体内外实验探讨千斤拔多糖对类风湿关节炎模型小鼠免疫调节的作用

doi:10.5246/jcps.2024.10.067

中国药学(英文版) ›› 2024, Vol. 33 ›› Issue (10): 932-942.DOI: 10.5246/jcps.2024.10.067

体内外实验探讨千斤拔多糖对类风湿关节炎模型小鼠免疫调节的作用

高艺文¹^,^#, 李聚仓²^,^#, 张楠¹, 李爽¹, 王莉莉¹^,^*(), 秦海洸³^,^*()

^1. 滨州医学院烟台附属医院, 山东烟台 264100
^2. 青岛滨海学院附属医院, 山东青岛 266400
^3. 山东中医药高等专科学校, 山东烟台 264100

收稿日期:2024-04-01 修回日期:2024-05-12 接受日期:2024-06-26 出版日期:2024-10-31 发布日期:2024-10-31
通讯作者: 王莉莉, 秦海洸

Clinical and experimental investigations into the immune modulatory effects of Qianjinba polysaccharides on a rheumatoid arthritis mouse model: in vivo and in vitro studies

Yiwen Gao¹^,^#, Jucang Li²^,^#, Nan Zhang¹, Shuang Li¹, Lili Wang¹^,^*(), Haiguang Qin³^,^*()

¹ Yantai?Affiliated?Hospital?of?Binzhou?Medical?College,?Yantai?264100,?Shandong, China
² The Affiliated Hospital of Qingdao Binhai University, Qingdao 266400,?Shandong, China
³ Shandong College of Traditional Chinese Medicine, Yantai?264100,?Shandong, China

Received:2024-04-01 Revised:2024-05-12 Accepted:2024-06-26 Online:2024-10-31 Published:2024-10-31
Contact: Lili Wang, Haiguang Qin
About author:
^# Yiwen Gao and Jucang Li contributed equally to this work.
Supported by:
Shandong Provincial Key Project of TCM Science and Technology (Grant No. 2021Z051), Shandong Medical and Health Science and Technology Development Program (Grant No. 202102040972), and the Binzhou Medical College Student Innovation and Entrepreneurship Training Program (Grant No. X202210440354).

摘要/Abstract

摘要：

千斤拔为豆科植物蔓性千斤拔的干燥根, 其成分植物多糖具有抗炎、镇痛、保护周围神经损伤等作用, 免疫调节的作用少有报道。本研究通过观察千斤拔多糖(QJBDT)对类风湿关节炎模型小鼠免疫调节的作用, 初步探讨其抑制类风湿关节炎的作用机制。体外实验将巨噬RAW264.7细胞经过细菌脂多糖(LPS)诱导, 用不同浓度的千斤拔多糖或白芍总苷(TGP)处理。细胞分为正常组, 模型组, QJBDT低、中、高剂量组, 阳性对照组, 检测各组细胞的增殖情况; 提取细胞蛋白, 检测细胞中丝裂原活化蛋白激酶p38与核因子NF-κB蛋白的表达情况。体内实验小鼠致炎造模, 造模成功后给予不同浓度千斤拔多糖和白芍总苷灌胃, 每天给药1次, 连续21天。小鼠分为正常组, 模型组, QJBDT低、中、高剂量组, 阳性对照组(TGP组), 每组10只。计算各组脏器指数; 检测血细胞中免疫指标; 流式细胞术检测T细胞17(T helper cell 17, Th17)和调节性T细胞Treg的变化。结果发现,与正常组相比, 各组细胞增值率明显提高(P < 0.05); 模型组、QJBDT低剂量组和QJBDT中剂量组细胞中p38、NF-κB蛋白表达增强(P < 0.05); 血细胞中系统性免疫性炎症指数(SII)、全身炎症反应指数(AISI)、血小板淋巴细胞比值(PLR)、中性粒细胞淋巴细胞比值(NLR)、单核细胞淋巴细胞比值(MLR)升高(P < 0.05); 模型组和千斤拔低剂量组的小鼠体重、胸腺指数、脾脏指数及肝脏指数降低(P < 0.05); 同时各组小鼠全血中Th17升高, 调节性T细胞Treg降低(P < 0.05), 模型组和千斤拔低剂量组Th17与Treg比值增高(P < 0.05)。与模型组相比, QJBDT中剂量、QJBDT高剂量组和TGP组抑制细胞增殖效果明显(P < 0.05); 同时降低蛋白P38、NF-κB的表达(P < 0.05); 血常规部分免疫指标不同程度降低(P < 0.05); 各免疫器官指数升高(P < 0.05); QJBDT低、中、高剂量组和阳性对照组小鼠Th17降低, Treg升高, Th17与Treg比值降低(P < 0.05), 且QJBDT高剂量组作用与TGP组无统计学差异(P > 0.05)。本研究发现千斤拔多糖对类风湿关节炎小鼠有较好的免疫调节作用且作用成剂量依赖性, 作用机制可能是通过抑制p38 MAPK信号通路抑制NF-κB活化发挥作用。

关键词: 千斤拔多糖, 类风湿关节炎, 细胞增值, p38, NF-κB, Th17, Treg

Abstract:

Qianjinba is derived from the dried root of a legume vine and is known for its plant polysaccharide, which has demonstrated anti-inflammatory, analgesic, and neuroprotective properties. Despite these known effects, its immunomodulatory potential remains underexplored. This study aimed to investigate the immunoregulatory effects of Qianjinba polysaccharide (QJBDT) in a murine model of rheumatoid arthritis (RA) and elucidate its mechanism of action in inhibiting the disease. To assess the immunomodulatory effects, RAW264.7 macrophage cells were stimulated with bacterial lipopolysaccharide (LPS) and subsequently treated with varying concentrations of QJBDT or total glucoside of paeonic acid (TGP). Cell proliferation and the expression levels of mitogen-activated protein kinase p38 and nuclear factor NF-κB proteins were evaluated. Following the successful modeling of RA in mice, different doses of QJBDT and TGP were administered via intragastric administration once daily for 21 d. Mice were divided into normal, model, QJBDT low-, medium-, and high-dose groups, along with a positive control group (TGP group), each comprising 10 mice. Organ coefficients were calculated, immune indexes in blood cells were determined, and alterations in T helper cell 17 (Th17) and regulatory T cells (Treg) were analyzed using flow cytometry. Compared to the normal group, cell proliferation rates significantly increased across all groups (P < 0.05). Elevated expressions of p38 and NF-κB proteins were observed in the model group, QJBDT low- and medium-dose groups (P < 0.05). Various systemic immune inflammation indexes, including systemic immune inflammation index (SII), total inflammation systemic index (AISI), and systemic inflammation response index (SIRI), platelet-to-lymphocyte ratio (PLR), neutrophil-to-lymphocyte ratio (NLR)，monocyte-to-lymphocyte ratio (MLR), showed increments (P < 0.05). Additionally, body weight and organ coefficients of the thymus, spleen, and liver decreased in both the model and low-dose groups (P < 0.05). Moreover, Th17 levels increased while Treg levels decreased across all groups (P < 0.05), resulting in a heightened Th17 to Treg ratio in the model and low-dose groups (P < 0.05). Notably, the QJBDT medium-dose, high-dose, and positive control groups demonstrated significant inhibitory effects on cell proliferation compared to the model group (P < 0.05), along with reduced expression levels of p38 and NF-κB (P < 0.05). Furthermore, various immune indicators from routine blood tests exhibited different degrees of decrease (P < 0.05), while immune indices displayed increases (P < 0.05). The low-, medium-, and high-dose groups of QJBDT, as well as the positive control group, showed decreased Th17 levels, elevated Treg levels, and diminished Th17 to Treg ratios (P < 0.05). Additionally, there was no statistically significant difference in efficacy between the QJBDT high-dose and TGP groups (P > 0.05). In conclusion, this study demonstrated that QJBDT exerted potent immunomodulatory effects on RA in mice in a dose-dependent manner. Its mechanism of action might involve the inhibition of NF-κB activation by suppressing the p38 MAPK signaling pathway.

Key words: Qianjin polysaccharide, Rheumatoid arthritis, Cell proliferation, p38, NF-κB, Th17, Treg

Supporting:

高艺文, 李聚仓, 张楠, 李爽, 王莉莉, 秦海洸. 体内外实验探讨千斤拔多糖对类风湿关节炎模型小鼠免疫调节的作用[J]. 中国药学(英文版), 2024, 33(10): 932-942.

Yiwen Gao, Jucang Li, Nan Zhang, Shuang Li, Lili Wang, Haiguang Qin. Clinical and experimental investigations into the immune modulatory effects of Qianjinba polysaccharides on a rheumatoid arthritis mouse model: in vivo and in vitro studies[J]. Journal of Chinese Pharmaceutical Sciences, 2024, 33(10): 932-942.

图/表 5

Figure 1. Effects of QJBDT on cell proliferation of RAW264.7 (x ± s, n = 10).*P < 0.05, compared with the normal group; #P < 0.05, compared with the model group; ∆P < 0.05, compared with the TGP group.

Figure 2. Effects of QJBDT on p38 and NF-κB protein expression (x ± s, n = 10). (A) Expression of p38 and NF-κB protein; (B) Pyroptotic protein immunoblotting bands of p38; (C) Pyroptotic protein immunoblotting bands of NF-κB, *P < 0.05, compared with the normal group; #P < 0.05, compared with the model group; ∆P < 0.05, compared with the TGP group.

Figure 3. Effects of QJBDT on immune indicators in mouse blood cells (x ± s, n = 10). (A) Expression of AISI; (B) Expression of SIRI; (C) Expression of SII; (D) Expression of NLR; (E) Expression of PLR; (F) Expression of MLR, *P < 0.05, compared with the normal group; #P < 0.05, compared with the model group; ∆P < 0.05, compared with the TGP group.

Figure 4. Effects of QJBDT on the organ coefficients of mice (x ± s, n = 10). (A) Expression of body weight; (B) Expression of thymus index; (C) Expression of spleen index; (D) Expression of liver index, *P < 0.05, compared with the normal group; #P < 0.05, compared with the model group; ∆P < 0.05, compared with the TGP group.

Figure 5. Effects of QJBDT on Th17 and Treg expression (mg/cm3) (x ± s, n = 10). (A) Expression of Th17; (B) Expression of Treg; (C) The ratio of Th17/Treg; (D) Th17 flow cytometry; (E) Treg flow cytometry, *P < 0.05, compared with the normal group; #P < 0.05, compared with the model group; ∆P < 0.05, compared with the TGP group.

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体内外实验探讨千斤拔多糖对类风湿关节炎模型小鼠免疫调节的作用

Clinical and experimental investigations into the immune modulatory effects of Qianjinba polysaccharides on a rheumatoid arthritis mouse model: in vivo and in vitro studies

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