柴胡皂苷A对小鼠髓源性抑制细胞的抑制作用及其机制研究

doi:10.3969/j.issn.1674-8115.2025.10.007

摘要/Abstract

摘要：

目的·研究柴胡皂苷A（saikosaponin A，SSA）对小鼠髓源性抑制细胞（myeloid-derived suppressor cells，MDSCs）分化、凋亡、免疫抑制功能的调控作用，并探讨其分子机制。方法·用重组小鼠粒细胞-巨噬细胞集落刺激因子（granulocyte-macrophage colony-stimulating factor，GM-CSF）诱导小鼠骨髓细胞（bone marrow cells，BMCs）分化为MDSCs，或磁珠分选荷瘤小鼠MDSCs。以不同浓度（0、2.5、5.0 mg/L）的SSA处理MDSCs后，采用流式细胞术（flow cytometry，FCM）检测MDSCs的分化、凋亡情况，以及肝X受体α（liver X receptor α，LXRα）、精氨酸代谢酶1（arginase-1，Arg-1）、活性氧（reactive oxygen species，ROS）的表达水平，同时检测MDSCs对T细胞增殖功能的影响，以及对核因子κB（nuclear factor κB，NF-κB）、信号转导及转录激活蛋白（signal transducer and activator of transcription 1，STAT1）信号通路的影响。利用实时荧光定量PCR（quantitative real-time PCR，qPCR）检测LXRα和Arg-1的mRNA水平。通过灌胃或腹腔注射给予小鼠SSA，给药结束后处死小鼠，测定其体质量、脾脏，计算脾指数。FCM检测小鼠脾脏内各免疫细胞的比例。结果·SSA可上调MDSCs的LXRα表达水平，减少M-MDSCs的分化，诱导MDSCs凋亡，降低MDSCs的Arg-1和ROS的表达水平，减轻MDSCs对T细胞增殖的抑制作用。SSA可抑制MDSCs中的NF-κB和STAT1的磷酸化水平。经SSA灌胃和腹腔注射处理的小鼠，其体质量以及脾指数均无明显变化。且这2种给药方式均可降低小鼠体内MDSCs及其亚群M-MDSCs的比例，但对其他免疫细胞有不同程度的调控作用。结论·SSA可调控MDSCs的分化、凋亡，并抑制其免疫抑制功能，这可能与SSA上调MDSCs的LXRα表达、抑制NF-κB和STAT1信号通路相关。SSA可通过LXRα逆转MDSCs的免疫抑制功能。

关键词: 柴胡皂苷A, 髓源性抑制细胞, 免疫抑制, 肝X受体α

Abstract:

Objective ·To study the regulatory effect of saikosaponin A (SSA) on the differentiation, apoptosis, and immunosuppressive function of myeloid-derived suppressor cells (MDSCs) in mice, and to explore their molecular mechanism. Methods ·Recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to induce the differentiation of mouse bone marrow cells (BMCs) into MDSCs, or magnetic beads were used to sort MDSCs from tumor-bearing mice. After treating MDSCs with different concentrations (0, 2.5, 5.0 mg/L), flow cytometry (FCM) was used to detect the differentiation and apoptosis of MDSCs, as well as the expression levels of liver X receptor α (LXRα), arginase-1 (Arg-1), and reactive oxygen species (ROS). At the same time, the effects of MDSCs on the proliferation function of T cells, and the effects on the nuclear factor κB (NF-κB), and signal transducer and activator of transcription 1 (STAT1) signaling pathways were also detected. The mRNA levels of LXRα and Arg-1 were detected by quantitative real-time PCR (qPCR). Mice were given SSA by gavage (ig) or intraperitoneal injection (ip), and the mice were sacrificed after administration; and body mass, spleen weight, and spleen index were calculated. FCM was used to detect the proportion of immune cells in the spleen of mice. Results ·SSA could up-regulate the expression level of LXRα in MDSCs, reduce the differentiation of M-MDSCs, induce apoptosis of MDSCs, reduce the expression levels of Arg-1 and ROS in MDSCs, and reduce the inhibitory effect of MDSCs on T cell proliferation. SSA inhibited the phosphorylation levels of NF-κB and STAT1 in MDSCs. The mice treated with SSA by gavage or intraperitoneal injection showed no significant changes in body weight and spleen index. Both modes of administration can reduce the proportion of MDSCs and their subset M-MDSCs in mice, but had different degrees of regulatory effects on other immune cells. Conclusion ·SSA could regulate the differentiation and apoptosis of MDSCs, and inhibit their immunosuppressive function, which may be associated with the up-regulation of LXRα expression, and down-regulation of the NF-κB and STAT1 signaling pathways in MDSCs.

Key words: saikosaponin A (SSA), myeloid-derived suppressor cell (MDSC), immunosuppression, liver X receptor α (LXRα)

中图分类号:

R392

秦雅含, 束雅婷, 彭美玉. 柴胡皂苷A对小鼠髓源性抑制细胞的抑制作用及其机制研究[J]. 上海交通大学学报（医学版）, 2025, 45(10): 1320-1332.

QIN Yahan, SHU Yating, PENG Meiyu. Inhibitory effect and mechanism of saikosaponin A on mouse myeloid-derived suppressor cells[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(10): 1320-1332.

图/表 8

表1 qPCR所用引物序列

Tab 1 Primer sequences used in qPCR

Primer name	Primer sequence (5'→3')
GAPDH	F: TGTCTCCTGCGACTTCAACA
	R: GGTGGTCCAGGGTTTCTTACT
ARG-1	F: TGTCCCTAATGACAGCTCCTT
	R: GCATCCACCCAAATGACACAT
NR1H3	F: AAGGGAGCACGCTATGTCTG
	R: TTGCGAAGTCGACACTCCTG

图1 SSA上调荷瘤小鼠MDSCs的 LXRα 表达Note： A—D. A typical flow cytometry pattern. E. Percentage of intracellular LXRα in MDSCs (n=5). F. mRNA expression levels of LXRα in MDSCs (n=5). ①P=0.036, ②P=0.001, ③P=0.038.

Fig 1 SSA upregulated the expression of LXRα in MDSCs of tumor-bearing mice

图2 SSA对MDSCs分化及凋亡的影响Note： A—D/H—J. A typical flow cytometry pattern. First, CD11b is gated. M-MDSCs: CD11b+Ly6ChighLy6G-; PMN-MDSCs:CD11b+Ly6ClowLy6G+. E. Percentage of total MDSCs. F. Percentage of M-MDSCs. G. Percentage of PMN-MDSCs (n=5). K. MDSCs total apoptotic proportion, early apoptosis proportion, and late apoptosis proportion (n=5). L. Absolute number of MDSCs (n=5). ①P=0.015, ②P=0.004, ③P=0.019, ④P=0.001, ⑤P<0.001, ⑥P=0.002, ⑦P=0.006, ⑧P<0.001.

Fig 2 Effect of SSA on the differentiation and apoptosis of MDSCs

图3 SSA抑制MDSCs的免疫抑制功能Note： A—D/G/I—M. A typical flow cytometry pattern. E. Arg-1 expression in MDSCs. F. mRNA expression levels of Arg-1 in MDSCs (n=3). H. MFI expression of ROS in MDSCs (n=5). N. T cell proliferation rate (n=5). ①P=0.006, ②P=0.027, ③P=0.029, ④P=0.013, ⑤P=0.001, ⑥P<0.001.

Fig 3 Immunosuppressive function of SSA in inhibiting MDSCs

图4 SSA对MDSCs 中STAT1和NF-κB信号通路的影响Note： A—D/F—I. A typical flow cytometry pattern. E. Expression level of p-STAT1 in MDSCs. J. Expression level of p-NF-κB p65 in MDSCs (n=5). ①P=0.012, ②P=0.036, ③P=0.028.

Fig 4 Effect of SSA on STAT1 and NF-κB signaling pathways in MDSCs

图5 SSA对体内MDSCs和Treg细胞的影响Note： A. Histogram of mouse body weight. B. Histogram of mouse spleen index. C. Lymphocyte gating. D. Exclusion of adhesion cells. E. CD11b gating. F‒H. A typical flow cytometry pattern. I. Percentage of total MDSCs. J. Percentage of M-MDSCs and PMN-MDSCs. K—M. A typical flow cytometry pattern. N. Percentage of Treg cells (n=6). ①P=0.014, ②P=0.019, ③P=0.013, ④P=0.016.

Fig 5 Effect of SSA on MDSCs and Treg cells in vivo

图6 SSA对体内T细胞、T细胞亚群和B细胞的影响Note： A—O. A typical flow cytometry pattern. P. Percentage of CD3+ cells in spleen lymphocytes (n=6). Q. Percentage of CD4+ T、CD8+ T, and γδ+ T cells in spleen CD3+ cells (n=6). R. Percentage of CD19+ cells in spleen lymphocytes (n=6). ①P=0.028, ②P=0.007, ③P=0.030, ④P=0.016.

Fig 6 Effect of SSA on T cells, T cell subsets and B cells in vivo

图7 SSA对体内NK细胞和单核/巨噬细胞的影响Note： A—C/E—G. A typical flow cytometry pattern. D. Percentage of NK cells in splenic lymphocytes (n=6). H. Percentage of CD11b+ F4/80+ cells in the spleen (n=6).①P=0.020.

Fig 7 Effect of SSA on NK cells and monocytes/macrophages in vivo

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