Objective

2

To explore the potential mechanism of

Jianpi Bushen Yiqi Formula

（健脾补肾益气方， JBYF） for myasthenia gravis （MG） from the perspective of the helper T cell （Th）/regulatory T cell （T

reg） immune axis.

Methods

2

A total of 104 female C57BL/6J mice were divided into a blank control group （n=12） and a model group （n=92）. Mice in the model group were subcutaneously injected with an immune emulsion composed of mouse-derived acetylcholine receptor （AChR）-α subunit 97-116 peptide segment， phosphate-buffered saline （PBS）， and complete Freund's adjuvant （CFA） to establish the experimental autoimmune myasthenia gravis （EAMG） mouse model. A total of 65 successfully modeled mice were further randomly divided into 5 groups （13 mice per group）， model control group， low-， medium-， and high-dose JBYF groups， and prednisone group. The low-， medium-， and high-dose JBYF groups were given 7.46， 14.92， and 29.84 g·kg⁻¹·d⁻¹ JBYF， respectively； the prednisone group was given prednisone tablets of 9.10 mg·kg⁻¹·d⁻¹； the blank control group and the model control group were given 0.30 ml of 0.9% sodium chloride solution. All groups received intragastric administration once a day for 5 consecutive weeks. Before and after intervention， the body weight and Lennon score of mice in each group were recorded， and a dynamometer was used to measure the grip strength of mice. After intervention， the spleen and thymus indices were calculated； HE staining was used to observe the histological morphology of the thymus and lymph nodes； flow cytometry was employed to detect the percentages of Th1， Th2， Th17， Treg， and follicular helper T cell （Tfh） in the splenic cell suspension； immunofluorescence was used to observe the morphology of AChR at the neuromuscular junctions （NMJ） of mouse muscle groups； ELISA was used to detect the serum levels of acetylcholine receptor antibody （AChR-Ab）， interleukin-6 （IL-6）， and tumor necrosis factor-α （TNF-α）.

Results

2

Compared with the model control group， the prednisone group showed decreases in body weight， Lennon score， thymus index， spleen index， number of lymph node germinal centers， Treg percentage， serum AChR-Ab level， and TNF-α level

， as well as increases in grip strength， thymic cortex-medulla area ratio， ocular muscle AChR area ratio， and IL-6 level （

P

＜0.05 or

P

＜0.01）； in all dose groups of JBYF， the number of lymph node germinal centers and the percentages of Th17 and Tfh decreased， while grip strength and AChR area ratio of multiple muscle groups increased； in the medium- and high-dose JBYF

group showed decreases in Lennon score and IL-6 level， and increases in thymic cortex-medulla area ratio and Treg percentage （

P

＜0.05 or

P

＜0.01）. Compared with the prednisone group， all dose groups of JBYF exhibited increases in body weight and thymus index， and decreases in the percentages of Th2， Th17， Tfh， and IL-6 level （

P

＜0.05 or

P

＜0.01）； the low- and medium-dose groups of JBYF showed increases in Lennon score and the number of lymph node germinal centers； the medium- and high-dose groups of JBYF had an increase in Treg percentage and a decrease in Th1 percentage； all dose groups of JBYF exerted an elevating effect on the AChR area ratio of different muscle groups （

P

＜0.05 or

P

＜0.01）.

Conclusion

2

JBYF can reshape the immune microenvironment by regulating the Th/Treg immune axis and increase the expression of AChR in muscle groups， thereby improving the myasthenic symptoms of EAMG mice.