莱菔硫烷诱导线粒体裂变抑制血管生成

目的 探讨莱菔硫烷对血管生成的影响及其可能机制。方法 体外实验以原代人脐静脉血管内皮细胞为研究模型，以1‰ 二甲基亚砜（dimethyl sulfoxide，DMSO）干预作为对照。应用WST-1试剂盒检测不同浓度莱菔硫烷（5～100μmol/L）对原代人脐静脉血管内皮细胞增殖活性的影响，采用小管形成实验和Transwell迁移实验分析莱菔硫烷干预（10μmol/L）对原代人脐静脉血管内皮细胞管腔生成、迁移能力的影响，采用Mito-Tracker Green FM染料标记线粒体结合共聚焦显微镜成像技术观察莱菔硫烷（10μmol/L）干预后线粒体形态学改变，以及蛋白免疫印迹法检测线粒体动态相关蛋白包括线粒体融合蛋白1/2、动力相关蛋白1表达水平，以及血管内皮生长因子的蛋白表达。结果 5μmol/L莱菔硫烷干预24h对人脐静脉血管内皮细胞增殖活性无显著影响，而 ≥ 10μmol/L 莱菔硫烷干预则抑制了人脐静脉血管内皮细胞活性，10、20、40、80、100μmol/L干预组抑制率分别为（17.82±5.80）%（P=0.009）、（35.33±6.26）%（P<0.001）、（65.29±4.26）%（P<0.001）、（66.82±3.94）%（P<0.001）及（68.05±2.54）%（P<0.001），IC50为38.15μmol/L。为避免过高干预剂量所致严重毒性效应，我们将选取10μmol/L作为主要干预剂量。与对照组相比，10μmol/L 莱菔硫烷干预显著抑制人脐静脉血管内皮细胞血管生成能力，其中迁移能力降低了（42.98±9.21）%（P=0.018），而管腔样结构形成能力，包括管腔数量、节点数和管腔总长度则分别降低了（83.94±18.36）%（P=0.011）、（59.22±25.60）%（P=0.021）及（50.49±23.44）%（P=0.025）。人脐静脉血管内皮细胞血管生成能力被莱菔硫烷抑制同时，伴有线粒体动态紊乱、线粒体裂变的发生。相对于对照组而言，10μmol/L 莱菔硫烷干预组线粒体网络数、纵横比显著减少（27.39±22.46）%（P=0.006）、（11.37±8.26）%（P<0.001），而圆度显著增加（11.97±12.07）%（P<0.001）。与此相对应的是，10μmol/L莱菔硫烷干预显著上调促裂变蛋白而抑制促融合蛋白表达，动力相关蛋白1表达相当于对照组的（1.71±0.039）倍（P<0.001），而线粒体融合蛋白1/2表达则分别相当于对照组的（59.30+1.50）%（P=0.006）和（74.75±11.84）%（P=0.031）。同时，血管内皮生长因子蛋白表达则相当于对照组的（65.66±9.49）%（P=0.003）。结论 莱菔硫烷具有抑制血管生成活性，其机制可能与其促进血管内皮细胞线粒体裂变有关。

Sulforaphane inhibits angiogenesis via induction of mitochondrial fission

Objective To investigate the effect of Sulforaphane(SFN)on angiogenesis and the underlying mechanism.Methods The primary human umbilical vein endothelial cells(HUVECs)were used as the study model in vitro,with 1‰DMSO incubation as control group.WST-1 kit was used to detect the proliferative activity of HUVECs after exposed to 5~100μmol/L SFN for 24h.The effects of SFN on angiogenesis were further examined by the tube formation assay and transwell migration assay after 10μmol/L treatment.Mito-Tracker Green FM dye-labeled mitochondria were captured by confocal microscopy imaging technology,and Western blot was applied to detect the expression level of mitochondrial dynamics related proteins,including mitofusin-1/2(Mfn1/2)and dynamin-related protein 1(Drp1),as well as vascular endothelial growth factor(VEGF)in HUVECs after 10μmol/L SFN intervention for 24h.Results SFN significantly inhibited the proliferative activity of HUVECs after intervention for 24h at concentrations higher than 5μmol/L with the IC5038.15μmol/L.The inhibition rates were 17.82%(P=0.009),35.33%(P<0.001),65.29%(P<0.001),66.82%(P<0.001)and 68.05%(P<0.001),respectively,at concentrations of 10,20,40,80,and 100μmol/L.Compared to control,the migration rate of HUVECs in the 10μmol/L SFN-treated group was reduced by 42.98%(P=0.018),and the formation capacity of the capillary-like structure quantified by the number of meshes,the number of nodes,and the total length,were reduced by 83.94%(P=0.011),59.22%(P=0.021)and 50.49%(P=0.025),respectively.Mechanistically,analysis of mitochondrial morphology and mitochondrial dynamics related protein expression showed that 10μmol/L SFN significantly promoted mitochondrial fission with the number of mitochondrial networks and the aspect ratio significantly decreased by 27.39%(P=0.006)and 11.37%(P<0.001),whereas circularity significantly increased by 11.97%(P<0.001).In line with mitochondrial morphology alteration,SFN at 10μmol/L significantly induced the protein expression of Drp1(1.71 folds)(P<0.001),while surpressed that of Mfn1/259.30%(P=0.006)and 74.75%(P=0.031)of control,accompanied by VEGF protein expression downregulation(65.66%of control)(P=0.003).Conclusion SFN possesses the anti-angiogenic activity and the potential mechanism may involve promotion of mitochondrial fission in vascular endothelial cell.