莲房原花青素通过线粒体介导的内源性Caspase途径诱导HepG2细胞凋亡

研究莲房原花青素(LSPCs)诱导肝癌Hep G2细胞凋亡的机制。利用MTT法检测LSPCs对肝癌Hep G2细胞的生长抑制作用,Hochest 33258染色观察凋亡细胞的核形态,Annexin V-FITC/PI双染流式细胞术检测细胞凋亡水平,彗星实验用来检测细胞DNA损伤,JC-1染色用来检测线粒体膜电位,Western blotting法检测细胞凋亡蛋白水平的表达。LSPCs作用细胞后,显著抑制Hep G2细胞的增殖;细胞凋亡征象明显,核染色体高度凝聚,细胞核碎裂,核固缩,染色质凝聚的细胞数从3.86%增至42.76%(p0.01);活细胞率急剧减少,而凋亡率从5.32%增至67.05%(p0.01);LSPCs诱发细胞产生DNA损伤,线粒体膜电位下降,导致Cytochromec、Caspase-3和Caspase-9蛋白的表达量增加,Bax蛋白的表达量上调,Bcl-2蛋白的表达量下调,Bax/Bcl-2的比值上升。而凋亡抑制剂Z-VAD能够削弱LSPCs对Hep G2细胞增殖的抑制作用,显著下调细胞核聚缩,抑制线粒体损伤及Caspase相关蛋白的表达量,最终阻断LSPCs的致凋亡作用。由此得出结论,LSPCs可通过线粒体介导的内源性Caspase途径诱导人肝癌细胞凋亡。

Procyanidins from Lotus Seedpod Induce Apoptosis in HepG2 Cells via Caspase-dependent Pathway

The possible mechanism of apoptosis induced by procyanidins from lotus seedpod (LSPCs) in human hepatoma HepG2 cells was investigated. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to study the inhibitory effect of LSPCs on HepG2 cell proliferation, morphological changes in apoptotic nuclei were observed via Hoechst 33258 staining, and the percentage of apoptotic cells was calculated by double-staining flow cytometry using Annexin V conjugated to fluorescein isothiocyanate and propidium iodide. DNA damage, mitochondrial membrane potential, and expression levels of apoptotic proteins were determined by comet assay, JC-1 staining, and western blot, respectively. The results indicate that after treatment with LSPCs, HepG2 cell proliferation was significantly inhibited; there were obvious signs of apoptosis, including high chromatin condensation level, nuclear fragmentation, and karyopyknosis; and the number of chromatin-condensed cells increased from 3.86% to 42.76% (p<0.01). The percentage of viable cells decreased significantly and the proportion of apoptotic cells increased from 5.32% to 67.05% (p<0.01). Treatment with LSPCs caused DNA damage, loss of mitochondrial membrane potential, an increase in the protein expression levels of cytochrome C, caspase-3, and caspase-9, in addition to upregulation of Bax protein expression and downregulation of Bcl-2 protein expression. However, the apoptosis inhibitor Z-VAD reduced the inhibitory effect of LSPCs on HepG2 cell proliferation, significantly reduced nuclear condensation, inhibited mitochondrial damage and caspase protein expression, and eventually inhibited LSPC-induced apoptosis. Thus, the results show that LSPCs trigger apoptosis in HepG2 cells via the mitochondria-mediated, endogenous caspase pathway.