消肿止痛合剂诱导缺氧内皮细胞再生效应观察及机制研究  

Observation on the effects of Xiaozhong Zhitong Mixture on the regeneration of hypoxic endothelial cells and its mechanism

作　　者：何志军[1] 刘涛 宋渊[1] 李岩[1] 陈文[1] 李金鹏[1] 姚兴璋[1] HE Zhi-jun;LIU Tao;SONG Yuan;LI Yan;CHEN Wen;LI Jin-peng;YAO Xing-zhang(Gansu Provincial Hospital of Traditional Chinese Medicine,Lanzhou 730050,China)

机构地区：[1]甘肃省中医院,兰州730050 [2]甘肃省中医院足踝骨科,兰州730050

出　　处：《中华中医药杂志》2020年第6期3123-3127,共5页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：国家自然科学基金项目(No.81660802,No.81860863)。

摘　　要：目的:研究消肿止痛合剂对大鼠皮瓣血管内皮细胞功能及其信号通路VEGF-Dll4/Notch中相关基因表达的影响。方法:以103、104、105、106、107倍稀释的消肿止痛合剂药物原液分别对大鼠皮瓣血管内皮细胞作用1、3、6、9d进行浓度筛选;大鼠皮瓣血管内皮细胞的分离与培养,将细胞分为对照组、缺氧组、缺氧+消肿止痛剂组(缺氧+消肿组)、缺氧+消肿止痛剂+Axitinib组(缺氧+消肿+AXI组)、缺氧+消肿止痛剂+MK-0752组(缺氧+消肿+MK组)。采用CCK-8法测定消肿止痛合剂对大鼠皮瓣血管内皮细胞的增殖抑制作用。按试剂盒方法检测细胞上清液乳酸脱氢酶(LDH)水平,RT-PCR检测VEGFA、Notch、Dll4 mRNA表达。结果:与对照组比较,缺氧组细胞凋亡显著增加,48、72h细胞活性下降(P<0.01),24、48h细胞上清中LDH含量显著增加(P<0.01),且24、48h VEGFA、Notch、Dll4 mRNA表达量显著增加(P<0.05,P<0.01);与缺氧组比较,消肿止痛合剂干预后,48、72h血管内皮细胞相对活性显著升高(P<0.01),细胞分泌的24、48h LDH水平显著降低(P<0.01),且24、48h VEGFA、Notch、Dll4 mRNA表达量显著升高(P<0.05)。与消肿止痛合剂组比较,VEGF阻断剂干预细胞后,消肿止痛合剂对缺氧诱导的血管内皮细胞损伤的保护作用减弱,48、72h细胞活性显著下降(P<0.01),48、72h LDH水平显著升高(P<0.05),且48、72h VEGFA、Notch、Dll4 mRNA表达量降低(P<0.01)。Notch阻断剂干预细胞后,48、72h细胞活性不变,24、48h LHD下降的趋势增加,24、48h VEGFA mRNA表达水平显著增加(P<0.05),Notch、Dll4 mRNA表达增加的趋势被有效抵制(P<0.01)。结论:消肿止痛合剂可改善大鼠皮瓣血管内皮细胞,其机制可能与影响VEGF-Dll4/Notch信号转导通路有关。Objective:To study the effects of Xiaozhong Zhitong Mixture on vascular endothelial cell function and the expression of related genes in vascular endothelial cell(VEC)VEGF-Dll4/Notch signaling pathway of rat skin flap.Methods:Rat skin flap vascular endothelial cells were isolated and cultured on 103,104,105,106,107 concentrations of Xiaozhong Zhitong Mixture for 1 day,3 days,6 days and 9 days,respectively,and the cells were divided into control group,hypoxia group,hypoxia+Xiaozhong Zhitong Mixture group(hypoxia+detumescence group),Anoxia+detumescence+AXItinib group(anoxia+detumescence+AXI group),anoxia+detumescence+MK-0752 group(anoxia+detumescence+MK group).CCK-8 method was used to determine the inhibitory effect of Xiaozhong Zhitong Mixture on the proliferation of vascular endothelial cells in rat skin flap.The level of LDH in supernatant was detected by kit method,and the expression of mRNA in VEGFA,Notch and Dll4 was detected by RT-PCR.Results:Compared with the control group,the apoptotic cells in hypoxia group increased significantly,the cell activity at 48 h and 72 h decreased(P<0.01),the LDH content in cell supernatant at 24 h and 48 h increased significantly(P<0.01),the mRNA expressions of VEGFA,Notch and Dll4 at 24 h and 48 h were significantly increased(P<0.05,P<0.01).Compared with the hypoxic group,the relative survival rate of vascular endothelial cells at 48 h and 72 h was significantly increased after the intervention of Xiaozhong Zhitong Mixture(P<0.01),the level of LDH secreted by the cells at 24 h and 48 h was significantly decreased(P<0.01),and the expressions of VEGFA,Notch and Dll4 mRNA at 24 h and 48 h were significantly increased(P<0.05).Compared with Xiaozhong Zhitong Mixture group,the protective effect of Xiaozhong Zhitong Mixture on hypoxia-induced vascular endothelial cell injury was weakened,the cell survival rate at 48 h and 72 h was significantly decreased(P<0.01),the level of LDH at 48 h and 72 h was significantly increased(P<0.05),and the expressions of VEGFA,Notch and Dll4 mRNA at

关 键 词：消肿止痛合剂 血管内皮细胞 VEGF-Dll4/Notch 皮瓣坏死 

分 类 号：R285.5[医药卫生—中药学]