背景:桂路二仙胶(GEG)和当归补血汤(DBT)是传统的中草药配方。根据中医理论,这两个公式的结合(修正桂路二仙胶,MGEG)具有补肾生血的作用,通常用于治疗骨髓衰竭疾病,包括再生障碍性贫血(AA)。
目的:T淋巴细胞在AA的发病及进展过程中发挥重要作用。我们的初步成果证实,GEG可以改良小鼠造血干细胞的毁伤,而DBT可以降低T淋巴细胞的增殖和分化,抑制IFN-γ的产生。我们假设这两种草药配方的组合可以通过多种机制抑制免疫攻击并恢复造血功能。在这项研究中,我们的目的是研究MGEG对信号淋巴细胞激活分子(SLAM)表达的调节作用,T淋巴细胞中的活化相关分子,从而抑制T细胞的免疫功能,减缓造血干细胞的损伤。
方法:高效液相色谱-电喷雾电离/质谱系统用于鉴定MGEG制剂的成分。BABL/c小鼠电离辐射后注射异基因淋巴细胞悬液诱导再生障碍性贫血小鼠模型。环孢菌素A(CsA)用作阳性对照药物。流式细胞术检测骨髓造血干细胞的数量和凋亡率。进行酶联免疫吸附测定以测量IFN-γ和TNF-α的水平。免疫荧光染色用于评估T-bet和SLAM-SAP的表达。WesternBlot检测T淋巴细胞活化相关分子和Fas信号通路相关蛋白的表达。进行苏木精-伊红染色以观察骨髓组织的病理变化。利用Wright-Giemsa染色来评估骨髓细胞(BMC)的细胞组成和基本结构的改变。透射电镜观察造血干细胞结构和形态的变化。血液分析仪用于检测外周血参数。
结果:在MGEG中鉴定出23种不同的成分。MGEG治疗后,在AA小鼠中Fyn和SLAM-SAP结合的表达水平增加,而T-bet的表达水平降低,IFN-γ的分泌显着减少。此外,MGEG还可以下调Fas的蛋白质水平,在AA小鼠中caspase-3和裂解的caspase-3。
结论:MGEG可以通过促进SLAM-SAP信号通路来调节T-bet在T细胞中的产生和分布,从而减弱IFN-γ的产生。此外,它通过干预Fas依赖性途径抑制HSC的凋亡,从而减轻对HSC的免疫介导的损伤。
BACKGROUND: Guilu Erxian Glue (GEG) and Danggui Buxue Tang (DBT) are traditional Chinese herbal formulas. According to the theory of traditional Chinese medicine, the combination of those two formulas (Modified Guilu Erxian Glue, MGEG) has the effects of tonifying the kidney and producing blood, was usually used to treat bone marrow failure diseases, including aplastic anemia (AA).
OBJECTIVE: T lymphocytes play a crucial role in the disease pathogenesis and progression of AA. Our preliminary results confirmed that GEG can improve the damage of hematopoietic stem cells in mice, while DBT can reduce the proliferation and differentiation of T lymphocytes and inhibit the production of IFN-γ. We hypothesized that the combination of those two herbal formulas could inhibit immune attack and restore hematopoietic function through multiple mechanisms. In this study, we aim to study the curative effect of MGEG on regulating the expression of Signal lymphocyte activating molecule (SLAM), an activation-related molecule in T lymphocytes, thereby suppressing the immune function of T cells and decelerating the damage to hematopoietic stem cells.
METHODS: High-performance liquid chromatography-electrospray ionization/mass spectrometry system was used to identify the components of the MGEG formulation. Induction of aplastic anemia mouse model by injecting allogeneic lymphocyte suspension into BABL/c mice after ionizing radiation. Cyclosporine A (CsA) was used as a positive control drug. Flow cytometry was used to detect the number and apoptosis rate of hematopoietic stem cells in the bone marrow. Enzyme-linked immunosorbent assay was performed to measure the levels of IFN-γ and TNF-α. Immunofluorescence staining was used to assess the expression of T-bet and SLAM-SAP. Western Blot was conducted to examine the expression of activation-related molecules in T lymphocytes and proteins related to the Fas signal pathway. Hematoxylin-eosin staining was performed to observe pathological changes in the bone marrow tissue. Wright-Giemsa staining was utilized to evaluate alterations in the cellular composition and basic structure of the bone marrow cells (BMCs). Transmission electron microscopy was employed to observe changes in the structure and morphology of hematopoietic stem cells. The hematology analyzer was used to detect peripheral blood parameters.
RESULTS: Twenty-three different components were identified in MGEG. After MGEG treatment, the expression levels of Fyn and SLAM-SAP binding were increased in AA mice, while the expression levels of T-bet were decreased and the secretion of IFN-γ was reduced significantly. Additionally, MGEG also could downregulate the protein levels of Fas, caspase-3, and cleaved caspase-3 in AA mice.
CONCLUSIONS: MGEG could attenuate the production of IFN-γ by promoting the SLAM-SAP signal pathway to regulate the generation and distribution of T-bet in T cells. Additionally, it suppresses apoptosis of HSCs through intervention in the Fas-dependent pathway, thereby mitigating immune-mediated damage to HSCs.