苦碟子注射液基于miR-21调控PDCD4/NF-κB通路保护急性脑梗死神经血管单元的机制研究

Our previous study have confirmed that acute cerebral infarction (ACI) patients usually present with heat toxin syndrome after the attack, this period in patient's condition and inflammatory response index change together and activated intracerebral inflammatory cascade reaction causes blood brain barrier disruption and neurovascular unit (NVU) damage, and respond well to treatment by clearing heat, detoxification, and dredging Chinese medicinal at the critical phase, with the important process adjusting miR-21 which regulates PDCD4/NF-κB pathway and inhibiting inflammatory response. Taking MCAO rat model as objects, our research takes advantage of miR-21agonist/antagomir thereby to observe the effect of Kudiezi Injection on neurobehavioral, cerebral infarction volume, pathological morphology, apoptosis, angiogenesis and blood brain barrier leakage in rats, for the comprehensive evaluation of protective effect of clearing heat, detoxification, and dredging Chinese medicinal on NVU. The molecular mechanism of clearing heat, detoxification, and dredging Chinese medicinal on the protection of NVU and its key process and target specific therapeutic effect are also illuminated by detecting the expression of miR-21 and its expression in neurons, astrocytes and microglia, PDCD4/NF- κB pathway and its downstream proteins.

脑梗死急性期出现的火毒证是病情加重、恶化乃至死亡的重要标志，前期工作发现此时期患者病情变化与炎症反应为核心的指标体系呈组群联动变化，脑内炎症级联反应激活，而炎症反应是血脑屏障破坏、神经血管单元功能损伤的重要原因。清热解毒通络对急性脑梗死神经血管单元具有保护作用，通过miR-21调控PDCD4/NF-κB通路抑制炎症反应是重要环节。课题拟在MCAO大鼠模型上，应用miR-21agomir/antagomir动态观察苦碟子注射液对大鼠神经行为学、脑组织梗死体积、病理形态、细胞凋亡、血管生成及血脑屏障渗漏的影响，全面评价清热解毒通络对神经血管单元的保护作用；通过检测脑组织miR-21及其在神经元、星形胶质细胞、小胶质细胞的原位表达、PDCD4/NF-κB通路及其下游蛋白的表达，阐明清热解毒通络保护神经血管单元的分子调控机制，明确其发挥疗效的关键环节和作用靶点。

炎症反应是血脑屏障破坏、神经血管单元功能损伤的重要原因，NF-κB是促炎反应的核心因子，PDCD4作为协助NF-κB激活的促炎蛋白，被miR-21调控。主要研究内容包括采用线栓法致左侧大脑中动脉闭塞建立缺血再灌注大鼠模型（MCAO），通过动态观察MCAO缺血再灌注6h、1d、3d、7d、14d的5个时间点，模型大鼠的神经行为学变化、脑梗死体积、脑组织病理形态学变化，选出敏感时间位点。发现神经功能缺损评分、脑梗死体积和病理形态学变化在缺血再灌注3d时变化最为明显，脑组织miR-21表达量也在缺血再灌注3d达到高峰（p＜0.01），随后逐渐下降，在缺血再灌注后14d降低到Sham水平。与模型组比较，苦碟子组的功能缺损评分、脑梗死面积和脑含水量均下降（p＜0.01），苦碟子组显著上调miR-21、MVD-CD31、ZO-1、Occludin、Bcl-2、Ang-1表达（p＜0.01），显著下调caspase-3、PDCD4、NF-κB、Bax、Ang-2表达（p＜0.01）。结论：苦碟子可明显改善MCAO大鼠神经行为功能，减轻脑组织缺血形态学改变，减小脑梗死面积，调节炎症反应，减轻细胞凋亡，促进急性脑梗死后脑组织的血管生成修复，保护神经血管单元。