基于PI3K/PDK1/nPKC/Rac1通路研究三焦针法改善SAMP8小鼠认知功能障碍的作用机制

Many clinical practice and experimental research show that the SanJiao (triple energizer) acupuncture treatment of AD have certain curative effect. Previous basic experiment in SAMP8 mice, proved that the acupuncture can improve cognitive dysfunction and inhibit cell apoptosis and neuron loss. But the study of the internal mechanism of acupuncture inhibiting cell apoptosis and neuron loss has not yet clear. The latest research shows that A beta 1-42 as dementia main nerve toxicity factor, by activating PI3K/PDK/PKC in the cell/Rac1 signaling pathways induced apoptosis. Based on this, we propose the hypothesis that acupuncture inhibited cell apoptosis may improve cognitive function by regulating the signal transduction pathway. To test this hypothesis, this project applicated specific inhibitors to block the expression of PI3K/PDK1, nPKC, Rac1, in ordor to set up signaling pathway block model, then respectively from neurological behavior and morphology, applying pathology, immunology and molecular biology technology, to analyse intervention effect of acupuncture on signal molecules before and after block. This research will deepen the understanding of mechanism of resisting brain aging by acupuncture, and provide a new theoretical basis for the prevention and treatment of acupuncture used in AD.

许多临床实践及实验研究表明，三焦针法治疗AD具有肯定的疗效。前期基础实验以SAMP8老化鼠作为研究对象，表明了该针刺方法能够改善老化鼠认知功能障碍，并抑制细胞凋亡及神经元的丢失。然该针刺方法如何抑制细胞凋亡及神经元丢失的内在机制的研究尚未清晰。最新的研究显示Aβ1-42作为痴呆主要的神经毒性因子，通过激活细胞内PI3K/PDK/PKC/Rac1信号通路诱导细胞凋亡。基于此，我们提出针刺抑制细胞凋亡改善认知功能可能是对该信号转导通路调控的假说。为了证实这一假说，本课题拟应用特异性抑制剂分别阻断PI3K/PDK1、nPKC、Rac1的表达，建立信号通路阻断模型，再分别从神经行为学和形态学上，利用病理学、免疫学、分子生物学等技术，探讨针刺对信号分子阻断前后的干预效应。本项目研究将深化针刺抗脑衰老机理的认识，为针刺应用于AD的防治提供新的理论依据。

目的：观察针刺治疗对快速老化痴呆模型（SAMP8）小鼠认知功能障碍及细胞超微结构影响，探讨针刺调控PI3K/PDK1/nPKC/Rac1信号通路及实现改善认知障碍的作用机制。方法：将SAMP8小鼠随机分成模型组、P8阻断剂组（OSU03012、Rottlerin及NSC23766）、P8针刺组、P8针刺非穴位组、P8阻断剂（OSU03012、Rottlerin及NSC23766）针刺组、P8阻断剂（OSU03012、Rottlerin及NSC23766）针刺非穴组，采用侧脑室注射阻断剂建立P8阻断剂模型小鼠，治疗后采用HE染色、TUNEL、电镜等观察细胞形态、细胞凋亡及细胞超微结构；Western blot技术检测PI3K、PDK1、nPKC及Rac1表达的变化。结果：与SAMR1正常组比较，其它各组均出现不同程度认知障碍、细胞凋亡及细胞超微结构的改变；与P8模型组比较，P8针刺组细胞凋亡减少，细胞结构改变损害明显减轻（P<0.01），且均与认知障碍密切相关；与P8阻断剂模型组比较，P8阻断剂针刺组细胞凋亡及细胞结构损害无显著差异（P>0.05）；与P8模型组比较，P8阻断剂模型组细胞凋亡及细胞结构损害有显著差异（P<0.05），结论：针刺通过调控细胞内PI3K/PDK1/nPKC/Rac1信号通路抑制细胞凋亡，从而起到改善SAMP8认知功能障碍的作用。