目的 探究桃红四物汤通过保护脑微血管内皮糖萼完整性与功能性减轻脑缺血再灌注损伤（cerebral ischemia-reperfusion injury，CIRI）的作用机制。方法 建立大脑中动脉闭塞再灌注（middle cerebral artery occlusion-reperfusion，MCAO/R）模型以模拟CIRI损伤，将SD大鼠随机分成假手术组、模型组及桃红四物汤低、中、高剂量（4.5、9.0、18.0 g/kg）组和尼莫地平（20 mg/kg）组，给予药物干预。造模7 d后，采用Longa分级评分法进行神经功能评分，采用2,3,5-氯化三苯基四氮唑（2,3,5-triphenyltetrazolium chloride，TTC）染色评估脑梗死体积，通过转角实验、平衡木实验评价运动功能；利用激光散斑、小动物超分辨率成像检测脑血流及脑血管密度，结合伊文思蓝渗漏实验、Western blotting实验评估血脑屏障完整性；采用透射电镜观察糖萼形态；采用免疫荧光、免疫组化、Western blotting及ELISA检测糖萼相关指标多配体蛋白聚糖-1（syndecan-1，SDC-1）、透明质酸（hyaluronic acid，HA）、硫酸乙酰肝素（heparan sulfate，HS）、硫酸软骨素（chondroitin sulfate，CS）及糖萼核心代谢酶乙酰肝素酶（heparanase，HPSE）、透明质酸酶2（hyaluronidase 2，Hyal2）、唾液酸酶1（neuraminidase 1，Neu1）、金属基质蛋白酶-9（matrix metalloproteinase-9，MMP-9）表达，并通过酶抑制剂验证HPSE、MMP-9的作用。结果 与模型组比较，桃红四物汤显著降低MCAO/R大鼠脑梗死体积（P＜0.05、0.001），改善神经功能及四肢协调能力（P＜0.05、0.01、0.001），缓解体质量减轻，改善梗死区脑血流及微血管生成，减少血脑屏障渗漏（P＜0.01、0.001），调节血管舒缩功能（P＜0.01、0.001）；同时桃红四物汤能显著减少糖萼核心成分（CS、HA、HS、SDC-1）脱落至血清（P＜0.01、0.001），通过抑制HPSE及MMP-9活性，保留糖萼完整性。结论 桃红四物汤通过抑制HPSE及MMP-9活性，减少糖萼脱落，维持血脑屏障完整性，减少脑卒中损伤。

Objective To explore the mechanism by which Taohong Siwu Decoction (桃红四物汤, THSWD) alleviates cerebral ischemia-reperfusion injury (CIRI) through protecting the integrity and functionality of glycocalyx of brain microvascular endothelial cells. Methods A model of middle cerebral artery occlusion-reperfusion (MCAO/R) was established to simulate CIRI injury. SD rats were randomly divided into sham group, model group, THSWD low-, medium-, high-dose (4.5, 9.0, 18.0 g/kg) groups and nimodipine (20 mg/kg) group, and drugs were given for intervention. After 7 d of modeling, Longa grading system was used for neurological function evaluation, 2,3,5-triphenyltetrazolium chloride (TTC) staining was used to assess cerebral infarction volume. The motion function was evaluated through corner experiments and balance beam experiments. Laser speckle and small animal super-resolution imaging were used to detect cerebral blood flow and cerebral vascular density, Evans blue leak assay combined with Western blotting were to evaluate blood-brain barrier integrity. Glycocalyx morphology was observed using transmission electron microscopy. The expression of glycocalyx-related indicator such as syndecan-1 (SDC-1), hyaluronic acid (HA), heparan sulfate (HS), chondroitin sulfate (CS) and core metabolic enzymes of glycocalyx such as heparanase (HPSE), hyaluronidase 2 (Hyal2), neuraminidase 1 (Neu1), matrix metalloproteinase-9 (MMP-9) were detected by immunofluorescence, immunohistochemistry, Western blotting and ELISA, and the effects of HPSE and MMP-9 were verified by enzyme inhibitors. Results Compared with model group, THSWD significantly reduced the cerebral infarction volume in MCAO/R rats (P < 0.05, 0.001), improved neurological function and limb coordination ability (P < 0.05, 0.01, 0.001), alleviated weight loss, improved cerebral blood flow and microvascular generation in the infarct area, reduced blood-brain barrier leakage (P < 0.01, 0.001), regulated vascular dilation and contraction function (P < 0.01, 0.001). Meanwhile, THSWD could significantly reduce the shedding of core components of glycocalyx (CS, HA, HS, SDC-1) into the serum (P < 0.01, 0.001), and maintain the integrity of glycocalyx by inhibiting the activities of HPSE and MMP-9. Conclusion THSWD reduces the shedding of glycocalyx by inhibiting the activity of HPSE and MMP-9, thereby maintaining the integrity of blood-brain barrier and minimizing stroke damage.

R285.5

国家自然科学基金资助项目（82074152）