电针预处理通过抑制AMPK-Beclin1/Vps34通路介导的细胞自噬减轻脑缺血再灌注损伤

Electroacupuncture pretreatment reduces cerebral ischemia/reperfusion injury through inhibiting the AMPK-Beclin1/Vps34 pathway-mediated autophagy

摘要:

目的：探讨电针预处理治疗对大鼠脑缺血再灌注（ischemia/reperfusion，I/R）损伤的影响及其相关机制。方法：将健康成年雄性SD大鼠75只随机分为Sham组，脑缺血再灌注组（I/R组），电针预处理并缺血再灌注组（EA+I/R组），脑纹状体感染AAV-VC（空载体病毒）后再给予电针预处理并缺血再灌注组（AAV-VC+EA+I/R组）和脑纹状体感染AAV-Beclin1（过表达Be－clin1病毒）后再给予电针预处理并缺血再灌注组（AAV-Beclin1+EA+I/R组），每组15只大鼠。对各组大鼠脑中动脉梗死（middle cerebral artery occlusion，MCAO）术后1、2、3 d进行神经功能评分；在MCAO术后7 d处死大鼠，TTC染色以测定脑梗死体积；TUNEL染色法检测脑细胞凋亡状况；免疫印迹法检测自噬相关分子LC3-Ⅰ、LC3-Ⅱ、AMPK和Beclin1的蛋白表达水平；免疫共沉淀检测Beclin1与Vps34的相互作用。结果：与Sham组相比，I/R组脑梗死体积与神经行为学评分明显增加（均P<0.05）。机制研究显示缺血再灌注后LC3-Ⅱ/LC3-Ⅰ比值增加（P<0.05），且自噬调控蛋白AMPK与Beclin1上调表达（均P<0.05），Beclin1与Vps34复合物形成增多；与此同时，细胞凋亡数目明显增加。给予电针预处理后，上述指标均明显改善（均P<0.05）。而在电针预处理之前对自噬关键蛋白Beclin1进行脑内过表达可以逆转电针预处理对缺血脑损伤的保护作用（P<0.05）。结论：电针预处理可能通过抑制AMPK-Beclin1/Vps34通路减少自噬，发挥神经保护作用。

Abstract:

Objective：To explore the effect of electroacupuncture（EA） pretreatment on cerebral ischemia/reperfusion（I/R） injury in rats and its relevant mechanism. Methods：Seventy-five healthy adult male Sprague-Dawley rats were randomly divided into Sham group，I/R group，EA+I/R group，corpus striatum infection with adeno-associated virus-vector control（AAV-VC，an empty virus vector）+EA+I/R group，and corpus striatum infection with adeno-associated virus-Beclin1（AAV-Beclin1，a Beclin1-overexpressing virus）+EA+I/R group，with 15 rats in each group. The neurological function scores of the rats in each group were evaluated 1，2，and 3 days after a middle cerebral artery occlusion（MCAO）；the rats were sacrificed 7 days after the MCAO，and their cerebral infarct volumes were measured by TTC staining；cerebral cell apoptosis was evaluated by TUNEL staining；the protein expression levels of the autophagy-related molecules LC3-Ⅰ，LC3-Ⅱ，AMPK，and Beclin1 were determined by Western blot；the interaction between Beclin1 and Vps34 was evaluated by co-immunoprecipitation assay. Results：Compared with the Sham group，the I/R group showed significant increases in cerebral infarct volume and neurobehavioral score（both P<0.05）. The mechanism research showed a significantly increased LC3-Ⅱ/LC3-Ⅰ ratio and up-regulat－ed expression of the autophagy regulatory proteins AMPK and Beclin1（all P<0.05） with increased formation of Beclin1 and Vps34 complex after I/R treatment；meanwhile，the number of apoptotic cells increased significantly. The above indices were significantly improved after EA pretreatment（all P<0.05）. However，intracerebral overexpression of the key autophagy-related protein Beclin1 prior to EA pretreatment reversed the protective effect of EA pretreat－ment against I/R injury（P<0.05）. Conclusion：EA pretreatment may play a neuroprotective role through inhibiting the AMPK-Beclin1/Vps34 pathway to reduce autophagy.