Guilu Erxian Glue Restores Immune Homeostasis in Aplastic Anemia Mice by Regulating Treg Lineage Stability via the miRNA-17/10a and FasL/Fas Signaling Pathways

Background: This study investigates the functional role and mechanisms of Guilu Erxian Glue (GEG) in regulating immune homeostasis to treat bone marrow (BM) failure in aplastic anemia (AA). Methods: Differentially expressed miRNAs of AA patients were identified through bioinformatics analysis of the Gene Expression Omnibus (GEO) database, followed by enrichment analysis of the identified miRNAs and their predicted target genes. The chemical profile of GEG was characterized by UPLC-MS/MS. An AA mouse model was established via 60 Co γ-ray irradiation combined with the infusion of an allogeneic lymphocyte suspension for immune induction. GEG + Immunosuppressive therapy (IST) was administered as the therapeutic intervention, with IST + Eltrombopag (EPAG) serving as the positive control. Bone marrow hematopoietic function was evaluated via hematological parameters, BM histopathology, and flow cytometry. CyTOF-2 mass cytometry was utilized to analyze the differentiation profile of CD4 + T-cell subsets, and flow cytometry was further used to detect the proliferation and differentiation of naïve T cells, Tregs, and follicular helper T cells (Tfh). The expression of genes and proteins involved in the miRNA-17/IKZF4, miRNA-10a/BCL6, and Fas pathways was quantified by RT-qPCR and Western blot, with serum cytokines measured by enzyme-linked immunosorbent assay (ELISA). Results: Bioinformatic analysis of the GSE82095 dataset identified significantly dysregulated miRNAs in T cells from AA patients, specifically miRNA-17-5p and miRNA-10a-5p. Preliminary enrichment analysis indicated that these miRNAs are primarily involved in hypoxic stress, T-cell differentiation, and apoptosis. Subsequently, the downstream target genes of these miRNAs were predicted, and further functional enrichment analysis corroborated that these target genes are critically associated with T-cell differentiation. HPLC-MS/MS identified 22 compounds from the aqueous extract of GEG. In vivo, GEG treatment significantly improved BM histopathology and restored peripheral blood counts. It also reduced the percentages of Tfh, Th1, Th17, and effector memory T cells (Tem) ( P < 0.01), while increasing the percentages of Tregs and central memory T cells (Tcm) ( P < 0.01). Molecular analysis revealed that GEG upregulated miRNA-10a and downregulated miRNA-17 in Tregs ( P < 0.01), leading to increased expression of the Foxp3 cofactor IKZF4 and decreased BCL6 expression ( P < 0.01). GEG elevated Foxp3 protein levels and inhibited cleaved caspase-3 expression in Tregs, and reduced the levels of pro-inflammatory cytokines (IFN-γ, TNF-α, and IL-6) ( P < 0.01),while enhancing the secretion of anti-inflammatory cytokines (IL-10 and IL-35) ( P < 0.01). Conclusion: Our findings suggest that GEG contributes to Treg lineage stability and survival through the potential modulation of the miRNA-17/IKZF4, miRNA-10a/BCL6, and Fas/FasL signaling axes. These synergistic effects appear to support the remodeling of T-cell homeostasis and restoration of cytokine balance, offering a promising, miRNA-targeted strategy for enhancing AA therapy that warrants further clinical investigation. The image illustrates an immune suppression pathway in the BALB/c AA model. It begins with radiation exposure at 3.5 Gy using cobalt 60 gamma rays, followed by injection of DBA/2 lymphocyte suspension. The process involves bone marrow cells and hematopoietic stem cells. The establishment period of immunosuppression is characterized by the interaction between TNF - β, IFN - γ, and IL-6 and their respective receptors, regulating the expression of cellular miRNA and affecting its function and stability. At the same time, FasL binds to Fas and activates FADD, which triggers caspase 8, cleaved caspase 8, caspase 3/7, and cleaved caspase 3/7, leading to cell apoptosis. Regulatory elements include miRNA10a affecting BCL6 mRNA, miRNA17 influencing IKZF4 mRNA and HIF1 alpha mRNA. T cell regulation involves TGF-beta, IL-35 and IL-10 affecting Tfh and Treg cells, which interact with Th1, Th2 and Th17 cells, leading to effector T cells and naïve T cells. BCL6 is involved in the regulation of these processes.Immune suppression pathway in BALB/c AA model showing radiation effects, apoptosis and T cell regulation.