加味当归贝母苦参丸对H22肝癌荷瘤小鼠抑瘤及T细胞免疫调节作用

Modified Danggui Beimu Kushen Pills Inhibit Tumor Growth and Regulates T Cell Subsets in H22 Hepatocellular Carcinoma-bearing Mice

目的:探讨加味当归贝母苦参丸对H22肝癌荷瘤小鼠抑瘤及T细胞免疫调节作用,为加味当归贝母苦参丸联合免疫检查点抗体治疗肝癌提供依据。方法:建立H22肝癌荷瘤小鼠模型,随机分为模型组、顺铂组、加味当归贝母苦参丸低、中、高剂量组(4、8、16 g·kg^(-1)·d^(-1)),连续给药14 d,第15天处死小鼠。给药第0、4、8、12、15天测量肿瘤体积;称取瘤重、计算胸腺指数和脾指数;将脾脏淋巴细胞与H22肝癌细胞共培养,细胞增殖与活性检测(CCK-8)法检测肿瘤细胞增殖抑制率;实时荧光定量聚合酶链式反应(Real-time PCR)检测脾脏和肿瘤组织中程序性细胞死亡蛋白-1(PD-1)、淋巴细胞活化基因-3(LAG-3)mRNA的表达情况;免疫组织化学法(IHC)检测脾脏和肿瘤组织中CD4^(+)、CD8^(+)T细胞数量及PD-1、LAG-3蛋白的表达情况。结果:给药后第8天,各给药组肿瘤体积较模型组均有不同程度下降,第15天肿瘤体积和瘤重均较模型组显著降低(P<0.01),其中顺铂组下降最为明显。与模型组、顺铂组比较,加味当归贝母苦参丸中、高剂量组胸腺指数显著升高(P<0.01);与模型组比较,各用药组脾脏指数均明显升高(P<0.05,P<0.01),其中顺铂组升高最为显著;与模型组、顺铂组比较,加味当归贝母苦参丸各组脾脏、肿瘤组织中CD4^(+)、CD8^(+)T细胞数量和肿瘤细胞杀伤率均显著增高(P<0.01),LAG-3 mRNA和蛋白表达均明显降低(P<0.05,P<0.01);加味当归贝母苦参丸高剂量组肿瘤组织中PD-1 mRNA表达较模型组、顺铂组显著降低(P<0.01)。结论:加味当归贝母苦参丸可能通过下调LAG-3表达,促进H22肝癌荷瘤小鼠CD4^(+)、CD8^(+)T细胞的增殖并向肿瘤微环境浸润,从而改善T细胞免疫活性、抑制肿瘤生长,为加味当归贝母苦参丸与免疫检查点抗体联用治疗肝癌提供实验依据。

Objective:To explore the effects of modified Danggui Beimu Kushen pills on tumor growth and T-cell subsets in H22 hepatocellular carcinoma-bearing mice and to provide an experimental basis for the treatment of hepatocellular carcinoma with modified Danggui Beimu Kushen pills combined with immune checkpoint antibodies.Method:A H22 hepatocellular carcinoma-bearing mouse model was established.The modeled mice were randomized into model,cisplatin,low-(4 g·kg^(-1)·d^(-1)),medium-(8 g·kg^(-1)·d^(-1)),and highdose(16 g·kg^(-1)·d^(-1))modified Danggui Beimu Kushen pills groups.After continuous administration for 14 days,the mice were sacrificed on day 15.The tumor volume was measured on days 0,4,8,12,15 of drug administration.Tumors were weighed and thymus index and spleen index were calculated.Spleen lymphocytes were co-cultured with H22 hepatoma cells,and the tumor cell-killing rate was detected by the cell counting kit-8(CCK-8).Real-time polymerase chain reaction was carried to determine the mRNA levels of programmed cell death protein-1(PD-1)and lymphocyte activation gene-3(LAG-3)in spleen and tumor tissues.The number of CD4^(+)and CD8^(+)T cells and the expression of PD-1 and LAG-3 were detected by immunohistochemistry(IHC).Result:On day 8 of drug administration,tumor volumes in all treatment groups decreased compared with that in the model group.On day 15,both tumor volume and tumor weight were significantly lower in the treatment groups than in the model group(P<0.01),with the cisplatin group showing the most pronounced reduction.Compared with the model and cisplatin groups,medium-and high-dose modified Danggui Beimu Kushen pills increased the thymus index(P<0.01).Compared with the model group,all treatment groups showed increased spleen index(P<0.05,P<0.01),with the cisplatin group showing the most significant increase.Compared with the model and cisplatin groups,all the groups of modified Danggui Beimu Kushen pills demonstrated increased number of CD4^(+)and CD8^(+)T cells and tumor cell-killing rate in the spleen and tumor tissues(P<0.01)and downregulated mRNA and protein levels of LAG-3(P<0.05,P<0.01).The high-dose group of modified Danggui Beimu Kushen pills had lower mRNA level of PD-1 in the tumor tissue than the model and cisplatin groups(P<0.01).Conclusion:Modified Danggui Beimu Kushen pills may promote the proliferation and tumor microenvironment infiltration of CD4^(+)and CD8^(+)T cells in H22 tumor-bearing mice by down-regulating LAG-3 expression,thereby improving T-cell immune activity and inhibiting tumor growth.This study provides an experimental basis for the combination of modified Danggui Beimu Kushen pills and immune checkpoint antibodies in the treatment of hepatocellular carcinoma.