钩藤总生物碱增强自噬降低氧化应激诱导的内皮细胞炎性反应

Rhynchophylla total alkaloids reduce endothelial cell inflammatory response induced by oxidative stress through enhancing autophagy

目的:探讨钩藤总生物碱调控细胞自噬对氧化应激诱导的内皮细胞炎性反应的影响。方法:将HUVECs随机分为6组:正常对照组、oxLDL组、DMSO组、雷帕霉素(Rap)组、钩藤总生物碱组、钩藤总生物碱+3-MA组。以oxLDL诱导氧化应激并进一步诱导产生炎性反应。透射电镜和免疫荧光进行自噬形态学检测,Western blot检测LC3Ⅱ/LC3Ⅰ、Beclin-1、p62以及核转录因子κB(NF-κB)、肿瘤坏死因子α(TNF-α)、C反应蛋白(CRP)表达,ELISA法检测培养上清中细胞间黏附分子1(ICAM-1)和血管细胞黏附分子1(VCAM-1)含量,流式细胞技术评价细胞凋亡情况。结果:oxLDL明显上调NF-κB、TNF-α、CRP表达和ICAM-1、VCAM-1含量(P<0.05)。同时oxLDL组自噬小体增多,LC3Ⅱ/LC3Ⅰ比值、Beclin-1的表达显著上调,p62表达下调(P<0.05)。与oxLDL组比较,钩藤总生物碱明显促进了自噬小体的产生,显著上调LC3Ⅱ/LC3Ⅰ比值、Beclin-1的表达,下调p62表达(P<0.05)。同时减少NF-κB、TNF-α、CRP的表达及ICAM-1、VCAM-1含量(P<0.05),减轻了细胞凋亡,3-MA在一定程度上抑制了其作用效果。结论:钩藤总生物碱能够通过增强细胞自噬而降低氧化应激诱导的内皮细胞炎性反应,减轻细胞凋亡。

Objective: To investigate the effect of rhynchophylla total alkaloids on endothelial cell inflammatory response induced by oxidative stress through regulating autophagy. Methods: HUVECs were randomly divided into six groups: normal control group, oxLDL group, DMSO group, rapamycin(Rap) group, rhynchophylla total alkaloids group, rhynchophylla total alkaloids +3-MA group. OxLDL was used to induce oxidative stress to further induce inflammatory response. The morphological characteristic of autophagy was detected by transmission electron microscope and immunofluorescence technique.The expressions of LC3Ⅱ/LC3Ⅰ, Beclin-1, p62, NF-κB, TNF-α and were detected by Western blot. The contents of ICAM-1 and VCAM-1 in supernatant were detected by ELISA. The apoptosis of the cells was evaluated by flow cytometry. Results: OxLDL significantly up-regulated the expressions of NF-κB, TNFα, CRP and increased the contents of ICAM-1 and VCAM-1(P<0.05). Meanwhile, oxLDL increased the LC3Ⅱ/LC3Ⅰ ratio and the expression of beclin-1,and reduced theexpression of p62, and autophagosomes increased in oxLDL group(P<0.05). Compared with oxLDL group, rhynchophylla total alkaloidssignificantly promoted the production of autophagosome, up-regulated the LC3Ⅱ/LC3Ⅰ ratio and the expression of Beclin-1, and down-regulated the expression of p62(P<0.05). At the same time, rhynchophylla total alkaloids could reduce the expressions of NF-κB, TNF-α, CRP and the contents of ICAM-1 and VCAM-1(P<0.05), which reduced cell apoptosis. And the effect wasinhibitedto a certain extent by 3-MA. Conclusion: Rhynchophylla total alkaloids can reduce endothelial cell inflammatory response induced by oxidative stress through enhancing autophagy, which also inhibit cell apoptosis.