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標題: | 蛇毒抗黏著蛋白 trimucrin 抑制脂多醣活化吞噬細胞及對抗心臟缺血再灌注損傷機轉之研究 Mechanism of action of a disintegrin, trimucrin in suppressing LPS-induced activation of phagocytes and attenuating myocardial ischemia-reperfusion injury |
作者: | Yu-Chun Hung 洪幼軍 |
指導教授: | 黃德富(Huang, Tur-Fu) |
關鍵字: | 蛇毒,抗黏著蛋白,醣蛋白IIb/IIIa,脂多醣,抗發炎,缺血再灌注損傷,心肌梗塞, snake venom,disintegrin,GPIIb/IIIa,LPS,anti-inflammation,ischemia-reperfusion injury,myocardial infarction, |
出版年 : | 2017 |
學位: | 博士 |
摘要: | 蛇毒蛋白目前已經廣泛的被研究與應用在生命科學各種不同的領域;這些成分能夠直接與細胞作用,並可參與不同的作用機轉進而造成不同的細胞反應導致活化或阻斷細胞生理功能。然而,毒蛇中有許多不同的毒性成份,這些蛇毒蛋白經由純化後可以專一性的結合到受體並調節細胞功能;研究這些蛇毒蛋白的結構和活性的關係,不只可以作為研究工具更可以發展成為新的應用與治療策略;例如,我們研究一種由臺灣龜殼花 (Trimeresurus mucrosquamatus) 分離出的 disintegrin,trimucrin,在調節吞噬細胞功能的作用與免疫反應;而本論文利用純化自臺灣龜殼花中具有抗血小板活性的蛇毒蛋白研究其抗發炎與減少心肌缺血性再灌注損傷之作用機轉,並期望在臨床應用上成為藥物的研究策略。
眾所週知,敗血症常會引起敗血性休克和多重器官衰竭並造成病人死亡,然而治療策略仍然有限;在抗發炎研究中,我們給予內毒素(endotoxin) 例如脂多醣 (LPS) 以誘發急性發炎反應,並且評估抗黏著蛇毒蛋白 trimucrin 在抗發炎反應以及對酯多醣 (LPS) 活化吞噬細胞的作用機轉;然而,抗黏著蛇毒蛋白 trimucrin 除了抗血小板活性的作用以外,目前已經發現具有抗發炎作用,但其作用機轉仍不清楚;在本研究中,我們報導 trimucrin,一種由臺灣龜殼花中純化的蛇毒蛋白,它可以有效的抑制經由脂多醣 (LPS) 刺激人類單核球細胞株 (THP-1) 以及巨噬細胞株 (RAW264.7) 所引發的發炎反應;trimucrin 也可以降低發炎前趨物質細胞激素 (cytokine),包括 TNF-α、IL-6、NO (Nitric oxide) 及 ROS 的釋放,並且抑制經由 LPS 活化的細胞附著 (adhesion) 與移行 (migration) 作用;同時,trimucrin 亦可以明顯的阻斷 NFκB 下游相關發炎物質包括 iNOS 及 COX-2 的表現;此外,我們發現 trimucrin 抗發炎之分子作用機轉是與 MAPK (包括 ERK1/2、JNK 和 p38) 磷酸化降低有關聯性;而 trimucrin 也可以呈現濃度相關性的抑制在 LPS 刺激下誘導產生的 FAK、PI3K、Akt 磷酸化、p65 核轉移、細胞質 IκB 釋放,而可以反轉 NFκB 的 DNA 結合活性;另外,在細胞與受體結合試驗方面,流式細胞儀分析顯示標示有螢光染劑 FITC 之 trimucrin 可以呈現濃度相關性的結合到細胞受體,而 anti-αVβ3 的單株抗體也可以專一性的抑制螢光染劑 FITC 與 trimucrin 形成之複合物結合到細胞 αVβ3 受體;這些結合試驗證實了在人類單核球細胞 (THP-1) 及巨噬細胞 (RAW264.7),αVβ3 是 trimucrin 的結合受體;總而言之,在 LPS 刺激吞噬細胞下,trimucrin 可以降低發炎反應是經由阻斷 MAPK 及 NFκB 的活化而抑制 iNOS 與 NO 的表現。 血小板引起之血栓在急性心肌梗塞之病理反應中扮演一種很重要的角色,在缺血性再灌注損傷之後,血小板在微循環中也可以引起冠狀動脈之血栓性阻塞並導致加速心肌缺血之傷害。眾所週知,disintegrins 是一種具有特定小分子胺基酸序列 RGD 之蛇毒蛋白的總稱。其藉由阻斷 αIIbβ3 integrin,進而抑制血小板的凝集作用;先前研究發現,出血性蛇毒蛋白在血管中經由影響血球,血管和血中蛋白而產生不同的病理反應;此外,disintegrins 能專一性的影響細胞與細胞以及細胞與細胞外間質的作用,某些蛇毒蛋白已經成功的被用來發展成抗血小板、抗血栓或抗血管新生藥物。在本研究中,我們利用血小板凝集器與流式細胞儀,我們發現蛇毒蛋白 trimucrin可以特異性的結合到血小板受體上並抑制血小板的活化與凝集;然而,trimucrin,這一種含有特定序列的 RGD ”小分子” disintegrin,目前已經證實經由阻斷血小板 αIIbβ3 及吞噬細胞的 αVβ3 integrin,並且具有抗血小板凝集及抗發炎作用;在本實驗中,我們給予 trimucrin 後由大鼠取出富含血小板之血漿,證實可以有效抑制在 ADP 刺激下的血小板凝集;此外,我們想證明在大鼠的心肌缺血性灌注傷害下,trimucrin 是否具有心臟保護作用,在大鼠接受了60分鐘的左側冠狀動脈梗塞,並經歷三小時的血流再灌注,大白鼠靜脈給予trimucrin,並且進行缺血性再灌注引發的心律不整以及心肌梗塞的大小比較,trimucrin 明顯的減少了缺血性再灌注引發的心律不整以及死亡率,並且降低了梗塞的體積、肌鈣蛋白 (troponin-I)及乳酸脫氫酶 (lactate dehydrogenase) 活性;此外,在缺血性再灌注損傷之後,trimucrin 也可以改善心臟功能並提升存活率;在體外試驗中,trimucrin 可以呈現濃度相關性的抑制血小板附著到覆蓋有膠質 (collagen) 及纖維蛋白原 (fibrinogen) 的表面;因此,在心臟組織中,trimucrin 可以有意義的降低了缺血性再灌注所引起之嗜中性白血球浸潤 (neutrophil infilitration);此外,給予 trimucrin 可以明顯的減少細胞凋亡蛋白包括 Bax、Caspase-3 的表現,以及增加抗凋亡蛋白 Bcl-2 的表現;這些結果證明了 trimucrin 可以降低心肌缺血性再灌注損傷,可能起因於其抗血小板、抗發炎、以及抗細胞凋亡作用,並進而改善心臟功能。 在本論文中,trimucrin 的發現為蛇毒蛋白在抑制血球細胞間的作用上提供一種新的角色,亦即未來它或許有潛力能應用為抗發炎或是心臟血管保護作用藥物;研究開發蛇毒抗發炎成分亦是未來努力的方向。總之,在探討抗血栓蛇毒蛋白對吞噬細胞以及心臟缺血性灌注損傷的影響之模式中,我們期望能開發以抗發炎理論為基礎的心臟血管保護作用劑或是抗發炎藥物可以提供一種具有潛力的藥理研究方向。 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68059 |
DOI: | 10.6342/NTU201704512 |
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