Thaliporphine保護天竺鼠內毒素血症對抗心臟缺血再灌流損傷之機轉

Abstract

Thaliporphine是由樟科植物分離出來的阿朴酚類生物鹼，對5HT7接受器有很高的親和力。在之前的研究得知，thaliporphine在心臟缺血再灌流具有保護作用，也具有降血糖作用，而且在敗血症動物可以減少多重器官衰竭發生，增加存活率。 敗血症是一個複雜性的發炎疾病。大量的細胞激素及發炎物質產生，像是TNFα、自由基及一氧化氮，會導致pro- 和 anti- 細胞凋零路徑不平衡。敗血症也會導致凝血功能異常，容易產生瀰漫性血管性凝血，減少血液灌流及器官氧氣供給，導致多重器官衰竭。目前對於敗血症充斥的發炎物質，是否影響心臟缺血再灌流時細胞存活，並不是很清楚。本篇研究當內毒素血症動物續發心臟缺血再灌流，影響細胞存活及死亡的訊息傳遞，並且探討thaliporphine是否具有心臟保護的作用。 本篇以LPS 1mg/kg腹腔注射誘導天竺鼠內毒素血症，並且有些動物在誘導的同時給予thaliporphine。給藥24小時候，天竺鼠以pentobarbital 40mg/kg腹腔注射麻醉，開胸後，心臟左主冠狀動脈結紮，模擬缺血三十分鐘，再放鬆灌流一小時後，取出心臟，以TTC染色看壞死面積，或是萃取心臟蛋白質。和控制組比較，內毒素血症動物續發心臟缺血再灌流會明顯增加壞死面積，並且延長心臟QTc數值，同時給予thaliporphine可以減少心臟壞死面積，但是此保護作用，不受pimozide (1mg/kg, i.p.) 5HT7接受器抑制劑影響。 心臟蛋白質分為正常及缺血區域，觀察其蛋白質表現變化。內毒素血症正常區域中，蛋白質Bax、被切斷PARP以及JNK磷酸化會明顯增加，缺血區域中，這些蛋白質會更顯著增加，並且caspase 9活性明顯增加。 Thaliporphine可以明顯減少正常及缺血區域中JNK磷酸化及PARP被切斷。缺血區域中所增加的Bax表現及caspase 9活性增加－，也都能被thaliporphine所抑制。 心臟細胞內的自由基及一氧化氮分別以CM-H2DCFDA和DAF-AM測得。在天竺鼠內毒素血症中，自由基及一氧化氮明顯增加，同時給予thaliporphine可以減少此現象。心臟中cGMP含量也跟著增加，但是此現象卻無法被thaliporphine明顯改善。 總之，在天竺鼠內毒素血症會活化pro-細胞凋零途徑，所以會導致心臟續發缺血再灌流時壞死面積增加。自由基及一氧化氮會產生大量的peroxynitrite，增加細胞凋零途徑活化。Thaliporphine可以減少心臟細胞中自由基及一氧化氮含量，減少了JNK磷酸化，所以可以減少細胞凋零路徑活化，使得內毒素血症續發心臟缺血再灌流時，壞死面積減少。Thaliporphine的心臟保護作用，並非經由5HT7接受器和抑制cGMP路徑而來。Thaliporphine是經由哪個分子層面，減少內毒素血症細胞凋零路徑活化，仍需進一步探討。

Thaliporphine is a phenolic aporphine alkaloid which is isolated from Lauraceae, and has high affinity to 5HT7 receptor. Our previous study found that thaliporphine exerted cardioprotection in ischemia-reperfused rat hearts, anti-hyperglycemic effect, and anti-septic effect with the benefit of increasing survival rate and attenuating multiple organ failure. Sepsis is a complicated inflammatory disease. Under pathophysiological conditions, the elevated plasma level of cytokines and inflammatory mediators, such as TNFα, reactive oxygen species, and NO, may cause imbalance between pro- and anti- apoptotic signalings in multiple organs. The imbalance of coagulation and anticoagulation system may increase the occurrence of disseminated intravascular coagulation, and decrease organ blood perfusion and oxygen supply, which result in multiple organ failure. It remains unclear whether cell death could be enhanced in ischemia-reperfused hearts under the circumstance of endotoxemia. The present study aimed to investigate the underlying signaling of cell survival or death in endotoxemic guinea pig hearts subjected to ischemia reperfusion injury, and to examine whether thaliporphine could have benefit in the cardioprotection against ischemia reperfusion injury. Endotoxemic guinea pigs were induced by intraparenteral injection of lipopolysaccaride (LPS) 1mg/kg with or without thaliporphine coadministration. After LPS injection for 24 hours, guinea pigs were anethetized by pentobarbital (40mg/kg, i.p.) to perform the thoracotomy, and the left anterior decending coronary artery of guinea pig hearts were ligated for 30 mins and followed by reperfusion for 1h before excising the heart for the measurement of infarct size by TTC stain or for the protein extraction. As compared with the sham group, the infarct size was markedly increased in association with a prolongation of QTc interval in endotoxemic heart subjected to ischemia reperfusion injury, which could be significantly attenuated in thaliporphine coadministrated endotoxemic guinea pigs. Thaliporphine mediated cardioprotective effect was not affected by pimozide (1mg/kg, i.p.) 5HT7 receptor inhibition. The alteration of protein expression was separatedly analyzed in the non-risk and the risk area of endotoxemic guinea pig hearts subjected to ischemic reperfusion injury. In the non risk area of endotoxemic heart, the protein level of Bax, cleaved PARP, and phospho-JNK was significantly elevated without altering the caspase 9 activity, which was further enhanced in risk area of myocardium in association with the pronounced increase of caspase 9 activity. Thaliporphine treatment significantly attenuated the increased level of phospho-JNK and the cleaved PARP in both non-risk area and risk area of ischemia reperfusion hearts. The suppression of Bax expression by thaliporphine in risk area was in parallel with the attenuation of caspase 9 activity. The intracellular level of ROS and nitric oxide was measured by loading the isolated ventricular cells of guinea pig with CM-H2DCFDA and DAF-AM, respectively. In endotoxemic guinea-pig hearts, the intracellular concentration of both ROS and NO were markedly increased, which was significantly attenuated in thaliporphine coadministrated group. The increased cGMP level was found in endotoxemic guinea pig hearts and was not significantly altered in thaliporphine coadminstrated endotoxemic guinea pig hearts. In conclusion, the pro-apoptotic signaling was initiated in endotoxemic guinea pig hearts, which had an impact on the tolerance of myocardium against ischemia reperfusion injury. The elevated oxidative stress in endotoxemic guinea pig hearts may increase the formation of oxidative mediators, such as peroxynitrite, to enhance the apoptotic pathway, which results in the aggravation of myocardial infarction after ischemia reperfusion. Thaliporphine treatment attenuated the intracellular ROS and NO in association with the inhibition of JNK phosphorylation which contributed to the anti-apoptotic action and the amelioration of myocardial infarction. Thaliporphine mediated cardioprotective effect was not mediated through the activation of 5HT7 receptor or inhibition cGMP- dependent pathway. The molecular targets of thaliporphine mediated anti-apoptosis in endotoxemic guinea pig hearts remains for further investigation.