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伊拉地平对Fe2+诱导MES23.5细胞毒性的作用

2021-07-08赵莎马泽刚

青岛大学学报(医学版) 2021年2期
关键词:帕金森病

赵莎 马泽刚

[摘要]目的 探讨L型Ca2+通道阻断剂伊拉地平对硫酸亚铁诱导的MES23.5细胞毒性的作用。方法 以MES23.5多巴胺能细胞系为观察对象,利用流式细胞仪筛选Fe2+最适浓度,伊拉地平则选用实验室前期筛选浓度5 μmol/L。应用免疫印迹法(Western blot)检测与凋亡相关蛋白Bcl-2和Bax的表达,初步评估伊拉地平对Fe2+诱导的细胞毒性的保护作用。结果 与对照组相比,40 μmol/L Fe2+组线粒体膜电位(ΔΨm)明显下降,差异具有统计学意义(F=68.190,q=8.898,P<0.001)。与对照组相比,单独加Fe2+组细胞Bcl-2/Bax蛋白表达比值明显降低(F=6.856,q=6.055,P<0.01),而伊拉地平预处理可改善由Fe2+造成的Bcl-2/Bax蛋白表达比值降低的现象,差异具有统计学意义(q=4.103,P<0.05)。结论 伊拉地平预处理可以抑制Bcl-2/Bax蛋白表达比值降低,對Fe2+诱导的细胞损伤可能具有保护作用。

[关键词]伊拉地平;钙通道阻滞药;铁;帕金森病;神经保护

[中图分类号]R338.2

[文献标志码]A

[文章编号]2096-5532(2021)02-0190-04

[ABSTRACT]Objective To investigate the effect of isradipine, a blocker of L-type Ca2+ channels, on cytotoxicity induced by ferrous sulfate heptahydrate in MES23.5 cells. Methods The MES23.5 dopaminergic cell line was selected for observation. Flow cytometry was used to screen out the optimal concentration of Fe2+, and a concentration of 5 μmol/L was determined for isradipine in previous laboratory analysis. Western blot was used to measure the expression of the apoptosis-related proteins Bcl-2 and Bax, and the protective effect of isradipine against Fe2+-induced cytotoxicity was evaluated. Results Compared with the control group, the 40 μmol/L Fe2+ group had a significant reduction in mitochondrial membrane potential (ΔΨm) (F=68.190,q=8.898,P<0.001). Compared with the control group, the Fe2+ alone group had a significant reduction in Bcl-2/Bax ratio (F=6.856,q=6.055,P<0.01), while pretreatment with isradipine significantly improved the reduction in Bcl-2/Bax ratio caused by Fe2+ (q=4.103,P<0.05). Conclusion Pretreatment with isradipine can inhibit the reduction in Bcl-2/Bax ratio and thus exert a protective effect against cell injury induced by Fe2+.

[KEY WORDS]isradipine; calcium channel blockers; iron; Parkinson disease; neuroprotection

帕金森病(PD)是一种常见的神经退行性老年疾病,其病理特征是黑质(SN)多巴胺(DA)能神经元进行性缺失以及由此引起的纹状体DA含量的耗竭[1]。病人表现出静止性震颤、肌僵直、运动迟缓、姿势平衡障碍等运动症状[2],以及嗅觉障碍、抑郁、睡眠质量差和认知功能障碍等非运动症状[3]。虽然PD的发病机制尚未完全明确,但越来越多的证据表明SN区过量的铁沉积是PD发病的关键因素之一[4-5]。在高铁作用下,大脑中过量的Fe2+会通过Fenton和Haber-Weiss反应产生大量活性氧,从而引起蛋白质的异常聚集,损伤神经元,导致PD等神经退行性疾病的发生[6]。正常情况下,铁主要是通过转铁蛋白和二价金属转运蛋白1(DMT1)等途径进入细胞[7],但在PD等病理条件下,转铁蛋白在脑脊液中处于饱和状态,表明非转铁蛋白结合铁发挥主要作用[8],即L型Ca2+通道(LTCC)可能参与了铁的异常聚集[9]。因此,本实验探究了LTCC阻断剂伊拉地平(ISR)对硫酸亚铁诱导的MES23.5多巴胺能细胞毒性的作用。

1 材料和方法

1.1 实验材料

本实验所用细胞为MES23.5多巴胺能细胞系(是由大鼠胚胎中脑神经元与小鼠神经母细胞瘤胶质瘤细胞融合而成的杂交瘤细胞),由大连医科大学附属医院乐卫东教授惠赠。胎牛血清(FBS),BI公司;DMEM/F12(1∶1)粉末,Gibco公司;多聚赖氨酸(poly-L),Sigma公司;青霉素/链霉素,索莱宝公司;二甲基亚砜(DMSO),Biosharp公司;硫酸亚铁,Sigma公司;ISR,上海杨帆公司。

1.2 细胞培养

MES23.5多巴胺能细胞系用DMEM/F12培养液培养,该培养液中添加了10 mL的FBS、2 mL的青霉素/链霉素和4.4 mL的佐藤成分(含0.5 g/L胰岛素和转铁蛋白)。实验前,将细胞以1×108/L的密度接种在涂有poly-L的培养瓶或者6孔培养皿中。

1.3 Fe2+最适浓度筛选

应用不同浓度的硫酸亚铁(0、20、40、60、80、100 μmol/L Fe2+)处理MES23.5细胞,通过流式细胞仪检测MES23.5多巴胺能细胞线粒体膜电位(ΔΨm)的变化。将细胞置于涂有poly-L的6孔板中培养2 d。以PBS洗涤3次后,使用1 mL罗丹明123(5 mg/L)在37 ℃培养箱中孵育30 min,然后通过300目的细胞滤网收集并制成单细胞悬液。上流式细胞仪检测,设置门控区域M1和M2作为标记,使用Cell Quest软件(BD Biosciences,美国)评估荧光强度的变化。

1.4 实验分组

在确定Fe2+最适浓度的基础上,后续实验分为4组:对照组、Fe2+组、Fe2++ISR组、ISR组。将细胞以1×108/L的密度种在6孔板中。第3天行分组处理细胞:对照组用酸性培养液孵育5 h;Fe2+组先用酸性培养液孵育1 h,再使用Fe2+孵育4 h;Fe2++ISR组用ISR预孵育1 h,再使用Fe2+孵育4 h;ISR组用酸性培养液孵育5 h。ISR选用实验室前期筛选浓度5 μmol/L。各组细胞置于37 ℃、体积分数0.05 CO2培养箱中培养。

1.5 免疫印迹法检测Bcl-2/Bax蛋白表达

细胞处理结束后,以PBS洗涤3次,每孔加入100 μL裂解液冰上裂解30 min。用刮板将细胞刮下,4 ℃下以12 000 r/min离心20 min。采用二辛可宁酸(BCA)法测定蛋白质浓度,计算十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)上样量。用SDS-PAGE(8 mL)分离每泳道包含40 μg蛋白样品的细胞裂解液,将蛋白转移到PVDF膜上。在室温下用50 g/L的脱脂奶粉封闭2 h后,再加入Bcl-2、Bax(1∶1 000)和β-actin(1∶10 000)一抗于4 ℃摇床过夜。次日,使用TBST洗涤3次,加入与辣根过氧化物酶(HRP)偶联的山羊抗兔IgG二抗(1∶10 000)在室温下孵育1 h。采用ECL法,通过化学发光的方式对抗原-抗体复合物进行可视化,然后使用Image J系统进行光密度分析。

1.6 统计学处理

使用Prism Graphpad 5.0软件进行统计学处理。实验所得结果以x2±s形式表示,多组比较采用单因素方差分析(ANOVA),组间两两比较采用Student-Newman-Keuls检验。P<0.05表示差异具有统计学意义。

2 结 果

2.1 不同浓度Fe2+对MES23.5细胞ΔΨm的影响

用不同浓度的Fe2+处理MES23.5细胞24 h后,0、20、40、60、80、100 μmol/L Fe2+处理组细胞的ΔΨm分别为0.01±1.66、-4.67±3.77、-10.17±3.34、-17.67±1.92、-21.50±2.29和-29.78±2.17(n=6)。与对照组细胞相比较,20、40、60、80、100 μmol/L Fe2+處理组ΔΨm均有不同程度的降低(F=68.190,P<0.01),且ΔΨm降低呈浓度依赖性。若Fe2+对细胞毒性太小则损伤不够,Fe2+对细胞毒性太大则造成细胞不可逆死亡,因此选取浓度40 μmol/L的Fe2+用于后续实验。

2.2 各组细胞Bcl-2/Bax蛋白表达比值的比较

对照组、Fe2+组、Fe2++ISR组和ISR组细胞Bcl-2/Bax蛋白表达比值分别为1.04±0.12、0.82±0.03、0.97±0.12和0.99±0.08(n=4)。与对照组相比较,Fe2+组Bcl-2/Bax蛋白表达比值明显降低(F=6.856,q=6.055,P<0.01);ISR预处理可以明显缓解由Fe2+诱导的Bcl-2/Bax蛋白表达比值降低,差异具有统计学意义(q=4.103,P<0.05);ISR组与对照组Bcl-2/Bax蛋白表达比值比较差异无显著性(P>0.05)。

3 讨 论

许多研究已经证实,铁的积累是PD的一个标志[6]。有研究显示,PD病人以及PD动物模型SN区DA能神经元内铁水平较正常人或动物异常增高[10-11]。但是,尚不清楚SN中铁蓄积的确切机制。众所周知,铁通过与转铁蛋白结合进入大脑[12]。但在铁过载的情况下,转铁蛋白饱和,非转铁蛋白途径显得尤为重要[13]。因此,包括DMT1和钙通道介导的铁转运在内的非转铁蛋白结合铁(NTBI)途径已引起更多关注[14]。有研究结果表明,铁可以通过LTCC进入心肌细胞[15-16],LTCC还介导铁流入其他可兴奋细胞,如胰腺β细胞[17]、腺垂体细胞[18]和神经元[19]。因此我们推测,在铁负载情况下,在DA能神经元上广泛表达的LTCC可能是铁过量进入DA能神经元的重要替代路径,而铁的过量积累可能导致DA能神经元的损伤甚至死亡[20]。因此,LTCC阻滞剂可能通过阻断钙通道来缓解神经元中的铁超负荷,成为PD的新治疗方法。目前,LTCC参与PD的确切机制仍不清楚,LTCC是否直接参与SN区铁的聚集也不清楚,因此深入探讨PD发病过程中LTCC与Fe2+聚集的相关性,对了解DA能神经元的损伤机制十分重要。

LTCC主要以Cav1.2和Cav1.3亚型的形式存在于神经元中[21-22],其相应的成孔亚基α1C以及α1D[23]在SN区DA能神经元中功能性表达[24]。研究表明,DA能神经元对Cav1.3钙通道的异常依赖导致胞质内Ca2+水平升高,Ca2+进入细胞会持续刺激线粒体氧化磷酸化,这可能是PD中SN区DA能神经元更易受损的原因之一[25]。而Cav1.2钙通道在平滑肌细胞[26]、神经元[27]、神经内分泌细胞[28]和感觉细胞等不同细胞中普遍表达[29-30]。有研究证实,在6-羟基多巴胺(6-OHDA)损伤的大鼠和1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)处理的PD模型小鼠SN中,Cav1.2 α1亚基的表达显著增加[23]。然而,迄今为止尚不清楚Cav1.2钙通道对PD发病机制中铁诱导的神经毒性的影响。而之前的研究报道,在铁负载条件下,LTCC可能是铁进入心肌细胞的主要途径[31-32]。

本次研究首先利用不同濃度的Fe2+作用于MES23.5细胞[33],通过流式细胞术观察细胞ΔΨm的变化,结果显示,Fe2+处理MES23.5细胞后,细胞△Ψm下降,提示线粒体功能受损。进一步的研究结果显示,Fe2+组Bcl-2/Bax蛋白表达比值较对照组明显降低,而5 μmol/L的ISR预处理部分逆转了Fe2+诱导的Bcl-2/Bax蛋白表达比值的下降,表明LTCC可能参与了铁积累引起的PD的发生。总之,以上结果表明,ISR可能会抑制Fe2+诱导的MES23.5细胞毒性,LTCC阻滞剂可能通过阻断钙通道来缓解神经元中的铁超负荷。

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(本文編辑 马伟平)

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