BACKGROUND MF6p/host defense molecules (HDMs) are a broad family of small proteins secreted by helminth parasites. Although the physiological role of MF6p/HDMs in trematode parasites is not fully understood, their potential biological function in maintaining heme homeostasis and modulating host immune response has been proposed. METHODS A gene encoding the MF6p/HDM of Clonorchis sinensis (CsMF6p/HDM) was cloned. Recombinant CsMF6p/HDM (rCsMF6p/HDM) was expressed in Escherichia coli. The biochemical and immunological properties of rCsMF6/HDM were analyzed. CsMF6p/HDM induced pro-inflammatory response in RAW 264.7 cells was analyzed by cytokine array assay, reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay. The structural feature of CsMF6p/HDM was analyzed by three-dimensional modeling and molecular docking simulations. RESULTS The CsMF6p/HDM shares a high level of amino acid sequence similarity with orthologs from other trematodes and is expressed in diverse developmental stages of the parasite. The rCsMF6p/HDM bound to bacteria-derived lipopolysaccharide (LPS), without effectively neutralizing LPS-induced inflammatory response in RAW 264.7 macrophage cells. Rather, the rCsMF6p/HDM induced pro-inflammatory immune response, which is characterized by the expression of TNF-α and IL-6, in RAW 264.7 cells. The rCsMF6p/HDM-induced pro-inflammatory immune response was regulated by JNK and p38 MAPKs, and was effectively down-regulated via inhibition of NF-κB. The structural analysis of CsMF6p/HDM and the docking simulation with LPS suggested insufficient capture of LPS by CsMF6p/HDM, which suggested that rCsMF6p/HDM could not effectively neutralize LPS-induced inflammatory response in RAW 264.7 cells. CONCLUSIONS Although rCsMF6p/HDM binds to LPS, the binding affinity may not be sufficient to maintain a stable complex of rCsMF6p/HDM and LPS. Moreover, the rCsMF6p/HDM-induced pro-inflammatory response is characterized by the release of IL-6 and TNF-α in RAW 264.7 macrophage cells. The pro-inflammatory response induced by rCsMF6p/HDM is mediated via NF-κB-dependent MAPK signaling pathway. These results collectively suggest that CsMF6p/HDM mediates C. sinensis-induced inflammation cascades that eventually lead to hepatobiliary diseases.

中文翻译：

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华支睾吸虫MF6p / HDM（CsMF6p / HDM）通过NF-κB依赖性MAPK途径在RAW 264.7巨噬细胞中诱导促炎性免疫反应。

背景技术MF6p /宿主防御分子（HDM）是蠕虫寄生虫分泌的一系列小蛋白。尽管尚未完全了解MF6p / HDM在吸虫性寄生虫中的生理作用，但已提出了它们在维持血红素稳态和调节宿主免疫反应中的潜在生物学功能。方法克隆华支睾吸虫MF6p / HDM基因（CsMF6p / HDM）。重组CsMF6p / HDM（rCsMF6p / HDM）在大肠杆菌中表达。分析了rCsMF6 / HDM的生化和免疫学特性。通过细胞因子阵列测定，逆转录聚合酶链反应和酶联免疫吸附测定，分析了CsMF6p / HDM诱导的RAW 264.7细胞的促炎反应。通过三维建模和分子对接模拟分析了CsMF6p / HDM的结构特征。结果CsMF6p / HDM与其他吸虫的直向同源物具有高度的氨基酸序列相似性，并在该寄生虫的不同发育阶段表达。rCsMF6p / HDM与细菌衍生的脂多糖（LPS）结合，而没有有效中和RAW 264.7巨噬细胞中LPS诱导的炎症反应。相反，rCsMF6p / HDM可以在RAW 264.7细胞中诱导促炎性免疫应答，其特征在于TNF-α和IL-6的表达。rCsMF6p / HDM诱导的促炎性免疫反应受JNK和p38 MAPK调节，并通过抑制NF-κB有效下调。CsMF6p / HDM的结构分析和与LPS的对接模拟表明CsMF6p / HDM无法充分捕获LPS，这表明rCsMF6p / HDM无法有效中和LPS诱导的RAW 264.7细胞中的炎症反应。结论尽管rCsMF6p / HDM与LPS结合，但结合亲和力可能不足以维持rCsMF6p / HDM和LPS的稳定复合物。此外，rCsMF6p / HDM诱导的促炎反应的特征是在RAW 264.7巨噬细胞中释放IL-6和TNF-α。rCsMF6p / HDM诱导的促炎反应是通过依赖NF-κB的MAPK信号通路介导的。这些结果共同表明，CsMF6p / HDM介导了中华绒螯蟹诱导的炎症级联反应，最终导致肝胆疾病。提示rCsMF6p / HDM无法有效中和LPS诱导的RAW 264.7细胞炎症反应。结论尽管rCsMF6p / HDM与LPS结合，但结合亲和力可能不足以维持rCsMF6p / HDM和LPS的稳定复合物。此外，rCsMF6p / HDM诱导的促炎反应的特征是在RAW 264.7巨噬细胞中释放IL-6和TNF-α。rCsMF6p / HDM诱导的促炎反应是通过依赖NF-κB的MAPK信号传导途径介导的。这些结果共同表明，CsMF6p / HDM介导了中华绒螯蟹诱导的炎症级联反应，最终导致肝胆疾病。提示rCsMF6p / HDM无法有效中和LPS诱导的RAW 264.7细胞炎症反应。结论尽管rCsMF6p / HDM与LPS结合，但结合亲和力可能不足以维持rCsMF6p / HDM和LPS的稳定复合物。此外，rCsMF6p / HDM诱导的促炎反应的特征是在RAW 264.7巨噬细胞中释放IL-6和TNF-α。rCsMF6p / HDM诱导的促炎反应是通过依赖NF-κB的MAPK信号通路介导的。这些结果共同表明，CsMF6p / HDM介导了中华绒螯蟹诱导的炎症级联反应，最终导致肝胆疾病。结合亲和力可能不足以维持rCsMF6p / HDM和LPS的稳定复合物。此外，rCsMF6p / HDM诱导的促炎反应的特征是在RAW 264.7巨噬细胞中释放IL-6和TNF-α。rCsMF6p / HDM诱导的促炎反应是通过依赖NF-κB的MAPK信号传导途径介导的。这些结果共同表明，CsMF6p / HDM介导了中华绒螯蟹诱导的炎症级联反应，最终导致肝胆疾病。结合亲和力可能不足以维持rCsMF6p / HDM和LPS的稳定复合物。此外，rCsMF6p / HDM诱导的促炎反应的特征是在RAW 264.7巨噬细胞中释放IL-6和TNF-α。rCsMF6p / HDM诱导的促炎反应是通过依赖NF-κB的MAPK信号传导途径介导的。这些结果共同表明，CsMF6p / HDM介导了中华绒螯蟹诱导的炎症级联反应，最终导致肝胆疾病。