Dose-Dependent Immunomodulation by Wheat-Grain Moxibustion at Guanyuan (CV4) and Zusanli (ST36) via JAK2/STAT3 Signaling in Cyclophosphamide-Induced Immunosuppressed Rats

Background Immunosuppression is a critical clinical challenge, and wheat-grain moxibustion (WGM) is a TCM therapy hypothesized to modulate immune function. However, the dose-dependent effects of WGM and its mechanistic link to the JAK2–STAT3 pathway remain understudied. Objectives To investigate the immunopotentiating effects of different dosages of WGM (3, 6, and 9 zhuang) at Guanyuan (CV4) and Zusanli (ST36) on the cyclophosphamide (CTX)-induced immunosuppressed rats, and elucidate its association with the JAK2–STAT3 signaling pathway. Methods Forty-five Sprague–Dawley (SD) rats were randomized into 5 groups: blank control (CON), MG (Model group), and WGM groups (LOW: 3 zhuang, MID: 6 zhuang, and HIGH: 9 zhuang). WGM was applied daily for 7 days. Immune function was assessed via peripheral blood counts (WBC and CD4+/CD8+), organ indices (spleen, thymus), histopathology (HE staining), and molecular markers (JAK2/STAT3 protein/mRNA, cytokines: IgG, IgM, TNF-α, IFN-γ, IL-6, and IL-10). Results Compared to MG, all WGM groups increased WBC counts ( p < 0.05), but the MID (6 zhuang) and HIGH (9 zhuang) groups showed a 1.8-fold and 2.1-fold higher WBC count ( p < 0.01), respectively. Flow cytometry revealed a 2.3-fold increase in CD4+/CD8+ ratio in the HIGH group ( p < 0.01) vs. MG. Histopathology showed restored spleen marginal zones and thymic corticomedullary structure in WGM groups, with the HIGH group exhibiting the least lymphocyte necrosis. Mechanistically, WGM upregulated JAK2/STAT3 phosphorylation (p-JAK2: p < 0.01 in HIGH vs. MG; p-STAT3: p < 0.05 in HIGH vs. MG; p < 0.01) and downstream cytokines (e.g., IFN-γ: in HIGH vs. MG; p < 0.01). Notably, IL-10 (anti-inflammatory) was downregulated in the HIGH group ( p < 0.01), suggesting a shift from immunosuppression to immune activation. Conclusions WGM at Guanyuan and Zusanli ameliorates MG-induced immunosuppression in a dose-dependent manner, with 9 zhuang (HIGH) being the most effective for restoring cellular (CD4+/CD8+) and molecular (JAK2–STAT3) immune function. These findings provide quantitative evidence for optimizing WGM dosage in clinical settings and reveal a novel mechanistic link between TCM therapy and modern immunology. Graphical abstract A graphical abstract that can encompass all the content of this article.