Myasthenia gravis (MG) is an autoimmune disorder characterized by autoantibody-mediated attack on proteins at the neuromuscular junction (NMJ), primarily targeting the acetylcholine receptor (AChR) and muscle-specific kinase (MuSK) [1]. This pathogenic process leads to impaired voluntary skeletal muscle function, significantly affecting patients' daily activities and quality of life. MG exhibits a bimodal age distribution, predominantly affecting young women and older men, with incidence rates varying by age, gender and ethnicity [2], [3]. Epidemiological studies report an incidence of 4.1–30 per million person-years and a prevalence of 150–200 cases per million, with a rising trend in recent decades [4], highlighting the growing clinical and socioeconomic burden of this disease.

Current therapeutic strategies for MG include acetylcholinesterase inhibitors, corticosteroids, immunosuppressants and thymectomy [5], [6]. However, these treatments often provide only symptomatic relief but rarely achieve sustained remission or cure. Althoughemerging biologics (e.g., complement inhibitors like eculizumab and FcRn blockers like efgartigimod) show promise, their clinical application is limited by high costs, antibody specificity, and adverse effects [7], [8]. While, novel approaches, including nipocalimab and CAR-T cells therapy, are under investigation [9] [10], their long-term efficacy remains unproven. Thus, the development of cost-effective, mechanism-based therapies remains an urgent priority.

The pathogenesis of MG involves a complex interplay of genetic susceptibility, thymic abnormalities, and immune dysregulation. Approximately 85% of patients harbor AChR autoantibodies, which impair NMJ function by promoting receptor degradation, complement activation, and postsynaptic membrane damage [11]. Subsets of patients also exhibit autoantibodies against MuSK or low-density lipoprotein receptor-related protein 4 (LRP4), further diversifying disease mechanisms [12], [13], [14]. CD4+ T cells drive pathogenesis by promoting B-cell activation and autoantibody production, while dysregulated cytokine networks (e.g., IFN-γ, IL-17) and impaired regulatory T cell (Treg) function exacerbate autoimmunity [15], [16]. Emerging evidence also implicates extrathymic mechanisms, particularly gut microbiota dysbiosis in MG progression. MG patients exhibit altered microbial composition (e.g., reduced Firmicutes, elevated Proteobacteria) and impaired intestinal barrier function, which may exacerbate systemic inflammation [17], [18]. Notably, fecal microbiota transplantation (FMT) from MG patients into germ-free mice induces motor deficits, confirming a causal role [19]. Additionally, metabolomic disturbances, including reduced short-chain fatty acids (SCFAs) impair immune homeostasis and Treg function [20]. Recent studies further highlight ferroptosis as critical pathways: iron dysregulation and mitochondrial dysfunction amplify oxidative stress at the NMJ [21], [22], [23].

Astragaloside IV (AS-IV), a bioactive saponin derived from Astragalus membranaceus, exhibits pleiotropic pharmacological activities including antioxidant, anti-inflammatory, anti-apoptotic and immunomodulatory actions [24]. Preclinical studies have demonstrated its therapeutic potential in diverse disease models, including cancer [25], cerebral ischaemia/reperfusion injury [26], [27], [28], cardiovascular disease [29], diabetic kidney disease [30] and ulcerative colitis [31]. Mechanistically, AS-IV has been shown to mitigate septic myocardial injury through DUSP1–Prohibitin-2-mediated mitochondrial quality control and ER-phagy [32], and attenuate inflammatory organ damage by suppressing multiple pro-inflammatory cascades [33]. In the context of autoimmune neuromuscular disorders, a single study reported that AS-IV alleviates experimental autoimmune myasthenia gravis (EAMG) by modulating gut microbiota composition [34]. However, the precise mechanisms underlying AS-IV’s therapeutic effects, particularly its interplay with immune regulation, metabolic profile, ferroptosis, remain incompletely understood and warrant systematic investigation.

In this study, we systematically evaluated the therapeutic effects of AS-IV in EAMG model through an integrated multi-omics approach combining 16S rRNA sequencing, metabolomics, and network pharmacology. Our results demonstrate that AS-IV restored immune homeostasis by significantly decreasing pro-inflammatory Th1 and Th17 cell populations while increasing immunosuppressive Treg cells. Additionally, AS-IV restrained T follicular helper (Tfh) cell expansion. Moreover, AS-IV reconfigured gut microbiota composition, modulated metabolic profiles, and inhibited ferroptosis. These findings collectively position AS-IV as a promising therapeutic candidate for MG.