Supplementary Material for: Role of Raf Kinase Inhibitor Protein in Modulating Echinococcal Cyst Fluid-induced Degranulation in Bone Marrow-Derived Mast Cells

Objective: This study aimed to investigate the role of Raf kinase inhibitor protein (RKIP) in degranulation induced by echinococcal cyst fluid (EgCF) in bone marrow-derived mast cells (BMMCs).
Methods: Primary BMMCs were isolated and cultured from the femurs and tibias of RKIP gene knockout (KO) and wild-type (WT) C57BL/6 mice. EgCF-induced degranulation models were established for both groups. Samples of cells and supernatant were collected for analysis. Surface expression levels of CD117 and Fc-epsilon Receptor Ⅰ α (FcεRIα) were assessed. Supernatant concentrations of β-hexosaminidase, IL-4, IL-6, and tumor necrosis factor (TNF-α) were measured. Cellular mRNA levels of IL-4, IL-6, and TNF-α were quantified, and changes in RKIP protein expression during degranulation in WT BMMCs were monitored.
Results: After 4 weeks of induction culture, the double-positive rates for CD117 and FcεRIα exceeded 98% in both KO and WT BMMCs. Following sensitization, BMMCs from the KO group demonstrated significantly higher degranulation rates compared to the WT group (p < 0.05). In WT BMMCs, surface RKIP protein expression progressively decreased at 1 hour, 2 hours, and 3 hours post-sensitization, corresponding with degranulation progression (p < 0.05). The KO group exhibited elevated release of BMMC-related cytokines, including IL-4, IL-6, and TNF-α, compared to the WT group after sensitization (p < 0.05). Similarly, transcription levels of cytokines IL-4, IL-6, and TNF-α were higher in the KO group than in the WT group following sensitization (p < 0.05).
Conclusion: RKIP gene knockout resulted in an increased EgCF-induced release of cytokines and bioactive substances by BMMCs, indicating that RKIP may suppress EgCF-induced BMMC degranulation. These findings suggest that RKIP could serve as a potential therapeutic target for managing allergic reactions associated with cystic echinococcosis.

研究目的：本研究旨在探讨Raf激酶抑制蛋白（Raf kinase inhibitor protein, RKIP）在细粒棘球蚴囊液（echinococcal cyst fluid, EgCF）诱导的骨髓源性肥大细胞（bone marrow-derived mast cells, BMMCs）脱颗粒过程中的作用。实验方法：从RKIP基因敲除（gene knockout, KO）与野生型（wild-type, WT）C57BL/6小鼠的股骨和胫骨中分离并培养原代骨髓源性肥大细胞，为两组建立EgCF诱导的脱颗粒模型。收集细胞及上清样本进行分析，检测CD117与Fcε受体Ⅰα（Fc-epsilon Receptor Ⅰ α, FcεRIα）的表面表达水平，测定上清中β-己糖胺酶（β-hexosaminidase）、IL-4、IL-6及肿瘤坏死因子（tumor necrosis factor, TNF-α）的浓度，定量检测IL-4、IL-6及TNF-α的细胞mRNA水平，并监测野生型BMMCs脱颗粒过程中RKIP蛋白的表达变化。实验结果：经4周诱导培养后，KO组与WT组BMMCs的CD117及FcεRIα双阳性率均超过98%。致敏处理后，KO组BMMCs的脱颗粒率显著高于WT组（p<0.05）。在WT组BMMCs中，致敏后1、2、3小时，表面RKIP蛋白表达水平逐渐降低，且与脱颗粒进程一致（p<0.05）。致敏后，KO组BMMCs相关细胞因子（包括IL-4、IL-6、TNF-α）的释放量显著高于WT组（p<0.05）。同样，致敏后KO组IL-4、IL-6及TNF-α的转录水平亦高于WT组（p<0.05）。研究结论：RKIP基因敲除可增强BMMCs经EgCF诱导的细胞因子与生物活性物质释放，提示RKIP可能抑制EgCF诱导的BMMCs脱颗粒。本研究结果表明，RKIP有望作为治疗细粒棘球蚴病相关过敏反应的潜在治疗靶点。