Effects Of The Jianpi Bushen Yiqi Formula On The Th/Treg Immune Axis In The Spleen And AChR At The Neuromuscular Junction In Skeletal Muscle Of EAMG Mice
Jan 29, 2026
Abstract
Objective:
To investigate the potential therapeutic mechanism of the Jianpi Bushen Yiqi (Spleen-Fortifying, Kidney-Tonifying, Qi-Boosting) formula in treating myasthenia gravis (MG) from the perspective of the T-helper/T-regulatory (Th/Treg) immune axis.
Methods:
A total of 104 female C57BL/6J mice were randomly divided into a blank control group (n=12) and a model group (n=92). The EAMG (Experimental Autoimmune Myasthenia Gravis) model was induced in the model group via subcutaneous injection of a mouse-derived acetylcholine receptor (AChR)-α97–116 peptide emulsified in phosphate-buffered saline (PBS) and complete Freund's adjuvant (CFA). After model validation, 65 successful EAMG mice were randomly assigned into five groups: model control, low-, medium-, and high-dose Jianpi Bushen Yiqi formula, and prednisone group (n=13 per group).
The formula was administered by gavage daily for 5 weeks at doses of 7.46, 14.92, and 29.84 g·kg⁻¹·d⁻¹, respectively. The prednisone group received 9.10 mg·kg⁻¹·d⁻¹ prednisone. Control groups received 0.30 ml of 0.9% NaCl.
Body weight, Lennon scores, and grip strength were recorded before and after intervention. Thymus and spleen indices were calculated. Histopathological changes in the thymus and lymph nodes were observed via HE staining. Flow cytometry assessed percentages of Th1, Th2, Th17, Treg, and Tfh cells in splenic suspensions. Immunofluorescence was used to evaluate AChR distribution at the neuromuscular junction (NMJ). ELISA measured serum AChR-Ab, IL-6, and TNF-α levels.
Results:
Compared to the model group, the prednisone group showed reduced body weight, Lennon scores, thymus and spleen indices, germinal center counts, Treg cell percentages, AChR-Ab, and TNF-α levels, while grip strength, thymic cortex/medulla ratio, extraocular muscle AChR area ratio, and IL-6 levels increased (P<0.05 or P<0.01). The Jianpi Bushen Yiqi formula groups showed reduced germinal center counts, Th17 and Tfh percentages, and increased grip strength and AChR expression in multiple muscle groups. Medium and high-dose groups showed decreased Th1 and AChR-Ab levels; the high-dose group improved Lennon scores and IL-6, increased Treg percentage and thymic cortex/medulla ratio.
Compared with the prednisone group, Jianpi Bushen Yiqi groups had higher body weight and thymus index, lower Th2, Th17, Tfh, and IL-6 levels (P<0.05 or P<0.01); Treg percentage was higher and Th1 lower in medium and high-dose groups; AChR area ratio increased across all doses (P<0.05 or P<0.01).
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Conclusion:
The Jianpi Bushen Yiqi formula may restore immune balance by modulating the Th/Treg axis, reshaping the immune microenvironment, and enhancing AChR expression at NMJs, thus alleviating myasthenic symptoms in EAMG mice.
Keywords:
Myasthenia gravis; Jianpi Bushen Yiqi formula; T helper cells; Regulatory T cells; Acetylcholine receptor; Immune microenvironment
Table 1. Comparison of Body Weight, Grip Strength, and Lennon Scores Among Mice in Each Group
| Group | n | Body Weight (g) Before | Body Weight (g) After | Grip Strength (N) Before | Grip Strength (N) After | Lennon Score Before | Lennon Score After |
|---|---|---|---|---|---|---|---|
| Blank Control Group | 12 | 22.41 ± 0.89 | 23.17 ± 1.10 | 153.91 ± 9.92 | 158.08 ± 7.70 | 0 (0, 0) | 0 (0, 0) |
| Model Control Group | 13 | 22.40 ± 0.97 | 21.68 ± 1.11 | 112.46 ± 11.8 a,b | 138.35 ± 11.41 a | 1.0 (1.0, 1.5) a | 1.5 (1.0, 2.0) a |
| Prednisone Group | 13 | 22.66 ± 0.95 | 20.48 ± 1.26 b | 112.62 ± 9.53 | 138.40 ± 10.4 b | 1.0 (1.0, 1.5) | 0.5 (0.0, 0.5) b |
| Jianpi Bushen Yiqi Low Dose Group | 13 | 22.31 ± 0.62 | 22.17 ± 0.93 c | 116.92 ± 9.71 | 137.77 ± 9.99 b | 1.0 (1.0, 1.5) | 1.0 (0.5, 1.5) b |
| Jianpi Bushen Yiqi Medium Dose Group | 13 | 22.69 ± 0.74 | 22.80 ± 1.13 c | 116.11 ± 11.94 | 138.85 ± 10.55 b,c | 1.0 (1.0, 1.5) | 0.5 (0.5, 1.0) b |
| Jianpi Bushen Yiqi High Dose Group | 13 | 22.88 ± 1.25 | 23.18 ± 1.05 c | 116.24 ± 9.35 | 136.62 ± 6.29 b | 1.0 (1.0, 1.5) | 0.5 (0.0, 1.0) b |
Notes:
a) P < 0.01 vs. blank control group at the same time point
b) P < 0.01 vs. model control group at the same time point
c) P < 0.05 vs. prednisone group at the same time point
d) P < 0.01 vs. prednisone group at the same time point
Introduction
Myasthenia gravis (MG) is a chronic autoimmune disorder primarily affecting the neuromuscular junction (NMJ), driven by autoantibodies targeting acetylcholine receptors (AChRs) [1]. In recent years, the incidence and severity of MG have been on the rise in China, prompting an urgent need for safer and more effective treatments [2].
While cholinesterase inhibitors and immunosuppressants like glucocorticoids are the current standard of care [3], their long-term use is associated with increased infection risk and reduced therapeutic efficacy. Immunologically, MG pathogenesis is strongly linked to the imbalance between CD4⁺ T helper (Th) subsets and regulatory T (Treg) cells. Dysfunction in these subsets contributes to the production of pathogenic AChR autoantibodies (AChR-Ab), complement activation, and ultimately, NMJ structural damage [4–6].
In Traditional Chinese Medicine (TCM), MG is categorized under "Wei Zheng (flaccidity syndrome)" and "jian fei (eyelid paralysis)." It is primarily attributed to deficiency of the spleen and kidney, with qi sinking and essence depletion as the core pathogenesis [7–8]. Accordingly, the therapeutic strategy emphasizes strengthening the spleen and kidney, raising yang, and replenishing marrow.
The Jianpi Bushen Yiqi formula is derived from the classical Buzhong Yiqi Tang and Guilu Erxian Jiao, formulated by Prof. Li Fazhi, a renowned national TCM expert. It exhibits spleen- and kidney-tonifying effects and has shown clinical efficacy in reducing AChR antibody levels and improving NMJ function in MG patients [9–10].
Importantly, one of the key components in this formula is Cistanche (肉苁蓉), a traditional herb widely recognized for its kidney-tonifying and essence-replenishing properties. Studies suggest that Cistanche extracts can enhance immune modulation, improve fatigue resistance, and support neuromuscular health, which aligns well with the pathophysiological needs of MG patients. For more details on the pharmacological actions of Cistanche, visit:
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This study investigates the effects of the Jianpi Bushen Yiqi formula on EAMG mice through the lens of the Th/Treg axis, aiming to provide experimental evidence for its clinical application.
