消肿止痛合剂通过VEGF-Dll4/Notch信号通路减轻大鼠血管内皮细胞缺氧损伤

目的:研究消肿止痛合剂对大鼠皮瓣血管内皮细胞功能及VEGF-Dll4/Notch信号通路中相关蛋白表达的影响。方法:体外分离并培养大鼠皮瓣血管内皮细胞,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(VEGF受体抑制剂)组及缺氧+消肿止痛剂+MK-0752(Notch通路阻断剂)组。采用ELISA法测定血清中VEGF含量,采用细胞calcein-AM和PI双染色法判断缺氧后1 d、2 d和3 d死活细胞数量,Western blot检测了24 h和48 h细胞内VEGF-A、Notch和Dll4蛋白的表达量。结果:与对照组相比,缺氧24 h和48 h后VEGF含量显著增加,细胞死亡率显著增加,且VEGF-A、Notch和Dll4的蛋白表达量显著增加(P<0.05);与缺氧组细胞相比,消肿止痛合剂干预后,VEGF的含量显著增加,细胞死亡率显著降低,且VEGF-A、Notch和Dll4的蛋白表达量显著升高(P<0.05)。与消肿止痛合剂组相比,VEGF受体抑制剂干预细胞后,消肿止痛合剂对缺氧损伤的血管内皮细胞的保护作用减弱,细胞死亡率明显增加,VEGF的含量降低,且VEGF-A、Notch和Dll4的蛋白表达量降低(P<0.05)。Notch通路阻断剂干预细胞后,细胞存活率不变,VEGF-A的蛋白表达水平增加,Notch和Dll4蛋白表达增加的趋势被有效抑制(P<0.01)。结论:消肿止痛合剂可改善大鼠皮瓣血管内皮细胞的功能,其机制与影响VEGF-Dll4/Notch信号转导通路有关。

Xiaozhong-Zhitong mixture attenuates hypoxic damage of vascular endothelial cells in rats via VEGF-Dll4/Notch signaling pathway

AIM:To study the effect of Xiaozhong(detumescence)-Zhitong(analgesia)mixture on the function of vascular endothelial cells of rat skin flaps and the expression of VEGF-Dll4/Notch signaling pathway-related proteins.METHODS:Vascular endothelial cells of rat skin flaps were isolated and cultured.The cells were divided into control group,hypoxia group,hypoxia+detumescence analgesia group,hypoxia+detumescence analgesia+axitinib(VEGF receptor inhibitor)group,and hypoxia+detumescence analgesia+MK-0752(Notch signaling pathway blocker)group.The serum levels of VEGF were measured by ELISA.The number of dead and living cells at 1 d,2 d and 3 d after hypoxia was determined by cell calcein-AM and PI double staining.The protein expression levels of VEGF-A,Notch and Dll4 in the cells at 24 h and 48 h were detected by Western blot.RESULTS:Compared with control group,the content of VEGF was increased significantly after 24 h and 48 h,and the protein expression of VEGF-A,Notch and Dll4 was increased significantly(P<0.05).Compared with hypoxia group,the content of VEGF was increased significantly after the intervention of Xiaozhong-Zhitong mixture,the death rate was decreased significantly,and the protein expression of VEGF-A,Notch and Dll4 was increased significantly(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 by VEGF receptor inhibitor,the cell mortality was significantly increased,the content of VEGF was decreased,and the protein expression of VEGF-A,Notch and Dll4 was decreased(P<0.05).After intervention with Notch signaling pathway blocker,the cell viability remained unchanged,the expression level of VEGF-A was increased,and the increased Notch and Dll4 protein expression was effectively resisted(P<0.01).CONCLUSION:Xiaozhong-Zhitong mixture improves the function of vascular endothelial cells of rat skin flaps,and its mechanism may be related to the influence of the signal transduction pathway of VEGF-Dll4/Notch.