陰電性低密度脂蛋白引起巨噬細胞發炎反應之探討

Study on electronegative LDL-induced inflammation in human macrophages

白若湘(Juo-Hsiang Pai)

指導教授 ： 呂紹俊

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201802245

摘要

心血管疾病是全球十大致死原因之一，而動脈粥狀硬化為造成心血管疾病的主因。研究認為發炎反應在動脈粥狀硬化發展中扮演重要的角色。
LDL(-)為帶負電的低密度脂蛋白，被發現存在於動脈粥狀硬化、高膽固醇血症、第二型糖尿病及心肌梗塞等患者的血液中。先前實驗室的研究顯示從ST-時段上升型心肌梗塞(STEMI)病患所分離的LDL(-)會造成巨噬細胞產生大量的促發炎激素IL-1β。而我的研究則是探討從飲食引起的高膽固醇血症兔子所分離出的LDL(-)是否也有引起發炎反應的作用。結果顯示，STEMI病人及兔子的LDL(-)皆會誘導巨噬細胞產生大量的促發炎激素IL-1β、TNF-α、IL-6。除此之外，LDL(-)誘導NF-κB、ERK1/2、p38 及 JNK的活化。我也比較了LDL(-)與銅離子氧化的oxLDL誘導的發炎反應，結果顯示LDL(-)誘導IL-1β、TNF-α、IL-6產生及NF-κB、ERK1/2、p38 及 JNK的活化皆明顯的高於Cu-oxLDL所誘導的。
　　先前學長的實驗指出，LDL(-)在非酒精脂肪肝炎的發展中也扮演重要的角色。而B28號藥為實驗室的學長姐所篩選出具有預防高脂高膽固醇飼料誘發的非酒精性脂肪肝炎作用的中藥材。本實驗探討，B28號藥是否能夠抑制LDL(-)在巨噬細胞誘導的發炎反應。B28號藥為B65號藥的加工製品；首先，我比較B65與B28號藥對於LDL(-)誘導發炎的影響，結果顯示兩者皆會抑制LDL(-)誘導之發炎反應，在B28號藥的效果較為顯著。另外，B28號藥能夠抑制巨噬細胞極化成M1型。接著，我們探討B28號藥是透過何種機制抑制LDL(-)誘導巨噬細胞之作用，結果顯示，B28號藥可以有效的抑制NF-κB、ERK的活化，降低促發炎激素的產生。之前的文獻顯示，LDL(-)透過LOX-1誘導下游的訊息傳遞，並引起發炎反應。而在B28號藥處理的巨噬細胞，LOX-1的表現明顯的被抑制。因此推測B28號藥，可能是透過抑制LOX-1的表現而減少了NF-κB及ERK的活化，使得促發炎激素產生降低。最後，為分離出B28號藥之有效抗發炎的成分，我們以乙酸乙酯萃取B28號藥，並比較B28號藥粗萃物、B28號藥水層萃取物及B28號藥乙酸乙酯層萃取物對LDL(-)引起發炎反應的影響。結果顯示，B28號藥粗萃物及B28號藥乙酸乙酯層萃取物有效抑制LDL(-)誘導的發炎反應，而B28號藥水層萃取物則沒有看到此作用，因此推測B28號藥的有效抗發炎成份在乙酸乙酯萃取物中。

關鍵字

動脈粥狀硬化 ； 高膽固醇血症 ； 陰電性低密度脂蛋白 ； 氧化低密度脂蛋白 ； 泡沫細胞 ； 發炎反應 ； 抗發炎

並列摘要

Cardiovascular disease (CVD) is the leading cause of mortality worldwide, and atherosclerosis is the major cause of CVD. Recent research has shown that inflammation plays a crucial role in the pathogenesis of atherosclerosis.
Higher levels electronegative LDL (LDL(-)) was found in plasma of patients with acute myocardial infarction, hypercholesterolemia and type II diabetes. We have recently reported that LDL(-) isolated from ST-segment elevation myocardial infarction (STEMI) induced production of IL-1β in human macrophages. Therefore, LDL(-) has inflammatory properties. In this study, we investigated whether LDL(-) isolated from hypercholesterolemic rabbits also induces inflammatory response, similar to that from STEMI patients. Our results showed that LDL(-) isolated from STEMI or hypercholesterolemic rabbits induced production of IL-1β, TNF-α and IL-6 in human macrophages. In addition, LDL(-) induced activation of NF-κB, ERK1/2, p38 and JNK. We also compared the inflammatory effects of LDL(-) with the copper-mediated oxidation of LDL (Cu-oxLDL). The results showed that LDL(-) induced higher levels of IL-1β, TNF-α and IL-6 and activation of NF-κB, ERK1/2, p38 and JNK than that induced by Cu-oxLDL.
Our previous studies showed that LDL(-) also played an important role in the development of nonalcoholic steatohepatitis (NASH), and identified a chinese medicinal herb, herb-B28, which ameliorated high fat/high cholesterol (HFC) diet-induced NASH in hamster. In the second part of this study, we investigated the effects of herb-B28 on LDL(-) induced inflammation in human macrophages. Herb-B28 is the processed product of herb-B65. First, we compared the anti-inflammatory effects of herb-B28 and herb-B65 on LDL(-) induced inflammation. The results show that both herb-B28 and herb-B65 inhibited LDL(-) induced production of inflammatory cytokines in human macrophages; In addition, the anti-inflammatory effect of herb-B28 is more potent than herb-B65. Moreover, herb-B28 inhibited LDL(-) induced polarization of macrophage into M1 phenotype. We also showed that herb-B28 suppressed the production of inflammatory cytokines through reducing LOX-1 expression and inhibiting the activation of NF-κB and ERK1/2. In order to search for the effective anti-inflammatory component of herb-B28, herb-B28 was extracted with ethyl acetate. The effects of ethyl acetate extracted B28 (EA28) and water soluble fraction of B28 (W28) on LDL(-) induced production of inflammatory cytokines were investigated. The results showed that the anti-inflammatory effects of EA28 was similar to that of herb-B28, but W28 had no effect. Therefore, the effective anti-inflammatory compounds of herb-B28 is mainly in the ethyl acetate fraction.

並列關鍵字

Atherosclerosis ； Hypercholesterolemia ； LDL(-) ； oxLDL ； Foam cell ； Inflammation ； Anti-inflammation

參考文獻

Ait-Oufella, H., Taleb, S., Mallat, Z., and Tedgui, A. (2011). Recent advances on the role of cytokines in atherosclerosis. Arterioscler Thromb Vasc Biol 31, 969-979.

Anstee, Q.M., Targher, G., and Day, C.P. (2013). Progression of NAFLD to diabetes mellitus, cardiovascular disease or cirrhosis. Nat Rev Gastroenterol Hepatol 10, 330-344.

Antonopoulos, A.S., Margaritis, M., Lee, R., Channon, K., and Antoniades, C. (2012). Statins as anti-inflammatory agents in atherogenesis: molecular mechanisms and lessons from the recent clinical trials. Curr Pharm Des 18, 1519-1530.

Bancells, C., Canals, F., Benitez, S., Colome, N., Julve, J., Ordonez-Llanos, J., and Sanchez-Quesada, J.L. (2010a). Proteomic analysis of electronegative low-density lipoprotein. J Lipid Res 51, 3508-3515.

Bancells, C., Sanchez-Quesada, J.L., Birkelund, R., Ordonez-Llanos, J., and Benitez, S. (2010b). HDL and electronegative LDL exchange anti- and pro-inflammatory properties. J Lipid Res 51, 2947-2956.