血栓素及血栓素受體訊息在短期與長期被動吸菸造成動脈血栓性疾病及肺部損傷之角色

Abstract

被動菸害，包含二手菸害及三手菸害，其中二手菸害又分成主流菸煙及側流菸煙， 被動菸害與主動菸害皆會造成心血管疾病及肺部疾病，本研究主要利用側流菸煙及三手菸探討心血管及肺部發炎性相關疾病。 香菸中的活性氧化物質會導致血小板過度活化，促進血栓形成，而花生四烯酸經血栓素合成酶 (Thromboxane A2 synthase, TXAS) 合成血栓素A2 (Thromboxane A2, TXA2) ，與血栓素受體 (Thromboxane prostanoid receptor, TP receptor) 結合會引起血小板活化，而血小板過度活化或內皮細胞受損，又會導致TXA2濃度增加，然而過去對於香菸及TXAS-TXA2-TP 訊息傳遞路徑的調控仍未釐清。 本研究利用TXAS、TP基因剔除小鼠及兩者基因皆剔除之小鼠，吸入側流菸煙及三手菸煙與動脈血栓性疾病的模式，探討被動菸害對動脈血栓形成及肺部發炎性變化。實驗分成TXAS+/+TP+/+、TXAS-/-TP+/+、TXAS+/+TP-/- 及TXAS-/-TP-/-小鼠並給予無菸煙暴露，菸煙暴露1週，菸煙暴露5週及菸煙暴露8週處理，共16組 （各組n=6），並利用氯化鐵誘發急性動脈血管栓塞以檢測血栓形成所需時間，再藉由病生理及分生結果了解香菸及TXAS-TXA2-TP引起肺部發炎性之關聯。我們利用菸煙暴露刺激以評估肺損傷與氯化鐵誘發之急性動脈血管栓塞在四種小鼠之作用。我們以化學發光放大方式偵測活性氧數量，以西方墨點和免疫染色法探究氧化壓力、細胞凋亡、細胞自噬、發炎性細胞凋亡和發炎之分生機轉。 研究結果指出利用氯化鐵誘發之急性動脈血管栓塞，其血栓形成時間之結果在不同基因老鼠與菸煙處理之間，並無顯著關聯性，表示各基因對於菸煙暴露時間的反應具有一致性，但菸害仍會影響血栓形成，因而利用血小板吸附模式探討不同基因鼠的結果，在無菸害暴露的老鼠裡發現TXAS+/+TP+/+鼠之血小板吸附性顯著顯著高於其他三個品系品的老鼠，未來會再加入抽煙處理的組別以繼續探討;在肺部發炎性疾病方面，由病生理結果發現，不同基因鼠與菸煙處理之間具顯著關聯，表示菸煙會藉由TXAS-TXA2-TP訊息傳遞路徑引起肺部發炎性疾病。Passive smoking contains second-hand smoke (SHS) and third-hand smoke (THS). SHS can be divided into mainstream smoke and sidestream smoke. Both active and passive smoking would cause cardiovascular disease (CVD) and pulmonary injuries. We used sidestream smoke and THS as our cigarette smoke (CS) model. Oxidants in cigarettes activate platelets consequently induce thrombus formation. It is well known that thromboxane A2 (TXA2) through its receptor—thromboxane prostanoid (TP) receptor — would activate platelets, and the overactivation of platelets or endothelial cells injury would further induce TXA2 generation. The relationship between CS and thromboxane A2 synthase (TXAS)-TXA2-TP signaling is still ambiguous. In order to investigate the influence of CS on TXAS-TXA2-TP signaling in thrombosis and pulmonary injuries, we used TXAS and TP gene knockout mice, inhaled sidestream and THS as whole body CS exposure model. Mice were divided into TXAS+/+TP+/+, TXAS-/-TP+/+, TXAS+/+TP-/- and TXAS-/-TP-/- treated with non-CS exposure (i.e. Control), acute cigarette smoke exposure group (ACE), prolonged cigarette smoke exposure 5 weeks group (PCE 5) and prolonged cigarette smoke exposure 8 weeks group (PCE 8) (16 groups, n=6 in each group). We evaluated the effects of CS exposure on lung injuries and FeCl3-induced thrombus formation in these four groups. We utilized the chemiluminescence amplification method to measure the amount of reactive oxygen species (ROS), western blot and immunohistochemistry to explore the underlying mechanisms including oxidative stress, apoptosis, autophagy, pyroptosis and inflammation in these animals. Our results showed CS exposure triggered the formation of thrombus by the index of time to occlusion (TTO). The response of CS shortened TTO was attenuated by blocking TXAS or TP receptors. CS exposure induced oxidative stress would cause pulmonary injuries including endothelial dysfunction, endoplasmic reticulum (ER) stress, inflammation, cell apoptosis, autophagy and pyroptosis. In conclusion, TXAS-TXA2-TP signaling plays an important role in CS induced pulmonary injury and FeCl3-induced thrombus formation.