NF-κB、p38 MAPK和PKC通路对Aβ_(1-42)诱导K_(ATP)亚基Kir6.2/SUR1蛋白表达影响的研究
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摘要
研究目的：
阿尔茨海默病(Alzheimer's disease, AD)是老年人最常见的神经变性性疾病,临床特征为隐袭起病,进行性智能衰退、认知障碍和行为异常,多伴有人格改变,症状持续进展,病程通常为5-10年。
在AD的发病机制研究中,研究最多的是神经细胞凋亡途径,而最常见的致凋亡因素是老年炎性斑(Senile plaque, SP)的主要成分p-淀粉样蛋白(Ap),尤其是容易发生聚集且难溶解的Aβ1-42。Aβ的神经毒性机制非常复杂,Aβ可诱发过度氧化应激,而过度的氧化应激导致自由基的产生与消除之间的动态平衡被打破,这一平衡打破加速了细胞的凋亡最终导致细胞死亡。氧自由基的增加可使细胞膜系统的脂质和蛋白被氧化修饰,活性氧增加,线粒体功能、ATP酶活性下降,从而导致细胞内ATP的降低。而细胞内ATP水平低于某一临界值时,ATP敏感性钾通道(KATP)被激活,其激活后可通过降低细胞的兴奋性来维持Na+和Ca2+的稳定以及保持ATP的水平,且已经有研究证明对ATP较敏感的KATP通道亚基是Kir6.2及SUR1亚基。
核转录因子NF-κB是一种普遍存在的核转录因子,在中枢神经系统中也广泛表达,并参与神经系统中多种基因调控、细胞凋亡的信号传导过程。p38MAPK是分裂原激活的蛋白激酶家族(mitogen-activated protein kinase, MAPK)的成员之一,而MAPK通路是细胞外信号引起细胞核反应的汇聚通路。蛋白激酶C(protein kinase C, PKC)是一组具有单一肽链结构的丝氨酸／苏氨酸蛋白激酶,是机体细胞信号传导通路的中心环节,广泛分布于中枢神经系统。这三种信号通道与AD的发生发展有着密切的不可分割的联系。本实验通过研究NF-κB、p38MAPK和PKC信号通路对KATP通道亚基Kir6.2/SUR1蛋白表达的影响,来探讨这三个通路在阿尔茨海默病中的可能作用。为NF-κB、P38MAPK及PKC信号通路对神经元的作用提供了新思路,同时也为研究防治AD药物提供了新视角。
研究方法：
取Wistar大乳鼠(出生24h内),原代培养皮层和海马胆碱能神经元。并采用免疫细胞化学的方法,对爬片7天的细胞,通过胆碱乙酰转移酶(ChAT)进行胆碱能神经元的鉴定。将培养至第7天的胆碱能神经元进行随机分组：空白对照组、Apt-42组、Aβ142+抑制剂组和抑制剂组。分组后进行药物处理：空白对照组予以等量的培养液和PBS；Aβ1-42组：予以2μM的Aβ1-42；Ap1-42+抑制剂组：首先分别给予NF-kB抑制剂SN50(0.5μM)/p38MAPK抑制剂SB203580(2μM)/PKC抑制剂Chelerythrine chloride(CTC)(2μM),30min后,再分别加入Aβ1-42(2μM)继续培养；抑制剂组：分别予以SN50(0.5μM)/SB203580(2μM)/Chelerythrine chloride(CTC)(2μM)。药物处理后每组均培养至72h。收集并裂解细胞,然后提取蛋白,用BCA法检测蛋白浓度。取40μg总蛋白上样行蛋白质的SDS-聚丙烯酰胺凝胶电泳,经电转移至PVDF膜上,5%脱脂牛奶封闭,加入一抗(兔抗大鼠Kir6.2多克隆抗体或兔抗大鼠SUR1多克隆抗体)和二抗杂交,用发光试剂(ECL发光液)显色,以GAPDH作内参照,用图像分析软件进行光密度值分析。最后用SPSS统计分析NF-κB,p38MAPK和PKC三条通路各自对Aβ1-2诱导的KATP通道亚基Kir6.2/SUR1蛋白表达的影响。
研究结果：
(1)ChAT免疫细胞化学染色：胆碱能阳性神经元胞浆内呈棕黄色着色,阳性细胞计数结果显示90%以上；
(2).Ap1-42作用72h后KATP通道亚基Kir6.2/SUR1蛋白表达均显著升高,具有统计学意义p<0.05或p<0.01)；
(3).与Aβ142组相比较,SN50+Aβ1-42组的KATP通道亚基SUR1和Kir6.2蛋白表达显著降低p<0.01)；与空白对照组、Aβ1-42组相比,单独SN50组的KATP通道亚基SUR1和Kir6.2蛋白表达也均显著降低p<0.05或<0.01)；但与SN50+Aβ1-42组相比,单独SN50组的KATP通道亚基SUR1蛋白表达显著降低p<0.01),而亚基Kir6.2蛋白表达差异无统计学意义p>0.05)；
(4).与Aβ1-42组相比,SB203580+Aβ1-42组的KATP通道亚基Kir6.2及SUR1蛋白表达均降低(p＜0.05或p<0.01);与空白对照、Aβ1-42组以及SB203580+Ap1-42组相比,SB203580组的KATP通道亚基Kir6.2蛋白表达是降低的p<0.05)；然而与SB203580+Aβ142组相比较,KATP通道亚基SURl蛋白表达没有差异(p>0.05)。
(5)与Aβ1-42组相比,CTC+Aβ1-42组的KATP通道亚基Kir6.2及SUR1蛋白表达均明显降低(p<0.01)；与空白对照组、Aβ1-42组相比,单独CTC组的KATP通道亚基SUR1和Kir6.2蛋白表达也均显著降低p<0.05或p<0.01),但与CTC+Aβ1-42组相比,单独CTC组的KATP通道亚基SURl蛋白表达显著降低(p<0.05),而亚基Kir6.2蛋白表达差异无统计学意义((p>0.05)。
研究结论：
(1).原代培养的细胞90%以上为胆碱神经元；
(2).Ap1-42作用72h后KATP通道亚基Kir6.2/SURl蛋白的表达升高可能原因是Aβ1-42诱发氧化应激导致线粒体功能失调,ATP下降,为维持细胞正常膜电位及电生理活动KATP通道被激活,从而起到防治Aβ1-42的细胞毒性作用；
(3).NF-κB抑制剂SN50抑制Kir6.2/SUR1蛋白的表达,可能原因是SN50通过抑制NF-κkB核移位干扰NF-κB的生成,从而影响了NF-kB诱导增加Aβl-42水平,进一步影响了KATP通道亚基蛋白的表达。因此,NF-κB通路参与了Ap增加神经细胞KATP亚基表达的作用,阻断此通路能够减少神经细胞KATP亚基的表达。提示与神经元内Aβ1-42沉积有关的NF-kB活化是一种细胞保护反应；
(4).p38MAPK抑制剂SB203580抑制Kir6.2/SURl蛋白的表达,SB203580与p38MAPK的特异性结合,使p38MAPK失去与ATP结合的能力,从而使其失去激酶活性,进一步影响了KATP通道亚基蛋白的表达,本实验也进一步证实了p38MAPK抑制剂能抑制或消除Ap诱导的培养的神经元的凋亡；
(5)PKC抑制剂CTC抑制Kir6.2/SURl蛋白的表达,可能原因是剂CTC抑制PKC的活化,从而抑制PKC由胞浆向细胞膜转移,进而影响离子通道蛋白中的丝氨酸或苏氨酸残基磷酸化,使通道蛋白构象和门控动力学改变,最终调控离子通道的开闭,从而影响KATP通道。
研究意义：
信号通路NF-κB、p38MAPK、PKC均部分参与Aβ1-42诱导的KATP通道亚基Kir6.2/SUR1蛋白的表达,提示以上信号通路可能参与阿尔茨海默病的发生发展,为研究防治AD药物提供了新的理论依据。
Objective:
Alzheimer's disease (AD) is the most common neurodegenerative disease in elderly people, clinical manifestations of insidious onset, progressive smart recession, accompanied by personality changes. General symptoms continued to progress, there is usually a course of5-10years. In the study of the pathogenesis of AD, neuronal apoptosis pathway is the most studied, especially the pathological features of senile plaque (SP) in the AD, the main component of beta-amyloid protein (Aβ) induced apoptosis, and Aβ1-42is the first precipitation and the most difficult to dissolve. Beta-amyloid protein (AP) neurotoxicity mechanism is very complex and is one of the key factors of AD formation and development, and excessive oxidative stress and free radicals toxic effects play an important role in the development of AD pathogenesis. KATP are activated when intracellular ATP levels fall below a certain threshold by reducing cell excitability to maintain the stability of Na+and Ca+as well as to maintain the ATP level, and especially for the subunits Kir6.2/SUR1. NF-κB is a nuclear transcription factor, is widespread in a variety of cells in the human body, is also widely expressed in the central nervous system, such as neurons. Via the inhibitor of p38MAPK (SB203580), a major role in the T160of ATP-binding active site, can make the p38MAPK lost the ability to bind with ATP, which lost a kinase activity. Protein kinase C (protein kinase C, PKC) is a serine/threonine protein kinase with a single peptide chain structure, which is the central link of the body cell signaling pathways, widely distributed in the central nervous system. The pathogenesis of Alzheimer's disease is very complex and also associated with a variety of signaling pathway. Study of the effect of NF-κB, p38MAPK and PKC pathways on the neural KATPsubunits Kir6.2/SUR1expression induced by Aβ1-42in this experiment, to explore each of the paths possible role in Alzheimer's disease, maybe providing a new thinking for targeted therapy of AD.
Methods:
Cultured Wistar rats (within24h of birth) cortical and hippocampal cholinergic neurons in primary cells cultured and identification of cholinergic neurons in choline acetyltransferase (ChAT) immuno cytochemistry methods. The cells were randomly divided into four groups:the control group, the Aβ1-42group, the Aβ1-42+SN50group and the SN50group. The cells were cultured for7days, the control group was treated with equal volume PBS and culture medium; the Aβ1-42group was treated with Aβ-42(2μM); Aβ1-42+SN50group was treated with SN50(0.5μM),after30min, then added Aβ1-42(2μM) continue to culture; SN50group was treated with SN50(0.5μM); and a collection of cells by drug effects of72h, and the protein concentration in the extracts was determined by BCA Protein Assay Kit. SDS-PAGE electrophoresis,40g total protein samples taken, and then transferred to PVDF, closed one hour with5%skim milk, and finally closed overnight with primary antibody (Kir6.2or SUR1) and anti-closed2h (horseradish peroxidase conjugated goat anti-rabbit IgG), the final color using the ECL, and GAPDH as an internal reference, and finally analysis of the integral value of the optical density using image analysis software.
Results:
(1).Identification of rat hippocampal and cortical cholinergic neurons:the cytoplasm of positive neurons were stained brown,positive cells in more than90%of ChAT positive neurons.
(2).The experimental results showed that Aβ1-42significantly increased the subunits Kir6.2and SUR1of KATP expression.
(3).The subunits Kir6.2/SUR1of KATP expression level for72h compared with the Aβ1-42group were significantly reduced in the Aβ1-42+SN50group (p<0.01); compared with the Aβ1-42group or control group, the subunits Kir6.2/SUR1of KATP expression were significantly reduced in the SN50group(p<0.01and p<0.05), but compared with Aβ1-42+SN50group, the only subunit SUR1of KATP expression was significantly reduced in the SN50group(p<0.01), while the subunit Kir6.2of KATP expression was not obvious (p>0.05).
(4).The subunits Kir6.2/SUR1of KATP expression level for72h compared with the Aβ1-42group were significantly reduced in SB203580+Aβ1-42group(p<0.05). Compared with the Aβ1-42group or the control group or SB203580+Aβ1-42group, the subunits Kir6.2of KATP expression were significantly reduced in SB203580group(p<0.05). However, compared with SB203580+Aβ1-42group, the subunit SUR1of KATP expression level of SB203580group has no difference.
(5).The subunits Kir6.2/SUR1of KATP expression compared with the Aβ1-42group were significantly reduced in the Aβ+CTC(p<0.01); compared with the Aβ1-42group or control group, the subunits Kir6.2/SUR1of KATP expression were significantly reduced in the CTC group(p<0.01andp<0.05), but compared with Aβ+CTC group, the only subunit SUR1of KATP expression was significantly reduced in the CTC group(p<0.01), while the subunit Kir6.2of KATP expression was not obvious (p>0.05).
Conclusion:
(1).More than90%of cholinergic neurons in the primary cultured cells.
(2).The experimental results showed that Aβ1-42significantly increased the subunits Kir6.2and SUR1of KTP expression in the primary cholinergic neurons for72h compared with control group. One of possible causes is the KATP each subunit sensitivity to oxidative stress, the persistence of oxidative stress leading to mitochondrial dysfunction, serious disorder of cell metabolism, the KATP of the activation and opening up can maintain normal cell membrane potential and spontaneous electrical activity, which play the prevention and treatment of Aβ1-42cytotoxicity.
(3).The NF-κB inhibitor SN50inhibit Kir6.2/SUR1protein expression, the possible reason is that SN50interferes with NF-κB by inhibiting NF-κB nuclear translocation, thereby affecting the transcription expression of the subunits Kir6.2and SUR1of KATP induced by NF-κB, further affecting the expression of KATP.Therefore, NF-κB signal pathway is involved in the subunits of KATP expression increased by Aβ in neurons, blocking NF-κB signal pathway can reduce the subunits of KATP expression in neurons.
(4).The p38MAPK inhibitor SB203580inhibit Kir6.2/SUR1protein expression, the possible reason is the surrounding glial cell proliferation activated by amyloid plaques, releasing inflammatory products, thereby activating p38MAPK, but SB203580occupy the ATP binding sites, making the p38MAPK lose the ability to bind with ATP, thereby weakening the Aβ toxicity,and further affecting the subunits Kir6.2/SUR1of KATP expression, this experiment also further confirmed that p38MAPK can suppress or eliminate the neuronal apoptosis induced by Aβ。
(5).The PKC inhibitor CTC inhibit Kir6.2/SUR1protein expression, the possible reason is the activation of the PKC from the cytoplasm to the cell membrane inhibited by CTC and remain on the membrane and to promote a variety of protein phosphorylation, including serine or threonine residues of ion channel protein,so that changing the channel protein conformation and gating kinetics, KATP channel protein,finally regulating opening and closing of ion channels, thereby affecting the expression of KATP channel protein.
Significance:
It is indicated that NF-κB or p38MAPK or PKC has a protective effect against excessive oxidative stress, against destruction of the mitochondrial membrane potential, maybe a new theoretical basis for research drugs of prevention and treatment AD.
阿尔茨海默病(Alzheimer's disease, AD)是老年人最常见的神经变性性疾病,临床特征为隐袭起病,进行性智能衰退、认知障碍和行为异常,多伴有人格改变,症状持续进展,病程通常为5-10年。
在AD的发病机制研究中,研究最多的是神经细胞凋亡途径,而最常见的致凋亡因素是老年炎性斑(Senile plaque, SP)的主要成分p-淀粉样蛋白(Ap),尤其是容易发生聚集且难溶解的Aβ1-42。Aβ的神经毒性机制非常复杂,Aβ可诱发过度氧化应激,而过度的氧化应激导致自由基的产生与消除之间的动态平衡被打破,这一平衡打破加速了细胞的凋亡最终导致细胞死亡。氧自由基的增加可使细胞膜系统的脂质和蛋白被氧化修饰,活性氧增加,线粒体功能、ATP酶活性下降,从而导致细胞内ATP的降低。而细胞内ATP水平低于某一临界值时,ATP敏感性钾通道(KATP)被激活,其激活后可通过降低细胞的兴奋性来维持Na+和Ca2+的稳定以及保持ATP的水平,且已经有研究证明对ATP较敏感的KATP通道亚基是Kir6.2及SUR1亚基。
核转录因子NF-κB是一种普遍存在的核转录因子,在中枢神经系统中也广泛表达,并参与神经系统中多种基因调控、细胞凋亡的信号传导过程。p38MAPK是分裂原激活的蛋白激酶家族(mitogen-activated protein kinase, MAPK)的成员之一,而MAPK通路是细胞外信号引起细胞核反应的汇聚通路。蛋白激酶C(protein kinase C, PKC)是一组具有单一肽链结构的丝氨酸／苏氨酸蛋白激酶,是机体细胞信号传导通路的中心环节,广泛分布于中枢神经系统。这三种信号通道与AD的发生发展有着密切的不可分割的联系。本实验通过研究NF-κB、p38MAPK和PKC信号通路对KATP通道亚基Kir6.2/SUR1蛋白表达的影响,来探讨这三个通路在阿尔茨海默病中的可能作用。为NF-κB、P38MAPK及PKC信号通路对神经元的作用提供了新思路,同时也为研究防治AD药物提供了新视角。
研究方法：
取Wistar大乳鼠(出生24h内),原代培养皮层和海马胆碱能神经元。并采用免疫细胞化学的方法,对爬片7天的细胞,通过胆碱乙酰转移酶(ChAT)进行胆碱能神经元的鉴定。将培养至第7天的胆碱能神经元进行随机分组：空白对照组、Apt-42组、Aβ142+抑制剂组和抑制剂组。分组后进行药物处理：空白对照组予以等量的培养液和PBS；Aβ1-42组：予以2μM的Aβ1-42；Ap1-42+抑制剂组：首先分别给予NF-kB抑制剂SN50(0.5μM)/p38MAPK抑制剂SB203580(2μM)/PKC抑制剂Chelerythrine chloride(CTC)(2μM),30min后,再分别加入Aβ1-42(2μM)继续培养；抑制剂组：分别予以SN50(0.5μM)/SB203580(2μM)/Chelerythrine chloride(CTC)(2μM)。药物处理后每组均培养至72h。收集并裂解细胞,然后提取蛋白,用BCA法检测蛋白浓度。取40μg总蛋白上样行蛋白质的SDS-聚丙烯酰胺凝胶电泳,经电转移至PVDF膜上,5%脱脂牛奶封闭,加入一抗(兔抗大鼠Kir6.2多克隆抗体或兔抗大鼠SUR1多克隆抗体)和二抗杂交,用发光试剂(ECL发光液)显色,以GAPDH作内参照,用图像分析软件进行光密度值分析。最后用SPSS统计分析NF-κB,p38MAPK和PKC三条通路各自对Aβ1-2诱导的KATP通道亚基Kir6.2/SUR1蛋白表达的影响。
研究结果：
(1)ChAT免疫细胞化学染色：胆碱能阳性神经元胞浆内呈棕黄色着色,阳性细胞计数结果显示90%以上；
(2).Ap1-42作用72h后KATP通道亚基Kir6.2/SUR1蛋白表达均显著升高,具有统计学意义p<0.05或p<0.01)；
(3).与Aβ142组相比较,SN50+Aβ1-42组的KATP通道亚基SUR1和Kir6.2蛋白表达显著降低p<0.01)；与空白对照组、Aβ1-42组相比,单独SN50组的KATP通道亚基SUR1和Kir6.2蛋白表达也均显著降低p<0.05或<0.01)；但与SN50+Aβ1-42组相比,单独SN50组的KATP通道亚基SUR1蛋白表达显著降低p<0.01),而亚基Kir6.2蛋白表达差异无统计学意义p>0.05)；
(4).与Aβ1-42组相比,SB203580+Aβ1-42组的KATP通道亚基Kir6.2及SUR1蛋白表达均降低(p＜0.05或p<0.01);与空白对照、Aβ1-42组以及SB203580+Ap1-42组相比,SB203580组的KATP通道亚基Kir6.2蛋白表达是降低的p<0.05)；然而与SB203580+Aβ142组相比较,KATP通道亚基SURl蛋白表达没有差异(p>0.05)。
(5)与Aβ1-42组相比,CTC+Aβ1-42组的KATP通道亚基Kir6.2及SUR1蛋白表达均明显降低(p<0.01)；与空白对照组、Aβ1-42组相比,单独CTC组的KATP通道亚基SUR1和Kir6.2蛋白表达也均显著降低p<0.05或p<0.01),但与CTC+Aβ1-42组相比,单独CTC组的KATP通道亚基SURl蛋白表达显著降低(p<0.05),而亚基Kir6.2蛋白表达差异无统计学意义((p>0.05)。
研究结论：
(1).原代培养的细胞90%以上为胆碱神经元；
(2).Ap1-42作用72h后KATP通道亚基Kir6.2/SURl蛋白的表达升高可能原因是Aβ1-42诱发氧化应激导致线粒体功能失调,ATP下降,为维持细胞正常膜电位及电生理活动KATP通道被激活,从而起到防治Aβ1-42的细胞毒性作用；
(3).NF-κB抑制剂SN50抑制Kir6.2/SUR1蛋白的表达,可能原因是SN50通过抑制NF-κkB核移位干扰NF-κB的生成,从而影响了NF-kB诱导增加Aβl-42水平,进一步影响了KATP通道亚基蛋白的表达。因此,NF-κB通路参与了Ap增加神经细胞KATP亚基表达的作用,阻断此通路能够减少神经细胞KATP亚基的表达。提示与神经元内Aβ1-42沉积有关的NF-kB活化是一种细胞保护反应；
(4).p38MAPK抑制剂SB203580抑制Kir6.2/SURl蛋白的表达,SB203580与p38MAPK的特异性结合,使p38MAPK失去与ATP结合的能力,从而使其失去激酶活性,进一步影响了KATP通道亚基蛋白的表达,本实验也进一步证实了p38MAPK抑制剂能抑制或消除Ap诱导的培养的神经元的凋亡；
(5)PKC抑制剂CTC抑制Kir6.2/SURl蛋白的表达,可能原因是剂CTC抑制PKC的活化,从而抑制PKC由胞浆向细胞膜转移,进而影响离子通道蛋白中的丝氨酸或苏氨酸残基磷酸化,使通道蛋白构象和门控动力学改变,最终调控离子通道的开闭,从而影响KATP通道。
研究意义：
信号通路NF-κB、p38MAPK、PKC均部分参与Aβ1-42诱导的KATP通道亚基Kir6.2/SUR1蛋白的表达,提示以上信号通路可能参与阿尔茨海默病的发生发展,为研究防治AD药物提供了新的理论依据。
Objective:
Alzheimer's disease (AD) is the most common neurodegenerative disease in elderly people, clinical manifestations of insidious onset, progressive smart recession, accompanied by personality changes. General symptoms continued to progress, there is usually a course of5-10years. In the study of the pathogenesis of AD, neuronal apoptosis pathway is the most studied, especially the pathological features of senile plaque (SP) in the AD, the main component of beta-amyloid protein (Aβ) induced apoptosis, and Aβ1-42is the first precipitation and the most difficult to dissolve. Beta-amyloid protein (AP) neurotoxicity mechanism is very complex and is one of the key factors of AD formation and development, and excessive oxidative stress and free radicals toxic effects play an important role in the development of AD pathogenesis. KATP are activated when intracellular ATP levels fall below a certain threshold by reducing cell excitability to maintain the stability of Na+and Ca+as well as to maintain the ATP level, and especially for the subunits Kir6.2/SUR1. NF-κB is a nuclear transcription factor, is widespread in a variety of cells in the human body, is also widely expressed in the central nervous system, such as neurons. Via the inhibitor of p38MAPK (SB203580), a major role in the T160of ATP-binding active site, can make the p38MAPK lost the ability to bind with ATP, which lost a kinase activity. Protein kinase C (protein kinase C, PKC) is a serine/threonine protein kinase with a single peptide chain structure, which is the central link of the body cell signaling pathways, widely distributed in the central nervous system. The pathogenesis of Alzheimer's disease is very complex and also associated with a variety of signaling pathway. Study of the effect of NF-κB, p38MAPK and PKC pathways on the neural KATPsubunits Kir6.2/SUR1expression induced by Aβ1-42in this experiment, to explore each of the paths possible role in Alzheimer's disease, maybe providing a new thinking for targeted therapy of AD.
Methods:
Cultured Wistar rats (within24h of birth) cortical and hippocampal cholinergic neurons in primary cells cultured and identification of cholinergic neurons in choline acetyltransferase (ChAT) immuno cytochemistry methods. The cells were randomly divided into four groups:the control group, the Aβ1-42group, the Aβ1-42+SN50group and the SN50group. The cells were cultured for7days, the control group was treated with equal volume PBS and culture medium; the Aβ1-42group was treated with Aβ-42(2μM); Aβ1-42+SN50group was treated with SN50(0.5μM),after30min, then added Aβ1-42(2μM) continue to culture; SN50group was treated with SN50(0.5μM); and a collection of cells by drug effects of72h, and the protein concentration in the extracts was determined by BCA Protein Assay Kit. SDS-PAGE electrophoresis,40g total protein samples taken, and then transferred to PVDF, closed one hour with5%skim milk, and finally closed overnight with primary antibody (Kir6.2or SUR1) and anti-closed2h (horseradish peroxidase conjugated goat anti-rabbit IgG), the final color using the ECL, and GAPDH as an internal reference, and finally analysis of the integral value of the optical density using image analysis software.
Results:
(1).Identification of rat hippocampal and cortical cholinergic neurons:the cytoplasm of positive neurons were stained brown,positive cells in more than90%of ChAT positive neurons.
(2).The experimental results showed that Aβ1-42significantly increased the subunits Kir6.2and SUR1of KATP expression.
(3).The subunits Kir6.2/SUR1of KATP expression level for72h compared with the Aβ1-42group were significantly reduced in the Aβ1-42+SN50group (p<0.01); compared with the Aβ1-42group or control group, the subunits Kir6.2/SUR1of KATP expression were significantly reduced in the SN50group(p<0.01and p<0.05), but compared with Aβ1-42+SN50group, the only subunit SUR1of KATP expression was significantly reduced in the SN50group(p<0.01), while the subunit Kir6.2of KATP expression was not obvious (p>0.05).
(4).The subunits Kir6.2/SUR1of KATP expression level for72h compared with the Aβ1-42group were significantly reduced in SB203580+Aβ1-42group(p<0.05). Compared with the Aβ1-42group or the control group or SB203580+Aβ1-42group, the subunits Kir6.2of KATP expression were significantly reduced in SB203580group(p<0.05). However, compared with SB203580+Aβ1-42group, the subunit SUR1of KATP expression level of SB203580group has no difference.
(5).The subunits Kir6.2/SUR1of KATP expression compared with the Aβ1-42group were significantly reduced in the Aβ+CTC(p<0.01); compared with the Aβ1-42group or control group, the subunits Kir6.2/SUR1of KATP expression were significantly reduced in the CTC group(p<0.01andp<0.05), but compared with Aβ+CTC group, the only subunit SUR1of KATP expression was significantly reduced in the CTC group(p<0.01), while the subunit Kir6.2of KATP expression was not obvious (p>0.05).
Conclusion:
(1).More than90%of cholinergic neurons in the primary cultured cells.
(2).The experimental results showed that Aβ1-42significantly increased the subunits Kir6.2and SUR1of KTP expression in the primary cholinergic neurons for72h compared with control group. One of possible causes is the KATP each subunit sensitivity to oxidative stress, the persistence of oxidative stress leading to mitochondrial dysfunction, serious disorder of cell metabolism, the KATP of the activation and opening up can maintain normal cell membrane potential and spontaneous electrical activity, which play the prevention and treatment of Aβ1-42cytotoxicity.
(3).The NF-κB inhibitor SN50inhibit Kir6.2/SUR1protein expression, the possible reason is that SN50interferes with NF-κB by inhibiting NF-κB nuclear translocation, thereby affecting the transcription expression of the subunits Kir6.2and SUR1of KATP induced by NF-κB, further affecting the expression of KATP.Therefore, NF-κB signal pathway is involved in the subunits of KATP expression increased by Aβ in neurons, blocking NF-κB signal pathway can reduce the subunits of KATP expression in neurons.
(4).The p38MAPK inhibitor SB203580inhibit Kir6.2/SUR1protein expression, the possible reason is the surrounding glial cell proliferation activated by amyloid plaques, releasing inflammatory products, thereby activating p38MAPK, but SB203580occupy the ATP binding sites, making the p38MAPK lose the ability to bind with ATP, thereby weakening the Aβ toxicity,and further affecting the subunits Kir6.2/SUR1of KATP expression, this experiment also further confirmed that p38MAPK can suppress or eliminate the neuronal apoptosis induced by Aβ。
(5).The PKC inhibitor CTC inhibit Kir6.2/SUR1protein expression, the possible reason is the activation of the PKC from the cytoplasm to the cell membrane inhibited by CTC and remain on the membrane and to promote a variety of protein phosphorylation, including serine or threonine residues of ion channel protein,so that changing the channel protein conformation and gating kinetics, KATP channel protein,finally regulating opening and closing of ion channels, thereby affecting the expression of KATP channel protein.
Significance:
It is indicated that NF-κB or p38MAPK or PKC has a protective effect against excessive oxidative stress, against destruction of the mitochondrial membrane potential, maybe a new theoretical basis for research drugs of prevention and treatment AD.
引文
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