基于OPA1调控线粒体稳态研究姜黄素对HUVECs增殖、迁移的双向调节作用

Bidirectional effects of curcumin on HUVECs proliferation and migration via OPA1 mediated mitochondrial homeostasis

目的:基于O P A1介导的线粒体稳态明确不同浓度姜黄素双向调节人脐静脉血管内皮细胞(HUVECs)增殖、迁移的药理作用及机制。方法:高糖DMEM完全培养基培养HUVECs,CD31荧光染色鉴定;不同浓度姜黄素干预HUVECs,CCK8检测增殖活性,结晶紫染色检测克隆增殖状态;细胞划痕实验及Transwell实验检测其迁移能力;RT-qPCR检测HUVECs的VEGFα、HIF-1αmRNA水平;ROS、JC-1试剂盒检测HUVECs ROS水平及线粒体膜电位;Western Blotting检测HUVECs OPA1及其下游蛋白表达。结果:20、30、50μmol/L姜黄素显示显著的HUVECs增殖抑制效果(P<0.01),1μmol/L姜黄素能够促进HUVECs的克隆增殖;与Ctrl组比较,1、10μmol/L姜黄素能够显著促进HUVECs迁移(P<0.01),20、30μmol/L姜黄素能够显著抑制HUVECs细胞迁移(P<0.01);20、30μmol/L姜黄素能够显著抑制HIF-1αmRNA水平(P<0.05);与Ctrl组比较,20μmol/L姜黄素显著上调HUVECsROS水平(P<0.05),20、30μmol/L姜黄素能够显著降低HUVECs线粒体活性(P<0.05);与Ctrl组比较,1、10μmol/L姜黄素均能够显著上调OPA1蛋白表达(P<0.01,P<0.05)。结论:以10μmol/L为区分,不同浓度的姜黄素对HUVECs的增殖、迁移发挥双向调节作用,并且这一作用与OPA1介导的线粒体稳态密切相关。

Objective:To investigate the pharmacological action and mechanism of curcumin in different concentrations in bidirectional regulation of HUVECs proliferation and migration based on OPA1-mediated mitochondrial homeostasis.Methods:HUVECs were cultured in H-DMEM and identified by CD31 fluorescence staining.HUVECs were treated with different concentrations of curcumin,the proliferation activity of HUVECs was detected by CCK8,and the clones proliferation status of HUVECs was detected by crystal violet staining.Cell scratch assay and Transwell assay were used to detect the migration of HUVECs.The mRNA levels of VEGFα and HIF-1α in HUVECs were detected by RT-qPCR.ROS and JC-1 kits were used to detect the ROS level and mitochondrial membrane potential of HUVECs.Western Bloting was used to detect HUVECs OPA1 protein expression.Results:20,30 and 50μmol/L curcumin showed significant inhibition of HUVECs proliferation and clonal proliferation(P<0.01).1μmol/L curcumin could promote the clonal proliferation of HUVECs.Compared with blank group,1 and 10μmol/L curcumin could significantly promote the migration of HUVECs(P<0.01),and 20 and 30μmol/L curcumin could significantly inhibit the migration of HUVECs(P<0.01).20,30μmol/L curcumin significantly inhibited HIF-1α mRNA level(P<0.05).Compared with the blank group,20μmol/L curcumin tended to up-regulate HUVECs ROS level(P<0.05).20,30μmol/L curcumin can significantly decrease the mitochondrial activity of HUVECs(P<0.05).Compared with the control group,1 and 10μmol/L curcumin significantly up-regulated OPA1 protein expression(P<0.01,P<0.05).Conclusion:Different concentrations of curcumin at 10μmol/L can regulate the proliferation and migration of HUVECs bidirectively,and this effect is closely related to OPA1-mediated mitochondrial homeostasis.