基于Cdk5介导的凋亡与自噬交互作用探讨补阳还五汤类方抗脑缺血作用机制

The thin recipe which was simplified from Buyang Huanwu Decoction based on the traditional medicine theory of Supplementing qi, removing blood stasis and dredging collateral for promoting tissue regeneration, present remarkable effects on cerebral ischemic injury. However, the mechanism remains unknown. Cdk5, as a key neuronal regulator, received extensively attention. In our previous experiments, we have observed lower expression of Cdk5 and Tau, as well as less Caspase 3 and the ratio of Bax/Bcl-2 in Buyang Huanwu associated prescriptions groups than model groups. There were signal transductions between Cdk5 and Akt kinase been reported, which could play double regulation on apoptosis and autophagy. Owing to the importance of the intercross relation between the two programmed cell death paths, we raised the view as follows: Apoptosis and autophagy which mediated by Cdk5 cleared away the impaired cells together at the initial stage of cerebral ischemia. While the abnormal activation of Cdk5 would inhibit the Akt kinase, that signal transduction could promote apoptosis and restrain autophagy, the intercross between apoptosis and autophagy performed as competition with the aggravation of cerebral damage. Buyang Huanwu associated prescriptions could play the neuroprotective effects by Cdk5 signal pathway to affect the co-regulators such as Akt、Bax、Caspase-3、Bcl-2 and Beclin1 after cerebral ischemia. In this research, we would reveal the effects of Cdk5 on Akt kinase dynamically, and discuss the double regulation mechanism of co-regulators between apoptosis and autophagy after cerebral ischemia, also illuminate the mechanism of Buyang Huanwu associated prescriptions by this signal path, which demonstrate the reasonability and effectiveness of this new recipe simplified from Buyang huanwu decoction.

基于益气祛瘀生新法建立的补阳还五汤类方抗脑缺血损伤功效显著，然其作用机制尚未明确。Cdk5作为神经元的关键调控子颇受关注，前期实验初步表明，补阳还五汤类方能有效干预Cdk5和凋亡因子的表达。据报道，Cdk5与Akt激酶间存在信号传导，且Akt激酶对凋亡和自噬具有双重调控作用，明确凋亡与自噬的关系对脑缺血后神经元的调控具有重要意义，故本项目提出：在脑缺血初期，Cdk5介导二者以合作方式共同清除受损细胞；随着脑缺血进程，Cdk5的异常活化将抑制Akt活性，从而促进凋亡、抑制自噬，二者的交互作用方式主要表现为对抗；补阳还五汤类方可通过调控Cdk5介导凋亡与自噬的共享调节子（如Akt、Bax、Caspase-3、Bcl-2和Beclin1等）进而干预二者的交互作用方式，有效防止脑损伤。本研究采用分子生物学方法，从体内体外两角度动态展开实验，从分子层面阐明补阳还五汤类方抗脑缺血的科学性和合理性。

前期研究已证实，基于益气祛瘀生新法建立的补阳还五汤类方抗脑缺血损伤功效显著，然其作用机制尚未明确，本项目分别以MCAO大鼠模型和氧化应激PC12模型细胞为研究对象，评价补阳还五汤类方对MCAO模型大鼠海马组织Cdk5的调控，并从凋亡与自噬的调控探讨该方提取物对PC12细胞氧化应激模型的保护机制。将雄性SD大鼠随机分为假手术组、MCAO模型组、补阳还五汤原方干预组及补阳还五汤精简方干预组，每组按1d、5d、7d再各分3小组。TTC染色、尼氏染色和电镜观测结果显示补阳还五汤类方能有效降低MCAO大鼠的脑梗死比、保护海马神经元的活性和改善海马神经细胞中的亚细胞器形态、降低自噬水平，免疫组化法、Western blot法和RT-PCR法的检测结果显示：MCAO模型大鼠海马组织中，脑缺血后1d即出现Cdk5上调趋势，并随着脑缺血的加剧，Cdk5的表达继续上调，其中mRNA及蛋白水平在7d到达最大，与各时间点假手术组比P<0.01，提示脑缺血损伤将激活Cdk5信号转导途径；补阳还五汤类方干预后均能显著降低Cdk5的表达，与各时间点模型组比P<0.05，且随着干预时间的延长，下调趋势越明显，提示补阳还五汤类方可下调Cdk5的异常表达，与补阳还五汤原方比，两方对Cdk5的调控无统计学差异（P>0.05）。体外细胞实验分别以0.5、2.0 mmol/L H2O2处理PC12细胞制备氧化应激损伤的初期模型和加剧期模型；与对照组比，模型组细胞凋亡率增加，自噬程度加剧，Bax/Bcl-2比例增加，Beclin1和LC3B表达上调，LC3A表达下调（P＜0.05）；与氧化应激损伤初期的模型1组比较，补阳还五汤类方提取物均能下调Bax/Bcl-2，抑制凋亡率，并一定程度上调Beclin1和LC3B/LC3A的表达，激活自噬（P＜0.05）；当氧化应激损伤程度加剧时，模型细胞中出现较高水平的凋亡与自噬，此时，两方则通过抑制凋亡和降低自噬，共同发挥保护作用。综上，补阳还五汤类方可能通过干预Cdk5信号转导途径对脑缺血海马组织发挥保护作用，并可通过对凋亡与自噬的交互动态调控对不同程度损伤的氧化应激模型细胞挥发保护作用，本研究进一步从分子层面阐明补阳还五汤类方抗脑缺血的科学性和合理性。