利用CMap及分化型HL-60細胞篩選與分析 抑制嗜中性球胞外網絡形成之天然化合物

Abstract

嗜中性球胞外網絡（Neutrophil extracellular traps, NETs）是由活化的嗜中性球釋放的網狀結構，主要由DNA與顆粒蛋白質組成，作為一種防禦機制。然而，過度的NETosis與多種疾病的發病機轉有關，包括血栓形成、自體免疫疾病、慢性發炎及癌症。在這些情況下，NETs可能促進血管阻塞、組織損傷及持續性發炎反應。因此，尋找能調節NETosis的化合物，已成為治療發展中日益受到關注的方向。 本研究利用Connectivity Map（CMap）資料庫進行反轉NETosis相關基因表現特徵的天然化合物篩選。根據此電腦模擬分析，選出十字花科蔬菜中所含的3,3'-雙吲哚基甲烷（3,3'-diindolylmethane, DIM）與ω-3脂肪酸之一的α-次亞麻油酸（alpha-linolenic acid, ALA）進行體外測試。 本研究使用分化後的HL-60細胞（dHL-60）作為實驗模型，並以PMA（phorbol 12-myristate 13-acetate）誘導NETosis。螢光影像定量分析顯示，PMA可有效誘導NET形成；而經DIM與ALA預處理後，與NETosis相關的參數（如細胞核面積與螢光比例）呈現下降趨勢，尤其在較低濃度時更為明顯。然而，在較高濃度下，PI陽性細胞比例增加，顯示可能存在細胞毒性作用。在所有測試條件中，以10 μM DIM的抑制效果最為穩定，且未觀察到明顯的細胞膜損傷現象。 本研究結果支持DIM與ALA作為潛在NETosis調節劑的可能性，並強調營養保健品在針對NET相關疾病中的應用潛力。此外，本研究突顯了整合電腦預測與影像實驗技術以尋找新型NETosis抑制劑的價值，未來可望應用於血栓、慢性發炎、自體免疫疾病及癌症等領域。

Neutrophil extracellular traps (NETs) are web-like structures composed of DNA and granular proteins released by activated neutrophils as a defense mechanism. However, excessive NETosis has been implicated in the pathogenesis of various diseases, including thrombosis, autoimmune disorders, chronic inflammation, and cancer. In these conditions, NETs can promote vascular occlusion, tissue damage, and sustained inflammation. Identifying compounds that modulate NETosis is of growing interest for therapeutic development. In this study, we employed the Connectivity Map (CMap) to screen for natural compounds capable of reversing NETosis-associated gene expression signatures. Based on this insilico analysis, 3,3'-diindolylmethane (DIM), a compound derived from cruciferous vegetables, and alpha-linolenic acid (ALA), an omega-3 fatty acid, were selected for in vitro testing. Using a differentiated HL-60 (dHL-60) cell model, NETosis was induced with phorbol 12-myristate 13-acetate (PMA). Quantitative fluorescence imaging indicated NET formation in response to PMA. Pre-treatment with DIM and ALA led to reductions in NETosis-related parameters, including nuclear area and fluorescence ratios, particularly at lower concentrations. However, higher doses were associated with increased PI-positive cells, indicating potential cytotoxic effects. Among all conditions tested, 10 μM DIM showed the most consistent inhibitory profile without signs of membrane damage. These findings support the potential of DIM and ALA as promising candidates for NETosis modulation and highlight the relevance of nutraceuticals in targeting NET-associated diseases. The study also emphasizes the value of integrating computational predictions with image-based experimental approaches to identify novel NETosis inhibitors, with possible applications in thrombosis, chronic inflammation, autoimmune diseases, and cancer.