| 摘要: | 大部分的心臟外科開心手術，在手術過程中需要有體外循環 (cardiopulmonary bypass, CPB) 以配合手術。除了外科手術本身對心臟造成的傷害外，體外循環過程也被認為可引起嚴重的發炎反應，甚至導致後續多重器官的損傷，產生嚴重的術後併發症。體外循環過程引起發炎的原因很多，包含缺氧-再灌注損傷、內毒素刺激與補體系統活化等，都曾經有過研究報導。然而，部份關於發炎的研究中觀察到，病人循環中的發炎物質會先上升然後快速下降，而這樣的現象在類似心臟手術但未接受體外循環患者身上卻未發現。因而，我們推測體內可能存在對抗發炎的保護機制，可以拮抗因應體外循環產生的發炎反應。
發炎體是一個多元的蛋白質複合體，在發炎反應中扮演重要的中介角色。其中NLRP3近來被探討得相當多，主要是作為產生caspase-1與IL-1的重要平台。IL-6與IL-8都是可被巨噬細胞分泌的發炎重要介質，在先天免疫作用中可以因應刺激白血球增加並趨化之目標組織發揮作用。近來許多的研究發現關於微型核糖核酸 (microRNA) 與外排體 (exosome) 在細胞間溝通的機制，以及在心血管疾病與發炎反應所扮演的角色。部分研究指出microRNA在體外循環時會有隨時間點變化的趨勢。我們藉由生物資訊與文獻回顧的方式選定microRNA-223作為研究標的。本研究旨在探討，接受心臟手術與體外循環時，體內外排體與其攜帶的微型核糖核酸，在發炎反應中可能存在的調節與保護的角色。
本研究共納入21位病人，在以下五個預定的時間點採血：麻醉誘導後 (pre-surgery)、開始體外循環前 (pre-CPB)、CPB後2小時、CPB後4小時、CPB後24小時。循環中TNF-α的表現自CPB後開始增加。而循環中的IL-8與IL-6則是在CPB後4小時達到峰值，但在CPB後24小時，則呈現下降現象。循環中的外排體 (exosome)在CPB後2小時 (55.1 ± 8.3%)，4小時 (63.8 ± 10.1%) 與24小時 (83.5 ± 3.72%) 明顯的比術前要增加 (42.8 ± 0.11%)。這些外排體 (exosome) 主要是來自於紅血球與血小板。另外，循環中的exosomal miR-223的濃度在CPB術後明顯比術前增加。而以CPB後患者血漿取得的exosomal miR-223可以降低單核球中IL-6與NLRP3的表現。
體外循環確實產生數中與術後相當明顯的發炎現象。從我們的研究結果得知外排體與其攜帶的微型微糖核酸可以達到細胞-細胞間的溝通，也藉此作用在體外循環手術中扮演發炎保護的調節角色。我們的研究也是第一次指出體外循環手術患者血液中的外排體主要來自紅血球與血小板的釋放。而miR-223的增加也是首次被發現，藉由抑制NLRP3與IL-6的產生，達到抑制發炎反應的拮抗效果。這個研究結果或許可以為體外循環產生嚴重發炎反應甚至併發症，找到可能的生物標記與治療方法。
BACKGROUND
Cardiopulmonary bypass had been widely used in cardiac surgery. In addition to surgical injury, CPB also leads to inflammatory responses, which could lead to systemic inflammatory response and subsequent clinical morbidities and even mortalities. There were several possible mechanisms reported for CPB related inflammation, included ischemia-reperfusion injury, endotoxin and complement activation. Some clinical studies noticed the inflammatory mediators peaked early in the CPB process and declined thereafter, which wasn’t observed in patients received cardiac surgery without CPB. We hypothesized certain protective endogenous homeostasis existed and should be important for preventing excessive hazardous systemic inflammation during CPB.
Inflammasome is a multi-meric protein complex and plays pivotal roles in inflammatory response. Among them, NLRP3 had been extensively studied for its important facilitation in formation of caspase and IL-1. IL-6 and IL-8 are secreted by macrophages and are key mediators during inflammation in terms of their enhancement of development and chemotaxis of leukocytes into the target sites. Recently, cell-cell communication with exosomes and microRNA had been reported. It had been shown that some microRNAs play pivotal roles in inflammatory response and cardiovascular disease. Some studies indicated the temporal change of circulating microRNA during CPB surgery. By using bioinformatics and literature review, we choose microRNA-223 as our study target. We assessed whether plasma exosomal microRNAs in patients undergoing cardiac surgery with CPB are involved in the regulation of inflammatory responses.
METHODS & RESULTS
We enrolled patients underwent cardiac surgery with CPB. Patients with autoimmunity disease, asthma, COPD, cancer and use of NSAIDs or steroids were excluded. Plasma samples were isolated from CPB patients (n = 21) at 5 specified time points: pre-surgery, pre-CPB and 2 hours (h), 4 h and 24 h after CPB began. Plasma TNF-α expression was increased after CPB began compared to that in the pre-surgery samples. Plasma IL-8 and IL-6 expression peaked at 4 h after CPB began but was downregulated at 24 h. The number of plasma exosomes collected at 2 h (55.1 ± 8.3%), 4 h (63.8 ± 10.1%) and 24 h (83.5 ± 3.72%) after CPB began was significantly increased compared to that in the pre-CPB samples (42.8 ± 0.11%). These exosomes had a predominantly parental cellular origin from RBCs and platelets. Additionally, the plasma exosomal miR-223 levels were significantly increased after CPB began compared to those in the pre-CPB samples. Further, exosomal miR-223 from plasma collected after CPB began downregulated IL-6 and NLRP3 expression in the monocytes.
CONCLUSIONS
Cardiopulmonary bypass (CPB) evokes a broad range of inflammatory responses during the intra- and post-operative periods. Here, we show the important role of exosomes and exosomal miRNA in cell-cell communication and their regulatory role in inflammatory responses during cardiac surgery with CPB. RBCs and platelets released predominant exosomes into circulation, and exosomal miR-223 that was significantly overexpressed after CPB began downregulated NLRP3 and IL-6 expression in monocytes. Our novel findings might provide a clue for using exosomal microRNA as a biomarker and even therapeutic target in the systemic inflammatory response in patients underwent CPB. |
