基于“自噬-凋亡交互作用”对血管内皮微环境稳态的影响探讨化痰通络法对急性脑梗死溶栓后脑侧支循环的调节效应与机制

Ischemia/reperfusion injury after thrombolysis for acute cerebral infarction could inhibit the building and opening of collateral circulation, leading to irreversible damage to nerve cells. The mechanism might be involved with endothelial cell injury and vascular microenvironment imbalance mediated by "crosstalk between autophagy and apoptosis", which lead to increase the cerebral microcirculation dysfunction. In previous study, we found that Huatan Tongluo method could reduce ischemia/reperfusion injury, and adaptively protect nerve cells by regulating endoplasmic reticulum steady-state. Based on that, we use MCAO rats as observed objects, with Huatan Tongluo method medicine, from the "overall macro" and "micro-elements" view, to observe the recovery of neurological defects in MACO rats; LASCA to observe cerebral artery microcirculation status; while to observe morphology changes of vascular endothelial cells, with HPLC, CoIp, and Western Blot to observe related factors of the steady-state of microenvironment and the key factor protein expression of " crosstalk between autophagy and apoptosis", to explore the mechanism of Huatan Tongluo method to regulate cerebral microcirculation after thrombolysis for acute cerebral infarction, to supply its experimental basis to promote nerve cell function recovery by protecting endothelial cells, and to provide evidence for the application of Chinese medicine in the treatment of acute critical illness.

急性脑梗死溶栓后易继发缺血再灌注损伤，可严重阻碍大脑侧支循环的建立与开放，进而影响神经功能的恢复。目前研究显示，其机制可能与“自噬—凋亡交互作用”所介导的血管内皮细胞损伤及其微环境稳态失衡，进而加重大脑微循环障碍有关。课题组前期发现，化痰通络法具有减轻缺血再灌注损伤，调控内质网稳态进而适应性保护神经细胞的作用。基于此，本研究拟采用MCAO大鼠，通过化痰通络法中药干预，从“宏观整体”与“微观分子”相结合角度，观察其促进神经功能缺损恢复情况，LASCA技术观察大脑侧支循环建立及血流状况，同时观察血管内皮细胞微观形态变化。进一步结合HPLC、WB和CoIp技术观察血管内皮微环境稳态相关因子及“自噬—凋亡交互作用”中关键蛋白表达，以期初步探讨化痰通络法调节急性脑梗死溶栓后侧支循环的作用机制，充实其通过保护血管内皮细胞以达到促进神经细胞功能恢复的实验依据，为中医药在急危重症治疗中的应用提供部分证据。

溶栓是治疗急性脑梗死的有效方法，但溶栓后再灌损伤限制了其使用，而改善侧支循环在溶栓后的治疗中具有重要意义。本研究通过复制MCAO大鼠，以化痰通络法中药干预，采用动物行为学、LASCA、EB染色检测侧枝循环变化；光镜、TEM观察内皮细胞（VEC）微观形态变化，ELISA、TUNEL染色、Western Blot和CoIp观察VEC微环境稳态，细胞凋亡、“自噬-凋亡交互作用”中关键蛋白表达。.行为学显示：与模型组相比，rt-PA 3d、7d时和中药联合 rt-PA 24h、3d、7d时相大鼠评分明显降低（P<0.05）。LASCA结果显示，与模型组相比，rt-PA组大鼠脑血流24h明显增加，3d和7d时血流增加更加明显，可见丰富的侧枝血管生成；与rt-PA组相比，中药组在7d时血流明显加快，微血管数量增多。EB结果显示：与模型组相比，rt-PA组与中药组在各个时点EB含量均明显降低（P<0.05），且中药组在6h，1d，3d时EB含量均低于rt-PA组。NO和ET结果显示，与模型组相比，7d时rt-PA组与中药组NO浓度明显升高，ET含量明显降低（P<0.05）。光镜和TEM结果显示，中药组VEC形态和细胞器恢复情况优于模型组，同时电镜下可见自噬体形成，TUNEL染色显示中药组VEC凋亡明显减轻。WB结果显示，中药干预后，可见自噬相关蛋白Akt、PI3KC3、Beclin-1、LC3Ⅱ明显升高，而凋亡途径Bcl-2、Bax、Caspase9、Caspase3蛋白呈现下降的趋势。CoIP结果显示Bcl-2与Beclin1蛋白存在交互作用，与假手术组相比，模型组各个时点Bcl-2与Beclin1蛋白结合增加，而与模型组相比，rt-PA和中药组各个时点Bcl-2与Beclin1蛋白结合降低。.研究结果表明：（1）化痰通络法可促进脑梗死溶栓后血脑屏障修复，提高缺血半暗带脑血流，保护和改善脑梗死后大脑侧枝循环，进而促进神经功能恢复。（2）化痰通络法可促进脑梗死溶栓后血管内皮细胞修复，维持ET、NO相对平衡，保护血管内皮微环境稳态。（3）化痰通络法可调节“自噬-凋亡交互作用”中关键靶点Beclin-1/Bcl-2及相关反应蛋白，促进自噬发生，发挥抗凋亡作用，进而保护血管内皮细胞。