山楂叶提取物及其制剂对人脐静脉内皮细胞缺氧复氧损伤中性粒细胞黏附的影响

目的 研究山楂叶提取物（牡荆素鼠李糖苷、牡荆素葡萄糖苷）及其制剂奥沙恩注射液对人脐静脉内皮细胞（HUVEC）缺氧复氧损伤时中性粒细胞（PMN）黏附的影响及其分子机制。方法以HUVEC为实验对象，复制缺氧复氧损伤模型，用酶标仪检测山楂叶提取物牡荆素鼠李糖苷、牡荆素葡萄糖苷及其制剂奥沙恩注射液对HUVEC缺氧60 min复氧30 min和复氧240 min时PMN黏附的影响；应用流式细胞学方法，观察牡荆素鼠李糖苷、牡荆素葡萄糖苷及奥沙恩注射液对PMN表面黏附分子CD11/CD18表达的影响。结果 缺氧60 min后可见HUVEC皱缩，变形；复氧30 min，模型组PMN HUVEC之间有明显的细胞黏附现象，正常组细胞之间黏附较少，各用药组与模型组比较，PMN-HUVEC黏附较少。复氧240 min处理的细胞各组情况与复氧30 min相似。山楂叶提取物牡荆素鼠李糖苷、牡荆素葡萄糖苷及其制剂奥沙恩注射液均可显著抑制复氧30 min和240 min时PMN-HUVEC之间的黏附,且呈现出一定的量效关系。牡荆素鼠李糖苷、牡荆素葡萄糖苷可显著抑制HUVEC缺氧复氧损伤导致的PMN表面CD11/CD18分子表达，且奥沙恩注射液和牡荆素葡萄糖苷均表现出良好的量效关系。结论 在静息状态下，PMN表面几乎不表达CD11/CD18分子，但缺氧复氧30 min和240 min处理的HUVEC上清液孵育中性粒细胞30 min，可增强PMN表面CD11/CD18分子表达的增加。且第1个时相即复氧后30 min时PMN HUVEC的黏附比第2个时相点更为明显。复氧时经牡荆素鼠李糖苷、牡荆素葡萄糖苷及其制剂奥沙恩注射液处理的HUVEC上清液可抑制PMN表面CD11/CD18分子表达的增加，降低PMN HUVEC黏附，这可能是山楂叶提取物及其制剂奥沙恩注射液保护心脏免于缺血再灌注损伤的重要分子作用机制之一。

Effect of Haw Leaf Extract and Its Preparation on Polymorphonuclear Leucocyte Adhesion during HUVEC Anoxia/Reoxygenation Injury

OBJECTIVE: To study the effect and molecular mechanism of two haw leaf extracts, Vitexin-rhamnoside (VR) and Vitexin-glucoside (VG), and their preparation, Aoshaen injection (AI), on the polymorphonuclear leucocyte (PMN) adhesion during human umbilical vein endothelial cell (HUVEC) anoxia/reoxygenation (A/R) injury. METHODS: The cell model of A/R injury duplicated by breaking off the oxygen supplying of HUVEC for 60 min followed with reoxygenating for 30 min (phase 1) or 240 min (phase 2) was taken as the experimental objective. The effects of testing drugs (VR, VG and AI) on PMN adhesion in the model cells were measured by enzyme immunoassay, and their effects on PMN superficial adhesion molecule CD11/CD18 expression were measured by flow cytometer respectively. RESULTS: After 60 min of anoxia, HUVEC was shrunk and deformed. The adhesion between PMN and HUVEC significantly revealed at phase 1 in the model group, but it was fewer in the normal cell group, and also lesser in the groups treated with various drugs. The condition of cell adhesion revealed at phase 2 was the similar to that at phase 1. All testing drugs, VR, VG and AI, showed inhibitory effect on the cell adhesion at either phase 1 or phase 2, showing a certain dose-effect relationship. The expression of CD11/ CD18 was also inhibited by the testing drugs, and a good dose-effect relation was shown by VG and AI. CONCLUSION: At the resting condition, there are almost no expression of CD11/CD18 molecule, but it could be enhanced by incubating PMN with supernate of A/R injured HUVEC culture, and more marked at phase 1. Adding the test drugs into the supernate could inhibit the enhancing of CD11/CD18 molecule expression and reduce the PMN-HUVEC adhesion, which may be one of the molecular mechanisms of haw leaf extracts and their preparation in protecting heart against A/R injury.