基于ERK介导C-Myc/PD-L1协同作用探讨参芪抑瘤方联合顺铂对H22肝癌荷瘤小鼠的抑瘤机制

ERK mediated C-Myc/PD-L1 synergy to investigate tumor suppression effect of Shenqi Yiliu decoction combined with cisplatin in combination with H22 hepatocellular carcinoma tumor bearing mice

目的:探讨参芪抑瘤方联合顺铂经ERK介导C-Myc/PD-L1相协途径对H22肝癌荷瘤小鼠的抑瘤作用及其机制。方法:60只SPF级雄性昆明小鼠,采用随机数字表法取10只小鼠作为空白组,其余50只小鼠复制H22肝癌荷瘤小鼠模型,模型复制成功后将模型小鼠随机分为模型组、顺铂组[2.5×10^(-3) g/(kg·3 d)]、参芪抑瘤方低[13.515 g/(kg·d)]、中[27.030 g/(kg·d)]、高剂量[54.060 g/(kg·d)]联合顺铂[2.5×10^(-3) g/(kg·3 d)]组,每组10只,治疗13 d,末次给药24 h后,麻醉处死小鼠,测定小鼠肿瘤抑制率和脾指数、胸腺指数;HE染色观察小鼠肿瘤组织病理学变化;ELISA试剂盒检测肿瘤组织匀浆液中EGF、IFN-γ含量;IHC法和Western blot法检测肿瘤组织中p-ERK1/2、C-Myc、PD-L1蛋白表达;RT-PCR法检测肿瘤组织中ERK、C-Myc、PD-L1mRNA表达水平。结果:与空白组相比,模型组小鼠平均体质量和脾脏指数均降低(P<0.05);与模型组相比,各治疗组肿瘤抑制效果明显,且参芪抑瘤方联合顺铂组以剂量依赖性方式抑制肝癌小鼠肿瘤生长,提高小鼠平均体质量和脾指数、胸腺指数,促进肿瘤细胞坏死,增加坏死面积,降低肿瘤组织中EGF和IFN-γ含量以及p-ERK1/2、C-Myc、PD-L1蛋白表达和ERK、C-Myc、PD-L1 mRNA表达(P<0.05);与顺铂组相比,参芪抑瘤方中、高剂量联合顺铂组治疗效果显著,差异具有统计学意义(P<0.05)。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,显著下调肿瘤组织中C-Myc与PD-L1蛋白表达,该机制可能通过调控ERK信号通路相关蛋白表达发挥抑瘤作用。

Objective:To investigate the tumor suppressing effect of Shenqi Yiliu decoction combined with cisplatin via ERK-mediated C-Myc/PD-L1 phase-coordinated pathway on H22 hepatocellular carcinoma tumor-bearing mice and its mechanism.Meth-ods:In 60 SPF-grade male Kunming mice,10 mice were taken as blank group by random number table method,and the other 50 mice were replicated as H22 hepatocellular carcinoma tumor-bearing mouse model.After successful replication of the model,the model mice were randomly divided into model group,cisplatin group[2.5×10^(-3) g/(kg·3 d)],Shenqi Yiliu decoction low[13.515 g/(kg·d)],me-dium[27.03 g/(kg·d-1)],and high dose[27.030 g/(kg·d)]combined with cisplatin group[2.5×10^(-3) g/(kg·3 d)],10 mice in each group were treated for 13 d.After 24 h of the last dose,the mice were anesthetized and sacrificed,and the tumor inhibition rate,spleen index and thymus index of each drug group were determined;HE staining was performed to observe the histopathological changes of tumor in mice;ELISA kit was used to detect the contents of EGF and IFN-γin tumor tissue homogenate;p-ERK1/2,C-Myc and PD-L1 protein expression in tumor tissue were detected by IHC and Western blot;ERK,C-Myc and PD-L1 mRNA expression levels in tumor tissue were detected by RT-PCR.Results:Compared with blank group,the average body mass and spleen index of mice in model group were decreased(P<0.05).Compared with model group,the tumor inhibition effect of each treatment group was obvious,and Shenqi Yiliu decoction combined with cisplatin group inhibited tumor growth in liver cancer mice in a dose-dependent way,im-proved the average body mass,spleen index and thymus index of mice,promoted the necrosis of tumor cells and increased the necrotic area.EGF and IFN-γcontents,P-ERK1/2,C-Myc,PD-L1 protein expressions and ERK,C-Myc,PD-L1 mRNA expression levels were decreased in tumor tissues(P<0.05).Compared with cisplatin group,the therapeutic effect of Shenqi decoction combined with cisplatin in medium and high dose groups was significant,and the difference was statistically significant(P<0.05).Conclusion:Shenqi Yiliu decoction combined with cisplatin effectively inhibited the tumor growth of H22 liver cancer tumor-bearing mice and significantly reduces the expression of C-Myc and PD-L1 proteins in the tumor tissues,which may be through the regulation of ERK signaling path-way-related protein expression to exert tumor suppressive effect.