基于自噬Akt-mTOR和AMPK信号通路探讨清热解毒法对AS的影响

Atherosclerosis (AS) plaque instability is the common basis of acute cardiocerebrovascular diseases. Moderate autophagy of macrophages can reduce macrophage apoptosis and necrosis, enhance the ability of macrophages to clean apoptosis and necrosis cells, reduce inflammation factor release in plaque, and finally, stable the plaques. In the progressed AS plaques, the autophagy of macrophages become dysfunctional, which is one of the main cause lead to unstable plaque. In previous research, we found that Qingrejiedu Chinese traditional medicine can reduce the macrophage apoptosis and necrosis in AS plaque, reduce the release of proinflammatory factor, and stable plaques. Based on the close relationship between apoptosis, inflammation, plaque stability and autophagy, we hypothesized that: the effect of Qingrejiedu Chinese medicine on anti-apoptosis, anti-inflammatory and stable plaques ,is due to the effect of Qingrejiedu Chinese medicine on repairing the autophagy of macrophage in progressed AS plaques. To this end, we proposed by: 1, determine the effect of Qingrejiedu Chinese medicine.on autophagy of macrophages in progressed AS plaque; 2, determine the relationship of the effect of Qingrejiedu Chinese medicine on autophagy of macrophages , and the degree of apoptosis or necrosis, ability of macrophages to remove apoptosis or dead cells, the level of inflammation and plaque stability ; 3, clarify the specific signaling pathways of Qingrejiedu Chinese medicine on regulating autophagy, which will provides theory basis in preventing and controling AS associated cerebrovascular disease with heat-clearing and detoxifying method .

动脉粥样硬化（AS）斑块不稳定是急性心脑血管疾病发生的共同基础。巨噬细胞适度的自噬，能减少斑块内巨噬细胞凋亡、坏死，增强巨噬细胞清除凋亡、坏死细胞的能力，减少炎症因子释放，从而稳定斑块。晚期AS斑块内，巨噬细胞自噬功能受损，是导致斑块不稳定的主要病因之一。我们前期研究发现，清热解毒中药能减少晚期AS斑块内巨噬细胞凋亡和坏死，减少炎症因子释放，稳定斑块。基于自噬与细胞凋亡、炎症和斑块稳定性的密切关系，我们推测：“清热解毒中药可能通过改善晚期AS斑块内巨噬细胞的自噬功能，从而发挥抗凋亡、抗炎和稳定斑块的作用”。为此，我们拟通过：1、确定清热解毒中药对晚期AS斑块内巨噬细胞自噬功能的影响；2、确定该影响与巨噬细胞凋亡坏死程度、巨噬细胞清除凋亡坏死细胞的能力、斑块内炎症水平和斑块稳定性等之间的关系；3、阐明清热解毒中药调节自噬的具体信号通路，从而为清热解毒法防治AS相关心脑血管疾病提供理论依据。

背景：动脉粥样硬化（AS）斑块不稳定是急性心脑血管疾病发生的共同基础。巨噬细胞适度的自噬能减少斑块内巨噬细胞凋亡、坏死，减少炎症因子释放，从而稳定斑块。晚期AS斑块内，巨噬细胞自噬功能受损，是导致斑块不稳定的主要病因之一。我们前期研究发现，清热解毒中药能减少晚期AS斑块内巨噬细胞凋亡和坏死，减少炎症因子释放，稳定斑块。基于自噬与细胞凋亡、炎症和斑块稳定性的密切关系，我们推测：“清热解毒中药可能通过改善晚期AS斑块内巨噬细胞的自噬功能，从而发挥抗凋亡、抗炎和稳定斑块的作用”。.主要研究内容：①黄连解毒汤对兔晚期动脉粥样硬化斑块稳定性影响 ②黄连解毒汤对兔晚期动脉粥样硬化炎症水平，凋亡水平和自噬水平的影响。③细胞实验探讨黄连解毒汤干预兔晚期动脉粥样硬化的可能机制。.重要结果：①黄连解毒汤能够改善兔晚期动脉粥样硬化斑块稳定性；②黄连解毒汤能够改善兔晚期动脉粥样硬化炎症和凋亡水平，增加自噬水平；③黄连解毒汤能够通过抑制PI3K-Akt-mTOR通路增加自噬从而改善兔动脉粥样硬化。.关键数据：①血管内超声提示黄连解毒汤能够增加兔晚期动脉粥样硬化斑块稳定性 ②TUNEL染色、ELISA检测分别提示黄连解毒汤能够改善兔晚期动脉粥样硬化炎症和凋亡水平，免疫组化及WB提示连解毒汤能够增加兔晚期动脉粥样硬化自噬水平；③细胞实验中，WB检测表明模型组较正常组P-PI3K/PI3K、P-Akt/Akt、P-mTOR/mTOR表达水平均明显升高，HL干预组与模型组相比P-PI3K/PI3K、P-Akt/Akt、P-mTOR/mTOR表达水平均降低；加入通路蛋白激动剂740Y-P、SC79、MHY后，其与HL组相比自噬蛋白表达水平均明显升高。.科学意义：清热解毒中药黄连解毒汤是能够改善晚期AS斑块中巨噬细胞的自噬功能，它的可能的分子机制为PI3K-Akt-mTOR通路；使用药物阻断巨噬细胞自噬后，清热解毒中药的“增强晚期AS斑块中巨噬细胞清除凋亡、坏死细胞的能力，减少巨噬细胞凋亡和坏死，减少炎症因子释放