抗凋亡蛋白Bcl-2在岗田酸诱导的神经细胞退化中的作用研究
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摘要
抗凋亡蛋白Bcl-2在岗田酸诱导的神经细胞退化中的作用研究
凋亡机制参与了AD中神经细胞退化过程。Bcl-2家族在凋亡信号转导通路中起重要调节作用。Bcl-2家族包括促凋亡和抑凋亡两类作用相反的蛋白，Bcl-2是起抑凋亡作用的代表性成员，其抗凋亡作用与家族中促凋亡蛋白Bax密切联系，Bcl-2与Bax之间的比例对细胞是否发生凋亡起重要作用。岗田酸(OA)是蛋白磷酸酶抑制剂，在整体或离体条件OA能使神经细胞微管相关蛋白tau异常高度磷酸化而使神经细胞退化，部分模拟了AD样病理特征。离体时OA所致神经细胞退化为凋亡，而整体时也发现OA能引起神经细胞发生凋亡样DNA双链断裂。为了研究Bcl-2相关凋亡调节机制在OA所致神经细胞退化中的影响，本课题首先采用大鼠脑额叶皮质定位注射OA的整体模型，观察OA致神经细胞退化时Bcl-2和Bax的表达变化规律，探讨Bcl-2在OA致神经细胞退化时的作用特征以及Bcl-2是否参与此过程中细胞自身代偿性保护机制；继而采用OA诱导体外培养神经细胞凋亡性退化的离体模型，研究Bcl-2过表达是否抑制OA所致神经细胞退化，以进一步确定Bcl-2在OA诱导的神经细胞退化过程中是否具有细胞保护作用。
整体实验部分Bcl-2在OA诱导大鼠脑神经细胞退化中的作用分析
为探讨Bcl-2相关凋亡调节机制与整体时OA致神经细胞tau蛋白高度磷酸化和退化之间的关系，采用大鼠额叶皮质定位注射OA的模型，应用免疫组织化学、免疫荧光三标结合激光共聚焦扫描的方法，检测OA致神经细胞退化时对Bcl-2表达和Bax表达的影响及二者与tau蛋白磷酸化的共存关系。结果如下：
1．在OA作用皮质，神经细胞高表达Bcl-2蛋白，OA作用皮质表达Bcl-2的细胞数明显增加，且具有时间和剂量反应特征，对各时间反应组的细胞计数显示，注射20ng OA 12h、1d、3d时Bcl-2免疫阳性细胞的数目显著高于相应时间点的生理盐水对照组(p＜0.001)，且OA 12h、1d、3d组与OA 3h、6h、7d组相比有显著性差异(12h，p＜0.01；1d、3d，p＜0.05)；对注射1d时各剂量反应组的细胞计数显示，20ng、50ng、100ng OA组均大量表达Bcl-2蛋白，与生理盐水对照组相比有显著差异(20ng，p＜0.01；50ng、100ng，p＜0.05)，20ng和100ng组与10ng组相比也有显著差异(p＜0.05)；
2．在OA作用皮质，神经细胞高表达Bax蛋白，且具有时间和剂量反应特征，对各时间反应组的细胞计数显示，注射20ng OA 12h、1d、3d时Bax免疫阳性细胞的数目显著高于相应时间点的生理盐水对照组(p＜0.01)；对注射1d时各剂量反应组的细胞计数显示，与生理盐水对照组和10ng组相比，20ng、100ng OA组的Bax免疫阳性细胞数有增多趋势，接近统计学显著性差异水平；
3．比较OA 20ng 1d组大鼠的相邻平面脑片AT-8、Bcl-2和Bax三种免疫组化染色，形态学观察显示，在OA注射皮质，三种免疫阳性细胞的分布位置大体一致，Bcl-2阳性细胞的分布范围和细胞数目明显大于或多于其他两种免疫阳性细胞，在三种免疫阳性细胞中，Bax阳性细胞的分布范围最小，阳性细胞数目最少，AT-8阳性细胞居中；对OA 20ng 1d组大鼠的AT-8、Bcl-2和Bax免疫荧光三标显示，在距OA注射位点较近的周边区域，Bcl-2表达与AT-8表达、Bax表达共存，或Bcl-2表达仅与AT-8表达共存；在距OA注射位点较远周边区域，仅见Bcl-2高表达，未见AT-8和Bax表达。
这部分结果提示，OA上调额叶皮质神经细胞中Bcl-2和Bax蛋白表达，且具有时程规律和剂量反应特征，并与OA所致tau蛋白磷酸化的时程规律和剂量反应特征较为一致，提示OA所致Bcl-2和Bax高表达与tau蛋白磷酸化之间可能存在内在联系；OA作用皮质区域，AT-8、Bcl-2、Bax表达三者共存或AT-8表达仅与Bcl-2表达二者共存，这提示Bcl-2家族活跃参与OA所致神经细胞退化过程，但Bcl-2、Bax高表达时的活性状态及其对OA所诱导细胞损伤的功能意义还有待证实。
离体实验部分Bel-2在OA诱导体外培养神经细胞退化中的作用分析
为了进一步探讨OA致神经细胞损伤过程中Bcl-2表达上调的功能意义，采用OA诱导人神经母细胞瘤SH-SY5Y细胞退化的离体模型，应用脂质体介导质粒DNA转染细胞的方法、DNA荧光染料Hoechst 33258染色法、MTT还原法，首先明确OA引起SH-SY5Y细胞凋亡性退化，鉴定质粒转染引起SH-SY5Y细胞外源性过表达功能性Bcl-2，进而研究Bcl-2过表达对OA所致体外培养神经细胞退化的影响。结果如下：
1．MTT还原法对细胞活力的检测显示，OA剂量依赖性地降低SH-SY5Y细胞活力，其中60nM／80nM／100nM OA作用24h显著降低细胞活力(60nM、80nM，p＜0.05；100nM，p＜0.01)；Hoechst 33258染色显示，80nM OA作用24h引起该种细胞出现多量核浓聚、核碎裂的凋亡样核型；
2．脂质体介导pEGFP-N1-Bcl-2和空质粒pEGFP-N1瞬时转染SH-SY5Y细胞，转染效率分别为2.01％和2.18％，细胞分别过表达Bcl-2-GFP和GFP蛋白，Bcl-2-GFP融合蛋白主要分布于近核的细胞胞体，而GFP蛋白则在胞质和胞核中均有分布；Hoechst 33258染色法显示，200nM促凋亡剂星形孢菌素(STS)作用细胞24h，Bcl-2重组质粒转染组的细胞凋亡率显著低于空质粒转染组的细胞凋亡率(p＜0.01)；
3．Hoechst 33258染色法显示，80 nM OA作用细胞24h，Bcl-2重组质粒转染组的细胞凋亡率显著低于空质粒转染组的细胞凋亡率(p＜0.05)。
这部分结果提示，OA剂量依赖性引起体外培养神经细胞SH-SY5Y凋亡性退化，Bcl-2重组质粒瞬时转染引起该种细胞过表达功能性融合蛋白Bcl-2-GFP，Bcl-2过表达能部分抑制OA所致SH-SY5Y细胞退化，这显示Bcl-2在OA所致神经细胞退化过程中可能起重要的调节作用，Bcl-2表达上调在OA损伤通路中具有细胞保护作用。
结论
1．大鼠脑额叶皮质定位注射OA能诱导该区神经细胞Bcl-2和Bax表达上调，且呈时间和剂量反应特征，二者的表达上调可能与OA致tau蛋白高度磷酸化有关，OA作用皮质区域AT-8、Bcl-2、Bax表达三者共存或AT-8表达仅与Bcl-2表达二者共存，这提示Bcl-2家族活跃参与OA所致神经细胞退化过程。
2．外源性Bcl-2高表达能部分抑制OA所致体外培养神经细胞SH-SY5Y凋亡性退化，提示Bcl-2相关凋亡调节机制参与了OA所致神经细胞退化过程，在该损伤通路中Bcl-2表达上调具有细胞保护的功能意义。
3．综合分析整体与离体实验的结果，提示Bcl-2相关凋亡调节机制参与OA所致神经细胞退化过程，OA引起的Bcl-2表达上调可能是机体神经细胞针对损伤信号的自身代偿性保护机制。
Apoptosis involves in progressive neurodegeneration in AD. Bcl-2 family plays important role in apoptotic signal transduction pathway. The family members are classified as two kinds according to their opposite functions, pro-apoptotic or anti-apoptotic activity. Bcl-2 and Bax are representative of anti-apoptotic and pro-apoptotic proteins, respectively. The anti-apoptotic activity of Bcl-2 associates closely with the pro-apoptotic activity of Bax. Occurrence of cell apoptosis is decided by the ratio of Bcl-2 versus Bax under certain physiological and pathological conditions. Okadaic acid (OA), a protein phosphatase inhibitor, induces microtubule-associated protein tau hyperphosphorylation both in vitro and in vivo, which is considered to associate with pathogenesis of neurodegeneration. Apoptosis is responsible for neurodegeneration induced by OA in vitro. It has also been found that apoptosis-like DNA double strand damage appeared in OA-induced neurodegeneration in vivo. In the present study, microinjection of OA into rat frontal cortex was used to explore the possible roles of Bcl-2-related apoptotic regulatory mechanism in OA-induced neurodegeneration process in vivo. Immunohistochemistry was applied to observe alteration of Bcl-2 and Bax expression following OA injection. Triple immuno-fluorescent labeling combined with cofocal laser scanning microscopic analysis was performed to illustrate the topographic relationship among tau hyperphosphorylation, Bcl-2 expression and Bax expression induced by OA. Furthermore, human SH-SY5Y neuroblastoma cells were applied to investigate the influence of exogenous Bcl-2 overexpression on apoptotic neuronal death induced by OA. The results are as follows:
Experiment in vivo Effect of Bcl-2 on OA-induced neurodegeneration in rat brain
Bcl-2 immunostaining showed that (1) the number of Bcl-2 positive staining cells significantly increased at 12 h, peaked at 1 d, then decreased at 3 d after injection of 20 ng OA compared with the number of Bcl-2-positive cells after vehicle
injection (p<0.001); 12 h, 1 d and 3 d of 20 ng OA treatment significantly increased the number of Bcl-2-positive cells compared with 3 h, 6 h and 7 d of 20 ng OA treatment (12 h, p<0.01; 1 d, 3 d, p<0.05); (2) compared with vehicle injection, injection of, 20 ng, 50 ng and 100 ng OA significantly increased the number of Bcl-2-positive cells at 1 d (20 ng,p<0.01; 50 ng, 100 ng,p<0.05); compared with 10 ng OA injection, injection of 20 ng and 100 ng OA also significantly increased the number of Bcl-2-positive cells at 1 d (p<0.05)
Bax immunostaining showed that (1) the number of Bax-positive cells also significantly increased at 12 h, peaked at 1 d, then decreased at 3 d after injection of 20 ng OA compared with the number of Bax-positive cells after vehicle injection (p<0.01); (2) injection of 50 ng and 100 ng OA tended to elevate the number of Bax-positive cells at 1 d, but not reach statistical level.
Immunohistochemistry of adjacent brain slices from the same rat delineated that the distributional profiles of AT-8-, Bcl-2- and Bax-positive cells were similar. However, the distributional range and the number of these positive cells were obviously different from one another. Among them, the range of Bcl-2-positive cells was most widely and the number was largest, the range of Bax-positive cells was least widely and the number was smallest and these two indices of AT-8-positive cells were in the middle.
Triple immuno-fluorescent staining delineated that Bcl-2 expression co-stained with AT-8 and Bax expression or only co-stained with AT-8 expression in the affected area more adjacent to OA injected site. But in the affected area less neighboring to OA injected site, only Bcl-2 immunoreactivity was observed, but neither AT-8 nor Bax immunoreactivity.
Experiment in vitro Effect of Bcl-2 overexpression on OA-induced cell death in SH-SY5Y cells
OA decreased cell viability of SH-SY5Y cells in dose dependent manner. 60 nM, 80 nM and 100 nM OA significantly reduced cell viability after 24 h of treatment (60 nM, 80 nM,p<0.05; 100 nM,p<0.01). Hoechst staining showed that a lot of chromatin condensation and fragmentation typical of apoptosis appeared following 24 h of 80 nM OA treatment.
The transfection efficiency of recombinant plasmid pEGFP-N1-Bcl-2 and empty plasmid pEGFP-N1 was 2.01% and 2.18% respectively. Exogenous Bcl-2-GFP induced by recombinant plasmid transfection mainly resided in peri-nucleus cell bodies, while Exogenous GFP induced by vector transfection located in cytosol, nuclei and processes. Hoechst staining showed that apoptotic Bcl-2 transfected cells were markedly less than apoptotic vector transfected cells after 24 h of 200 nM staurosporine (STS) exposure (p<0.01).
Hoechst staining further revealed that apoptotic Bcl-2 transfected cells were markedly less than apoptotic vector transfected cells after 24 h of 80 nM OA exposure (p<0.05).
Conclusion
1. Microinjection of OA into rat frontal cortex upregulated Bcl-2 and Bax protein expression in affected brain area. Moreover, induction of Bcl-2 and Bax expression by OA had temporal change and dose response feature. The upregulation of these two apoptotic regulatory protein probably attributed to tau hyperphosphorylation stimulated by OA. In affected cortex by OA, AT-8, Bcl-2 and Bax expression co-stained or AT-8 and Bcl-2 expression co-stained, suggestive of Bcl-2 family actively participated in OA-induced neurodegeneration process.
2. Exogenous Bcl-2 overexpression partially inhibited OA-induced apoptosis in SH-SY5Y neuronal cells, suggesting that Bcl-2 related apoptotic regulatory mechanism involved in OA-induced neurodegenerative pathway and that Bcl-2 had neuronal protective role in it.
3. Taken together, the results in vivo and in vitro indicated that Bcl-2 related apoptotic regulatory mechanism participated in OA-induced neurodegeneration and that the upregulation of Bcl-2 protein expression probably represented neuronal compensatory mechanism in response to OA toxicity to neuronal cytoskeleton.
凋亡机制参与了AD中神经细胞退化过程。Bcl-2家族在凋亡信号转导通路中起重要调节作用。Bcl-2家族包括促凋亡和抑凋亡两类作用相反的蛋白，Bcl-2是起抑凋亡作用的代表性成员，其抗凋亡作用与家族中促凋亡蛋白Bax密切联系，Bcl-2与Bax之间的比例对细胞是否发生凋亡起重要作用。岗田酸(OA)是蛋白磷酸酶抑制剂，在整体或离体条件OA能使神经细胞微管相关蛋白tau异常高度磷酸化而使神经细胞退化，部分模拟了AD样病理特征。离体时OA所致神经细胞退化为凋亡，而整体时也发现OA能引起神经细胞发生凋亡样DNA双链断裂。为了研究Bcl-2相关凋亡调节机制在OA所致神经细胞退化中的影响，本课题首先采用大鼠脑额叶皮质定位注射OA的整体模型，观察OA致神经细胞退化时Bcl-2和Bax的表达变化规律，探讨Bcl-2在OA致神经细胞退化时的作用特征以及Bcl-2是否参与此过程中细胞自身代偿性保护机制；继而采用OA诱导体外培养神经细胞凋亡性退化的离体模型，研究Bcl-2过表达是否抑制OA所致神经细胞退化，以进一步确定Bcl-2在OA诱导的神经细胞退化过程中是否具有细胞保护作用。
整体实验部分Bcl-2在OA诱导大鼠脑神经细胞退化中的作用分析
为探讨Bcl-2相关凋亡调节机制与整体时OA致神经细胞tau蛋白高度磷酸化和退化之间的关系，采用大鼠额叶皮质定位注射OA的模型，应用免疫组织化学、免疫荧光三标结合激光共聚焦扫描的方法，检测OA致神经细胞退化时对Bcl-2表达和Bax表达的影响及二者与tau蛋白磷酸化的共存关系。结果如下：
1．在OA作用皮质，神经细胞高表达Bcl-2蛋白，OA作用皮质表达Bcl-2的细胞数明显增加，且具有时间和剂量反应特征，对各时间反应组的细胞计数显示，注射20ng OA 12h、1d、3d时Bcl-2免疫阳性细胞的数目显著高于相应时间点的生理盐水对照组(p＜0.001)，且OA 12h、1d、3d组与OA 3h、6h、7d组相比有显著性差异(12h，p＜0.01；1d、3d，p＜0.05)；对注射1d时各剂量反应组的细胞计数显示，20ng、50ng、100ng OA组均大量表达Bcl-2蛋白，与生理盐水对照组相比有显著差异(20ng，p＜0.01；50ng、100ng，p＜0.05)，20ng和100ng组与10ng组相比也有显著差异(p＜0.05)；
2．在OA作用皮质，神经细胞高表达Bax蛋白，且具有时间和剂量反应特征，对各时间反应组的细胞计数显示，注射20ng OA 12h、1d、3d时Bax免疫阳性细胞的数目显著高于相应时间点的生理盐水对照组(p＜0.01)；对注射1d时各剂量反应组的细胞计数显示，与生理盐水对照组和10ng组相比，20ng、100ng OA组的Bax免疫阳性细胞数有增多趋势，接近统计学显著性差异水平；
3．比较OA 20ng 1d组大鼠的相邻平面脑片AT-8、Bcl-2和Bax三种免疫组化染色，形态学观察显示，在OA注射皮质，三种免疫阳性细胞的分布位置大体一致，Bcl-2阳性细胞的分布范围和细胞数目明显大于或多于其他两种免疫阳性细胞，在三种免疫阳性细胞中，Bax阳性细胞的分布范围最小，阳性细胞数目最少，AT-8阳性细胞居中；对OA 20ng 1d组大鼠的AT-8、Bcl-2和Bax免疫荧光三标显示，在距OA注射位点较近的周边区域，Bcl-2表达与AT-8表达、Bax表达共存，或Bcl-2表达仅与AT-8表达共存；在距OA注射位点较远周边区域，仅见Bcl-2高表达，未见AT-8和Bax表达。
这部分结果提示，OA上调额叶皮质神经细胞中Bcl-2和Bax蛋白表达，且具有时程规律和剂量反应特征，并与OA所致tau蛋白磷酸化的时程规律和剂量反应特征较为一致，提示OA所致Bcl-2和Bax高表达与tau蛋白磷酸化之间可能存在内在联系；OA作用皮质区域，AT-8、Bcl-2、Bax表达三者共存或AT-8表达仅与Bcl-2表达二者共存，这提示Bcl-2家族活跃参与OA所致神经细胞退化过程，但Bcl-2、Bax高表达时的活性状态及其对OA所诱导细胞损伤的功能意义还有待证实。
离体实验部分Bel-2在OA诱导体外培养神经细胞退化中的作用分析
为了进一步探讨OA致神经细胞损伤过程中Bcl-2表达上调的功能意义，采用OA诱导人神经母细胞瘤SH-SY5Y细胞退化的离体模型，应用脂质体介导质粒DNA转染细胞的方法、DNA荧光染料Hoechst 33258染色法、MTT还原法，首先明确OA引起SH-SY5Y细胞凋亡性退化，鉴定质粒转染引起SH-SY5Y细胞外源性过表达功能性Bcl-2，进而研究Bcl-2过表达对OA所致体外培养神经细胞退化的影响。结果如下：
1．MTT还原法对细胞活力的检测显示，OA剂量依赖性地降低SH-SY5Y细胞活力，其中60nM／80nM／100nM OA作用24h显著降低细胞活力(60nM、80nM，p＜0.05；100nM，p＜0.01)；Hoechst 33258染色显示，80nM OA作用24h引起该种细胞出现多量核浓聚、核碎裂的凋亡样核型；
2．脂质体介导pEGFP-N1-Bcl-2和空质粒pEGFP-N1瞬时转染SH-SY5Y细胞，转染效率分别为2.01％和2.18％，细胞分别过表达Bcl-2-GFP和GFP蛋白，Bcl-2-GFP融合蛋白主要分布于近核的细胞胞体，而GFP蛋白则在胞质和胞核中均有分布；Hoechst 33258染色法显示，200nM促凋亡剂星形孢菌素(STS)作用细胞24h，Bcl-2重组质粒转染组的细胞凋亡率显著低于空质粒转染组的细胞凋亡率(p＜0.01)；
3．Hoechst 33258染色法显示，80 nM OA作用细胞24h，Bcl-2重组质粒转染组的细胞凋亡率显著低于空质粒转染组的细胞凋亡率(p＜0.05)。
这部分结果提示，OA剂量依赖性引起体外培养神经细胞SH-SY5Y凋亡性退化，Bcl-2重组质粒瞬时转染引起该种细胞过表达功能性融合蛋白Bcl-2-GFP，Bcl-2过表达能部分抑制OA所致SH-SY5Y细胞退化，这显示Bcl-2在OA所致神经细胞退化过程中可能起重要的调节作用，Bcl-2表达上调在OA损伤通路中具有细胞保护作用。
结论
1．大鼠脑额叶皮质定位注射OA能诱导该区神经细胞Bcl-2和Bax表达上调，且呈时间和剂量反应特征，二者的表达上调可能与OA致tau蛋白高度磷酸化有关，OA作用皮质区域AT-8、Bcl-2、Bax表达三者共存或AT-8表达仅与Bcl-2表达二者共存，这提示Bcl-2家族活跃参与OA所致神经细胞退化过程。
2．外源性Bcl-2高表达能部分抑制OA所致体外培养神经细胞SH-SY5Y凋亡性退化，提示Bcl-2相关凋亡调节机制参与了OA所致神经细胞退化过程，在该损伤通路中Bcl-2表达上调具有细胞保护的功能意义。
3．综合分析整体与离体实验的结果，提示Bcl-2相关凋亡调节机制参与OA所致神经细胞退化过程，OA引起的Bcl-2表达上调可能是机体神经细胞针对损伤信号的自身代偿性保护机制。
Apoptosis involves in progressive neurodegeneration in AD. Bcl-2 family plays important role in apoptotic signal transduction pathway. The family members are classified as two kinds according to their opposite functions, pro-apoptotic or anti-apoptotic activity. Bcl-2 and Bax are representative of anti-apoptotic and pro-apoptotic proteins, respectively. The anti-apoptotic activity of Bcl-2 associates closely with the pro-apoptotic activity of Bax. Occurrence of cell apoptosis is decided by the ratio of Bcl-2 versus Bax under certain physiological and pathological conditions. Okadaic acid (OA), a protein phosphatase inhibitor, induces microtubule-associated protein tau hyperphosphorylation both in vitro and in vivo, which is considered to associate with pathogenesis of neurodegeneration. Apoptosis is responsible for neurodegeneration induced by OA in vitro. It has also been found that apoptosis-like DNA double strand damage appeared in OA-induced neurodegeneration in vivo. In the present study, microinjection of OA into rat frontal cortex was used to explore the possible roles of Bcl-2-related apoptotic regulatory mechanism in OA-induced neurodegeneration process in vivo. Immunohistochemistry was applied to observe alteration of Bcl-2 and Bax expression following OA injection. Triple immuno-fluorescent labeling combined with cofocal laser scanning microscopic analysis was performed to illustrate the topographic relationship among tau hyperphosphorylation, Bcl-2 expression and Bax expression induced by OA. Furthermore, human SH-SY5Y neuroblastoma cells were applied to investigate the influence of exogenous Bcl-2 overexpression on apoptotic neuronal death induced by OA. The results are as follows:
Experiment in vivo Effect of Bcl-2 on OA-induced neurodegeneration in rat brain
Bcl-2 immunostaining showed that (1) the number of Bcl-2 positive staining cells significantly increased at 12 h, peaked at 1 d, then decreased at 3 d after injection of 20 ng OA compared with the number of Bcl-2-positive cells after vehicle
injection (p<0.001); 12 h, 1 d and 3 d of 20 ng OA treatment significantly increased the number of Bcl-2-positive cells compared with 3 h, 6 h and 7 d of 20 ng OA treatment (12 h, p<0.01; 1 d, 3 d, p<0.05); (2) compared with vehicle injection, injection of, 20 ng, 50 ng and 100 ng OA significantly increased the number of Bcl-2-positive cells at 1 d (20 ng,p<0.01; 50 ng, 100 ng,p<0.05); compared with 10 ng OA injection, injection of 20 ng and 100 ng OA also significantly increased the number of Bcl-2-positive cells at 1 d (p<0.05)
Bax immunostaining showed that (1) the number of Bax-positive cells also significantly increased at 12 h, peaked at 1 d, then decreased at 3 d after injection of 20 ng OA compared with the number of Bax-positive cells after vehicle injection (p<0.01); (2) injection of 50 ng and 100 ng OA tended to elevate the number of Bax-positive cells at 1 d, but not reach statistical level.
Immunohistochemistry of adjacent brain slices from the same rat delineated that the distributional profiles of AT-8-, Bcl-2- and Bax-positive cells were similar. However, the distributional range and the number of these positive cells were obviously different from one another. Among them, the range of Bcl-2-positive cells was most widely and the number was largest, the range of Bax-positive cells was least widely and the number was smallest and these two indices of AT-8-positive cells were in the middle.
Triple immuno-fluorescent staining delineated that Bcl-2 expression co-stained with AT-8 and Bax expression or only co-stained with AT-8 expression in the affected area more adjacent to OA injected site. But in the affected area less neighboring to OA injected site, only Bcl-2 immunoreactivity was observed, but neither AT-8 nor Bax immunoreactivity.
Experiment in vitro Effect of Bcl-2 overexpression on OA-induced cell death in SH-SY5Y cells
OA decreased cell viability of SH-SY5Y cells in dose dependent manner. 60 nM, 80 nM and 100 nM OA significantly reduced cell viability after 24 h of treatment (60 nM, 80 nM,p<0.05; 100 nM,p<0.01). Hoechst staining showed that a lot of chromatin condensation and fragmentation typical of apoptosis appeared following 24 h of 80 nM OA treatment.
The transfection efficiency of recombinant plasmid pEGFP-N1-Bcl-2 and empty plasmid pEGFP-N1 was 2.01% and 2.18% respectively. Exogenous Bcl-2-GFP induced by recombinant plasmid transfection mainly resided in peri-nucleus cell bodies, while Exogenous GFP induced by vector transfection located in cytosol, nuclei and processes. Hoechst staining showed that apoptotic Bcl-2 transfected cells were markedly less than apoptotic vector transfected cells after 24 h of 200 nM staurosporine (STS) exposure (p<0.01).
Hoechst staining further revealed that apoptotic Bcl-2 transfected cells were markedly less than apoptotic vector transfected cells after 24 h of 80 nM OA exposure (p<0.05).
Conclusion
1. Microinjection of OA into rat frontal cortex upregulated Bcl-2 and Bax protein expression in affected brain area. Moreover, induction of Bcl-2 and Bax expression by OA had temporal change and dose response feature. The upregulation of these two apoptotic regulatory protein probably attributed to tau hyperphosphorylation stimulated by OA. In affected cortex by OA, AT-8, Bcl-2 and Bax expression co-stained or AT-8 and Bcl-2 expression co-stained, suggestive of Bcl-2 family actively participated in OA-induced neurodegeneration process.
2. Exogenous Bcl-2 overexpression partially inhibited OA-induced apoptosis in SH-SY5Y neuronal cells, suggesting that Bcl-2 related apoptotic regulatory mechanism involved in OA-induced neurodegenerative pathway and that Bcl-2 had neuronal protective role in it.
3. Taken together, the results in vivo and in vitro indicated that Bcl-2 related apoptotic regulatory mechanism participated in OA-induced neurodegeneration and that the upregulation of Bcl-2 protein expression probably represented neuronal compensatory mechanism in response to OA toxicity to neuronal cytoskeleton.
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