参芪抑瘤方联合顺铂对H22肝癌荷瘤小鼠抑瘤作用的研究

Study on the anti-tumor effect of Shenqi Yiliu decoction combined with cisplatin on H22 liver cancer-bearing mice

目的探讨参芪抑瘤方联合顺铂对H22肝癌荷瘤小鼠的抑瘤作用及其机制。方法用雄性昆明小鼠复制H22肝癌荷瘤小鼠模型,另取10只正常小鼠作为空白组。将模型鼠随机分为模型组(0.9%NaCl)、顺铂组(2.5×10^(-3)g·kg^(-1)·3 d^(-1)顺铂)及低、中、高剂量(13.52、27.03和54.06 g·kg^(-1)·d^(-1)参芪抑瘤方)联合顺铂(顺铂2.5×10^(-3)g·kg^(-1)·3 d^(-1))组,干预治疗13 d。用苏木精-伊红(HE)染色和细胞凋亡染色(TUNEL)法观察肿瘤病理变化及凋亡情况;用实时荧光定量聚合酶链反应法和蛋白质印迹法检测肿瘤组织中磷酸化c-Jun氨基末端蛋白激酶(p-JNK)、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、B淋巴细胞瘤-2基因相关启动子(Bad)、半胱氨酸蛋白酶-9(Caspase-9)mRNA及蛋白的表达水平。结果模型组、顺铂组、参芪抑瘤方高剂量联合顺铂组肿瘤组织细胞凋亡率分别为(6.99±0.34)%、(33.18±3.50)%和(64.32±2.21)%,p-JNK蛋白相对表达水平分别为0.21±0.02、0.43±0.05和1.04±0.05,Bcl-2蛋白相对表达水平分别为1.60±0.01、1.36±0.05和0.47±0.01,Bax蛋白相对表达水平分别为0.31±0.03、0.45±0.03和0.99±0.02,Bad蛋白相对表达水平分别为0.34±0.02、0.66±0.03和1.10±0.03,Caspase-9蛋白相对表达水平分别为0.39±0.04、0.50±0.03和0.85±0.06;模型组的上述指标与顺铂组比较,差异均有统计学意义(均P<0.01);模型组和顺铂组的上述指标与参芪抑瘤方高剂量联合顺铂组比较,差异均有统计学意义(均P<0.01)。结论参芪抑瘤方联合顺铂能有效抑制H22肝癌荷瘤小鼠的肿瘤生长,该作用机制可能与调控JNK信号通路及相关凋亡蛋白的表达有关。

Objective To explore the tumor suppressing effect of Shenqi Yiliu(SQYL)decoction combined with cisplatin(CDDP)on H22 liver cancer tumor-bearing mice and its mechanism.Methods The H22hepatocellular carcinoma tumor-bearing mouse model was replicated using male Kunming mice,and another 10 normal mice were taken as blank group.The model mice were randomly divided into model(0.9%Na Cl),CDDP(2.5×10^(-3)g·kg^(-1)·3 d^(-1)CDDP),and SQYL-L,-M,-H+CDDP(13.52,27.03,54.06 g·kg^(-1)·d^(-1)SQYL+2.5×10^(-3)g·kg^(-1)·3 d^(-1)CDDP).Interventions were administered for 13 d.Tumor pathological changes and apoptosis were observed by hematoxylin-eosin staining(HE)and apoptosis staining(TUNEL);real-time fluorescence quantitative polymerase chain reaction and protein blotting methods were used to detected the mRNA and protein expression levels of phosphorylated c-Jun amino-terminal protein kinase(p-JNK),B lymphocytoma-2(Bcl-2),Bcl-2-associated X protein(Bax),B lymphocytoma-2 gene-associated promoter(Bad),and duck cysteine protease-9(Caspase-9)in tumor tissues.Results The apoptosis rates of tumor tissues in the model group,CDDP group and SQYL-H+CDDP group were(6.99±0.34)%,(33.18±3.50)%and(64.32±2.21)%,respectively;the relative protein expression levels of p-JNK were 0.21±0.02,0.43±0.05 and1.04±0.05,respectively;the relative protein expression levels of Bcl-2 were 1.60±0.01,1.36±0.05,0.47±0.01,respectively;the relative protein expression levels of Bax in the three groups were 0.31±0.03,0.45±0.03 and 0.99±0.02,respectively;the relative protein expression levels of Bad in the three groups were0.34±0.02,0.66±0.03 and 1.10±0.03,respectively;the relative protein expression levels of Caspase-9 were0.39±0.04,0.50±0.03 and 0.85±0.06,respectively.The above indexes in the model group were significantly different from those in the CDDP group(all P<0.01).The above indexes in the model group and CDDP group were significantly different from those in the SQYL-H+CDDP group(all P<0.01).Conclusion Shenqi Yiliu decoction combined with cisplatin effectively inhibited tumor growth in H22 hepatoma-bearing mice,and the mechanism of action may be related to the regulation of JNK signaling pathway and the expression of related apoptotic proteins.