益气通阳活血和清热解毒通络两种治法对脑梗死后神经元自噬动态调节作用的比较研究

Cerebral infarction is characterized by high morbidity, mortality and disability rate, which cannot be treated effectively by western medicine besides thrombolytic therapy. However, traditional Chinese medicine has certain advantages in treating cerebral infarction with the effect of YiqiTongyangHuoxue（invigorating qi, and activating yang and blood circulation）and QingreJieduTongluo（clearing heat, removing toxicity and dredging collaterals）. The therapies of invigorating qi and clearing heat seem to have opposite efficacy, but both of them work. Then what is the underlying mechanism of the different therapies in treating the same disease？Is it related to the dynamic bidirectional regulation on autophagy? The answers of the above questions will help to understand the mechanism of traditional Chinese medicine in treating cerebral infarction, and has significance to improve the efficacy. The present subject choosesSailuotongcapsule and Tongluoqingnao injection, standing for the therapy of YiqiTongyangHuoxue and QingreJieduTongluo respectively. Through cerebral ischemic animal model and cell experiments in vitro, their intervention effects on neuronal autophagy are observed after ischemia and anoxiaat different time points. Autophagy inhibitor and inducer are used jointly to analyze the relevance between the regulation effect of traditional Chinese medicine on autophagy and the efficacy against cerebral ischemia. The related protein expression is tested to partly reveal the specific pathways of autophagy regulation. Thus, it is possible to explain the correlation between the efficacy against cerebral ischemia and the regulation effect of the medicine, to discuss the underlying mechanism of the different therapies in treating cerebral infarction, and to provide valuable reference for optimizing TCM treatment of cerebral infarction.

脑梗死具有发病率高、死亡率高、致残率高的特点，除溶栓外，尚无有效西药。以益气通阳活血或清热解毒通络为主要治法的中药，对脑梗死治疗具有一定优势。然而，两种治法，一者助阳为主，一者清热为主，看似相反，却都能起效，其同病异治的内在机制是什么？自噬是脑梗死的重要病理机制之一，对自噬的动态双向调节是抗脑缺血药理研究的热点方向，上述两种治法代表性中药对自噬表现出不同的调节作用。于是提出假说：不同时间点对神经细胞自噬的双向调节可能是两种治法对脑梗死同病异治的内在机制。本课题采用脑缺血大鼠和氧糖剥夺神经细胞模型，选用3种中药注射液代表上述两种治法，比较其在缺血/缺氧后不同时间点对神经细胞自噬的调节作用；联用自噬抑制剂和诱导剂，分析其调节自噬与抗脑缺血药效的关联性；检测相关通路蛋白，揭示其调节自噬的具体通路。由此可围绕自噬调节途径阐明两种治法对脑梗死同病异治的科学内涵，为脑梗死临床方案优化提供参考。

背景：脑梗死又称缺血性中风，缺血性中风占总中风患者的80%，其高致死致残率给社会及家庭带来沉重负担。自噬是近来关于脑缺血研究的热点，研究者发现不同功效中药对脑缺血后自噬具有抑制或促进不同的作用，益气活血通阳、清热解毒通络是中医临床治疗脑梗死的常用两种治疗方法，这两种几乎相反的两种的治法同对脑梗死具有显著疗效。主要研究内容：本课题采用脑缺血大鼠和氧糖剥夺PC12细胞模型，选用3种中药注射液代表上述两种治法，比较其在缺血/缺氧后不同时间点对神经细胞自噬的调节作用，并分析其调节自噬与抗脑缺血药效的关联性；通过检测相关通路蛋白，揭示其调节自噬的具体通路。关键数据：与模型组比较，HQXST和TLQN组脑梗死范围均显著减少(P<0.05，P<0.01)，神经行为学评分明显改善(P<0.01，P<0.05)。自噬发生在缺血后6h，在缺血12、24h时，与模型组比较，益气活血组，自噬水平显著升高。而清热解毒组从自噬起始阶段，显著抑制自噬。重要结果：经研究发现，益气活血通阳法不仅促进自噬小体的形成，还促进自噬小体的代谢，即整体自噬通量增加，这有助于受损细胞器的清除，同时避免了大量自噬小体的堆积，避免了自噬小体堆积导致的脑损伤。而清热解毒通络法从源头抑制自噬，整体自噬水平低，抑制了过度、致损伤的自噬。黄芪血塞通在缺血再灌注脑损伤中对神经元自噬及相关蛋白表达的影响结果与自噬诱导剂RAPA一致；通络清脑在缺血再灌注脑损伤中对神经元自噬及相关蛋白表达的影响结果与自噬抑制剂3-MA一致。科学意义：本课题证明了两种不同治法的代表中药对缺血再灌注大鼠的脑保护作用，发现了益气活血通阳与清热解毒通络两种治法对不同缺血时间神经细胞自噬的动态调节作用，并明确其抗脑梗死药效与调节自噬作用的相关性。从自噬水平阐释益气活血通阳与清热解毒通络两种治法治疗脑梗死的作用机制，通过分析两种治法对脑缺血后自噬调节作用的异同，部分解释中医对脑梗死同病异治的内在机制。