Ningxue Shengban decoction containing serum alleviates immune thrombocytopenia by modulating CD4+T cells balance via BMSCs-Exo-miR-199a-5p

Abstract

Background The abnormal immune response mediated by CD4+T cells is a key factor in Immune thrombocytopenia(ITP) progression. While Ningxue Shengban Decoction (NXSBD) is an effective therapeutic, its underlying mechanism and targets remain obscure. Aim This study aims to clarify the role of exosomal miR-199a-5p derived from bone marrow mesenchymal stem cells (BMSCs) in immune homeostasis, and to explore the therapeutic effects of exosomes from BMSCs(BMSCs-Exo) pretreated with NXSBD containing serum on ITP. Method We co-cultured CD4+T cells with BMSCs or pre-treated BMSCs-Exo. The proliferation and differentiation of CD4+T cells were then assessed using CFSE staining and flow cytometry (FCM). Additionally, an active ITP murine model was employed to assess the therapeutic efficacy of pre-treated BMSCs-Exo. Platelet counts were measured and organ indices were calculated. Serum autoantibody levels were measured by FCM and ELISA, changes in CD4⁺T cells subsets in the spleen were analyzed by FCM, megakaryocyte number and morphology in bone marrow tissues were examined by H&E staining, and key cytokine levels in mouse serum were quantified by ELISA. Results Our results indicate that the immunomodulatory effect of BMSCs-Exo on CD4+T cells is mediated by miR-199a-5p, and that NXSBD containing serum enhances this effect by increasing miR-199a-5p levels. In an active ITP murine model, BMSCs-Exo treatment significantly ameliorated the pathological features of ITP, as evidenced by increased peripheral platelet counts, reduced spleen and thymus indices, and decreased levels of autoantibodies. Immunophenotypic analysis revealed that an increased percentage of splenic Treg and Th2 cells, and a decreased percentage of Th17 and Th1 cells, were observed after BMSCs-Exo treatment. Additionally, BMSCs-Exo enhanced the production of mature megakaryocytes. Following BMSCs-Exo treatment, the levels of pro-inflammatory cytokines were sharply lowered, whereas anti-inflammatory cytokine levels were markedly elevated. BMSCs-Exo pretreated with NXSBD containing serum exert superior therapeutic efficacy compared with those derived from untreated BMSCs.

1. Introduction

Immune thrombocytopenia (ITP) is an acquired autoimmune disorder in which immune-mediated platelet destruction and impaired production result in isolated thrombocytopenia [1]. The clinical presentation can range from asymptomatic conditions to skin and mucosal bleeding, or even fatal intracranial hemorrhage [2]. ITP pathophysiology is multifactorial and heterogeneous, with dysfunction and imbalanced ratios of T cell subgroups being a critical factor. T cell imbalance is a well-recognized feature of ITP pathogenesis, characterized by a shift toward Th1 and Th17 responses at the expense of the Th2 and Treg subsets [3]. The impairment in both the function and number of Tregs disrupts immune balance, with consequent loss of peripheral tolerance and induction of autoimmune responses to autoantigens [4]. It has been shown that bone-marrow mesenchymal stem cells (BMSCs) derived from individuals diagnosed with ITP exhibit functional impairments, including defective immunosuppression and a reduced capacity to induce Tregs [5]. However, the precise process by which defective BMSCs cause peripheral CD4+T cells immune dysregulation in ITP remains to be clarified.

On the other hand, mesenchymal stem cells (MSCs) have widespread applications in various fields owing to their immunomodulatory capacities [6]. It has been established that MSC-based therapies and their derived extracellular vesicles (EVs) hold promising therapeutic potential for treating ITP [7–9]. Exosomes are EVs that are loaded with genetic molecules, including mRNAs, miRNAs, and proteins. These molecules are delivered to recipient cells, thereby mediating intercellular communication [10, 11]. MSCs-derived exosomes are novel and promising therapeutic agents that inherit the innate immunomodulatory and drug delivery functions of MSCs; their significant advantage lies in retaining the therapeutic effects of the parental cells while exhibiting a reduced risk of side effects [12, 13]. BMSCs are regarded as among the most effective cells for exosome production.