馬來亞蝮蛇蛇毒蛋白F3-5抑制LPS引起巨噬細胞釋放發炎性細胞激素機轉之探討

Abstract

巨噬細胞在內生性免疫反應以及發炎反應上扮演相當重要的角色。透過其細胞表面上的受體，能夠辨認外來的因子，引發免疫反應，同時巨噬細胞的附著功能在免疫系統上也是很重要的關鍵，使其能夠快速的聚集、移動和穿透，而發揮他們的功能。在馬來亞蝮蛇(Calloselasma rhodostoma)蛇毒蛋白中已純化出許多功能的蛋白，其中包括了抑制細胞附著的分子，並且也有抑制發炎反應之研究報告。本實驗中將CRV原毒進行純化，以DEAE-Sephadex A-50管柱分離出的第一分劃，再經CM-Sephadex C-50管柱收取第三分劃部分，最後以FPLC Superdex G75膠質過濾管柱純化而得到最終具有抑制巨噬細胞發炎性細胞激素釋放的CRV F3-5蛇毒蛋白，其分子量為22,186 Da，它不具有蛋白質分解酶之活性，亦不具有PLA2之酵素活性；而在抑制巨噬細胞受到LPS活化後釋放發炎性細胞激素之測試中，F3-5蛇毒蛋白10 μg/ml (約0.45μM)能夠抑制LPS 200 ng/ml在原生性小鼠巨噬細胞引發之IL-6釋放，同時也能夠有效的抑制低濃度LPS (100 ng/ml)在原生性巨噬細胞以及細胞株RAW巨噬細胞引發之TNF-α釋放反應。在貼附性試驗當中，CRV F3-5蛇毒蛋白(10μg/ml)具有降低巨噬細胞受到LPS刺激活化之後所提高的附著性作用。對於LPS刺激活化巨噬細胞之後所產生的下游訊息傳遞鏈之中，我們檢視了MAPK路徑中的三種重要激酶：p-38、ERK以及JNK的被活化(磷酸化)現象，結果發現到此三種激酶的活化在給予F3-5蛇毒蛋白之後都有被抑制的現象發生。最後再經由流式細胞儀分析原生性巨噬細胞表面上TLR4的表現，發現F3-5蛇毒蛋白(10μg/ml)存在時，可抑制TLR4 mAb對原生性小鼠巨噬細胞上TLR4的結合反應。綜上所述，由馬來亞蝮蛇蛇毒中所純化出的CRV F3-5蛇毒蛋白，具有對抗LPS所引起的巨噬細胞釋放發炎性細胞激素作用，而此抑制性作用，至少有部分，是透過對巨噬細胞表面上TLR4受體的影響，進而減少下游包括p-38、ERK以及JNK的磷酸化而來，詳細的作用機轉則有待更多的實驗證明。

Macrophages play a pivotal role in inflammation and innate immune responses. Foreign substances recognized by macrophages trigger a serial of signaling cascades upon ligand–receptor stimulation through receptors expressed on cell surface resulting in release of several pro-inflammatory and anti-inflammatory cytokines, and the activation of other immune cells. In the meanwhile, macrophages adhesion is a key event within the immune system. Relying on the receptors or integrins function, macrophages perform recruitment, migration and infiltration to the infected site from the bloodstream. Among the Malaysia viper (Calloselasma rhodostoma) venom, many types of functional snake venom proteins have been discovered, including the anti-adhesive molecules reported in anti-inflammatory literatures. The fraction 3-5 (F3-5) protein isolated from Calloselasma rhodostoma crude venom (CRV), has a molecular weight 22,186 Da, devoid of fibrinogenolytic, azocaseinolytic and PLA2 activities. F3-5 10μg/ml (0.45μM) inhibited the LPS (200ng/ml)-induced IL-6 release of primary mice macrophages, and also the release of TNF-α of primary macrophages and RAW macrophages stimulated by lower concentration (100ng/ml) LPS. In the cell adhesion assay, F3-5 (10μg/ml ) significantly reduced the augmented adhesive activity of macrophages caused by LPS stimulation. We also observed that F3-5 significantly inhibited the phosphorylation of p-38, ERK and JNK of macrophages stimulated by LPS, assayed by Western blot. Furthermore, F3-5 (10μg/ml) as well as LPS (1μg/ml), significantly inhibited the binding of TLR4 mAb to macrophages as evaluated by flow cytometric analysis. Taken together, the purified active component CRV F3-5 exhibits inhibitory effect on LPS-stimulated IL-6 and TNF-α release of primary macrophages and RAW cells at least partly through TLR4 blockade and the inhibition on the subsequent phosphorylation of p-38, ERK and JNK. However, the detailed mechanism of its action needs further investigations.