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±0.11、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.
目的 觀察神經型尼古丁受體α3亞單位(α3nAChR)及絲裂原活化蛋白激酶(MAPK)信號通路中細胞外調節蛋白激酶(ERK1/2)、c-Jun氨基末耑激酶(JNK)、p38激酶在氟暴露神經母細胞瘤細胞繫SH-SY5Y中的錶達,探討過量氟暴露對細胞形成損害的信號傳遞機製.方法 以α3nAChR基因沉默的SH-SY5Y細胞作為α3nAChR沉默組,以正常SH-SY5Y細胞作為對照組,採用蛋白印跡法和實時熒光定量PCR法檢測α3nAChR基因沉默效果.分彆用0.000、0.005、0.050、0.500、1.000、2.500、5.000 mmol/L氟化鈉(NaF)處理正常SH-SY5Y細胞,用四甲基偶氮唑鹽(MTT)法檢測細胞存活率,篩選染氟安全濃度.根據染氟安全濃度篩選結果,選擇4.000 mmol/L NaF處理α3nAChR沉默組和對照組SH-SY5Y細胞0、4、8、12、24、36、48 h,用蛋白印跡法檢測細胞中ERK1/2、JNK、p38蛋白錶達.結果 基因沉默效果檢測結果顯示,在α3nAChR沉默組,α3nAChRmRNA(0.04±0.03)和蛋白(12.0±2.5)錶達明顯低于對照組(1.00±0.11、100.0±11.3,t=24.58、28.80,P均＜ 0.05).篩選染氟安全濃度結果顯示,在染氟5.000 mmol/L組,SH-SY5Y細胞存活率(0.53±0.15)明顯低于0.000 mmol/L組(1.05±0.05,P＜0.05),而其他染氟劑量組未見明顯改變(P均＞0.05).隨著染氟時間延長,α3nAChR沉默組和對照組燐痠化ERK1/2錶達逐漸升高,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)與同組0 h(100.00±0.00、100.00±0.00)比較,差異有統計學意義(P均＜ 0.05),且24、36、48 h α3nAChR沉默組明顯高于對照組(t=9.26、7.63、5.72,P均＜0.05).隨著染氟時間延長,α3nAChR沉默組和對照組燐痠化JNK錶達逐漸升高,其中12、24、36、48 h α3nAChR沉默組(154.00±6.25、149.00±5.57、156.00±6.08、141.67±2.52)和8、12、24、36、48 h對照組(133.33±10.69、173.00±4.00、175.00±11.79、200.67±11.93、200.33±18.58)與同組0 h(100.00±0.00、100.00±0.00)比較,差異有統計學意義(P均＜ 0.05),且8、12、24、36、48 h α3nAChR沉默組明顯低于對照組(t=-428、-5.02、-2.89、-8.33、-6.18,P均＜0.05).24、36、48 h對照組燐痠化p38激酶錶達(120.33±4.51、122.00±7.55、119.67±7.57)明顯高于0h(100.00±0.00,P均＜0.05),且高于同時間點α3nAChR沉默組(93.33±9.61、94.00±5.01、98.33±5.69,t=-4.01、-6.73、-5.59,P均＜0.05).兩組細胞中總ERK1/2、總JNK、總p38激酶錶達隨染氟時間延長未見明顯改變(P均＞ 0.05).結論 在過量氟暴露條件下,ERK1/2信號通路的激活不完全依賴于α3nAChR的錶達;而JNK和p38兩條通路的激活在一定程度上依賴于α3nAChR.
목적 관찰신경형니고정수체α3아단위(α3nAChR)급사렬원활화단백격매(MAPK)신호통로중세포외조절단백격매(ERK1/2)、c-Jun안기말단격매(JNK)、p38격매재불폭로신경모세포류세포계SH-SY5Y중적표체,탐토과량불폭로대세포형성손해적신호전체궤제.방법 이α3nAChR기인침묵적SH-SY5Y세포작위α3nAChR침묵조,이정상SH-SY5Y세포작위대조조,채용단백인적법화실시형광정량PCR법검측α3nAChR기인침묵효과.분별용0.000、0.005、0.050、0.500、1.000、2.500、5.000 mmol/L불화납(NaF)처리정상SH-SY5Y세포,용사갑기우담서염(MTT)법검측세포존활솔,사선염불안전농도.근거염불안전농도사선결과,선택4.000 mmol/L NaF처리α3nAChR침묵조화대조조SH-SY5Y세포0、4、8、12、24、36、48 h,용단백인적법검측세포중ERK1/2、JNK、p38단백표체.결과 기인침묵효과검측결과현시,재α3nAChR침묵조,α3nAChRmRNA(0.04±0.03)화단백(12.0±2.5)표체명현저우대조조(1.00±0.11、100.0±11.3,t=24.58、28.80,P균＜ 0.05).사선염불안전농도결과현시,재염불5.000 mmol/L조,SH-SY5Y세포존활솔(0.53±0.15)명현저우0.000 mmol/L조(1.05±0.05,P＜0.05),이기타염불제량조미견명현개변(P균＞0.05).수착염불시간연장,α3nAChR침묵조화대조조린산화ERK1/2표체축점승고,기중24、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)여동조0 h(100.00±0.00、100.00±0.00)비교,차이유통계학의의(P균＜ 0.05),차24、36、48 h α3nAChR침묵조명현고우대조조(t=9.26、7.63、5.72,P균＜0.05).수착염불시간연장,α3nAChR침묵조화대조조린산화JNK표체축점승고,기중12、24、36、48 h α3nAChR침묵조(154.00±6.25、149.00±5.57、156.00±6.08、141.67±2.52)화8、12、24、36、48 h대조조(133.33±10.69、173.00±4.00、175.00±11.79、200.67±11.93、200.33±18.58)여동조0 h(100.00±0.00、100.00±0.00)비교,차이유통계학의의(P균＜ 0.05),차8、12、24、36、48 h α3nAChR침묵조명현저우대조조(t=-428、-5.02、-2.89、-8.33、-6.18,P균＜0.05).24、36、48 h대조조린산화p38격매표체(120.33±4.51、122.00±7.55、119.67±7.57)명현고우0h(100.00±0.00,P균＜0.05),차고우동시간점α3nAChR침묵조(93.33±9.61、94.00±5.01、98.33±5.69,t=-4.01、-6.73、-5.59,P균＜0.05).량조세포중총ERK1/2、총JNK、총p38격매표체수염불시간연장미견명현개변(P균＞ 0.05).결론 재과량불폭로조건하,ERK1/2신호통로적격활불완전의뢰우α3nAChR적표체;이JNK화p38량조통로적격활재일정정도상의뢰우α3nAChR.
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 ± 0.03) and protein (12.0 ± 2.5) in α3nAChR silence group was decreased significantly compared with those of control group (1.00 ± 0.11,100.0 ± 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 ± 0.15) was decreased significantly compared with that of SH-SY5Y cell treated with 0.000 mmol/L fluoride (1.05 ± 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 ± 7.33,200.00 ± 10.01,213.33 ± 11.55;125.33 ± 5.69,136.00 ± 4.52,155.33 ± 6.51) compared to 0 h in the same groups (100.00 ± 0.00,100.00 ± 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 ± 6.25,149.00 ± 5.57,156.00 ± 6.08,141.67 ± 2.52) and in control group at 8,12,24,36,48 h (133.33 ± 10.69,173.00 ± 4.00,175.00 ± 11.79,200.67 ± 11.93,200.33 ± 18.58) was compared to those at 0 h in the same groups (100.00 ± 0.00,100.00 ± 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 ± 4.51,122.00 ± 7.55,119.67 ± 7.57) compared to 0 h in the same groups (100.00 ± 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 ± 9.61,94.00 ± 5.01,98.33 ± 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.
