摘要:Lipopolysaccharide (LPS)是革蘭氏陰性菌外膜的主要成份,可啟動受感染宿主之多樣性反應。LPS在動物實驗中可引發極類似臨床敗血症之特徵:增加促發炎物質如C-反應蛋白(CRP)、氧化自由基(ROS)和high mobility group box (HMGB)-1蛋白等細胞激素的分泌。LPS所誘發之CRP可調高糖化最終產物受體(receptor for advanced glycation end-products, RAGE)的表現。此外LPS亦可造成壞死的組織和發炎細胞釋出HMGB-1蛋白。HMGB-1是RAGE的配合基之一。RAGE和配合基的作用可導致ROS的生成和NFκB的活化,繼而強化RAGE的表現和促發炎媒介物的產生以延續和擴大發炎反應。另一方面,LPS可能部份經由NFκB的活化而調高血管和心臟細胞之誘導型一氧化氮合成酶(inducible nitric oxide synthase, iNOS)的生成。iNOS的活化所生成大量的一氧化氮(nitric oxide, NO)能與過氧化物(superoxide)作用而產生更具毒性的peroxinitrite。本二年期研究計畫之主要目的是探討LPS所引發活體動物(Wistar鼠)之發炎事件的活化與心臟收縮力學的改變之間的關係。使用彈性-阻力模型(elastance-resistance model)來分析左心室血壓和升主動脈血流訊號,產生評估心臟收縮力學之參數如最大收縮彈性(maximal systolic elastance, Emax)和理論最大血流(theoretical maximum flow, Qmax)。Emax可用來評估左心室的收縮力而Qmax是與左心室的內部阻力成反比關係。為進一步界定發炎反應在調控心臟收縮力學所扮演的角色,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(探計慢性發炎對心臟收縮力學的影響)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左心血壓和升主動脈血流訊號。4. 使用彈性-阻力模型來分析所量測的左心室血壓和升主動脈血流以產生評估心臟收縮力學之參數如Emax和Qmax。5. 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、HMGB1、NO和peroxynitrite。6. 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左心室肌肉所含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左心血壓和升主動脈血流訊號。4. 使用彈性-阻力模型來分析所量測的左心室血壓和升主動脈血流以產生評估心臟收縮力學之參數如Emax和Qmax。5. 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、HMGB1、NO和peroxynitrite。6. 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左心室肌肉所含RAGE、AGE、HMGB1和iNOS的表現。
Abstract: Lipopolysaccharide (LPS), the principal component found in the outer membrane of various Gram-negative bacteria, is the prototypical example of endotoxin to cause septic shock. In animal models, the LPS-induced systemic inflammation mimics many of the initial clinical features of sepsis, increasing the release of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, C-reactive protein (CRP), reactive oxygen species (ROS) and high mobility group box (HMGB)-1 protein. 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 HMGB-1, which was identified as a ligand for RAGE. Activation of RAGE by its ligands results in the generation of ROS and activation of NF-κB, and thereby enhances 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-κB. High levels of nitric oxide (NO) produced by iNOS elicit detrimental effects because NO can react with superoxide to generate highly toxic compounds such as peroxynitrite. Although septic cardiomyopathy is a well-described complication of the disease, the association between the activation of inflammatory events and the cardiodynamic changes has not been fully explored in intact animals. Therefore, this two-year project is designed to examine the hypothesis that the LPS-induced chronic inflammation may cause a detrimental effect on cardiac systolic mechanics in male Wistar rats. Left ventricular (LV) pressure and ascending aortic flow signals are measured to generate the maximal systolic elastance (Emax) and the theoretical maximum flow (Q.max), using the elastance-resistance model. Emax serves in a given heart to quantify the myocardial contractility of the left ventricle and Q.maxis inversely related to the ventricular internal resistance. To further define the role of systemic inflammation in the regulation of cardiac 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 inflammatory effects of LPS on the cardiac systolic mechanics in rats)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. To measure LV pressure and aortic flow signals on rats with a high-fidelity Millar pressure catheter and an electromagnetic flow probe respectively.4. To determine the inflammatory effects of LPS on the systolic mechanical behavior of the ventricular pump in terms of Emax and Qmax, using the elastance-resistance model.5. To quantify the plasma levels of LBP, IL-6, CRP, HMGB1, NO and peroxinitrite using commercially available enzyme-linked immunosorbent assay kits in strict accordance with the manufacturer’s instructions.6. Immunohistochemical staining for RAGE, AGE, HMGB1, and iNOS in the cardiac muscles is performed using their respective antibody.The second-year project (defining the role of inflammation in the regulation of cardiac systolic mechanics in the LPS-treated rats)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. To measure LV pressure and aortic flow signals on rats with a high-fidelity Millar pressure catheter and an electromagnetic flow probe respectively.4. To determine the anti-inflammatory effects of MP on the cardiac systolic mechanics in the LPS-treated rats, using the elastance-resistance model.5. To quantify the plasmalevels of LBP, IL-6, CRP, HMGB1, NO and peroxinitrite using commercially available enzyme-linked immunosorbent assay kits in strict accordance with the manufacturer’s instructions.6. Immunohistochemical staining for RAGE, AGE, HMGB1, and iNOS in the cardiacmuscles is performed using their respective antibody.