Modified Guilu Erxian Glue regulates Treg immune function to suppress bone marrow failure in aplastic anemia mice

Aplastic anemia (AA) is an autoimmune disorder characterized by impaired immunosuppressive function and abnormal differentiation of regulatory T (Treg) cells. Immunosuppressive treatment (IST) is a primary treatment for AA. Our previous studies have suggested that Modified Guilu Erxian Glue (MGEG) could improve hematopoietic function through immune modulation. These results indicated that it should serve as an adjunct therapy in boosting the efficacy of IST for AA treatment. Nevertheless, the regulatory mechanisms of MGEG on the Treg cells were unclear. In this study, we aimed to investigate the mechanisms of the combination therapy of IST and MGEG on the function and differentiation of Treg cells, contributing to alleviate the hematopoietic dysfunction in immune-mediated AA mice.

An AA mouse model was established using 3.5 Gy ⁶⁰Coγ irradiation followed by allogeneic lymphocyte infusion via the tail vein. The combination of IST + MGEG was used as therapeutic treatment. The combination of IST + EP was used as positive control. The chemical composition of MGEG was analyzed by HPLC-ESI/MS. Hematological parameters, histopathological staining, and flow cytometry were used to evaluate bone marrow hematopoiesis. The differentiation of CD4⁺ T and Treg cell subsets were analysed by CyTOF-2 mass cytometry. Inflammatory factor levels and Fas/FasL pathway protein expression were measured by ELISA and Western blot. Flow cytometry was also used to examine proliferation and differentiation of naïve T, effector T, and Treg cells. The regulatory effects of IST combined with MGEG on the IL-2/STAT5 and miR-17-5p/Eos signaling pathways were verified by qPCR and Western blot.

HPLC-ESI/MS identified 30 compounds from the aqueous extract of MGEG, including amentoflavone, berberine, and ononin. The combined treatment of IST + MGEG improved the hematopoietic function of AA mice, as indicated by restored blood cell counts and reduced bone marrow adiposity. It also rebalanced the Th1/Th2 and Th17/Treg ratios, increased the proportion of Treg B cells, and ameliorated bone marrow inflammatory status. Furthermore, the combination treatment could inhibit Treg cell apoptosis through downregulating the expression of Fas and levels of Cleaved-Caspase-3/8 while upregulating p-Bcl-2. It also enhanced p-STAT5 and Foxp3 protein levels, contributed to promoting naïve T cell differentiation into Treg cells. Additionally, IST combined with MGEG reduced miR-17-5p and HIF-1α expression in CD4⁺ T cells, accompanied by the increase in the protein expression of Eos.

Compared with the IST + EP or IST alone, the combination treatment of IST + MGEG further improved hematopoietic function in AA mice. These effects should involve regulating the differentiation of Treg cells through intervening in the activation of IL-2/STAT5 signaling pathway, improving the immune regulatory function of Treg cells by intervening in the miR-17-5p/Eos signaling pathway, and inhibiting Fas/FasL mediated apoptosis.