基于Cofilin线粒体转位探讨电针改善脑缺血大鼠运动功能障碍的机制研究

Our previous studies combing the ancient literature have found that acupuncture at Zusanli (ST 36) and Quchi (LI 11) can effectively improve motor dysfunction after cerebral ischemia. The recent fMRI research also demonstrated EA at ST 36 and LI 11 could enhance the neural activity of motor function-related brain regions in cerebral ischemia and MCAO rats, and have a significant anti-apoptotic effect. Cofilin mitochondrial translocation-mediated apoptosis is an important pathological mechanism of cerebral ischemia. It is widely accepted that saving penumbral cells is the key to current clinical treatment of cerebral ischemia. Based on these, we suppose that EA could improve the recovery of motor dysfunction through regulating mitochondrial translocation of cofilin after cerebral ischemia. Therefore, the current study intends to reveal the possible molecular mechanism of EA against apoptosis in MCAO rats from different aspects of the whole body, tissues, cells and molecules by using small animal MRI live imaging combined with brain stereotaxic injection, behavioral test, immunofluorescence, transmission electron microscopy, western blot and Q-PCR techniques. By using the “Cofilin mitochondrial translocation” as breakthrough point, we will partially elucidate the mechanism of EA to improve motor dysfunction after cerebral ischemia and provide a new theoretical basis for the clinical application of EA, so as to give full play to the advantages of traditional Chinese medicine in the treatment of cerebral ischemia.

团队在前期临床筛选和文献梳理中发现，针刺“曲池”、“足三里”穴能够有效改善脑缺血后运动功能障碍。我们最新fMRI研究也显示，电针“曲池”、“足三里”穴可增强脑缺血患者和MCAO大鼠运动相关脑区的神经功能活动，同时具有显著的抗凋亡作用。Cofilin线粒体转位介导的细胞凋亡是脑缺血的重要病理机制，而挽救缺血半暗带细胞凋亡是目前临床治疗脑缺血的关键。这些提示电针可能通过调控Cofilin线粒体转位介导的细胞凋亡来改善MCAO大鼠的运动功能障碍。因此，本研究拟采用小动物MRI活体成像结合脑立体定位注射、行为学检测、免疫荧光、透射电镜、Western blot与Q-PCR等技术，从动物整体、组织、细胞和分子等不同层面，以Cofilin线粒体转位为切入点揭示电针对MCAO大鼠的抗凋亡分子机制，部分阐明其改善脑缺血后运动功能障碍的作用机制，为电针的临床应用提供理论新依据，充分发挥中医药治疗该病的优势。

Cofilin线粒体转位介导的细胞凋亡是脑缺血引起运动功能障碍的重要病理机制。我们前期研究表明电针曲池、足三里穴能够增强脑缺血患者和MCAO大鼠运动相关脑区的功能活动，且具有显著的抗凋亡作用。电针该组穴治疗脑缺血后运动功能障碍，既符合祖国传统医学“治痿独取阳明”的理论学说，又有确切的临床疗效和扎实的研究基础。因此，电针曲池和足三里穴可能通过调控Cofilin线粒体转位介导的细胞凋亡改善MCAO大鼠的运动功能障碍。本项目采用小动物MRI活体成像结合脑立体定位病毒注射、细胞转染、行为学检测、免疫荧光、电镜、Western blot、Q-PCR和全细胞膜片钳等技术，从动物整体、组织、细胞和分子等不同层面，以Cofilin线粒体转位为切入点进一步揭示了脑缺血的病理机制并部分阐明电针改善脑缺血后运动功能障碍的作用机制。.我们研究发现：（1）Cofilin对脑缺血后Rod形成和细胞凋亡具有重要作用，可作为脑缺血治疗靶点。①体外（ATP耗竭诱导原代海马神经元）和体内（MCAO）的缺血/缺氧损伤均可诱导Cofilin rod结构形成和神经元细胞凋亡；②In vitro实验结果显示：利用Rho激活剂（CN03）增强Limk1活性，可抑制原代海马神经元Cofilin过表达及ATP耗竭引起的Cofilin rod形成，甚至消融过表达已产生的Cofilin rod，而Rock抑制剂（Y-27632）抑制Limk1活性则可逆转上述情况。另外，RFP-Limk1过表达能够显著改善ATP耗竭引起的GFP-Cofilin神经元mEPSCs频率下降。③In vivo实验结果显示：Cofilin rod在MCAO大鼠脑梗死核心区2h和半暗带8h开始生成，24h达到峰值，然后在MCAO后7d减少。而脑梗死半暗带定位注射RFP-Limk1过表达病毒抑制Cofilin过度激活，可显著减少脑缺血后皮质半暗带Rod形成、MAP2降解和Cofilin线粒体转位介导的细胞凋亡。.（2）电针通过调控Limk1/SHH1抑制Cofilin的线粒体转位，减少Cofilin rod形成和MAP2降解，减轻缺血性卒中的脑损伤和细胞凋亡，从而促进MCAO大鼠运动功能修复。这可能是电针疗法的新机制，将为电针的临床应用提供理论依据。