前列環素於缺氧性腎臟與心臟疾病之關係及 其治療性應用

Abstract

前列環素I2（PGI2）為前列腺素的代謝產物之一，主要作用於血管並於臨床上證明具血管擴張之功效。而前列環素I2主要是經由其受體（IP receptor）促使環單磷酸腺苷（cyclic adenosine monophosphate, cAMP）合成，同時增加細胞內第二訊息傳遞而進行廣泛的生理作用，其中包括擴張血管、抗血小板凝集、抑制嗜中性白血球及血管平滑肌細胞之增生等能力；此外另有文獻指稱，其亦具有降低組織之因缺氧再灌流（ischemia/reperfusion, I/R）所致傷害；惟其作用之詳實機制目前未有明確之定論。 職是之故，本研究論文乃分為兩部份進行；首先包括脂締素（adiponectin, APN）治療小鼠經缺氧再灌流之腎臟。試驗結果顯示，業經缺氧再灌流後導致腎臟功能損傷之小鼠，其血液中之脂締素有顯著下降，並伴隨有肌酸酐（creatinine）及尿素氮（urea nitrogen）濃度上升之情事；此外，彼等小鼠且常因發炎反應的誘發，導致嗜中性白血球浸潤（infiltrating neutrophils）、骨髓過氧化酵素活性（myeloperoxidase activity, MPO）上升及腎臟細胞凋亡（apoptosis）等現象。然若經由腹腔額外給予脂締素，從而提高彼等業經腎臟缺氧再灌流小鼠之血液中APN濃度，則可有效降低前述包括嗜中性白血球浸潤、MPO活性上升及腎臟細胞凋亡等情事之發生。進一步藉由分子與生化分析策略，探討脂締素之作用機制，結果顯示脂締素可經由過氧化酶增殖劑，激活受體 (PPAR-α)調控環氧合酶二型（cox-2）及前列環素I2 之表現，從而有效提高原血紅素氧化酵素第一型(HO-1) 之活性。此外，藉由染色質免疫沉澱（Chromatin immune-precipitation, ChIP）策略，進一步證實PPAR-α確可結合於HO-1起動子之PPAR-α反應調控區域（PPAR-α response element, PPRE）從而調控該基因之表現。同時，業經PPAR-α基因剔除小鼠證明脂締素無法顯著提高HO-1於腎臟之蛋白質表現量。綜合所述，本研究之結果提供了脂締素經由前列環素- PPAR-α-HO-1訊息傳導途徑活化，進而提高腎臟於缺氧再灌流後之保護成效。 第二部份則是旨在探討利用前列環素合成酶（PGI2 synthase, PGIS）結合骨髓間葉幹細胞（bone marrow deriver-mesenchymal stem cells, BM-MSCs）後所參與的旁泌因子（paracrine factors）之作用，並同時增加幹細胞修復受損組織的能力、免疫調節的提升、血管新生作用以及抑制心臟細胞凋亡。職是之故，本研究即利用慢病毒（lentivirus）感染使間葉幹細胞過度表現（overexpression）前列環素合成酶（MSCPGIS）。且經流式細胞儀分析MSCPGIS之表面抗原，指其並無改變幹細胞原有之特性。同時，於此等細胞之培養液內亦可測得高量之6-酮前列環素F1α（6-keto-PGF1α），並顯著降低因過氧化氫（H2O2）所造成之細胞凋亡。然若將MSCPGIS與脾臟細胞（splenocytes）共培養後，其具顯著抑制脾臟細胞因concanavalin-A誘發增殖之情事，證明MSCPGIS具免疫調節之能力。此外，將MSCPGIS以細胞數5x104移植於急性心肌梗塞（acute myocardium infarction）14天的小鼠並以心臟超音波觀察；結果顯示不僅改善心臟功能且彼等各項數值皆優於對照組。有鑑於此，同時再藉由組織之分析並證實，經移植MSCPGIS之心臟可明顯的降低纖維化的產生、心肌細胞的凋亡以及提高間葉幹細胞分化為血管及心肌細胞的可能性。綜合上述，本研究所提供之前列環素合成酶修飾間葉幹細胞之模式或可有效的於早期抑制由心臟梗塞所引起之心肌損傷及病變。

Prostaglandin I2 (PGI2) is major prostanoids in the vascular system and a clinically proven vasodilator. The PGI2 up regulates the intracellular production of cAMP by binding to its receptor (IP), which is postulated to be responsible for the multiple effects of PGI2, such as vasodilation, antiplatelet aggregation, inhibition neutrophils and smooth muscle proliferation. In addition, the PGI2 also reported to attenuate tissue ischemia/reperfusion (I/R) injury; however, the mechanisms underlying the reported therapeutic effects are poorly understood. Therefore, this study is comprised of two parts. In the first part of studies, investigates were need to evaluate the protective roles of adiponectin (APN) in the kidney I/R injury. Results from those studies appeared that the serum concentration of APN was decreased significantly after mice had been suffering from renal ischemia reperfusion (I/R) injury. Moreover, when comparison were mode to those of control mice, it was found that several features including I/R-induced renal dysfunction (elevated serum creatinine and urea levels), inflammation (number of infiltrating neutrophils and myeloperoxidase activity), and apoptotic responses (apoptotic cell number and caspase-3 activation) etc. were all appeared to be attenuated in those of APN-treated mice. Further, molecular and biochemical analyses comfirmed that APN up-regulates heme oxygenase-1 (HO-1) via peroxisome-proliferator-activated-receptor-α (PPAR-α) dependent pathway mediated by the enhancement expression of COX-2 and 6-keto PGF1α. Chromatin immune-precipitation assay demonstrated that APN increases the binding activity of PPAR-α to the PPRE region of HO-1 promoter. Furthermore, APN induced HO-1 expression only found in wild type but not in PPAR-α gene deleted mice. This provides in vivo evidence that APN mediated HO-1 expression depends on PPARα regulation. In conclusion, this study results provide a novel APN mediated prostacyclin-PPAR-α- HO-1 signaling pathway in protecting renal I/R injury. The second part of the investigation provides evidence for a novel mechanism that involves the secretion of paracrine cytoprotective factor(s) from bone marrow deriver-stem cells. The aim of this study attempts to explore how PGI2 synthase (PGIS) can beneficially modulate stem cell therapy, immunomudulatory, angiogenesis, and protecting the myocardium from apoptosis. This study genetically enhanced PGIS expression within mesenchymal stem cells (MSCPGIS). In vitro, the MSCPGIS did not change MSCs surface markers by flow-cytometry. In addition, the MSCPGIS could secrete 6-keto-PGF1α in a culture medium and showed decrease damage in hypoxia/re-oxygenation and H2O2 treatment. Furthermore, splenocytes proliferation was significantly suppressed under co-culture with MSCPGIS. In vivo, this study use mouse AMI model and then intra-myocardial injected 5x104 cells. Echocardiography shows improved cardiac function at 14 days post-AMI in MSCPGIS group compared to other three groups. Histological analysis on MSCPGIS treated heart demonstrated with decreased myocardial fibrosis, apoptotic cells and elevated levels on angiogenesis and cardiogenesis in the infracted region. To conclude, this study importance in explaining the role of PGIS-modified MSCs therapy in an AMI model during the early stages of disease and may have values for mediating limited process of inflammation and cardiac remodeling.