关键词: 黄芪, 多糖类, 肺肿瘤, CD8阳性T淋巴细胞, 基因表达调控, 干扰素调节因子类

Abstract:

Objective: To investigate the regulatory effect of Astragalus Polysaccharides (APS) on lung cancer tissue infiltrating PD-1^hiCD8⁺T cell subsets in mice. Methods: A tumor-bearing mouse model was constructed using 3LL lung cancer cells. 24 hours after model being established, 10 mice were randomly assigned into two groups. Control group mice were given 100 μl of sterile water for injection by gavage daily, while APS group were given APS (300 mg/kg) dissolved in 100 μl of sterile water for injection by gavage daily. Both groups were administered for 21 consecutive days. From day 5, the long and short diameters of abdominal tumors in mice were measured every 3 days. Mice were sacrificed when the tumor volume reached 2000 mm³. The experiment was repeated for 3 times to compare subcutaneous tumor volume and survival time between the two groups. Another 10 mice were similarly grouped, sacrificed 14 days after continuous administration following modeling. Tumor tissues were collected to prepare single-cell suspensions for examining the proportions of tumor CD8⁺ T cells, PD-1 expressions, IFN-γ secretions, as well as infiltrating mitochondrial metabolism (MTG: for mitochondrial structure; Rhod-2: for mitochondrial Ca²⁺ overload; TMRE: for mitochondrial membrane potential; Mito-SOX: for mitochondrial superoxide) and their correlation with IFN-γ secretion. The experiment was repeated 3 times, and flow cytometry analysis was performed using results from the latter 2 experiments. Results: Compared with the control group, the APS group significantly inhibited tumor growth from day 11 ((135.81±42.98) mm³ vs. (92.13±22.53) mm³;t=3.583, P=0.001) and significantly prolonged the survival time of tumor-bearing mice (χ²=12.180, P<0.001). Flow cytometry showed that the levels of PD-1⁺CD8⁺ and PD-1^hiCD8⁺ in APS group ((78.81±2.61) % and (21.52±1.94) %) were significantly lower than those in the control group ((83.41±1.95) % and (31.16±5.84) %, t=4.467, P<0.001; t=4.597, P<0.001), while the proportion of PD-1^intCD8⁺ ((57.48±2.84) %) was significantly higher than that in the control group ((52.20±5.83) %, t=2.542, P=0.020). The Rhod-2 level in PD-1^intCD8⁺ T cells ((57.10±6.25) %), and the expression of Rhod-2 and Mito-SOX in PD-1^hiCD8⁺ T cells ((69.25±5.56) % and (14.78±3.14) %) in APS group were significantly lower than those in the control group ((68.18±10.47) %, (78.95±9.48) %, and (24.66±5.54) %, t=2.873, P=0.010; t=2.791, P=0.012; t=4.907, P<0.001), while the MTG expression in PD-1^hiCD8⁺ T cells ((65.15±8.89) %) was significantly higher than that in the control group ((51.43±6.34) %, t=3.975, P<0.001). The secretion levels of IFN-γ in PD-1⁺CD8⁺, PD-1^hiCD8⁺, and PD-1^intCD8⁺ T cells in the APS group ((50.55±4.08) %, (70.48±3.62) %, (40.76±4.32) %) were significantly higher than those in the control group ((41.56±6.25) %, (58.87±6.54) %, (36.97±3.30) %, t=3.810, P=0.001; t=4.910, P<0.001; t=2.204, P=0.041). The IFN-γ secreted by PD-1^hiCD8⁺ T cells was significantly correlated with the expression of mitochondrial MTG, Rhod-2, and Mito-SOX (r=0.728, P<0.001; r=-0.618, P=0.004; r=-0.657, P=0.002), while the IFN-γ secreted by PD-1^intCD8⁺ T cells was only moderately associated with MTG and Rhod-2 expression (r=0.448, P=0.048; r=-0.550, P=0.012). Conclusion: APS can inhibit tumor growth in mice with lung cancer, significantly reduce the infiltration of PD-1^hiCD8⁺ T cells, remarkably enhance IFN-γ secretion in PD-1^hiCD8⁺ T cells, and regulate the anti-tumor effect of PD-1^hiCD8⁺ T cells through the mitochondrial metabolic pathway.

Key words: Astragalus membranaceus, Polysaccharides, Lung neoplasms, CD8-positive T-lymphocytes, Gene expression regulation, Interferon regulatory factors