四君子汤含药血清联合PD-1抗体调节肺癌肿瘤浸润CD4^(+)T淋巴细胞免疫功能和作用机制  

Effect and mechanism of Sijunzi Decoction containing serum combined with PD-1 antibody regulating immune function of tumor-infiltrating CD4^(+)T lymphocytes in lung cancer

作　　者：毛启远 王学谦[1] 林飞[1] 蒋伶俐 张楚楚 谭滢 蔡瑞娟 李道睿[1] 林洪生[1] MAO Qiyuan;WANG Xueqian;LIN Fei;JIANG Lingli;ZHANG Chuchu;TAN Ying;CAI Ruijuan;LIDaorui;LIN Hongsheng(Department of Oncology,Guang'anmen Hospital,China Academy of Chinese Medical Sciences,Beijing 100053,China;Institute of Information on Traditional Chinese Medicine,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]中国中医科学院广安门医院肿瘤科,北京100053 [2]中国中医科学院中医药信息研究所,北京100700

出　　处：《药物评价研究》2024年第11期2508-2516,共9页Drug Evaluation Research

基　　金：国家自然科学基金面上项目(82074405)。

摘　　要：目的通过体外实验探究四君子汤含药血清(SJZDCS)联合PD-1抗体对肺癌肿瘤浸润CD4^(+)T淋巴细胞免疫功能的影响和机制。方法取雄性SD大鼠,连续ig四君子汤6 d,麻醉取血,制备含药血清。取雄性C57BL/6J小鼠,建立Lewis肺癌荷瘤小鼠模型,饲养第18天后处死小鼠,提取肿瘤并分离肿瘤浸润淋巴细胞,磁珠分选肿瘤浸润CD4^(+)T淋巴细胞。建立Lewis细胞与肿瘤浸润CD4^(+)T淋巴细胞共培养体系,CCK-8法检测Lewis细胞的增殖,筛选SJZDCS的最佳作用浓度和时间。建立Lewis细胞与肿瘤浸润CD4^(+)T淋巴细胞间接共培养体系,分为对照组、PD-1抗体(20μg·mL^(−1))组、SJZDCS(20%)组、SJZDCS(20%)+PD-1抗体(20μg·mL^(−1))组,干预72 h;酶联免疫吸附实验(ELISA)检测共培养细胞上清中免疫功能相关细胞因子乳酸脱氢酶(LDH)、转化生长因子-β1(TGF-β1)、肿瘤坏死因子-α(TNF-α)、干扰素-γ(IFN-γ)、白细胞介素(IL)-2、IL-4、IL-6和IL-10表达水平,Western blotting检测肿瘤浸润CD4^(+)T淋巴细胞中PD-1和Ras-MEK-ERK通路蛋白表达水平。结果20%SJZDCS干预肿瘤浸润CD4^(+)T淋巴细胞72 h对抑制共培养Lewis细胞的增殖效果最佳。与对照组相比,PD-1抗体组TNF-α、IFN-γ、IL-2和TGF-β1水平显著升高(P<0.01),IL-4、IL-6、IL-10和LDH水平显著降低(P<0.05);SJZDCS组IL-2水平显著升高(P<0.01),IL-4、IL-6、LDH和TGF-β1水平显著降低(P<0.05、0.01);SJZDCS+PD-1抗体组TNF-α、IFN-γ和IL-2水平显著升高(P<0.01),IL-4、IL-6、LDH和TGF-β1水平降低(P<0.01)。与PD-1抗体组相比,SJZDCS+PD-1抗体组TNF-α、IFN-γ、IL-2升高水平和IL-4、IL-6和TGF-β1的降低水平更显著(P<0.01)。与SJZDCS组相比,SJZDCS+PD-1抗体组TNF-α、IFN-γ、IL-2和IL-6的水平更高(P<0.01)。与对照组相比,PD-1抗体组、SJZDCS组、SJZDCS+PD-1抗体组CD4^(+)T淋巴细胞中PD-1蛋白表达显著降低(P<0.01),p-MEK1/2和MRK1/2蛋白表达显著升高(P<0.05、0.01);PD-1抗体组、SJZDCS+PD-1抗体组RObjective The objective of this study was to investigate the impact and underlying mechanisms of Sijunzi Decoction containing serum(SJZDCS)in combination with a PD-1 antibody on the immune function of CD4^(+)T lymphocytes infiltrating lung cancer tumors through in vitro experiments.Methods Male SD rats were administered Sijunzi Decoction via continuous gavage for 6 d,after which blood was collected under anesthesia to prepare serum containing the medication.Lewis lung cancer tumor-bearing mouse model was established using male C57BL/6J mice.After 18 d,the mice were sacrificed,tumors were excised,and tumorinfiltrating lymphocytes were isolated.CD4^(+)T lymphocytes were sorted using magnetic beads,and a co-culture system with Lewis cells was established,and divided into control group,PD-1 antibody(20μg·mL^(−1))group,SJZDCS(20%)group,SJZDCS(20%)+PD-1 antibody(20μg·mL^(−1))group,intervention for 72 h.The proliferation of Lewis cells was assessed using the CCK-8 assay to determine the optimal concentration and exposure time of the medicated serum.Immune function-related cytokines,including lactate dehydrogenase(LDH),transforming growth factor-β1(TGF-β1),tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ),interleukin-2(IL-2),IL-4,IL-6,and IL-10,were measured in the supernatant of the co-cultured cells using an enzyme-linked immunosorbent assay(ELISA).The expression levels of PD-1 and proteins involved in the Ras-MEK-ERK pathway in CD4^(+)T lymphocytes were evaluated using Western blotting.Results The intervention of 20%SJZDCS on tumor infiltrating CD4^(+)T lymphocytes for 72 h showed the best inhibitory effect on the proliferation of co cultured Lewis cells.Compared with the control group,the levels of TNF-α,IFN-γ,IL-2 and TGF-β1 were significantly increased(P<0.01)and the levels of IL-4,IL-6,IL-10 and LDH were significantly decreased(P<0.05)in the PD-1 antibody group.The level of IL-2 was significantly increased(P<0.01)and the levels of IL-4,IL-6,LDH and TGF-β1 were significantly decreased(P<0.05,0.01

关 键 词：四君子汤 含药血清 抗PD-1免疫治疗 肿瘤浸润CD4^(+)T淋巴细胞 免疫细胞因子 Ras-MEK-ERK信号通路 

分 类 号：R285.5[医药卫生—中药学]