芒柄花黄素对小鼠H22肝癌移植瘤的抗肿瘤作用

Formononetin enhances the antitumor effect of H22 hepatoma transplanted mice

目的 揭示芒柄花黄素对H22肝癌移植瘤小鼠免疫的影响。方法 雄性C57BL/6小鼠皮下接种H22细胞(4×10^(5)个)建立荷瘤小鼠模型,用[10 mg/(kg·d)]或[50 mg/(kg·d)]的芒柄花黄素治疗小鼠28 d,然后计算抑瘤率。卡瑞利珠单抗作为阳性对照药物。免疫组织化学染色分析肝癌组织中CD8、颗粒酶B(granzyme B)和叉头盒P3(FOXP3)的表达,实时定量PCR或Western blot法检测肝癌组织中的程序性细胞死亡蛋白1(PD-1)及其配体1(PD-L1)的mRNA和蛋白表达,ELISA检测血清中白细胞介素10(IL-10)和转化生长因子β(TGF-β)的水平。结果 芒柄花黄素提高荷瘤小鼠的抑瘤率,增加肝癌组织中CD8和granzyme B染色阳性率。各组小鼠肿瘤组织中的FOXP3染色阳性率均无显著差异。芒柄花黄素降低的荷瘤小鼠血清中IL-10和TGF-β的水平,降低荷瘤小鼠肿瘤组织中PD-1和PD-L1的mRNA和蛋白相对表达量。结论 芒柄花黄素通过阻断PD-1/PD-L1通路激活CD8^(+)T细胞数量并减弱调节性T细胞(Treg)中免疫抑制因子的释放,从而发挥抗肿瘤作用。

Objective To explore the effect of formononetin on immunity of mice with transplanted H22 hepatocarcinoma.Methods Male C57BL/6 mice were subcutaneously inoculated with H22 cells(4×10^(5))to establish a tumor-bearing mouse model.The mice were treated with formononetin[10 mg/(kg·d)]or[50 mg/(kg·d)]for 28 days,and then the tumor inhibition rate was calculated.Carrilizumab was used as a positive control drug.The expressions of CD8,granzyme B and forkbox transcription factor 3(FOXP3)in HCC tissues were analyzed by immunohistochemical staining.The mRNA and protein expression of programmed cell death protein 1(PD-1)and its ligand 1(PD-L1)in HCC tissues were detected by real-time PCR or Western blot analysis,respectively.The serum levels of interleukin-10(IL-10)and transforming growth factor-β(TGF-β)were detected by ELISA.Results Formononetin increased the tumor inhibition rate and the positive rate of CD8 and granzyme B staining in tumor-bearing mice.There was no significant difference in the positive rate of FOxP3 staining in tumor tissues of mice in each group.Formononetin decreased the levels of IL-10 and TGF-β in serum of tumor-bearing mice,and decreased the relative expression of mRNA and protein of PD-1 and PD-L1 in tumor tissue of tumor-bearing mice.Conclusion Formononetin can activate CD8t T cells and reduce the release of immunosuppressive factors in regulatory T cells by blocking PD-1/PD-L1 pathway and play an antitumor role.