Investigate the regulatory effect of Astragalus Polysaccharides on lung cancer tissue infiltrating PD-1hiCD8+T cell subsets in mice

doi:10.19983/j.issn.2096-8493.20250023

Abstract

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

CLC Number:

R392
R734

Fan Guiqin, Lyu Hong, Qu Qiuxia. Investigate the regulatory effect of Astragalus Polysaccharides on lung cancer tissue infiltrating PD-1^hiCD8⁺T cell subsets in mice[J]. Journal of Tuberculosis and Lung Disease , 2025, 6(4): 393-400. doi: 10.19983/j.issn.2096-8493.20250023

Figures/Tables 8

时间(d)	对照组(15只) (mm³, $x ˉ$ ±s)	黄芪多糖组(15只) (mm³, $x ˉ$ ±s)	t值	P值
5	31.01±11.25	31.82±9.16	0.217	0.830
8	66.79±25.44	57.72±15.18	1.185	0.246
11	135.81±42.98	92.13±22.53	3.583	0.001
14	269.66±96.39	169.55±49.91	3.572	0.001
17	441.24±129.35	285.25±71.80	4.084	<0.001
20	690.56±184.61	484.68±142.61	3.572	0.001
23	1139.76±323.36	756.18±129.28	4.266	<0.001
26	1624.85±305.09	1199.75±206.12	4.522	<0.001
29	2059.54±167.77	1738.34±493.74	3.118	0.005

变量	对照组(10只) (%, $x ˉ$ ±s)	黄芪多糖组(10只) (%, $x ˉ$ ±s)	t值	P值
CD8⁺/CD45⁺	16.92±3.29	16.84±2.87	0.059	0.954
PD-1⁺CD8⁺/CD8⁺T	83.41±1.95	78.81±2.61	4.467	<0.001
PD-1^hiCD8⁺/CD8⁺ T	31.16±5.84	21.52±1.94	4.597	<0.001
PD-1^intCD8⁺/CD8⁺ T	52.20±5.83	57.48±2.84	2.542	0.020

组别	PD-1^intCD8⁺T细胞(%, $x ˉ$ ±s)				PD-1^hiCD8⁺T细胞(%, $x ˉ$ ±s)
组别	MTG	Rhod-2	Mito-SOX	TMRE	MTG	Rhod-2	Mito-SOX	TMRE
对照组(10只)	56.97±8.83	68.18±10.47	11.83±4.51	12.06±4.09	51.43±6.34	78.95±9.48	24.66±5.54	10.88±2.59
黄芪多糖组(10只)	63.42±8.04	57.10±6.25	9.41±1.38	14.71±5.32	65.15±8.89	69.25±5.56	14.78±3.14	13.29±4.41
t值	1.709	2.873	1.620	1.250	3.975	2.791	4.907	1.491
P值	0.105	0.010	0.124	0.227	<0.001	0.012	<0.001	0.153

变量	对照组(10只) (%, $x ˉ$ ±s)	黄芪多糖组(10只) (%, $x ˉ$ ±s)	t值	P值
INF-γ⁺/PD-1⁺CD8⁺	41.56±6.25	50.55±4.08	3.810	0.001
INF-γ⁺/PD-1^hiCD8⁺	58.87±6.54	70.48±3.62	4.910	<0.001
INF-γ⁺/PD-1^intCD8⁺	36.97±3.30	40.76±4.32	2.204	0.041

References 27

[1]	Siegel RL, Miller KD, Wagle NS, et al. Cancer statistics, 2023. CA Cancer J Clin, 2023, 73(1):17-48. doi:10.3322/caac.21763.
[2]	Han B, Zheng R, Zeng H, et al. Cancer incidence and mortality in China, 2022. J Natl Cancer Cent, 2024, 4(1):47-53. doi:10.1016/j.jncc.2024.01.006.
[3]	Song P, Zhang J, Shang C, et al. Real-world evidenceand clinical observations of the treatment of advanced non-small cell lung cancer with PD-1/PD-L1 inhibitors. Sci Rep, 2019, 9(1):4278. doi:10.1038/s41598-019-40748-7. pmid: 30862891
[4]	Okiyama N, Tanaka R. Immune-related adverse events in various organs caused by immune checkpoint inhibitors. Allergol Int, 2022, 71(2):169-178. doi:10.1016/j.alit.2022.01.001. pmid: 35101349
[5]	陈佳骏, 邱磊, 王蕾, 等. 中药在治疗非小细胞肺癌中的潜在作用研究进展. 中成药, 2024, 46(1):204-210. doi:10.3969/j.issn.1001-1528.2024.01.035.
[6]	Kong FM, Chen TQ, Li XJ, et al. The Current Application and Future Prospects of Astragalus Polysaccharide Combined with Cancer Immunotherapy:A Review. Front Pharmacol, 2021, 12:737674. doi:10.3389/fphar.2021.737674.
[7]	Franco F, Jaccard A, Romero P, et al. Metabolic and epigenetic regulation of T-cell exhaustion. Nat Metab, 2020, 2(10):1001-1012. doi:10.1038/s42255-020-00280-9. pmid: 32958939
[8]	Bamodu OA, Kuo KT, Wang CH, et al. Astragalus polysaccharides (PG2) Enhances the M1 Polarization of Macrophages, Functional Maturation of Dendritic Cells, and T Cell-Mediated Anticancer Immune Responses in Patients with Lung Cancer. Nutrients, 2019, 11(10): 2264. doi:10.3390/nu11102264.
[9]	Wang J, Zhang T, Cheng X. Regulatory Effect of Astragalus Polysacharin on Expresion of PD-1/PD-Ls Molecules in Melanoma Mice. Acta Universitatis Traditionis Medicalis Sinensis, 2014, 28 (5): 74-79. doi:10.16306/j.1008-861x.2014.05.019.
[10]	Gong Q, Yu H, Ding G, et al. Suppression of stemness and enhancement of chemosensibility in the resistant melanoma were induced by Astragalus polysaccharide through PD-L1 downregulation. Eur J Pharmacol, 2022, 916:174726. doi:10.1016/j.ejphar.2021.174726.
[11]	Zhang H, Huang H, Wu S, et al. Single-cell RNA sequencing reveals the effects of anti-PD-L 1 therapy on 3LL lung cancer model and its tumor microenvironment. Med Oncol, 2023, 40(10):285. doi:10.1007/s12032-023-02156-w.
[12]	Lv LL, Zhai JW, Wu JJ, et al. High CD38 expression defines a mitochondrial function-adapted CD8⁺ T cell subset with implications for lung cancer immunotherapy. Cancer Immunol Immunother, 2025, 74(2):49. doi:10.1007/s00262-024-03881-5.
[13]	Zhao LC, Zhong YT, Liang J, et al. Effect of Astragalus Polysaccharide on the Expression of VEGF and EGFR in Mice with Lewis Transplantable Lung Cancer. J Coll Physicians Surg Pak, 2019, 29(4): 392-394. doi:10.29271/jcpsp.2019.04.392.
[14]	Lim SM, Park HB, Jin JO. Polysaccharide from Astragalus membranaceus promotes the activation of human peripheral blood and mouse spleen dendritic cells. Chin J Nat Med, 2021, 19(1):56-62. doi:10.1016/S1875-5364(21)60006-7.
[15]	Liao CH, Yong CY, Lai GM, et al. Astragalus Polysaccharide (PG2) Suppresses Macrophage Migration Inhibitory Factor and Aggressiveness of Lung Adenocarcinoma Cells. Am J Chin Med, 2020, 48(6): 1491-1509. doi:10.1142/S0192415X20500731.
[16]	Phacharapiyangkul N, Wu LH, Lee WY, et al. The extracts of Astragalus membranaceus enhance chemosensitivity and reduce tumor indoleamine 2, 3-dioxygenase expression. Int J Med Sci, 2019, 16(8): 1107-1115. doi:10.7150/ijms.33106. pmid: 31523173
[17]	Chang FL, Tsai KC, Lin TY, et al. Astragalus membranaceus-Derived Anti-Programmed Death-1 Monoclonal Antibodies with Immunomodulatory Therapeutic Effects against Tumors. Biomed Res Int, 2020, 2020:3415471. doi:10.1155/2020/3415471.
[18]	Zhang Y, Wang K, Qin C, et al. Mitochondria dysfunction in CD8⁺ T cells as an important contributing factor for cancer development and a potential target for cancer treatment: a review. J Exp Clin Cancer Res, 2022, 41(1):227. doi:10.1186/s13046-022-02439-6. pmid: 35864520
[19]	Hashimoto M, Kamphorst AO, Im SJ, et al. CD8 T Cell Exhaustion in Chronic Infection and Cancer: Opportunities for Interventions. Annu Rev Med, 2018, 69:301-318. doi:10.1146/annurev-med-012017-043208. pmid: 29414259
[20]	Zehn D, Thimme R, Lugli E, et al. ‘Stem-like’ precursors are the fount to sustain persistent CD8⁺ T cell responses. Nat Immunol, 2022, 23(6):836-847. doi:10.1038/s41590-022-01219-w.
[21]	Scharping NE, Rivadeneira DB, Menk AV, et al. Mitochondrial stress induced by continuous stimulation under hypoxia rapidly drives T cell exhaustion. Nat Immunol, 2021, 22(2): 205-215. doi:10.1038/s41590-020-00834-9. pmid: 33398183
[22]	Yu YR, Imrichova H, Wang H, et al. Disturbed mitochondrial dynamics in CD8⁺ TILs reinforce T cell exhaustion. Nat Immunol, 2020, 21(12):1540-1551. doi:10.1038/s41590-020-0793-3.
[23]	Soto-Heredero G, Desdín-Micó G, Mittelbrunn M. Mitochondrial dysfunction defines T cell exhaustion. Cell Metab, 2021, 33(3):470-472. doi:10.1016/j.cmet.2021.02.010. pmid: 33657392
[24]	Kumar A, Chamoto K, Chowdhury PS, et al. Tumors attenua-ting the mitochondrial activity in T cells escape from PD-1 blockade therapy. Elife, 2020, 9:e52330. doi:10.7554/eLife.52330.
[25]	Ma Q, Xu Y, Tang L, et al. Astragalus Polysaccharide Attenuates Cisplatin-induced Acute Kidney Injury by Suppressing Oxidative Damage and Mitochondrial Dysfunction. Biomed Res Int, 2020, 2020:2851349. doi:10.1155/2020/2851349.
[26]	Thibaut R, Bost P, Milo I, et al. Bystander IFN-γ activity promotes widespread and sustained cytokine signaling altering the tumor microenvironment. Nat Cancer, 2020, 1(3):302-314. doi:10.1038/s43018-020-0038-2.
[27]	Hoekstra ME, Bornes L, Dijkgraaf FE, et al. Long-distance modulation of bystander tumor cells by CD8⁺ T cell-secreted IFNγ. Nat Cancer, 2020, 1(3):291-301. doi:10.1038/s43018-020-0036-4.