Lumbrokinase藉由抑制TLR4訊息路徑降低心肌缺血再灌注損傷

Lumbrokinase attenuates myocardial ischemia/reperfusion injury by inhibiting TLR4 signaling

王筱婷(Hsiao-Ting Wang)

指導教授 ： 蔡菁華 ； 黃相碩

中山醫學大學/醫學院/醫學研究所/碩士(2014年)

https://doi.org/10.6834/CSMU.2014.00203

摘要

Lumbrokinase是一種血栓溶解酵素，萃取自紅蚯蚓(Lumbricus rubellus)，臨床上已被應用於預防及治療中風和心血管疾病，有文獻指出lumbrokinase具有抗發炎、抗氧化、抗血栓及抗細胞凋亡等藥理活性，這些生物活性可能可以幫助心臟對抗缺血再灌注的傷害，但至目前為止尚無研究發表lumbrokinase是否具有心臟保護作用對抗心肌缺血再灌注損傷，因此本研究擬探討lumbrokinase對於大鼠心肌缺血再灌注損傷中是否具有心臟保護作用及其藥理作用機轉。
研究將雄性Sprague-Dawley大鼠以urethane (1.25 g/㎏)麻醉，進行六十分鐘左前降枝冠狀動脈(the left anterior descending, LAD)結紮及三小時再灌注，造成心肌缺血再灌注損傷(myocardial ischemia/reperfusion injury, myocardial I/R injury)。研究結果發現，在左冠狀動脈缺血前十五分鐘經由靜脈注射投予lumbrokinase 10 μg/kg，相較於投予溶劑的對照組會明顯地減少心肌梗塞區域(infract size)大小以及血液中乳酸去氫酶(lactate dehydrogenase, LDH)之活性，達統計差異，此外，亦有意義的降低心律不整(arrhythmia)的發生率及死亡率。證明lumbrokinase能夠保護心臟對抗心肌缺血再灌注損傷。進一步研究發現，心肌缺血再灌注損傷會有意義的增加toll-like receptor (TLR)-4、phosphorylation-c-Jun N-terminal kinase (p-JNK)、phosphorylation-nuclear factor kappa-light-chain-enhancer of B cell (p-NF-κB)、inducible nitric oxide synthase (iNOS)及cyclooxygenase (COX)-2之蛋白質表現量，以及有意義地增加metalloproteinase (MMP)-9的活性；然而，投予lumbrokinase會有意義的減少因心肌缺血再灌注損傷所誘發TLR4、p-JNK、p-NF-κB、iNOS及COX-2之蛋白質表現量以及MMP-9之活性。此外，利用病理組織染色發現，遭受缺血再灌注損傷的心臟組織中TLR4之蛋白質表現量增加並引起發炎細胞浸潤，而投予lumbrokinase會減少心肌缺血再灌注損傷增加的TLR4蛋白質表現量及發炎細胞浸潤之現象。因此，本研究證明lumbrokinase可能是具有潛力之心臟保護劑，可以保護心臟對抗心肌缺血再灌注損傷，其心臟保護作用可能是藉由抑制TLR4/NF-κB/MMP-9及JNK MAPK訊息傳遞路徑達到保護心臟對抗心肌缺血再灌注損傷。

關鍵字

心肌缺血再灌注損傷 ； lumbrokinase ； TLR4 ； MMP-9

並列摘要

Lumbrokinase, an antithrombotic agent, extracted from Lumbricus rubellus, has been used to prevent and treat stroke and cardiovascular diseases in clinical. It has been reported that lumbrokinase has some pharmacological properties, including anti-inflammatory, antioxidant, anti-thrombotic and anti-apoptotic. These biological activities might protect heart against myocardial ischemia/reperfusion (I/R) injury. However there is no research reported about whether lumbrokinaset is capable of cardioprotective effect against myocardial I/R injury. This study aim to investigate whether lumbrokinase is capable of cardioprotective effect against myocardial I/R injury in rats and its potential pharmacological mechanisms.
Myocardial I/R injury were established by 60 minutes occlusion of the left anterior descending (LAD) coronary artery and followed by three hours reperfusion in anesthetized male Sprague-Dawley rats. Lumbokinase was administered intravenously 15 minutes before LAD occlusion. We found that lumbrokinase (10 μg/kg) significantly decreased myocardial infarct size, lactate dehydrogenase (LDH) activity, and also significantly reduced the incidence of arrhythmia and mortality than the vehicle group. These results demonstrated that lumbrokinase protected the heart against myocardial I/R injury. To further, we found that toll-like receptor (TLR)-4, phosphorylation- c-Jun N-terminal kinase (p-JNK), phosphorylation-nuclear factor kappa-light-chain-enhancer of B cell (p-NF-κB), inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 expression and metalloproteinase (MMP)-9 activity were significantly increased during myocardial I/R injury. However, lumbrokinase not only significantly attenuated the increased of I/R-induced the expression of TLR4, p-JNK, p-NF-κB, iNOS and COX-2, but also significantly decreased the increased of I/R-induced the activity of MMP-9. Moreover, the results demonstrated that myocardial I/R injury could increased the expression of TLR4 and cause the inflammatory cell infiltration by histopathological stain. And lumbrokinase decreased the expression of TLR4 and the phenomenon of inflammatory cell infiltration in the heart from rats subjected to myocardial I/R injury. Therefore, this study suggested that lumbrokinase was a potential cardioprotective agent to against myocardial I/R injury, and lumbrokinase could against myocardial I/R injury via TLR4/NF-κB/MMP-9 and JNK MAPK signaling pathway.

並列關鍵字

myocardial ischemia/reperfusion injury ； lumbrokinase ； TLR4 ； MMP-9

參考文獻

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