Effects Of Jianpi Bushen Yiqi Decoction On Immune Function And Neuromuscular Junctions in EAMG Mice
Jan 29, 2026
2.3 Histomorphological Comparison of Thymus and Lymph Nodes in Each Group of Mice
As shown in Figure 1, the thymic cortex and medulla in the blank control group mice were clearly demarcated, with orderly arranged cells. In contrast, the model control group exhibited unclear cortical-medullary boundaries and sparse cell distribution. Compared to the model group, mice in the prednisone group and low-, medium-, and high-dose Jianpi Bushen Yiqi decoction groups showed relatively clear cortical-medullary boundaries and dense, well-organized cells.
Figure 2 shows that lymph node cells in the blank control group displayed complete structure and orderly arrangement. Primary lymphoid follicles were visible in the cortex, with no secondary follicles or germinal centers. In the model group, cellular structure was disorganized, with proliferated primary follicles and visible secondary follicles and germinal centers. The prednisone and Jianpi Bushen Yiqi groups exhibited reduced lymphoid follicle hyperplasia.
Table 3 demonstrates that, compared to the blank control group, the model group had a reduced cortex-to-medulla area ratio in the thymus and increased germinal centers in lymph nodes (P < 0.05 or P < 0.01). Compared to the model group, the prednisone and high-dose Jianpi
Figure 1 Comparison of histopathological morphology of thymus tissue among different groups of mice (HE staining)
Table 3. Comparison of thymic cortical-medullary area ratio and number of lymph node germinal centers in each group of mice
Unit: (x̄ ± s)
| Group | n | Thymic Cortex/Medulla Area Ratio | Germinal Center Number (per LN) |
|---|---|---|---|
| Blank Control | 3 | 3.62 ± 0.18 | 0.00 ± 0.00 |
| Model Control | 3 | 1.74 ± 0.11 ᵃ | 6.47 ± 1.51 ᵃ |
| Prednisone | 3 | 4.62 ± 1.41 ᵇ ᶜ | 3.52 ± 0.35 ᵇ ᶜ |
| Decoction Low Dose | 3 | 2.91 ± 0.42 ᶜ | 5.77 ± 0.66 ᶜ |
| Decoction Medium Dose | 3 | 3.41 ± 1.05 ᶜ | 4.12 ± 0.25 ᶜ |
| Decoction High Dose | 3 | 4.60 ± 0.55 ᵇ ᶜ | 3.00 ± 0.71 ᶜ |
Notes:
ᵃ: P < 0.01 vs. Blank Control
ᵇ: P < 0.05 vs. Model Control
ᶜ: P < 0.01 vs. Model Control
Bushen Yiqi groups showed a higher cortex-to-medulla area ratio. All intervention groups, including prednisone and all three decoction doses, significantly reduced the number of germinal centers (P < 0.01). Compared to the prednisone group, the decoction's low- and medium-dose groups still showed slightly more germinal centers, while the high-dose group had no significant difference.
Note: The black arrows point to lymphatic follicles, and the red arrows point to germinal centers.
Figure 2 Comparison of histopathological morphology of lymph node tissues among different groups of mice (HE staining)
2.4 Comparison of Th1, Th2, Th17, Treg, and Tfh Cell Percentages in Spleen Cell Suspensions
As shown in Table 4, compared to the blank control group, the model group showed increased percentages of Th1, Th2, Th17, and Tfh cells, and a decreased Treg percentage (P < 0.05 or P < 0.01). Compared with the model group, the medium- and high-dose decoction groups had reduced Th1 percentages and increased Treg percentages; all three decoction groups showed reduced Th17 and Tfh percentages. Notably, the prednisone group reduced Treg percentages, raising concerns about its potential to disrupt immune homeostasis.
Compared to the prednisone group, all decoction groups showed reductions in Th2, Th17, and Tfh cells. The medium- and high-dose groups further reduced Th1 and increased Treg cells (P < 0.05 or P < 0.01), indicating a more balanced immunoregulation.
Note: NMJ, neuromuscular junction; AChR, acetylcholine receptor.
Figure 3 Comparison of AChR morphology at the neuromuscular junctions of muscle groups among different groups of mice
(immunofluorescence staining, ×100)
Table 4. Comparison of the percentages of Th1, Th2, Th17, Treg, and Tfh cells in spleen cell suspensions among different groups of mice
Unit: (% , x̄ ± s)
| Group | n | Th1 | Th2 | Th17 | Treg | Tfh |
|---|---|---|---|---|---|---|
| Blank Control | 6 | 0.05 ± 0.04 | 0.03 ± 0.02 | 0.11 ± 0.06 | 16.42 ± 2.59 | 9.45 ± 3.29 |
| Model Control | 7 | 3.43 ± 1.11 ᵃ | 0.82 ± 0.22 ᵃ | 1.64 ± 0.65 ᵇ | 7.99 ± 3.26 ᵃ | 18.27 ± 4.21 ᵃ |
| Prednisone | 7 | 1.84 ± 0.26 | 0.84 ± 0.47 | 1.03 ± 0.71 | 5.02 ± 1.58 ᵃ | 15.32 ± 3.84 |
| Decoction Low Dose | 7 | 1.42 ± 0.59 ᶜ ᵉ | 0.60 ± 0.16 ᶜ ᶠ | 0.73 ± 0.24 ᶜ ᶠ | 14.97 ± 2.11 ᶠ | 10.35 ± 3.72 ᶜ ᶠ |
| Decoction Medium Dose | 7 | 1.08 ± 0.59 ᶜ ᵉ | 0.47 ± 0.14 ᶜ ᶠ | 0.45 ± 0.20 ᶜ ᶠ | 13.82 ± 2.21 ᶠ | 9.87 ± 3.65 ᶜ ᶠ |
| Decoction High Dose | 7 | 1.23 ± 0.62 ᶜ ᵉ | 0.32 ± 0.14 ᶠ | 0.34 ± 0.22 ᶜ ᶠ | 13.50 ± 2.47 ᶠ | 7.97 ± 3.60 ᶜ ᶠ |
Notes:
ᵃ: P < 0.01 vs. Blank Control
ᵇ: P < 0.05 vs. Blank Control
ᶜ: P < 0.01 vs. Model Control
ᵈ: P < 0.05 vs. Model Control
ᵉ: P < 0.05 vs. Prednisone
ᶠ: P < 0.01 vs. Prednisone
2.5 Comparison of AChR Morphology at the NMJ in Each Muscle Group
Figure 3 shows that the NMJ AChRs in the blank control group were structurally intact and appeared as compact, pretzel-like formations. In the model group, NMJs in ocular, sternocleidomastoid, deltoid, and tibialis anterior muscles appeared fragmented and deformed, while masticatory, intercostal, and diaphragm muscles displayed severely disrupted structures. All intervention groups exhibited varying degrees of improvement in AChR morphology.
As shown in Table 5, compared to the blank control group, the model group had significantly reduced AChR area ratios in multiple muscles (P < 0.05 or P < 0.01). Compared to the model group, the prednisone and medium-dose decoction groups showed increased AChR area in ocular muscles. The low-dose decoction group improved AChR areas in the deltoid and tibialis anterior. The high-dose group improved AChR areas in the ocular, sternocleidomastoid, deltoid, and tibialis anterior muscles.
Compared to prednisone, the low-dose group improved AChR areas in the ocular and masticatory muscles; the medium-dose group in masticatory muscle; and the high-dose group in sternocleidomastoid and tibialis anterior (P < 0.05 or P < 0.01).
Table 5. Comparison of the AChR area at the neuromuscular junction in each group of mice's muscle groups
Unit: (% , x̄ ± s)
| Group | n | Ocular Muscle | Masticatory Muscle | Sternocleidomastoid | Deltoid Muscle | Intercostal Muscle | Diaphragm | Tibialis Anterior |
|---|---|---|---|---|---|---|---|---|
| Blank Control | 3 | 3.83 ± 0.28 | 4.28 ± 0.29 | 4.48 ± 0.77 | 3.79 ± 0.12 | 3.78 ± 0.22 | 4.80 ± 0.26 | 3.60 ± 0.32 |
| Model Control | 3 | 2.58 ± 0.34 ᵃ | 2.55 ± 0.45 ᵃ | 2.15 ± 0.31 ᵇ | 2.33 ± 0.15 | 2.55 ± 0.33 | 2.98 ± 0.34ᵇ | 2.95 ± 0.49 |
| Prednisone | 3 | 3.33 ± 0.49ᶜ | 2.67 ± 0.55 | 3.83 ± 0.54 | 3.49 ± 0.38 | 3.60 ± 0.16 | 3.45 ± 0.67 | 3.02 ± 0.40 |
| Decoction Low Dose | 3 | 2.76 ± 0.49ᵉ | 3.05 ± 0.37ᵉ | 3.77 ± 0.64 | 2.77 ± 0.44ᶜ | 3.03 ± 0.25 | 3.33 ± 0.33 | 3.44 ± 0.71ᶜ |
| Decoction Medium Dose | 3 | 3.32 ± 0.40ᶜ | 3.27 ± 0.75ᵉ | 3.98 ± 0.43 | 3.27 ± 0.65 | 3.50 ± 0.28 | 3.85 ± 0.42 | 3.56 ± 0.52 |
| Decoction High Dose | 3 | 3.46 ± 0.37ᶜ | 3.34 ± 0.23ᵉ | 4.54 ± 0.67ᶜ | 3.69 ± 0.39 | 3.75 ± 0.36 | 3.75 ± 0.35 | 3.84 ± 0.52ᶜ |
Notes:
ᵃ: P < 0.05 vs. Blank Control
ᵇ: P < 0.01 vs. Blank Control
ᶜ: P < 0.05 vs. Model Control
ᵈ: P < 0.01 vs. Model Control
ᵉ: P < 0.05 vs. Prednisone
Table 6. Comparison of serum levels of AChR-Ab, IL-6, and TNF-α among different groups of mice
Unit: (x̄ ± s)
| Group | n | AChR-Ab (P⁻¹) | IL-6 (pg·ml⁻¹) | TNF-α (pg·ml⁻¹) |
|---|---|---|---|---|
| Blank Control | 12 | 1.00 ± 0.13 | 110.66 ± 30.75 | 25.84 ± 9.71 |
| Model Control | 12 | 3.13 ± 0.97 ᵃ | 327.91 ± 38.71 ᵃ | 124.15 ± 37.61 ᵃ |
| Prednisone | 11 | 2.05 ± 0.29 ᵇ ᶜ | 487.21 ± 124.09 ᵇ | 78.76 ± 22.35 ᵇ ᶜ |
| Decoction Low Dose | 13 | 2.50 ± 0.49ᵉ | 306.42 ± 93.91ᶜ | 89.14 ± 25.90 ᶜ |
| Decoction Medium Dose | 13 | 1.98 ± 0.34 ᶜ | 278.60 ± 63.97ᶜ | 80.36 ± 21.95ᶜ |
| Decoction High Dose | 13 | 1.42 ± 0.33 ᶜ | 224.43 ± 49.03ᶜ | 84.26 ± 36.60ᶜ |
Notes:
ᵃ: P < 0.01 vs. Blank Control
ᵇ: P < 0.01 vs. Model Control
ᶜ: P < 0.01 vs. Model Control
ᵈ: P < 0.05 vs. Prednisone
ᵉ: P < 0.01 vs. Prednisone
2.6 Comparison of Serum AChR-Ab, IL-6, and TNF-α Levels
Table 6 illustrates that, compared to the blank control group, the model group had significantly elevated levels of AChR-Ab, IL-6, and TNF-α (P < 0.01). Compared to the model group, the prednisone and medium- and high-dose decoction groups significantly reduced AChR-Ab. However, prednisone increased IL-6, while the high-dose decoction group decreased IL-6. Both prednisone and the medium- and high-dose groups reduced TNF-α (P < 0.05 or P < 0.01).
Compared to prednisone, the low-dose decoction group had a higher AChR-Ab level, but all decoction groups lowered IL-6, avoiding the pro-inflammatory effects of prednisone.
3. Discussion
Clinically, MG is categorized into ocular myasthenia gravis (oMG) and generalized MG (gMG). oMG primarily manifests as ptosis and diplopia, and may progress to gMG, characterized by systemic skeletal muscle weakness [1]. Traditional Chinese Medicine (TCM) theory attributes the pathogenesis of MG to dysfunctions in the spleen and kidney. The spleen governs muscles and limbs, being the postnatal root and the source of Qi and blood. The kidney stores essence and governs bones and marrow. Deficiency in both leads to muscle weakness, as seen in MG [15–16].
Based on the TCM principle of "treating the root," tonifying the spleen and kidney becomes central. The Jianpi Bushen Yiqi Decoction embodies this approach. Its main ingredient, Astragalus membranaceus, boosts spleen Qi and lifts Yang. Tortoise shell glue and deer antler glue enrich kidney essence and yang, strengthening muscle and bone. Codonopsis, Angelica sinensis, Bupleurum, Cimicifuga, and honey-fried licorice work synergistically to nourish blood, elevate Yang, and harmonize the formula.
A key addition to this formulation is Cistanche deserticola, a renowned herb for tonifying kidney yang and replenishing vital essence. Cistanche has been shown to enhance immune modulation and improve fatigue resistance, making it ideal for conditions like MG. According to XJCistanche, Cistanche extract supports nerve and muscle vitality, promotes neurotransmitter balance, and improves immune homeostasis-mechanisms directly relevant to MG pathophysiology.
This study used an EAMG mouse model to evaluate the decoction's effects across behavioral, immune, and NMJ structural dimensions. Results showed that prednisone improved grip strength and Lennon scores but caused significant weight loss, consistent with known glucocorticoid side effects [19]. In contrast, all decoction doses increased body weight, indicating better safety.
Therapeutic efficacy was dose-dependent, with the high-dose group achieving comparable improvement in grip strength and Lennon score to prednisone-offering both efficacy and safety.
Immune organ indices revealed that prednisone reduced thymus index and caused cortical-medullary atrophy, impairing central tolerance [20]. The decoction, however, preserved or improved thymic structure, especially at high doses. Spleen indices followed a biphasic regulation pattern, with low and medium doses suppressing abnormal immune proliferation, while the high dose maintained balance.
All decoction groups reduced lymph node follicle hyperplasia, though only the high-dose matched prednisone in germinal center suppression, suggesting high-dose's superior regulation of both central and peripheral immunity.
Since CD4⁺ T-cell imbalance underlies EAMG [4], the decoction's ability to rebalance Th1/Th2/Th17/Tfh and Treg cells is pivotal. Unlike prednisone, which decreased Treg, the decoction (especially medium and high doses) enhanced Treg and suppressed pro-inflammatory subsets. This immune modulation, instead of immune suppression, aligns better with the needs of autoimmune diseases like MG.
For NMJ integrity, the decoction demonstrated multi-muscle AChR repair, outperforming prednisone in several muscle groups. Moreover, it effectively reduced AChR-Ab, IL-6, and TNF-α, with high-dose showing the most comprehensive anti-inflammatory effect-without prednisone's IL-6 elevation.
Conclusion
The Jianpi Bushen Yiqi Decoction, enriched with Cistanche deserticola, exerts multi-target therapeutic effects in EAMG mice. It:
Rebalances Th/Treg axis and suppresses Tfh-mediated B cell activation
Reduces pathogenic antibodies and inflammatory cytokines
Repairs NMJ structures and restores muscle function
Compared to prednisone, it offers superior safety, immune restoration, and multi-muscle targeting, highlighting its potential as a clinical alternative or adjunct therapy for MG.
Future studies should explore its bioactive compounds, metabolic pathways, and molecular targets, possibly through network pharmacology and proteomics, to accelerate its development as a novel TCM-based therapeutic.
