巨噬細胞中鉀離子通道與脂筏蛋白在脂多醣刺激下棕櫚酸誘導IL-1β 生成路徑的角色

Abstract

在肥胖患者血液中，棕櫚酸（Palmitic acid, PA）會活化發炎反應，並使血液中IL-1β 增加，最終導致心血管疾病（Cardiovascular disease, CVD）。先前的研究表明，巨噬細胞中脂多醣（Lipopolysaccharide, LPS）和棕櫚酸對 IL-1β 的產生具有協同作用，其涉及電壓門控鉀離子通道（Voltage-gated potassium channel，Kv channel）和脂筏（Lipid raft）的參與。然而，目前仍不清楚是哪種 Kv 離子通道或是脂筏上蛋白參與此過程。在本研究中，第一部分的實驗顯示， LPS 預處理並用 PA-BSA 活化會增加 Kv1.3 、 Kv3.3 和 Kv11.1 的表現，並排除了 Kv1.3 的影響。全細胞膜片鉗實驗表明，在LPS和PA-BSA處理後，部分細胞向外電流增加，顯示出有鉀離子外流的現象，但目前無法排除細胞膜破裂的可能性。第二部分的研究中，我們使用蔗糖濃度梯度分離出脂筏上蛋白，並進行蛋白質體學和生物路徑分析（Ingenuity Pathway Analysis, IPA）。結果發現在 LPS 預處理的巨噬細胞中， PA-BSA 會活化脂筏中的發炎反應路徑。儘管未觀察到脂筏中 TLR4 的增加，但有發現脂筏中發炎相關膜蛋白 integrin β2 和 TLR2 的增加。然而，通過RNA干擾（RNAi）確認這些蛋白的重要性後，發現PA-BSA 可能並非透過脂筏中 integrin β2 誘導 IL-1β 的產生。另外，由於我們尚未成功沉默 TLR2 基因，因此目前無法確定是否透過脂筏中 TLR2 誘導 IL-1β 產生，未來研究將繼續探討這個問題。總體而言，我們期望這些研究成果能有助於人類了解飽和脂肪酸（Saturated Fatty Acid，SFA）如何引起發炎反應，並為心血管疾病的治療靶點提供更多的資訊。

Palmitic acid (PA) activates inflammatory responses, leading to an increase in serum IL-1β levels, ultimately contributing to cardiovascular disease in obese individuals. Previous studies have indicated a synergistic effect of lipopolysaccharide (LPS) and palmitic acid on IL-1β production within macrophages. More importantly, voltage-gated potassium channels (Kv channels) and lipid rafts participate in this pathway. However, it remains unclear which specific Kv channels or lipid raft proteins are involved in this process. In this study, the first part of the experiments demonstrated that PA-BSA stimulation increases the expression of Kv1.3, Kv3.3, and Kv11.1 in LPS-primed macrophages. Subsequently, we excluded the influence of Kv1.3. Whole-cell patch clamp experiments indicated an increase in outward current in some cells following LPS and PA-BSA treatment, suggesting potassium efflux after activation, although the possibility of cell membrane rupture cannot be ruled out at present. In the second part of the experiments, we isolated lipid raft proteins using a sucrose density gradient, followed by proteomics and Ingenuity Pathway Analysis (IPA). Results revealed that PA-BSA activates inflammatory pathways within lipid rafts in LPS-primed macrophages. Although an increase in TLR4 within lipid rafts was not observed, an elevation of inflammatory-associated membrane proteins integrin β2 and TLR2 was detected. However, upon confirming the role of these proteins through RNA interference (RNAi), it was found that PA-BSA might not induce IL-1β production via integrin β2. Additionally, due to the unsuccessful gene silencing of TLR2, it remains inconclusive whether IL-1β production is induced through TLR2 within lipid rafts. Further experiments are needed to explore the roles of TLR2 in this pathway. Overall, we anticipate that these findings will contribute to a better understanding of how saturated fatty acids trigger inflammatory responses and provide additional insights into potential therapeutic targets for cardiovascular disease.