馬兜鈴酸腎症在純系小鼠的確立與綠茶、 (+)-catechin 和 pravastatin sodium 在此腎炎模型的藥效評估

1. 馬兜鈴酸腎症在純系小鼠的確立與綠茶、(+)-catechin和pravastatin sodium在此腎炎模型的藥效評估 • 中文摘要 • 近年來發生許多馬兜鈴酸腎病變 (aristolochic acid nephropathy, AAN)的案例，其原因是誤用含馬兜鈴酸的植物。腎臟受損部位侷限於近曲小管，有明顯的細胞浸潤、腎小管萎縮，以及間質纖維化的情形。本研究藉由長期投予純系小鼠低劑量aristolochic acid (AA) 成功誘發與人類AAN相似的腎炎，並評估綠茶(green tea, GT)、(+)-catechin (CAT)與pravastatin (P)對AAN的改善效果。 • 純系小鼠C3H/He (6weeks, males) 連續56天飲水給予3.0 μg/mL AA，之後停藥恢復正常飲用水觀察14天；對照組在實驗期間投予正常飲用水。藥效評估實驗依前述方法給予56天AA後，連續14天經口投予GT 75, 150, 300 mg/kg，CAT 25, 50, 100 mg/kg，P，CAT 50 mg/kg 併用pravastatin (CAT+P)，對照組則給予蒸餾水。 • 測定尿蛋白，N-acetyl-beta-D-glucosaminidase (NAG) 與血中尿素氮 (blood urea nitrogen, BUN)，來評估小鼠腎功能；腎組織使用PAS染色觀察病理組織改變，並進行免疫螢光染色，包括：巨噬細胞，乙型轉型生長因子(TGF-β)，基質金屬蛋白分解?(MMP-9)，甲型腫瘤壞死因子(TNF-α)，以辨識損傷部位之特異性抗原。 • 實驗結果顯示，藉由飲用水連續給予56天AA會造成尿蛋白、NAG、BUN及血糖值輕微上昇；腎組織鏡檢發現腎小管萎縮、間質有細胞浸潤和纖維化的情形，免疫螢光染色發現腎小管間質巨噬細胞浸潤、TGF-β、TNF-α沉積明顯增加，MMP-9也有些許沉積，此外，腎臟受損的情形在停止投予AA後不但沒有恢復，反而惡化。在投予GT 150 mg/kg，CAT 50 mg/kg，pravastatin，CAT+P等五組之尿蛋白(GT 150 mg/kg除外)、NAG、BUN、血糖值都有降低；組織學觀察發現腎小管萎縮、間質細胞浸潤和纖維化的情形皆有緩解，根據以上結果發現本研究中的療效依序是P > CAT+P > CAT 50 mg/kg，其中P, CAT+P療效相近。故本研究之結論，pravastatin單獨使用或(+)-catechin與pravastatin併用的治療方法，皆會改善AAN病人腎臟功能。

2. Establishment of the aristolochic acid nephropathy in inbred mice and the effect of green tea, (+)-catechin and pravastatin sodium on the nephritis • 英文摘要 • Many cases about aristolochic acid nephropathy (AAN) have been identified in recent years. The reason is that people mistaken the herb that contains aristolochic acid (AA). AAN is a unique type of nephropathy, which was characterized by extensive cell infiltration, tubular atrophy, and interstitial fibrosis. We established a chronic interstitial fibrosis model of AAN in inbred mice and investigated the efficacy of green tea (GT), (+)-catechin (CAT), pravastatin (P) on AAN. • AA was dissolved in distilled water (3μg/ml) and as drinking water ad libitum to C3H/He mice (6 weeks, male) for 56 days, the control group was administered to distilled water. In the second experiment, they were administered orally with GT (75, 150, 300 mg/kg), CAT (25, 50, 100 mg/kg), P, CAT 50 mg/kg combined with pravastatin (CAT+P) once daily for 14 days. The control group was administered to distilled water. • Urinary protein (UP), urinary N-acetyl-beta-D-glucosaminidase (NAG), blood urea nitrogen (BUN), blood glucose were determined. Renal tissues were served to histological examination (PAS stain and immunofluorescence staining). The antibodies, including murine F4/80 macrophage, TGF-β (transforming growth factor-β), MMP-9 (matrix metalloproteinase-9), TNF-α (tumor necrosis factor-α) were chosen to recognize the specific antigens, which deposited in injury sites. • All animals treated with AA developed increased in UP, NAG, BUN, and blood glucose. In the histological examination, we observed tubular atrophy, interstitial infiltration and fibrosis in the typical AAN. In the immunofluorescence stain assay, macrophage, TGF-β, MMP-9, and TNF-α were localized in the renal tissue. After discontinuation of AA, renal function did not improve. • The amount of urinary protein, NAG, BUN, and blood sugar were decreased in the GT 150 mg/kg, CAT 50 mg/kg , P, CAT+P-treated groups compared with the control group (except for the UP of GT 150 mg/kg ). In the histological examination, we observed the alleviation in all treatment groups. The staining intensity of macrophage, TGF-β, TNF-α were decreased in all groups, and MMP-9 in the interstitium was significantly increased. Our results demonstrate the therapeutics efficacy of the treatment as follow: P > CAT+P > CAT 50 mg/kg. The groups of pravastatin, CAT 50 mg/kg combine with pravastatin have similar therapeutics efficacy. The conclusion of our study is that both pravastatin alone use and pravastatin combined with CAT 50 mg/kg can improve the renal function of AAN patients.