.00 ＆#177; 0.11,100.0 ＆#177; 11.3,t =24.58,28.80,all P 〈 0.05）.The viability of SH-SY5Y cell treated with 5.000 mmol/L fluoride （0.53 ＆#177; 0.15） was decreased significantly compared with that of SH-SY5Y cell treated with 0.000 mmol/L fluoride （1.05 ＆#177; 0.05,P 〈 0.05）.The increased expression of phospho-ERK1/2 was found in α3nAChR silence group and control group incubated with fluoride with time prolonged,and the expression of phospho-ERK1/2 increased significantly at time points 24,36 and 48 h （188.33 ＆#177; 7.33,200.00 ＆#177; 10.01,213.33 ＆#177; 11.55;125.33 ＆#177; 5.69,136.00 ＆#177; 4.52,155.33 ＆#177; 6.51） compared to 0 h in the same groups （100.00 ＆#177; 0.00,100.00 ＆#177; 0.00,all P 〈 0.05）,and the expression of phospho-ERK1/2 was higher significantly in α3nAChR silence group than those of control group （t =9.26,7.63,5.72,all P 〈 0.05）;no change of expression of total-ERK1/2 in the two groups was found with the passage of time.The gradually increased expression of phospho-JNK was found in α3nAChR silence group and control group,among which,the expression of phospho-JNK in o3nAChR silence group at time points 12,24,36 and 48 h （154.00 ＆#177; 6.25,149.00 ＆#177; 5.57,156.00 ＆#177; 6.08,141.67 ＆#177; 2.52） and in control group at 8,12,24,36,48 h （133.33 ＆#177; 10.69,173.00 ＆#177; 4.00,175.00 ＆#177; 11.79,200.67 ＆#177; 11.93,200.33 ＆#177; 18.58） was compared to those at 0 h in the same groups （100.00 ＆#177; 0.00,100.00 ＆#177; 0.00）,and the difference was significant （all P 〈 0.05）;the higher expression of phospho-JNK was found in α3nAChR silence group other than control group at 8,12,24,36,48 h （t =-4.28,-5.02,-2.89,-8.33,-6.18,all P 〈 0.05）;no change of expression of total-JNK was found in the two groups （P 〉 0.05）.The increased expression of phospho-p38 was detected in control group at time points 24,36 and 48 h （120.33 ＆#177; 4.51,122.00 ＆#177; 7.55,119.67 ＆#177; 7.57） compared to 0 h in the same groups （100.00 ＆#177; 0.00,all P 〈 0.05）,and the expression of phospho-p38 was significantly higher than that in α3nAChR silence group at the same time points （93.33 ＆#177; 9.61,94.00 ＆#177; 5.01,98.33 ＆#177; 5.69,t =-4.01,-6.73,-5.59,all P 〈 0.05）;no change of expression of total-p38 was found in the two types of SH-SY5Y cells treated with fluoride （P 〉 0.05）.Conclusion When SH-SY5Y cells are exposed to fluoride;activation of ERK1/2 may be not depend on α3nAChR;α3nAChR may have protected the cell from apoptotic injury caused by activation of JNK pathway,and the activation of p38 may be depend on nAChRα3.
出处
《中华地方病学杂志》
CAS
CSCD
北大核心
2015年第8期553-558,共6页
Chinese Journal of Endemiology
基金
国家自然科学基金（81260417）
国家科技部科技支撑计划（2013BAl05803）
贵州省科技厅贵阳医学院联合基金（黔科合LG[2012]006号）
贵州省2011协同创新中心（[2014]06）
关键词
神经型尼古丁受体
丝裂原活化蛋白激酶
人神经母细胞瘤细胞系
Neural nicotinic acetylcholine receptor
Mitogen-activated protein kinase
Human neuroblastoma cell line
