基于PI3K/AKT/mTOR/自噬通路研究回药药对“胡椒+荜茇”干预缺血性脑卒中的作用机制

At present, stroke is one of the most serious diseases that have been harming to human beings. Some studies have shown that the autophagy pathway of PI3K/Akt/mTOR is the key target for ischemic brain damage. Our previous studies showed that the effective part of “Piper nigrum L + Piper longum L” has a strong protective effect on cerebral ischemic injury in rats (the National Natural Science Foundation, No. 81360666). On the basis of this, the present study suggested that the effective part of “Piper nigrum L + Piper longum L” could inhibit the autophagy of cerebral ischemia after activation of PI3K/Akt/mTOR signaling pathway, so as to achieve neuroprotective hypothesis. Firstly, the HPLC-Q-TOF-MS technique was used to characterize the chemical components of the effective part. At the same time, a variety of column chromatography techniques were used to separate the typical active components. Then, the permanent focal cerebral ischemia (pMCAO) model of middle cerebral artery in rats will be used to explore the protective effect of effective part and typical active components on cerebral neurons. Observation of the ultrastructure of autophagy by transmission electron microscopy. Western Blot and immunofluorescence staining are applied to detect the expression of autophagy in different time and different cells. The expression of key protein and its phosphorylation level in PI3K/AKt/mTOR signaling pathway will be detected by immunohistochemistry,Western Blot and RT-PCR. This program will discuss the molecular mechanism of the effective part and typical active components, and provide a powerful data support for the potential treatment of new drug development in stroke.

脑卒中是目前危害人类最严重的疾病之一，研究表明PI3K/Akt/mTOR/自噬通路是缺血性脑损伤的关键靶点。前期研究发现“胡椒+荜茇”有效部位对大鼠缺血性脑损伤具有较强的保护作用（国家自然科学基金：81360666）。基于此本研究提出“胡椒+荜茇”有效部位及典型活性成分通过激活PI3K/Akt/mTOR通路，抑制脑缺血损伤后的细胞自噬达到神经保护作用。首先运用HPLC-Q-TOF-MS技术表征有效部位化学成分，同时分离制备典型活性成分；之后采用pMCAO动物模型，探讨有效部位及典型活性成分对大鼠大脑神经元的保护作用；透射电镜观察自噬的超微结构；Western Blot等方法检测自噬标志蛋白在不同时间点、不同细胞中的表达；多种方法检测PI3K/AKt/mTOR通路关键蛋白及其磷酸化水平的表达，探讨有效部位及典型活性成分神经保护的作用机制，为潜在的治疗脑卒中新药研发提供数据支撑。

项目最终结论如下：.本项目分离了药对“胡椒+荜茇”有效部位及典型活性成分；明确了药对“胡椒+荜茇”有效部位及典型活性成分的神经保护作用；证实了“胡椒+荜茇”有效部位、胡椒碱和四氢胡椒碱通过激活 PI3K/Akt/mTOR信号通路，抑制过度自噬，从而对缺血性脑卒中起到神经保护的作用。具体如下：.1. “胡椒+荜茇” 有效部位富集、化学成分表征及典型活性成分制备方法，可得到实验所需“胡椒+荜茇”二氯甲烷有效部位、单体化合物胡椒碱和四氢胡椒碱。.2.“胡椒+荜茇”二氯甲烷有效部位、胡椒碱和四氢胡椒碱可降低神经功能分数，有效缓解行为学障碍，增加身体协调性和运动能力及力量，抑制脑组织损伤，减轻大鼠脑梗死体积，改善细胞形态汇和神经元结构，对缺血性脑卒中后，可抑制脑组织损伤，加速了组织修复，具有神经保护及修复作用。.3.“胡椒+荜茇”有效部位通过上调PI3K、p-mTOR蛋白表达，下调p-AKT、蛋白表达，并可显著增加mTOR活性，下调mTOR下游自噬蛋白Atg7、Atg5、Atg12、LC3、Beclin1的蛋白表达；胡椒碱通过下调p-PI3K、p-AKT及p-mTOR蛋白表达，抑制LC3-I向LC3-II的转化，下调Beclin-1的蛋白表达；四氢胡椒碱及阻断剂上调P-mTOR/mTOR蛋白表达，下调自噬蛋白Atg7表达。“胡椒+荜茇”有效部位、胡椒碱和四氢胡椒碱具有神经保护作用，其神经保护作用与激活PI3K/AKT/mTOR通路，抑制过度自噬，改善脑缺血损伤有关。.4.这一结果为“胡椒+荜茇”典型活性成分治疗脑卒中作用靶点的研究提供了新的见解，并对其潜在机制提供了思路。