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（20 μg·mL^-1）组、SJZDCS（20%）组、SJZDCS（20%）＋PD-1抗体（20 μg·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Ras和P-ERK1/2蛋白表达显著升高（P＜0.01）。与PD-1抗体组相比，SJZDCS＋PD-1抗体组ERK1/2、p-MEK1/2和p-ERK1/2蛋白表达显著升高（P＜0.01），Ras蛋白表达显著降低（P＜0.01）。与SJZDCS组相比，SJZDCS＋PD-1抗体组Ras、p-MEK1/2、MEK1/2和p-ERK1/2蛋白表达显著升高（P＜0.05）。结论 SJZDCS单用或与PD-1抗体联用，可在体外调节肺癌肿瘤浸润CD4⁺T淋巴细胞免疫功能，其机制与调控PD-1及下游Ras-MEK-ERK通路表达有关。

Objective 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 (20 μg·mL^-1) group, SJZDCS (20%) group, SJZDCS (20%)+ PD-1 antibody (20 μg·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) in the SJZDCS group. The levels of TNF-α, IFN-γ and IL-2 were significantly increased (P < 0.01) and the levels of IL-4, IL-6, LDH and TGF-β1 were decreased (P < 0.01) in the SJZDCS+PD-1 antibody group. Compared with the PD-1 antibody group, the increase in the levels of TNF-α, IFN-γ, IL-2 and the decrease in the levels of IL-4, IL-6 and TGF-β1 were more significant (P < 0.01). Compared with the SJZDCS group, the levels of TNF-α, IFN-γ, IL-2 and IL-6 were higher (P < 0.01) in the SJZDCS+PD-1 antibody group. Compared with the control group, the expression of PD-1 protein in CD4⁺T lymphocytes was significantly decreased (P < 0.01) in the PD-1 antibody group, SJZDCS group and SJZDCS+ PD-1 antibody group, and the expression of p-MEK1/2 and MRK1/2 proteins was significantly increased (P < 0.05, 0.01). The expression of ERK1/2, p-MEK1/2 and p-ERK1/2 proteins was significantly increased (P < 0.01) in the PD-1 antibody group and SJZDCS+PD-1 antibody group, and the expression of Ras protein was significantly decreased (P < 0.01). Compared with the PD-1 antibody group, the expression of ERK1/2, p-MEK1/2, and p-ERK1/2 proteins was significantly increased (P < 0.01) in the SJZDCS+PD-1 antibody group, while the expression of Ras protein was significantly decreased (P < 0.01). Compared with the SJZDCS group, the expression of Ras, p-MEK1/2, MEK1/2, and p-ERK1/2 proteins was significantly increased (P < 0.05) in the SJZDCS+PD-1 antibody group. Conclusions Sijunzi Decoction medicated serum, whether used alone or in combination with PD-1 antibodies, can modulate the immune function of CD4⁺T lymphocytes infiltrating lung cancer tumors in vitro. This effect is associated with the regulation of PD-1 expression and the Ras-MEK-ERK signaling pathway.

R285.5

国家自然科学基金面上项目(82074405)