Mechanism of action of a disintegrin, trimucrin in suppressing LPS-induced activation of phagocytes andattenuating myocardial ischemia-reperfusion injury

• Main Authors: Yu-Chun Hung, 洪幼軍
• Other Authors: Huang, Tur-Fu
• Format: Others
• Language: en_US
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/ng447e

博士 === 國立臺灣大學 === 藥理學研究所 === 106 === Snake venom proteins have been broadly investigated and applied in the numerous areas of life science. These compounds can directly interact with cells in eliciting cellular responses including the activation or blockade of cellular physiological functions. However, some snakes contain some specific components which bind to their respective receptors in regulating cellular biological functions. According to the studies of their structure and activity relationship, we can not only use them as the research tool but also develop the new application and therapeutic strategy. In this study, we investigated the effect of trimucrin, a disintegrin isolated from the venom of Trimeresurus mucrosquamatus which originally was found to inhibit platelet aggregation. We further study its mechanisms of anti-inflammation and attenuating myocardial I-R injury. As we know, sepsis always causes lots of death due to septic shock and multiple organs failure, but the development of therapeutic strategy for sepsis has been restricted. In the anti-inflammatory study, we used endotoxin such as LPS to induce the acute inflammatory responses, and evaluated the effects of trimucrin in anti-inflammatory responses and investigated the mechanism on LPS-stimulated phagocytes. However, in addition to antiplatelet activity, disintegrin has been demonstrated to exert the anti-inflammatory effect but the possible mechanism is still unclear. In this study, we investigated if trimucrin exhibits anti-inflammatory activity on LPS-induced responses in THP-1 and RAW 264.7 cells. Trimucrin decreases the release of pro-inflammatory cytokines including TNF-α, IL-6, IL-1β and IL-8, nitric oxide, reactive oxygen species (ROS) as well as inhibiting adhesion and migration of LPS-stimulated phagocytes. Trimucrin also significantly attenuates the expression of NFκB-related downstream inducible enzymes like iNOS and COX-2. In addition, we observed that the molecular mechanism of trimucrin-mediated anti-inflammation is associated with decreasing phosphorylation of MAPK molecules including ERK1/2, JNK and p38. Furthermore, trimucrin also inhibits LPS-induced phosphorylation of FAK, PI3K and Akt in a concentration- dependent manner. Trimucrin also reverses NF-κB DNA binding activity via suppressing LPS-induced translocation of p65 into nucleus and cytosolic release of IκB. In cellular binding assay, the flowcytometric analysis showed that FITC-trimucrin bound to cells in a concentration-dependent manner. The anti-αVβ3 mAb also specifically reduced the binding of FITC-conjugated trimucrin. Binding assays demonstrated that integrin αVβ3 was the binding site for trimucrin on THP-1 and RAW 264.7 cells. In conclusion, we showed that trimucrin reduces the inflammatory reaction through inhibition of iNOS expression and NO production by blockade of MAP Kinase and NF-kB activation in LPS-stimulated phagocytes. Platelet-induced thrombus formation plays an important role in the pathological responses of acute myocardial infarction (AMI). Platelets also induce thrombotic occlusion of the coronary artery in microcirculation leading to exacerbating myocardial ischemia following ischemia reperfusion (I-R) injury. Disintegrins are a group of R(K)GD-containing snake venom proteins which inhibit platelet aggregation by blocking αIIbβ3 integrin. The previous reports found that hemorrhagic venom proteins have various pathological responses by influencing blood cells, plasma proteins and vessel wall in cardiovascular system. Furthermore, disintegrins can specifically affect cell-cell and cell-matrix interactions, some disintegrin mimetics have been applied for the anti-platelet, anti-thrombotic and anti-angiogenic agents. However, trimucrin, a novel small-mass RGD-containing disintegrin, has been demonstrated to possess anti-platelet and anti-inflammatory effect through blockade of platelet αIIbβ3 and phagocyte αVβ3 integrin. In this study, we found that the platelet-rich plasma prepared from trimucrin-treated rats showed to diminish platelet aggregation in response to ADP. We tried to determine whether trimucrin is cardioprotective in rats subjected to myocardial ischemia-reperfusion (I-R) injury. The left anterior descending coronary artery of rats was subjected to 1 h occlusion and 3 h reperfusion. The animals were treated with trimucrin intravenously, and the severities of I-R-induced arrhythmia and infarction were compared. Trimucrin significantly reduced I-R-induced arrhythmias and reduced mortality, as well as infarct volume, troponin-I levels, creatine kinase, and lactate dehydrogenase activity. Trimucrin also improved cardiac function and survival rates after I-R injury. In addition, trimucrin concentration-dependently inhibited platelet adhesion on collagen- and fibrinogen-coated surfaces. Trimucrin also significantly reduced neutrophil infiltration into heart tissues after I-R injury. Furthermore, trimucrin treatment caused significant downregulation of Bax, Caspase-3 apoptotic proteins and upregulation of anti-apoptotic Bcl-2 protein. These results demonstrate that trimucrin exerts cardioprotective property against myocardial I-R injury mediated through antiplatele, anti-inflammatory, anti-apoptotic mechanism, as well as improvements in cardiac function. In summary, based on the anti-inflammatory effects of anti-thrombotic venom proteins on phagocytes and myocardial I-R injury, these findings may provide a potentially pharmacological approach for the development of anti-inflammatory and cardiovascular protective agents.