益气活血软坚解毒方含药血清诱导人肝癌细胞系Bel-7402细胞凋亡过程中部分凋亡调控基因变化

Yiqi Huoxue Ruanjian Jiedu decoction regulates expression and activity of apoptosis-regulated genes in human hepatocellular carcinoma cell line BEL-7402

目的:研究益气活血软坚解毒方(YHRJ)含药血清对人肝癌细胞系Bel-7402细胞凋亡调控基因Fas,FasL,Bcl-2,Bax,P53,NF-kB表达影响.方法:将细胞分为对照组(NS)组、NS+DDP(顺氯氨铂)组、YHRJ等剂量组(YHRJD)、YHRJD+DDP组、YHRJ高剂量组(YHRJG)、YHRJG+DDP组,应用流式细胞术、免疫组化、原位杂交、RT-PCR等方法对用药24,48h的Bel-7402细胞凋亡的主要调控基因Fas,FasL,Bcl-2,Bax,P53,NF-kBmRNA与蛋白表达进行检测.结果:流式细胞术检测显示:与NS组相比,NS+DDP、YHRJD、YHRJD+DDP及YHRJG+DDP组Fas蛋白表达均明显提高(30.12%±22.94%,10.50%±8.41%,30.35%±22.98%,32.61%±26.87%vs8.77%±6.93%,P<0.01),YHRJG组效果不明显(P>0.05);NS+DDP、YHRJD+DDP组FasL蛋白表达也升高(16.40%±7.168%,8.41%±6.74%vs4.12%±2.60%,P<0.01),而YHRJG组FasL蛋白表达降低(3.05%±2.53%vs4.12%±2.60%,P<0.01).免疫组化结果显示:除YHRJG+DDP组外,其余各组突变型P53蛋白表达明显降低(30.2%,14.6%,19.8%,17.3%vs60.0%,P<0.05);各加药组Bax蛋白表达明显增高(40.7%,40.4%,72.1%,68.9%,42.2%vs30.0%,P<0.05);NS+DDP组与YHRJG组Bcl-2蛋白表达明显降低(26.3%,24.4%vs30.5%,P<0.05),而YHRJD、YHRJG+DDP、YHRJG+DDP组Bcl-2表达明显升高(41.8%,39.3%,45.6%vs30.5%,P<0.05);各加药组NF-kB蛋白表达明显降低(15.9%,13.3%,14.1%,7.8%,14.6%vs24.2%,P<0.05).原位杂交结果显示:各加药组NF-kBmRNA表达明显降低(30.5%,13.3%,21.4%,17.4%,53.2%vs58%,P<0.05).RT-PCR结果显示:YHRJ等效剂组凋亡调控基因Bcl-2mRNA表达明显降低(0.717±0.198vs1.327±0.097,P<0.001);DDP、YHRJD、YHRJG组凋亡调控基因BaxmRNA表达明显增高(46.22±6.22,56.19±7.36,62.32±11.06vs35.22±4.38,P<0.05).结论:YHRJ含药血清诱导人肝癌细胞系Bel-7402细胞凋亡可能的基因调控机制在于通过抑制凋亡信号转导基因FasL基因蛋白表达,促进凋亡调控基因Bax基因蛋白表达,抑制NF-kB基因mRNA及蛋白表达来实现的.

AIM： To investigate the effects of Yiqi Huoxue Ruanjian Jiedu （YHRJ） decoction on the expression of apoptosis-related genes Fas, FasL, Bcl-2, Bax, P53 and NF-kB in human hepatocellular carcinoma cell line BEL-7402. METHODS： Human hepatocellular carcinoma BEL-7402 cells were divided into 6 groups, and treated with normal saline （NS）, NS plus cisplatin （DDP）, equal-dose YHRJ decoction, YHRJ decoction plus DDP group, high-dose YHRJ decoction, high-dose YHRJ decoction plus DDP, respectively. Flow cytometry, immunohisto-chemistry, in situ hybridization, and reverse transcription-polymerase chain reaction （RT- PCR） were used to analyze the expression of Fas, FasL, Bcl-2, Bax, P53 and NF-kB. RESULTS： Flow cytometry revealed a dramatic increase of Fas expression in the cells treated with NS plus DDP, equal-dose YHRJ and equaldose YHRJ plus DDP, and high-dose YHRJ plus DDP in comparison with that in the control cells （30.12% ± 22.94%, 10.50% ± 8.41%, 30.35% ± 22.98%, 32.61% ± 26.87% vs 8.77% ± 6.93%, P 〈 0.01）, but not in the cells treated with high-dose YHRJ （P 〉 0.05）. The expression of FasL was significantly higher in NS＋DDP and YHRJD＋DDP treatment group than that in the normal controls （16.40% ± 7.168%, 8.41% ± 6.74% vs 4.12% ± 2.60%, P 〈 0.01）, but lower in high-dose YHRJ group （3.05% ± 2.53% vs 4.12% ± 2.60%, P 〈 0.01）. Immunohistochemistry showed a significant de- crease of P53 expression in all the drug-treated cells （30.2%, 14.6%, 19.8%, 17.3% vs 60.0%, P 〈 0.05） except high-dose YHRJ plus DDP treated ones; The expression of Bax protein in all the drug-treated cells was higher significantly than that in the controls （40.7%, 40.4%, 72.1%, 68.9%, 42.2% vs 30.0%, P 〈 0.05）; There was a notable decrease of Bcl-2 expression in NS plus DDP and high-dose YHRJ treated cells （26.3%, 24.4% vs 30.5%, P 〈 0.05） as well as of NF-kB expression in all the drug-treated cells （40.7%, 40.4%, 72.1%, 68.9%, 42.2% vs 30.0%, P 〈 0.05）. In situ hybridization also demonstrated that the mRNA expression of NF-kB was markedly decreased in all the drug-treated groups as compared with that in the control group （30.5%, 13.3%, 21.4%, 17.4%, 53.2% vs 58%, P 〈 0.05）. RT-PCR revealed a significant decrease of Bcl-2 mRNA expression in equal-dose YHRJ treated cells （0.717 ± 0.198 vs 1.327 ± 0.097, P 〈 0.001） as well as a dramatic increase of Bax mRNA expression in DDP, YHRJD, and YHRJG treated cells （46.22 ± 6.22, 56.19 ± 7.36, 62.32 ± 11.06 vs 35.22 ± 4.38, P 〈 0.05）. CONCLUSION： YHRJ decoction may induce apoptosis in human hepatocellular carcinoma by down-regulating the expression of FasL, upregulating the expression of Bax and inhibiting the activation of NF-kB.