[关键词]
[摘要]
目的 基于细胞自噬探讨钩藤碱对肿瘤坏死因子-α(TNF-α)诱导的人脐静脉内皮细胞(HUVECs)损伤时血栓前状态的影响。方法 采用TNF-α诱导HUVECs制备损伤模型,透射电镜和免疫荧光技术观察HUVECs自噬水平的变化,Western blotting法检测自噬标记蛋白人微管相关蛋白轻链3Ⅱ(LC3Ⅱ)/LC3I、Beclin-1、p62和凝血相关因子核转录因子-κB(NF-κB)、血管假性血友病因子(vWF)和纤溶酶原活化物抑制剂-1(PAI-1)的蛋白表达水平,ELISA法检测细胞培养上清中前列环素(PGI2)和内皮素-1(ET-1)的量。结果 TNF-α上调了HUVECs中凝血相关因子NF-κB、vWF、PAI-1蛋白的表达水平,降低了细胞培养上清液中ET-1和PGI2的量(P < 0.05),使细胞中出现自噬小体,上调LC3Ⅱ/LC3I、Beclin-1蛋白表达水平,下调p62蛋白表达水平(P < 0.05);钩藤碱能够促进HUVECs中自噬小体的产生,并在一定程度上调LC3Ⅱ/LC3I、Beclin-1蛋白表达水平,下调p62蛋白表达水平(P < 0.05),下调凝血相关因子NF-κB、vWF、PAI-1蛋白的表达水平,上调细胞培养上清液中ET-1和PGI2的量(P < 0.05);自噬抑制剂3-甲基腺嘌呤(3-MA)能够抑制钩藤碱的作用效果(P < 0.05)。结论 钩藤碱能够通过增强自噬降低TNF-α介导的HUVECs细胞凝血相关因子的表达,抑制血管内皮炎性损伤时血栓前状态的发生。
[Key word]
[Abstract]
Objective To investigate the effect of rhynchophylline on HUVECs prethrombotic state induced by TNF-α through regulating autophagy. Methods TNF-α was used to establish the HUVECs damage model. The change of autophagy was detected by transmission electron microscope and immunofluorescence technique. Western blotting was used to detect the LC3Ⅱ/LC3I ratio and the expression of Beclin-1, p62, NF-κB, vWF, and PAI-1. The content of ET-1 and PGI2 in cultural supernatant was detected by ELISA. Results TNF-α up-regulated the expression of NF-κB, vWF, and PAI-1in HUVECs, and decreased the content of ET-1 and PGI2 in cell culture supernatant (P < 0.05). In addition, more autophagosomes were observed in HUVECs, and the LC3Ⅱ/LC3I ratio and beclin-1 expression were increased, as well as the expression of p62 was decreased (P < 0.05). Rhynchophylline promoted the production of autophagosome, increased the LC3Ⅱ/LC3I ratio and Beclin-1 expression, and reduced the expression of p62 (P < 0.05). Moreover, the results showed that rhynchophylline down-regulated the expression of NF-κB, vWF, and PAI-1, as well as reduced the content of ET-1 and PGI2 in cultural supernatant (P < 0.05). 3-MA, an inhibitor of autophagy, abolished the effect of rhynchophylline to a certain extent (P < 0.05). Conclusion Rhynchophylline reduces the expression of coagulative factors induced by TNF-α through enhancing autophagy, and inhibits the prethrombotic state when vascular endothelial cells have inflammatory damage.
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[基金项目]
国家自然科学基金资助项目(81473653)