桃红四物汤对脑缺血再灌注损伤大鼠血脑屏障通透性的保护作用及机制

目的 观察桃红四物汤对脑缺血再灌注损伤的治疗作用，以及其对血脑屏障的保护作用和机制。方法 建立左侧大脑中动脉阻塞模型，将大鼠随机分为假手术组、模型组、桃红四物汤组和尼莫地平组。采用Longa评分法观察大鼠神经功能损伤程度，行为学实验观察大鼠的运动神经功能，2,3,5-氯化三苯基四氮唑（triphenyltetrazolium chloride，TTC）染色法观察大鼠脑梗死面积，苏木精—伊红染色观察大鼠缺血侧脑部皮质组织病理变化，干湿法测定脑含水率，伊文思蓝（Evans blue,EB）灌注染色法检测血脑屏障通透性，透射电子显微镜观察血脑屏障紧密连接超微结构，免疫组织化学法检测脑组织紧密连接蛋白occludin、胞质附着蛋白-1(zona occludens-1, ZO-1)和基质金属蛋白酶-9(matrix metalloprotein-9, MMP-9)表达水平。结果 大鼠神经功能损伤程度与运动神经功能经桃红四物汤给药后呈现改善的趋势。TTC结果显示，与模型组比较，桃红四物汤与尼莫地平组大鼠脑梗死面积明显减少（P＜0.05）。苏木精—伊红染色显示，与模型组比较，桃红四物汤与尼莫地平组大鼠脑皮质组织神经元细胞质浓缩，核固缩、分裂或溶解，细胞排列紊乱，层次不清晰的情况得到改善。与模型组比较，桃红四物汤与尼莫地平组大鼠脑含水率、脑出血性转化程度以及EB渗出量显著降低（P＜0.05）。免疫组织化学检测结果显示，与模型组比较，桃红四物汤组大鼠脑梗死区occludin、ZO-1蛋白表达水平显著升高，MMP-9表达水平显著降低（P＜0.05）。透射电子显微镜观察发现，模型组大鼠血脑屏障紧密连接明显受损，桃红四物汤组和尼莫地平组大鼠血脑屏障紧密连接损伤明显改善。结论 桃红四物汤可改善脑缺血再灌注损伤大鼠神经功能，缩小梗死面积及减轻脑水肿。其作用机制可能与改善血脑屏障紧密连接超微结构，减少紧密连接相关蛋白表达，进而降低血脑屏障通透性有关。

Protective Effect of Taohong Siwu Decoction on Blood-Brain Barrier Permeability in Rats with Cerebral Ischemia/Reperfusion Injury and Its Mechanism

Objective To investigate the therapeutic effect of Taohong Siwu Decoction on cerebral ischemia/reperfusion injury, its protective effect on the blood-brain barrier, and related mechanisms.Methods A model of left middle cerebral artery occlusion (MCAO) was established, and then rats were randomly divided into sham-operation group, model group, Taohong Siwu Decoction group (THSWD group), and nimodipine group. The Longa scoring method was used to observe the degree of nerve function damage, and behavioral experiments were used to observe motor nerve function; TTC staining was used to measure cerebral infarct volume; HE staining was used to observe pathological changes of brain cortex at the ischemic side; the wet-dry weight method was used to measure brain water content; Evans blue perfusion staining was used to observe blood-brain barrier permeability; a transmission electron microscope was used to observe the ultrastructure of the tight junction of the blood-brain barrier; immunohistochemistry was used to measure the expression of the tight junction proteins occludin, ZO-1, and matrix metalloproteinase-9 (MMP-9) in brain tissue.Results The degree of nerve function damage and motor nerve function showed a trend of improvement after the administration of Taohong Siwu Decoction. TTC staining showed that compared with the model group, the THSWD group and the nimodipine group had a significant reduction in cerebral infarct volume (P<0.05). HE staining showed that compared with the model group, the THSWD group and the nimodipine group showed improvements in cytoplasm concentration, karyopyknosis, division or lysis, disordered arrangement of cells, and unclear layers. Compared with the model group, the THSWD group and the nimodipine group had significant reductions in brain water content, cerebral hemorrhage volume and EB exudation (P <0.05). Immunohistochemistry showed that compared with the model group, the THSWD group had significant reductions in the expression levels of occludin, ZO-1, and had a significant increase in the level of MMP-9(P<0.05). Transmission electron microscopy showed that the model group had marked damage of the tight junction, while the THSWD group and the nimodipine group had significant improvement in such damage.Conclusion Taohong Siwu Decoction can improve neurological function, reduce infarct volume, and alleviate brain edema in ischemic stroke, possibly by improving the ultrastructure of the tight junction of blood-brain barrier, reducing the expression of tight junction proteins, thereby reducing blood-brain barrier permeability.