摘要:導致休克的嚴重敗血症在加護病房是造成病患死亡的主要原因。嚴重敗血症是促發炎媒介物(pro-inflammatory mediators)如腫瘤壞死因子(tumor necrosis factor, TNF)-、C-反應性蛋白(reactive protein, CRP)和反應性氧自由基(reactive oxygen species, ROS)過量釋出所造成的全身性發炎反應,這些促發炎媒介物可由革蘭氏陰性菌外膜的主要成份內毒素(endotoxin)誘發而產生。Lipopolysaccharide (LPS)是革蘭氏陰性菌外膜的主要成份,它在啟動對抗細菌感染所引起的宿主多樣性反應扮演著樞軸的角色。LPS可與血清蛋白(LPS-結合蛋白,LPS-binding protein, LBP)結合,並轉換到細胞表面的CD14。它隨之與橫跨細胞膜的訊息受體toll-like receptor 4 (TLR4)和附件蛋白MD-2所形成的複合体產生交互作用,進一步導致核因子(nuclear factor) B (NFB)的活化與促發炎基因的表現。此外,LPS可強化CRP的釋出,而CRP可調高糖化最終產物受體(receptor for advanced glycation end-products, RAGE)的表現。LPS亦可造成受損的組織細胞(parenchymal cells)和發炎細胞的壞死(necrosis),而細胞的壞死可引起high mobility group box (HMGB)-1蛋白的釋出。HMGB-1是RAGE的配合基之一。RAGE和配合基的作用可導玫ROS的生成和NFB的活化,繼而加強RAGE的表現和促發炎媒介物的產生。值得注意的是,NFB的活化和RAGE的表現之間存在著正向迴饋(positive feedback)的關係,這個正向迴饋在延續性的發炎反應上扮演著非常重要的角色。另一方面,LPS在血管和心臟細胞可向上調控可誘導的一氧化氮合成酶(inducible nitric oxide synthase, iNOS)的生成,此一作用可能部份是經由LPS所誘發NFB的活化而來。iNOS的活化能生成大量的一氧化氮(nitric oxide, NO) ,而一氧化氮能與過氧化物(superoxide)作用而產生高毒性的化合物如peroxinitrite和hydroxyl radicals。過多NO的釋放本身就可減低血管對血管收縮劑的反應。雖然LPS所造成的氧化還原反應的失衡(redox unbalance)和全身性發炎反應的持續與放大似乎與心血管系統的生理病理學有相當程度的牽涉,但是吾人對活體動物的發炎事件的活化與血行力學的改變並不甚清楚。因此,本三年期的研究計畫主要是要檢驗如下之假說:(1)LPS所誘發之慢性發炎可能對Wistar鼠之動脈管管壁的物理性質造成傷害,所使用的方法為主動脈輸入阻抗頻譜分析(aortic input impedance spectral analysis);(2)LPS所誘發之慢性發炎可能對Wistar鼠之左心室與動脈管間交互作用有所傷害,所使用的方法為收縮末期血壓-心搏出量分析法(end-systolic pressure-stroke volume analysis, Pes-SV analysis)。為進一步界定全身性發炎反應在心血管力學的調控上所扮演的角色,吾人(1)使用methylprednisolone (MP)當作抗發炎藥劑;(2)撤除LPS以觀察LPS所誘發全身性發炎反應的復原狀況。第一年研究計畫(探計慢性發炎對動脈力學的影響)1. 採用皮下包埋的方式植入緩慢釋放1 mg LPS的ALZET osmotic pump以誘發全身性發炎反應。2. 雄性Wistar鼠以隨機的方式分為以下組別:(i)正常控制組、(ii)2個禮拜的sham組、(iii)4個禮拜的sham組、(iv)2個禮拜的LPS組和(v)4個禮拜的LPS組。3. 使用高傳真米拉血壓導管和電磁血流探頭以量測老鼠之脈態升主動脈血壓與血流訊號。4. 使用阻抗分析技術以計算主動脈輸入阻抗頻譜和特徵阻抗。5. 使用脈衝分析技術以計算血壓波和血流波之波傳輸時間(Wave transit time)。6. 嚴格遵循商用enzyme-linked immunosorbent assay kits的使用方法來量化血漿LBP、IL-6、CRP和HMGB1。7. 分別使用RAGE、AGE、HMGB1和iNOS的抗體以組織免疫染色法量測老鼠之主動脈管壁所含RAGE、AGE、HMGB1和iNOS的表現。第二年研究計畫(探計慢性發炎對動脈力學的影響)1. 採用皮下包埋的方式植入緩慢釋放1 mg LPS的ALZET osmotic pump以誘發全身性發炎反應。2. 雄性Wistar鼠以隨機的方式分為以下組別:(i)使用MP(5 mg/kg/day, i.p.)治療的2個禮拜的sham組、(ii)使用MP(5 mg/kg/day, i.p.)治療的4個禮拜的sham組、(iii)使用MP(5 mg/kg/day, i.p.)治療的2個禮拜的LPS組和(iv)使用MP(5 mg/kg/day, i.p.)治療的4個禮拜的LPS組、(v)從4個禮拜的LPS組撤除LPS兩個禮拜和(vi)從4個禮拜的LPS組撤除LPS四個禮拜。3. 使用高傳真米拉血壓導管和電磁血流探頭以量測老鼠之脈態升主動脈血壓與血流訊號。4. 使用阻抗分析技術以計算主動脈輸入阻抗頻譜和特徵阻抗。5. 使用脈衝分析技術以計算血壓波和血流波之波傳輸時間(Wave transit time)。6. 嚴格遵循商用enzyme-linked immunosorbent assay kits的使用方法來量化血漿LBP、IL-6、CRP和HMGB1。7. 分別使用RAGE、AGE、HMGB1和iNOS的抗體以組織免疫染色法量測老鼠之主動脈管壁所含RAGE、AGE、HMGB1和iNOS的表現。第三年研究計畫(探計慢性發炎對左心室動脈管交互作用的影響)1. 採用皮下包埋的方式植入緩慢釋放1 mg LPS的ALZET osmotic pump以誘發全身性發炎反應。2. 雄性Wistar鼠以隨機的方式分為以下組別:(i)正常控制組、 (ii)2個禮拜的sham組與使用MP(5 mg/kg/day, i.p.)治療的2個禮拜的sham組、(iii)4個禮拜的sham組與使用MP(5 mg/kg/day, i.p.)治療的4個禮拜的sham組、(iv)2個禮拜的LPS組與使用MP(5 mg/kg/day, i.p.)治療的2個禮拜的LPS組和(v)4個禮拜的LPS組與使用MP(5 mg/kg/day, i.p.)治療的4個禮拜的LPS組、(vi)從4個禮拜的LPS組撤除LPS兩個禮拜和(vii)從4個禮拜的LPS組撤除LPS四個禮拜。3. 使用高傳真米拉血壓導管和電磁血流探頭以量測老鼠之脈態左心室血壓與升主動血流訊號。4. 使用心室與動脈的收縮末期血壓-心搏出量的關係曲線以計算左心室收縮末期彈性(LV end-systolic elastance,Ees)和等效動脈體積彈性(effective arterial volume elastance,Ea)。5. 以Ees 和Ea 來計算最佳後負荷(optimal afterload,Qload)。Qload可用以評估由左心室轉換至動脈系統之力學的效率。6. 嚴格遵循商用enzyme-linked immunosorbent assay kits的使用方法來量化血漿LBP、IL-6、CRP和HMGB1。7. 分別使用RAGE、AGE、HMGB1和iNOS的抗體以組織免疫染色法量測老鼠之主動脈管壁所含RAGE、AGE、HMGB1和iNOS的表現。
Abstract: ABSTRACTSevere sepsis leading to shock is the principal cause of death in intensive care unit. It is a systemic inflammatory response caused by excessive secretion of pro-inflammatory mediators, such as tumor necrosis factor (TNF)-, C-reactive protein (CRP), and reactive oxygen species (ROS), mainly induced by endotoxin (a major component of the Gram-negative bacterial outer membrane). Lipopolysaccharide (LPS) is the principal component of the outer membrane of Gram-negative bacteria and plays a pivotal role in the initiation of a variety of host responses. LPS binds to the serum protein LPS-binding protein (LBP) and is transferred to the CD14 at the cell surface. LPS then interacts with the transmembrane signaling receptor toll-like receptor 4 (TLR4) and its accessory protein MD-2, leading to nuclear factor B (NFB) activation and pro-inflammatory gene expression. The enhanced release of CRP by LPS has been reported to upregulate the receptor for advanced glycation end-products (RAGE) expression. In addition, the LPS-induced necrosis in damaged parenchymal cells and inflammatory cells promotes the release of high mobility group box (HMGB)-1 protein, which was identified as a ligand for RAGE. Activation of RAGE by its ligands results in the generation of ROS and activation of NF-B, and thereby enhances RAGE expression and pro-inflammatory mediator production. It is important to note that there exists a positive feedback between the NF-B activation and RAGE expression, which plays an important role in prolonged inflammation. On the other hand, LPS challenge upregulates induction of inducible nitric oxide synthase (iNOS) in blood vessels and cardiac muscles, in part, through the activation of NF-B. High levels of nitric oxide (NO) produced by iNOS elicit detrimental effects because NO reacts with superoxide to generate highly toxic compounds such as peroxynitrite and hydroxyl radicals. The increased NO release has also been implicated in the diminished response to vasoconstrictors. Although the redox unbalance and the maintenance and amplification of systemic inflammation after LPS seems to participate in the pathophysiology of cardiovascular system, the activation of inflammatory events and hemodynamic changes has not been fully understood in intact animals. Therefore, this three-year project is designed to examine (i) the hypothesis that the LPS-induced chronic inflammation may cause a detrimental effect on the physical properties of the arterial system in male Wistar rats, using the aortic input impedance analysis and (ii) the hypothesis that the LPS-induced chronic inflammation maycause a detrimental effect on the ventricular-arterial coupling in male Wistar rats, using the ventricular and arterial end-systolic pressure-stroke volume (Pes-SV) analysis. To further define the role of systemic inflammation in regulation of cardiovascular dynamics, (i) methylprednisolone (MP) is used as the anti-inflammatory agent in the animals administered LPS and (ii) the LPS-infused rats are recovered from the systemic inflammation after LPS withdrawal.The first-year project (determining the effects of chronic inflammation on arterial mechanics)1.Systemic inflammation is induced by implanting slow-release ALZET osmotic pump of 1 mg LPS subcutaneously.2.Male Wistar rats are randomly assigned to the following categories: (i) normal controls, (ii) 2-week sham group, (iii) 4-week sham group, (iv) 2-week LPS group, and (v) 4-week LPS group.3.Pulsatile pressure and flow signals are measured in the ascending aorta using a high-fidelity Millar pressure catheter and an electromagnetic flow probe respectively.4.The aortic input impedance spectra are obtained by making use of the vascular impedance analysis technique.5.The wave transit time in the path along arteries is obtained by making use of the filtered impulse response technique.6.Quantification of plasma LBP, IL-6, CRP, and HMGB1 is performed using commercially available enzyme-linked immunosorbent assay kits in strict accordance with the manufacturer’s instructions.7.Immunohistochemical staining for RAGE, AGE, HMGB1, and iNOS in the rat aortic ring is performed using their respective antibody.The second-year project (determining the effects of chronic inflammation on arterial mechanics)1.Systemic inflammation is induced by implanting slow-release ALZET osmotic pump of 1 mg LPS subcutaneously.2.Male Wistar rats are randomly assigned to the following categories: (i) 2-week sham group treated with MP (5 mg/kg/day, i.p.), (ii) 4-week sham group treated with MP (5 mg/kg/day, i.p.), (iii) 2-week LPS group treated with MP (5 mg/kg/day, i.p.), (iv) 4-week LPS group treated with MP (5 mg/kg/day, i.p.), (v) 2 weeks LPS withdrawal from the 4-week LPS group, and (vi) 4 weeks LPS withdrawal from the 4-week LPS group.3.Pulsatile pressure and flow signals are measured in the ascending aorta using ahigh-fidelity Millar pressure catheter and an electromagnetic flow probe.4.The aortic input impedance spectra are obtained by making use of the vascular impedance analysis technique.5.The wave transit time in the path along arteries is obtained by making use of the filtered impulse response technique.6.Quantification of plasma LBP, IL-6, CRP, and HMGB1 is performed using commercially available enzyme-linked immunosorbent assay kits in strict accordance with the manufacturer’s instructions.7.Immunohistochemical staining for RAGE, AGE, HMGB1, and iNOS in the rat aortic ring is performed using their respective antibody.The third-year project (determining the effects of chronic inflammation on left ventricular-arterial interaction)1.Systemic chronic inflammation is induced by implanting slow-release ALZET osmotic pump of 1 mg LPS subcutaneously.2.Male Wistar rats are randomly assigned to the following categories: (i) normal controls, (ii) 2- and 4-week sham groups treated with or without MP (5 mg/kg/day, i.p.), and (iii) 2- and 4-week LPS groups treated with or without MP (5 mg/kg/day, i.p.). The 4-week LPS-infused animals are recovered from the systemic inflammation after 2 and 4 weeks withdrawal of LPS.3.Pulsatile left ventricular (LV) pressure and ascending aortic flow signals are measured using a high-fidelity Millar pressure catheter and an electromagnetic flow probe.4.The LV end-systolic elastance (Ees) and the effective arterial volume elastance (Ea) are obtained by making use of the ventricular and arterial Pes-SV analysis technique.5.The optimal afterload (Qload) determined by the ratio of Ea to Ees is obtained to evaluate the efficiency of mechanical energy transferred from the left ventricle to the arterial system.6.Quantification of plasma LBP, IL-6, CRP, and HMGB1 is performed using commercially available enzyme-linked immunosorbent assay kits in strict accordance with the manufacturer’s instructions.7.Immunohistochemical staining for RAGE, AGE, HMGB1, and iNOS in the rat cardiac muscle is performed using their respective antibody.