逆针通过PI3K/Akt通路影响NCX时空应答增加脑缺血耐受性的细胞电生理机制

Stroke poses a severe threat to public health due to its high morbidity, disability, morbidity and recurrence rate, and prevention is the best way to ease such burden. Preventive acupuncture could prevent disease before its onset, for instance, electroacupuncture pretreatment could induce cerebral ischemic tolerance via adaptive regulation. Unbalanced ion homeostasis is one of the key factors leading to post-ischemia cerebral damage. However, mechanism of preventive acupuncture in cerebral ischemia has never been explored through the aspect of ion homeostasis regulation. Cytomembrane Na+/Ca2+ exchanger (NCX) could promote calcium efflux and therefore protect neuron through spatial and sequential response to ischemia attack. NCX calcium current could be monitored using advanced visible brain slice whole-cell patch-clamp in real time..With the purpose of illustrating role and regulation mechanism of NCX response in cerebral protection of preventive acupuncture, MCAO and OGD will be used as in vivo and in vitro model of cerebral ischemia, firstly, to evaluate effect of preventive acupuncture from perspectives of neurological behavior, histomorphology and pathology through 18-points scale, TTC staining, and double-staining flow cytometry; to analyze whether preventive acupuncture promote post-ischemia calcium efflux via NCX regulation though whole-cell patch-clamp and calcium image techniques; to analyze whether preventive acupuncture regulates post-ischemia NCX response to protect cerebral function though stimulating PI3K/Akt pathway though shRNA interference, IHC and WB techniques; and furthermore, to provide laboratory evidence for preventive acupuncture.

脑卒中发病率、致残率、致死率和复发率高，严重危害人类健康，预防是减轻疾病负担的最佳途径。逆针可未病先防，通过电针预处理诱导适应性调节增加脑缺血耐受性。离子稳态失衡是致缺血性脑损伤的关键因素，但从这一角度探讨逆针脑保护效应的研究尚属空白。质膜钠钙交换体NCX可通过应激性时空应答加速钙外排保护神经元，利用可视化脑片全细胞膜片钳技术可实现NCX钙电流的动态监测。.为明确细胞膜NCX时空应答在逆针脑保护效应中的作用及调节机制，研究将以MCAO和OGD为在体、离体脑缺血模型，以电针预处理为干预手段，利用双染流式细胞术等技术从病理学等方面评估逆针的脑保护效应；利用脑片全细胞膜片钳和钙离子成像技术从细胞电生理角度分析逆针是否通过NCX加速缺血后钙外排；利用shRNA干扰、IHC和WB等技术，分析逆针是否通过激活PI3K/Akt通路调节缺血后NCX时空应答，发挥脑保护作用，为逆针的应用提供实验依据。

背景：脑卒中严重危害人类健康，预防是减轻疾病负担的最佳途径。逆针可未病先防，通过电针预处理诱导适应性调节增加脑缺血耐受性。离子稳态失衡是致缺血性脑损伤的关键因素，但从这一角度探讨逆针脑保护效应的研究尚属空白。质膜钠钙交换体NCX可通过应激性时空应答加速钙外排保护神经元。因此，本研究拟探讨细胞膜NCX时空应答在逆针脑保护效应中的作用及调节机制。.内容：以MCAO和OGD为在体、离体脑缺血模型，以电针预处理为干预手段，利用双染流式细胞术等技术从病理学等方面评估逆针的脑保护效应；利用脑片全细胞膜片钳技术从细胞电生理角度分析逆针是否通过NCX加速缺血后钙外排；利用shRNA干扰、IHC和WB等技术，分析逆针是否通过激活PI3K/Akt通路调节缺血后NCX时空应答，发挥脑保护作用，为逆针的应用提供实验依据。.结果：电针预处理可减轻缺血后神经功能缺损，减轻脑梗死容积百分比，降低神经细胞凋亡率；电针预处理可以维持脑缺血后细胞膜NCX通道的总体钙外排特性，促进缺血后钙离子外排；缺血后电针预处理可上调脑区NCX1和NCX3蛋白表达，而这一调节效应呈现区域特异性和时间特异性；而NCX1或NCX3沉默均可阻断电针预处理的脑保护作用；抑制PI3K/Akt信号通路可阻断电针预处理对NCX表达的影响及其脑保护作用。.意义：创新性地将脑片全细胞膜片钳技术及shRNA干扰技术应用于针刺研究，发现电针预处理通过激活PI3K/Akt通路，特异性上调脑区细胞膜主要钙外排通道蛋白NCX的表达，促进缺血后钙离子外排，减轻细胞钙超载，增加脑缺血耐受性。本研究是首次从钙离子稳态角度探讨电针预处理的脑保护机制，为后续从离子稳态角度系统性认识针刺预防性脑保护效应的作用机制奠定基础。