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目的:观察益气养阴解毒方的含药血清与化疗药5-FU体外共同作用于肝癌HepG2细胞后,对细胞Survivin的表达和Caspase-3活性的影响,从而探讨本益气养阴解毒方增强5-FU诱导HepG2细胞凋亡的相关机制。方法:运用血清药理学方法制备大鼠的益气养阴解毒方的含药血清,分为中药含药血清组、化疗药物5-FU组和中药含药血清+5-FU组,并设不含药的空白大鼠血清为对照组,分别作用于HepG2细胞,应用SP免疫细胞化学染色法及western-blot检测各组细胞24h、48h、72h后Survivin蛋白的表达变化;应用DEVD-pNA降解法检测各组细胞24h、48h、72h后Cas-pase-3活性变化。结果:(1)各组作用24h、48h、72h后,中药含药血清组HepG2细胞Survivin蛋白的表达量无明显变化(与对照组相比,P>0.05);5-FU组HepG2细胞Survivin蛋白的表达量逐渐升高(与对照组相比,P<0.05),且具有时间依赖性;而中药含药血清+5-FU组细胞Survivin蛋白的升高水平与5-FU组相比明显降低(P<0.05)。(2)作用24h、48h、72h后含药血清组HepG2细胞Caspase-3的活性无明显变化(与对照组相比,P>0.05);作用48h、72h后,5-FU组HepG2细胞Caspase-3的活性逐渐升高(与对照组相比,P<0.05);而中药含药血清+5-FU组细胞Caspase-3的活性升高更显著(与5-FU组相比,P<0.05)。结论:益气养阴解毒方含药血清可能是通过降低5-FU诱导的凋亡抑制蛋白Survivin的表达,减弱Survivin对Caspase-3活性的抑制,从而促进细胞凋亡的,这可能是本方与化疗合用增效的重要机制之一。
Objective: To observe the effects of Yiqi Yangyinjiedu prescription drug-containing serum and chemotherapeutic agent 5-FU in hepatocellular carcinoma HepG2 cells on the expression of survivin and Caspase-3 activity in order to explore the effect of Benqi Yangyin Jiedu Fang Enhances the mechanism of apoptosis induced by 5-FU in HepG2 cells. METHODS: Rat serum of Yiqi Yangyin Jiedu Decoction was prepared by serum pharmacology and divided into traditional Chinese medicine drug-containing serum group, chemotherapeutic drug 5-FU group, and Chinese herbal drug-containing serum +5-FU group. The drug-containing blank rat serum served as a control group and were treated with HepG2 cells respectively. SP immunocytochemical staining and western-blot were used to detect the expression of survivin protein at 24h, 48h and 72h in each group. The DEVD-pNA degradation method was used. The activity of Cas-pase-3 was detected after 24h, 48h and 72h in each group of cells. RESULTS: (1) After 24h, 48h and 72h in each group, the expression of Survivin protein in HepG2 cells without TCM serum had no significant change (P>0.05 compared with the control group); Survivin protein in HepG2 cells in 5-FU group The expression level gradually increased (P<0.05 compared with the control group) and was time-dependent; while the elevated levels of Survivin protein in the serum of the Chinese medicine-containing serum+5-FU group was significantly lower than that of the 5-FU group. (P<0.05). (2) After 24h, 48h and 72h, the activity of Caspase-3 in HepG2 cells without sera was significantly changed (P>0.05 compared with the control group); after 48h and 72h, the 5-FU HepG2 cells Caspase- The activity of 3 increased gradually (P<0.05 compared with the control group); while the activity of Caspase-3 increased more significantly in the serum of the Chinese medicine-containing serum +5-FU group (P<0.05 compared with the 5-FU group. ). Conclusion: The blood serum of Yiqi Yangyin Jiedufang may reduce the expression of Survivin induced by 5-FU, and inhibit the inhibition of Caspase-3 activity by Survivin, thus promoting apoptosis. One of the important mechanisms for synergy with chemotherapy is synergy.