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
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 tumor-infiltrating 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.
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