棕櫚酸促進陰電性低密度脂蛋白在巨噬細胞中引起 IL-1β 產生透過增加鉀離子流出

Abstract

近年來，由於飲食習慣改變，肥胖率越來越高，肥胖者為代謝症候群的高危險族群，容易罹患第二型糖尿病、心血管疾病，如動脈粥狀硬化與心肌梗塞等。這些病人血液中，陰電性低密度脂蛋白(LDL(-))、游離脂肪酸及介白激素1β (IL-1β) 都明顯提高，巨噬細胞分泌的 IL-1β 在這些疾病扮演重要的角色，因此在這個研究我們探討LDL(-) 與游離脂肪酸在巨噬細胞引起之IL-1β 產生。 IL-1β 的產生需要兩條訊號：第一，啟動訊號為 NF-κB 活化，接著 NF-κB 作為轉錄因子促進 pro-IL-1β 和 NLRP3的表現；第二，激活訊號為 NLRP3 發炎小體活化，導致 Caspase-1 被切割且活化後，將 pro-IL-1β 切割轉變為成熟型 IL-1β。先前實驗室研究指出，LDL(-) 在巨噬細胞能誘導 NF-κB及Caspase-1 活化，但Caspase-1活化程度並不高。另外，游離脂肪酸被認為可以促進 NLRP3 發炎小體活化。因此，我們想探討 LDL(-)、游離脂肪酸和 IL-1β 在發炎反應之間的交互作用，且是否游離脂肪酸能提高 LDL(-) 在巨噬細胞誘導之 IL-1β 產生。 我們從餵食高膽固醇脂飲食之兔子的血漿分離到 LDL(-)，將其與飽和或不飽和游離脂肪酸一起處理人類單核球細胞 (THP-1) 分化之巨噬細胞，分析分泌到細胞培養液中的 IL-1β 含量。結果顯示，棕櫚酸單獨不能造成 IL-1β 產生，且其沒有誘導 NF-κB 活化之能力；但能促進 LDL(-) 誘導之 IL-1β 產生。而LDL(-) 會誘導清道夫受體 LOX-1 表現增加，LOX-1 也被指出和發炎反應有密切關係，然而棕櫚酸並不會進一步透過促進 LOX-1 表現，而增加 IL-1β 產生。將細胞培養在無鉀離子之培養基下可以使LDL(-) 誘導之 IL-1β達到 LDL(-) 與棕櫚酸一起處理的程度。相對的，幾種非專一性的鉀離子通道抑制劑會抑制 LDL(-) 與棕櫚酸一起所誘導之 IL-1β。這些結果顯示，棕櫚酸可能是透過促進鉀離子流出細胞之作用，造成 NLRP3發炎小體與caspase-1進一步活化，而使IL-1β 產生增加。 總結，在我們的研究發現LDL(-) 和棕櫚酸對於 IL-1β 產生有協同效應，並且發現棕櫚酸會提高LDL(-) 誘導 IL-1β 產生之機制可能是增加鉀離子排出。因此我們認為病人血液中若是 LDL(-) 和飽和游離脂肪酸升高，可能會引起IL-1β大量產生，是引起發炎的關鍵因素。但我們並不清楚棕櫚酸是影響那個鉀離子通道，其機制有待進一步探討。

Recently, prevalence of obesity is increasing worldwide because of changing of diet preference. And obesity is a major risk factor for metabolic diseases, which usually progresses to type 2 diabetes mellitus and cardiovascular diseases; such as atherosclerosis, myocardial infarction. Inflammation is associated with these diseases, and macrophages are the predominant contributor of inflammation. Moreover, electronegative LDL (LDL(-)), free fatty acids and interleukin-1β (IL-1β) are increased significantly in the blood of these patients. Production of IL-1β requires two signals. Firstly, the priming signal is to activate NF-κB signaling. NF-κB as a transcription factor that promotes pro-IL-1β and NLRP3 expression. Secondly, the activating signal is to activate NLRP3 inflammasome and caspase-1. Then activated caspase-1 proteolytically cleaves pro-IL-1β into mature IL-1β. Our previous study showed that LDL(-) could induce NF-κB activation in macrophage, and weakly promote caspase-1 activation. Evidence has shown that palmitic acid could promote NLRP3 inflammasome/caspase-1 activation. Thus, in this study we investigated whether there is an interaction between LDL(-) and palmitic acid in inducing IL-1β production in macrophages. LDL(-) was isolated from the plasma of the rabbits fed with high-fat/cholesterol diet. THP-1 macrophages were treated with LDL(-) and BSA bound saturated or unsaturated free fatty acids, then the levels of IL-1β in culture medium were analyzed by ELISA. The results show that palmitate, a saturated fatty acid, alone is unable to induce NF-κB activation and IL-1β production in macrophages. However, palmitate enhances LDL(-)-induced IL-1β production. Palmitate did not further activate NF-κB or increase the levels of pro-IL-1β mRNA and protein when treated with LDL(-). Our data also show that palmitate did not further increase LDL(-)-induced increase of LOX-1, a scavenger receptor for LDL(-). These results suggest that palmitate enhances LDL(-)-induced IL- 1β production was not through activation of signal one (NF-κB signaling) or increase levels of LOX-1. Cells treated with LDL(-) under a potassium free medium, and the level of IL-1β in the medium is close to that of LDL(-) and palmitate co-treated cells. Treated potassium channel blockers significantly lower LDL(-) and palmitate-induced levels of IL-1β. The results suggest that palmitate enhances LDL(-)-induced IL-1βproduction is likely through increase potassium efflux. In conclusion, our study show that palmitate enhances LDL(-)-induced IL-1β production, the results is physiological relevant since the concentrations of LDL(-) and palmitate used in this study are under physiological ranges in plasma of patients with AMI and diabetes. However, we do not know which potassium channel is activated by palmitate. The mechanism underlie deserved further investigation.