槲芪散及槲寄生提取物对肝癌细胞内游离钙离子浓度的影响

目的探讨方剂槲芪散及其君药槲寄生提取物多糖、总碱对人肝癌细胞HepG2生长的抑制作用及其对细胞内游离钙离子浓度的影响。方法采用MTT法测定槲芪散对细胞增生的抑制作用;利用流式细胞仪分析细胞周期、凋亡;利用激光共聚焦显微镜测定细胞内游离钙离子浓度。结果槲芪散及槲寄生提取物有较好的抑制肿瘤细胞增生的作用,高剂量组除了槲芪散和槲寄生总碱48 h时间点外,OA值均较对照组明显降低(P<0.001),呈现出时间-剂量依赖性;经药物作用48 h后,槲寄生多糖及总碱组G1期细胞比例增加,S期细胞和G2期细胞比例降低,槲芪散组G1期细胞比例无明显变化,S期比例降低,G2期比例增加,3者均使细胞凋亡增加;加入不同浓度的槲芪散、槲寄生多糖及总碱,低浓度时荧光增强不明显,而高浓度均导致细胞内荧光强度瞬间增强,升高到一定水平后,基本维持在一高水平,除槲寄生总碱组有略微下降外,其余2组未见明显下降趋势。结论槲芪散、槲寄生多糖及总碱均能抑制肿瘤细胞增生,促进细胞凋亡,且这3种药物引起胞内钙超载可能是诱导HepG2凋亡的机制之一。

Influence of Huqi San and its principal drug Ramulus Visci extract on intracellular free Ca2+ levels

Objective To probe the inhibitory effect of human hepatocarcinoma cell line HepG2 and the influence of intracellular free Ca²⁺ levels induced by Chinese herbs, Huqi San(HQS) and its principal drug Ramulus Visci Extract. Methods The proliferation of HepG2 were observed by MTT colormetric assay. The cell cycle and the rate of apoptosis of HepG2 were examined by using flow cytometry. The fluorescent intensity of intracellular free Ca²⁺ was observed by laser scanning confocal microscopy.Results Both HQS and Ramulus Visci extract could inhibit the proliferation of HepG2 in a time and dose-dependent manner, the numeric value of OD in high dose group decreased markedly compared to the control group(P<0.001), expect the high dose group of HQS and total Alkaloid of Viscum at the time of 48 h. After treatment with the drugs for 72 h, the cell population in the group of Ramulus Visci Polysaccharides and total Alkaloid of Viscum increased in G1 phase and G2 phase and decreased it in S phase; Comparatively, in the group of HQS, the proportion of the cells in S phase decreased, while cells at G2 phase increased, but there was no a significant change in the G1 phase. In addition, all of the three drugs could induce the apoptosis of HepG2. The results showed with laser scanning confocal microscopy that as different dose of the three drugs were put into the Petri dishes respectively, and we found that the fluorescent intensity did not change obviously when the cells were treated with the low dose drugs. But as we put into three high dose drugs into the Petri, dishes the fluorescent intensity increased instantly and maintained at a high level for periods of time without fluctuating nearly except a little decrease with total alkaloid of Viscum. ConclusionHuqi San, Ramulus Visci Polysaccharides and total alkaloid of Viscum all could inhibit the proliferation of cancer cells and promoted their apoptosis. The calcium overload maybe a possible mechanism of the apoptosis of HepG2 induced by Huqi San and Ramulus Visci Extract.