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目的:探讨julibroside J12抑制人微血管内皮细胞(human microvascular endothelial cells,HMEC-1)凋亡的机制。方法:体外培养HMEC-1,在不同浓度julibroside J12干预不同时间的条件下,采用磺基罗丹明(SRB)染色法测定细胞存活率,同时结合DAPI荧光染色,TUNEL染色法检测细胞凋亡情况;流式细胞仪分析DNA含量、细胞周期以及细胞线粒体跨膜电位(△ψ)的变化。酶标仪检测HMEC-1中凋亡相关蛋白酶Caspase-3,9的含量。结果:合欢皂苷julibroside J12可显著抑制HMEC-1生长(抑制率可达75.6%),并与作用时间和剂量正相关;细胞周期分析表明,julibroside J12作用48 h后,HMEC-1被阻滞在G2/M期,并有较大的凋亡峰(19.49%);julibroside J12作用24 h后,细胞线粒体跨膜电位ΔΨM没有明显改变,Caspase-3,9的含量也未随时间发生相应变化。结论:julibroside J12可以通过诱导HMEC-1凋亡而抑制其增殖,其诱导HMEC-1凋亡的机制可能与线粒体通路无关。
Objective: To investigate the mechanism of julibroside J12 inhibiting apoptosis of human microvascular endothelial cells (HMEC-1). Methods: HMEC-1 cells were cultured in vitro, and cell survival rate was determined by SRB staining with different concentrations of julibroside J12 in different time. DAPI staining and TUNEL staining were used to detect cell apoptosis. Flow cytometry analysis of DNA content, cell cycle and mitochondrial transmembrane potential (△ ψ) changes. The content of Caspase-3, 9 in HMEC-1 was detected by microplate reader. Results: Juribroside J12 significantly inhibited the growth of HMEC-1 cells (75.6% inhibition rate) and was positively correlated with the time and dose of HMEC-1. Cell cycle analysis showed that HMEC-1 was arrested in Juribroside J12 cells for 48 h G2 / M phase, and larger apoptotic peak (19.49%). After treated with julibroside J12 for 24 h, the mitochondrial transmembrane potential ΔΨM did not change significantly, and the content of Caspase-3,9 did not change with time. CONCLUSION: Julibroside J12 can inhibit the proliferation of HMEC-1 cells by inducing the apoptosis of HMEC-1. The mechanism of apoptosis induced by HMEC-1 may not be related to the mitochondrial pathway.