马钱子总碱治疗重症肌无力大鼠模型的机制研究

Investigating the mechanisms of total alkaloids of Strychnos against myasthenia gravis in a rat model

摘要

目的: 探讨马钱子总碱对实验性自身免疫性重症肌无力(experimental autoimmune myasthenia gravis,EAMG)大鼠的疗效及其作用机制。方法: Lewis雌性大鼠采用R97-116肽段免疫法构建EAMG模型,马钱子总碱(0.35、0.7、1.4 mg·kg^-1)和泼尼松(4.2 mg·kg^-1)分别对其进行分组给药治疗。通过记录大鼠Lennon评分和跌落时的转棒速度,测定血清抗乙酰胆碱受体抗体(anti-acetylcholine receptor antibody,AChR-Ab)、免疫球蛋白G(immunoglobulin G,IgG)、免疫球蛋白G1(immunoglobulin G1,IgG1)、免疫球蛋白 G2b(immunoglobulin G2b,IgG2b)、干扰素-γ(interferon-γ,IFN-γ)、白细胞介素-17(interleukin 17,IL-17)和转化生长因子-β(transforming growth factor-β,TGF-β)水平,检测外周血淋巴细胞CD4⁺/CD8⁺比例等相关指标,考察其药效并探究可能的作用机制。结果: 马钱子总碱能显著降低Lennon评分,显著升高跌落时的转棒速度,显著降低血清AChR-Ab、IgG和IgG2b亚型水平。马钱子总碱显著降低了血清IL-17和IFN-γ含量,升高血清TGF-β含量,显著降低外周血淋巴细胞CD4⁺/CD8⁺比例。结论: 马钱子总碱能明显改善EAMG大鼠肌无力的症状,其作用机制可能与抑制AChR-Ab的生成、降低CD4⁺/CD8⁺比例、调节免疫功能紊乱和机体异常炎性状态有关。

Abstract

Objective: To investigate the therapeutic effect of MQZZJ (total alkaloids of Strychnos) on experimental autoimmune myasthenia gravis (EAMG) rats and to elucidate its underlying mechanisms. Methods: The EAMG rat model was established by immunizing female Lewis rats with the R97-116 peptide fragment. Rats were assigned to different groups and treated with MQZZJ (0.35, 0.7, and 1.4 mg·kg^-1) or prednisone (4.2 mg·kg^-1). The therapeutic effects and potential mechanisms were evaluated by monitoring the Lennon score and rotarod falling speed, measuring serum levels of acetylcholine receptor antibody (AChR-Ab), IgG, IgG1, IgG2b, IFN-γ, IL-17, and TGF-β, and detecting the CD4⁺/CD8⁺ ratio in peripheral blood lymphocytes. Results: MQZZJ significantly reduced the Lennon score, increased the rotarod falling speed, and reduced serum levels of AChR-Ab, IgG, and the IgG2b subtype. It also significantly decreased serum IL-17 and IFN-γ levels, increased serum TGF-β levels, and reduced the CD4⁺/CD8⁺ ratio. Conclusion: MQZZJ can markedly ameliorate myasthenic symptoms in EAMG rats. The mechanism may be related to inhibiting AChR-Ab production, reducing the CD4⁺/CD8⁺ ratio, modulating immune dysfunction, and alleviating abnormal inflammation.

关键词

重症肌无力 / 马钱子总碱 / 抗乙酰胆碱受体抗体 / CD4⁺/CD8⁺ / 细胞因子

Key words

myasthenia gravis / total alkaloids of Strychnos / AChR-Ab / CD4⁺/CD8⁺ / cytokines

[1] BERRIH-AKNIN S. Myasthenia gravis: paradox versus paradigm in autoimmunity[J]. J Autoimmun, 2014, 52: 1-28.
[2] CARR AS, CARDWELL CR, MCCARRON PO, et al. A systematic review of population based epidemiological studies in Myasthenia Gravis[J]. BMC Neurol, 2010, 10(1): 46.
[3] HUDA R. Inflammation and autoimmune myasthenia gravis[J]. Front Immunol, 2023, 14: 1110499.
[4] 常婷. 中国重症肌无力诊断和治疗指南(2020版)[J]. 中国神经免疫学和神经病学杂志, 2021, 28(1): 1-12.
[5] VANOLI F, MANTEGAZZA R. Current drug treatment of myasthenia gravis[J]. Curr Opin Neurol, 2023, 36(5): 410-415.
[6] KAMINSKI HJ, SIKORSKI P, CORONEL SI, et al. Myasthenia gravis: the future is here[J]. J Clin Investig, 2024, 134(12): e179742.
[7] 罗婷, 张艺凡, 楚兰, 等. 重症肌无力患者外周血淋巴细胞亚群水平及其临床意义[J]. 贵州医科大学学报, 2021, 46(6): 712-718.
[8] CONTI-FINE BM, MILANI M, KAMINSKI HJ. Myasthenia gravis: past, present, and future[J]. J Clin Invest, 2006, 116(11): 2843-2854.
[9] 王艳君, 孟庆芳, 王思, 等. 青蒿素对实验性自身免疫性重症肌无力大鼠R97-116抗体及细胞因子的影响[J]. 中国神经免疫学和神经病学杂志, 2016, 23(3): 167-171.
[10] SAOUDI A, BERNARD I, HOEDEMAEKERS A, et al. Experimental autoimmune myasthenia gravis may occur in the context of a polarized Th1- or Th2-type immune response in rats[J]. J Immunol, 1999, 162(12): 7189-7197.
[11] LOSEN M, MARTINEZ-MARTINEZ P, MOLENAAR PC, et al. Standardization of the experimental autoimmune myasthenia gravis (EAMG) model by immunization of rats with Torpedo californica acetylcholine receptors: Recommendations for methods and experimental designs[J]. Exp Neurol, 2015, 270: 18-28.
[12] 黎明全, 王洪峰, 张炜煜, 等. 马钱子治疗痿病的的现状分析及思考[J]. 时珍国医国药, 2009, 20(4): 954-955.
[13] 邹莹, 裘涛, 杨峰. 炙马钱子对实验性自身免疫性重症肌无力大鼠免疫调节机制研究[J]. 中华中医药杂志, 2015, 30(8): 2994-2998.
[14] 李凤霞, 胡致平, 聂富意, 等. 马钱子治疗神经系统疾病研究进展[J]. 新中医, 2020, 52(13): 17-20.
[15] 吴攀锋, 方居正. 马钱子药理毒理作用及减毒机理研究进展[J]. 江苏中医药, 2024, 56(3): 81-85.
[16] 谢阳, 伍淳操, 杨宗发, 等. 马钱子药理和毒性机制的研究进展[J]. 华西药学杂志, 2022, 37(1): 102-107.
[17] 王小冬, 孙博, 王菁华, 等. 辅助性CD4⁺ T细胞亚群失衡在实验性自身免疫性重症肌无力大鼠发病机制中的作用[J]. 中国生物制品学杂志, 2010, 23(9): 930-934.
[18] 朱洁, 程杨, 许骏尧, 等. 升陷汤对实验性自身免疫性重症肌无力大鼠免疫机制研究[J]. 中华中医药学刊, 2017, 35(3): 717-720.
[19] JUTEL M, AKDIS CA. T-cell subset regulation in atopy[J]. Curr Allergy Asthma Rep, 2011, 11(2): 139-145.
[20] WANG Z. Role of IFN-g in induction of Foxp3 and conversion of CD4⁺CD25-T cells to CD4⁺ Tregs[J]. J Clin Investig, 2006, 116(9): 2434-2441.
[21] 王炜, 南振鸿, 凌振芬, 等. 干扰素-γ对重症肌无力患者自身抗体的调节作用及相关性分析[J]. 中国神经免疫学和神经病学杂志, 2000(4): 234-236, 262.
[22] 李潇, 张艳华. 免疫检查点抑制剂致肌炎、心肌炎和重症肌无力重叠综合征4例临床特征分析[J]. 中国新药杂志, 2025, 34(23): 2570-2576.
[23] DALAKAS MC. Novel future therapeutic options in Myasthenia Gravis[J]. Autoimmun Rev, 2013, 12(9): 936-941.
[24] WANG CC, LI H, ZHANG M, et al. Caspase-1 inhibitor ameliorates experimental autoimmune myasthenia gravis by innate dendric cell IL-1-IL-17 pathway[J]. J Neuroinflam, 2015, 12(1): 118.
[25] SCHAFFERT H, PELZ A, SAXENA A, et al. IL-17-producing CD4⁺ T cells contribute to the loss of B-cell tolerance in experimental autoimmune myasthenia gravis[J]. Eur J Immunol, 2015, 45(5): 1339-1347.
[26] KONG QF, SUN B, BAI SS, et al. Administration of bone marrow stromal cells ameliorates experimental autoimmune myasthenia gravis by altering the balance of Th1/Th2/Th17/Treg cell subsets through the secretion of TGF-β[J]. J Neuroimmunol, 2009, 207(1-2): 83-91.

基金资助

江苏省基础研究计划自然科学基金资助项目:前沿引领技术基础研究专项资助项目(BK20232014);中药制药过程控制与智能制造技术全国重点实验室目标资助项目(SKL2023D01001)