- 學位論文
和厚朴酚減輕急性腎絲球腎炎嚴重度及介白素18在慢性腎衰竭患者角色
Honokiol Alleviates Experimental Mesangial Proliferative Glomerulonephritis and Roles of Interleukin-18 in Chronic Renal Failure Patients
摘要
第一部份 傳統中藥成份-和厚朴酚(honokiol)對實驗性急性腎膈細胞增生性腎炎的腎臟保護研究 腎絲球腎炎(glomerulonephritis，簡稱GN)仍為最常見的末期腎病變(end-stage renal disease，簡稱ESRD)致病原因之ㄧ。腎絲球腎炎的病理變化明顯觀察到腎絲球巨噬細胞的聚集、腎膈細胞的增生及細胞外間質 (extracellular matrix，簡稱ECM)蛋白堆積。藥物治療以抑制上述病理變化，對腎絲球腎炎的惡化可有明顯減緩效果。和厚朴酚為萃取自Magnolia officinalis的小分子量純化配體，在許多動物實驗中証實它擁有抗氧化、抗發炎及抑制細胞增生的作用。然而，在腎臟疾病的療效從未被報導過，本研究應用抗Thy1腎絲球腎炎動物模式探討和厚朴酚對腎絲球腎炎的療效。吾人以小鼠抗大鼠Thy1抗體(mouse anti-rat Thy1 antibodies)靜脈注射Wistar 大鼠，實驗腎炎大鼠接受腹腔注射和厚朴酚(2.5mg/kg, 每天兩次)或對照溶劑(vehicle-phosphate-buffered saline，簡稱PBS)治療，在不同時間點犧牲大鼠以利研究。記錄尿蛋白(urine protein)排出量、腎絲球組織病理及免疫組織病理變化。以西方墨點法分析細胞增生相關指標；以北方墨點法分析細胞粘連因子(adhesion molecules)、趨化細胞激素(chemokine)及細胞外間質蛋白基因表現。結果顯示：和厚朴酚可有效減少尿蛋白排出量並減輕腎絲球細胞總數及腎絲球硬化現象；腎絲球腎炎大鼠的腎絲球內細胞增生核抗原(proliferating cell nuclear antigen，簡稱PCNA)及Akt磷酸化(Akt phosphorylation)，可經過和厚朴酚治療後減少表現量。腎絲球腎炎大鼠的腎絲球內單核球趨化蛋白-1(monocyte chemoattractant protein-1，簡稱MCP-1)、細胞間粘連因子-1(intercellular adhesion molecule-1，簡稱ICAM-1)、第一型膠原蛋白(α1)[type I (α1) collagen]及fibronectin mRNA 表現量有受到和厚朴酚抑制。上述結果顯示：和厚朴酚可能擁有治療腎膈細胞增生型腎炎 (mesangial proliferative glomerulonephritis) 的療效。 第二部份 生物標記-介白素18在透析患者預後的角色及尿毒素和血液透析過程對它的影響 末期腎病變(end-stage renal disease，簡稱ESRD)患者必需接受腎臟替代療法(renal replacement therapy，簡稱RRT)以維持生命。目前，血液透析或腹膜透析治療仍為末期腎病變患者的主要選擇；血液透析患者，其血液經由透析管路引流至透析器(dialyser)與透析液(dialysate)做毒素交換，治療頻率約每星期二至三次；腹膜透析患者，經由植入腹膜腔導管，灌注高糖透析液，利用滲透壓原理，達到排除尿毒素及水分。由於經常性的免疫和腹膜細胞與外來生物材質及高糖溶液接觸，透析治療患者長期處於慢性發炎狀態及免疫系統趨向Th1分化。介白素18(interleukin-18，簡稱IL-18)為近來新發現的前趨發炎細胞激素(proinflammatory cytokine)，原始命名為interferon gamma inducing factor，簡稱IGIF。在免疫系統分化上，與介白素12共同促進interferon-γ分泌，以驅使Th1分化；在發炎反應中，與其它前趨發炎細胞激素，如：介白素6及腫瘤壞死因子α(tumor necrotic factor-α，簡稱TNF-α)互為網路，媒介慢性發炎反應。介白素18在慢性發炎反應及免疫趨向Th1分化的角色與末期腎病患者所面臨的病理變化不謀而合；的確，在末期腎病變患者也觀察到介白素18有明顯升高現象。但是升高的介白素18在臨床上的意義仍不清楚。研究指出慢性發炎反應可能與末期腎病患者的高罹病率(morbidity)及死亡率(mortality)關係密切，近年來心臟血管疾病一直是透析患者的罹病及死亡的主要原因，介白素18在心臟血管疾病的預後角色，已為數篇重要文獻證明。基於上述觀察，吾人首先要探討介白素18在血液透析患者預後的角色；其次，要探討血液透析的過程中，因為血球與透析器的生物相容交互作用，對透析患者血漿介白素18的影響及與其他前趨發炎反應細胞激素的關係；最後，要研究慢性腎衰竭尚未接受透析治療患者，血漿介白素18與腎臟廓清率的關係；腹膜透析的過程中，因為高糖透析液與腹膜細胞的交互作用，對腹膜透析患者血漿介白素18的影響及與其他前趨發炎反應細胞激素的關係。 首先，為了確立介白素18在血液透析患者預後的角色，吾人收案184位接收血液透析治療的末期腎病變患者，其中男性佔62%，平均年齡為58.5±1.0歲。根據開始觀察點的介白素18值，將病患分成高、中及低三組，前瞻性追蹤觀察12個月，以Cox 迴歸存活分析以預測未來住院狀態，同時調節病患年齡、體質量指數(body mass index ，簡稱BMI)、接受血液透析時間(HD duration)、營養、發炎反應指標、合併症、透析廓清適量指標(Kt/Vurea)及血脂肪；以Kaplan-Meier存活曲線分析無住院事件(hospitalization-free events)的累計差異。根據介白素18值，將病患分成高、中及低三組，發現住院天數及頻率皆有顯著差異 (P<0.05)；以Kaplan-Meier 存活分析病患住院狀態與血漿介白素18值的關係，亦發現血漿介白素18值最高組的住院比例明顯高於其他兩組(p Log Rank = 0.027)；以Cox proportional hazard model經過校正相關因子後，每增加一個對數值介白素18(pg/ml)，會增加血液透析患者的相對危險率(relative risk，簡稱RR)達1.709 (95% CI, 1.114 to 2.620; P=0.014)。 其次，血液透析患者上升的介白素18可成為預後的指標，促使吾人進一步探討血液透析過程中對介白素18血液濃度的影響，以及介白素18與其他前趨發炎細胞激素，如：介白素6及腫瘤壞死因子α的關係，以釐清生物不相容性對介白素18的影響。研究方法以酵素結合免疫吸附法(Enzyme-linked immunosobent assay，簡稱ELISA)，測量血漿介白素18、介白素6及腫瘤壞死因子α在正常控制組(healthy controls，簡稱NC)，血液透析前(pre-dialysis)及血液透析後(post-dialysis)的濃度；紀錄透析治療及檢驗相關資料。結果顯示，血漿介白素18在血液透析患者的濃度明顯高於正常控制組(p <0.001)；當比較血液透析前及血液透析後的血漿介白素18變化，發現血液透析後的濃度明顯高於血液透析前(p = 0.032)，且與介白素6及腫瘤壞死因子α明顯相關。血漿介白素18在以纖維素為基礎的血液透析過程中，可以明顯增加。 最後，血液透析患者血漿介白素18升高時預後較差，血液透析過程(hemodialysis process)可以刺激血中濃度增加，並與其他前趨發炎細胞激素相關。對慢性腎衰竭(chronic renal failure，簡稱CRF)尚未接受透析治療及接受連續可活動性腹膜透析(continuous ambulatory peritoneal dialysis，簡稱CAPD)治療病患，吾人擬探討尿毒素及腎臟廓清率對血漿介白素18的影響，並討論介白素18與介白素6及腫瘤壞死因子α在CRF及CAPD患者的關係。血漿介白素18、介白素6及腫瘤壞死因子α在15位正常控制組(healthy normal controls，簡稱NC)，27位CRF組及15位CAPD組患者的濃度以酵素結合免疫吸附法測量。結果顯示，血漿介白素18 值在CRF組為572.5 ± 41.9 pg/ml、在CAPD組為479.2 ± 47.4 pg/ml，明顯高於正常控制組(NC)的263.6 ± 20.0 pg/ml，但CRF與CAPD 患者間並沒有差異；血漿介白素18與肌酸酐廓清率(creatinine clearance，簡稱Ccr)呈現明顯負相關；然而，血漿介白素18與接受腹膜透析時間、標準化蛋白質氮表現值(normalized protein nitrogen appearance，簡稱nPNA)、腹膜透析病患的透析廓清率指標：每週Kt/Vurea及每週肌酸酐廓清率(weekly Cr clearance，簡稱WCCR)無關。在CRF組及CAPD組的患者的血漿介白素18與腫瘤壞死因子α呈現正相關，但與介白素6無關。 綜合上述研究結果，吾人首度證實介白素18可當作血液透析患者未來住院的獨立預測因子，並提供發炎反應會造成血液透析患者不良預後的重要文獻報告。其次，前活化(pre-activation)免疫細胞，可能可以解釋血漿介白素18與其他發炎細胞激素的相關性。最後，尿毒症相關變化為造成腎臟衰竭患者血漿介白素18濃度上升的最主要因素；介白素18濃度上升可能與腎臟衰竭患者免疫系統趨向Th1分化有關，並引發前趨發炎細胞激素升高。
並列摘要
Part 1: Honokiol, a small molecular weight natural profuct, alleviates experimental mesangial proliferative glomerulonephritis Glomerulonephritis is still the most common cause of end-stage renal disease. Accumulation of glomerular macrophages, proliferation of mesangial cells, and deposition of extracellular matrix proteins are pathobiological hallmarks of glomerulonephritis. Pharmacological interventions that can inhibit these insults may be beneficial in the retardation of the progression of glomerulonephritis. Honokiol, a small molecular weight lignan, originally isolated from Magnolia officinalis, shows anti-oxidative, anti-inflammatory, and anti-proliferative activities in a variety of inflammation models. However, the therapeutic effect of honokiol on renal disease has never been reported. In this study, we investigated the in vivo effects of honokiol on rat anti-Thy1 nephritis. Anti-Thy1 nephritis was induced in Wistar rats by injecting mouse anti-rat Thy1 antibodies intravenously. Nephritic rats were randomly assigned to receive honokiol (2.5mg/kg, twice a day) or vehicle (phosphate-buffered saline) and were sacrificed at various time points. Urine protein excretion was determined. Glomerular histology and immunohistopathology were studied. Western blotting was conducted for markers of proliferation. Adhesion molecules, chemokine and extracellular matrix gene expression were evaluated by Northern blotting. Honokiol-treated nephritic rats excreted less urinary protein and had lower glomerular cellularity and sclerosis. The increased intraglomerular proliferating cell nuclear antigen and Akt phosphorylation in nephritic rats could be abolished by the treatment of honokiol. Honokiol also alleviated glomerular monocyte chemoattractant protein-1 and intracellular adhesion molecule-1, so as type I (α1) collagen and fibronectin mRNA levels of nephritic rats. These results indicate that honokiol may have therapeutic potential in mesangial proliferative glomerulonephritis. Part 2: Biomarker Interleukin-18 predicts dialysis outcome and influences by uremic toxin and hemodialysis process As the declining of renal function, patients with end-stage renal disease (ESRD) have to receive renal replacement therapy (RRT) to maintain their life. Currently, hemodialysis and peritoneal dialysis are still the most common dialytic modalities among patients with ESRD. In hemodialysis patients, they receive maintenance dialysis via the vascular access to draw out blood to dialytic tube and to exchange uremic toxins and water two to three times a week. In peritoneal dialysis patients, peritoneal catheters were implanted into peritoneal cavity. Uremic toxin and excess fluid was exchanged under the principle of semi-permeable membrane of peritoneum. Because of continuously exposed to uremic toxins and high concentrated dialysate with 1.5% dextrose, which led to a state of chronic inflammation in the local peritoneum and systemic immune system. Interleukin (IL)-18 is a proinflammatory cytokine that is a potent co-stimulator of interferon-γ (IFN-γ) release from peripheral immune cells, such as T helper type 1 (Th1) and natural killer cells, in combination with factors such as IL-12. IL-18 and other pro-inflammatory cytokines, such as IL-6 and tumor necrotic factor-α (TNF-α) established a complete network to mediate the chronic inflammation status. Roles of IL-18 in the chronic inflammation and the immune system differentiation are similar to the pathophysiologic mechanisms of the uremic patients. In agreement, the activity of IL-18 has been markedly up-regulated in ESRD patients. However, it has not been established whether elevated plasma IL-18 predicts outcome in hemodialysis patients. Recently, cardiovascular-related morbidity and mortality are high among patients with ESRD, and recent evidence suggests that this may be linked to inflammation. Elevated IL-18 has been demonstrated as a poor outcome predictor in cardiovascular diseases in the general population. Based on the above observation, we first hope to discuss the role of IL-18 in the outcome of hemodialysis patients. Second, we investigated the influence of biocompatibility on the changes of IL-18 levels before and after dialysis, and the possible association with other pro-inflammatory cytokines. Finally, we studied the influences of uremic toxin and renal clearance in the plasma IL-18 levels of chronic renal failure (CRF) and continuous ambulatory peritoneal dialysis (CAPD) patients. Furthermore, the relationship between IL-18 and other proinflammatory cytokines, such as IL-6, and tumor necrosis factor-alpha (TNF-alpha) were also investigated. First, to determine whether plasma IL-18 predicts overall hospitalization, we studied 184 ESRD patients (62% males, 58.5+/-1.0 years of age) undergoing maintenance HD treatment. The patients were followed for 12 months and were stratified by the tertiles of plasma IL-18 levels. Classic factors, such as age, body mass index, HD duration, nutritional and inflammatory parameters, co-morbidity, dialysis adequacy, and lipid status were entered into a Cox regression model to predict hospitalization. The Kaplan-Meier method was used to analyse the cumulative proportion of hospitalization-free events. Significantly different hospitalization days and frequencies (P<0.05) were observed when patients were divided according to tertiles of plasma IL-18 levels. Patients were stratified according to IL-18 tertiles and analysed separately according to the hospitalization-free period. In the Kaplan-Meier model, the upper tertile of IL-18 had the highest probability of a hospitalization event during the entire follow-up period (P log rank = 0.027). In the Cox proportional hazard model, the relative risk for first hospital admission for each increase in Ln IL-18 (pg/ml) concentration was associated with a 1.709 (95% CI, 1.114 to 2.620; P = 0.014) increase in the risk for future hospitalization events. Second, we based on the findings that elevated plasma IL-18 predicted poor outcomes in hemodialysis patients. In this study investigated changes of IL-18 levels before and after dialysis, and the possible association with other pro-inflammatory cytokines, such as IL-6 and TNF-alpha. Plasma IL-18 of healthy controls, and pre- and post-dialysis of uremic patients undergoing hemodialysis (HD) were evaluated by ELISA methodology. Plasma IL-18 levels were significantly increased in patients with maintenance HD (p <0.001) compared to its level in normal subjects. When compared to pre-dialysis levels, a significant increase in plasma IL-18 was measured at the end of HD (p = 0.032). There was a significant correlation among plasma IL-18, IL-6 and TNF-alpha levels in HD patients. Plasma IL-18 concentration was significantly higher in HD patients and was significantly elevated by cellulose-based HD processes. Finally, because of pending reports discussing the role of IL-18 in CAPD patients, we investigated IL-18, IL-6, and TNF-alpha in patients with CAPD. We also studied the influence of uremic toxins and renal clearance in plasma IL-18 levels on CRF patients. Plasma was evaluated by ELISA methodology in 15 healthy controls, 27 CRF and 15 CAPD patients. Plasma IL-18 levels in CRF (572.5 +/- 41.9 pg/ml) or CAPD (479.2 +/- 47.4 pg/ml) were significantly higher than normal (263.6 +/- 20.0 pg/ml), but there was no difference in IL-18 between CRF and CAPD patients. The IL-18 concentrations negatively correlated with creatinine clearance (Ccr). However, the duration of dialysis, normalized protein nitrogen appearance, weekly Ccr, and Kt/V(urea) were not correlated with plasma IL-18 in CAPD. The plasma IL-18 concentration was positively correlated with TNF-alpha but not with IL-6 in renal failure patients with or without CAPD. The present study demonstrated a strong predictive value of elevated IL-18 levels for poor outcome in HD patients. Pre-activation of immunologically active cells may contribute to the association between pre-dialysis IL-18 and post-dialysis IL-6 and TNF-alpha levels. Uremia, per se, is the principal origin of increased plasma IL-18 in these patients. Increased IL-18 levels may be associated with Th1 differentiation and elevated TNF-alpha.