馬兜鈴酸腎症在純系小鼠的確立與柴胡在此腎炎模型的藥效評估

Establishment of the aristolochic acid nephropathy in inbred mice and the effect of Bupleuri Radix on the nephritis

楊文淇(Yang Wen-Chi)

指導教授 ： 陳世銘

臺北醫學大學/藥學院/藥學研究所/碩士(2005年)

摘要

近年來中藥減肥藥引起腎衰竭的報導非常的頻繁，其中原因之一是誤用含馬兜鈴酸的植物造成馬兜鈴酸腎病變 (aristolochic acid nephropathy, AAN)。其受損部位侷限於近曲小管，除了明顯的間質纖維化，腎小管萎縮，還有輕微細胞浸潤的情形。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，並評估柴胡 (Bupleuri Radix) 及prednisolone等對AAN的改善效果。 純系小鼠C3H/He (6weeks, males) 連續28天飲水給予3.0 μg/mL AAs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28天AAs後，連續14天經口投予柴胡濃縮劑 (BR) 150, 300, 450 mg/kg，柴胡成分saikosaponin A, C, D, prednisolone (P), 柴胡濃縮劑300 mg/kg 併用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，及進行免疫螢光染色 (巨噬細胞，TGF-β，MMPs) 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28天AA會造成尿蛋白、NAG及BUN、血糖值輕微上昇；腎組織鏡檢發現腎小管萎縮、間質有細胞浸潤和輕微纖維化的情形，免疫螢光染色發現腎小管間質巨噬細胞、TGF-β表現明顯增加，MMP-9也有些許表現，此外，腎臟受損的情形在停止投予AA後不但沒有恢復，反而有惡化的情形。在投予柴胡濃縮劑300 mg/kg, saikosaponin A, D, P，柴胡濃縮劑併用P等五組之尿蛋白，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柴胡濃縮劑併用P, P, 柴胡濃縮劑300 mg/kg, saikosaponin D與saikosaponin A。因此利用生藥製劑與化學藥品的合併療法可以提供未來臨床上治療的新方向。

關鍵字

馬兜鈴酸腎病變 ； 柴胡 ； 巨噬細胞 ； 乙型轉型生長因子 ； 基質金屬蛋白分解?

並列摘要

Nephrotoxicity induced by weight-control Chinese medicine was reported frequently in recent years. One of the factors is mistaken the herb that contains aristolochic acid (AA). Aristolochic acid nephropathy (AAN) is a unique type of nephropathy, which was characterized by an extensive interstitial fibrosis with rare cell infiltration and tubular atrophy. We tried to establish a chronic interstitial fibrosis model of AAN in inbred mice and investigated the impact of Bupleuri Radix, prednisolone on AAN. Aristolochic acid (AA) dissolved in distilled water (3μg/ml) were administered orally to C3H/He mice (6 weeks, male) as drinking water ad libitum for 4 weeks, control group were treated with distilled water. In the second experiment, they were administered orally with Bupleuri Radix extract (BR) (150, 300, 450 mg/kg), saikosaponin A, C, D, prednisolone (P), BR 300 mg/kg combine with P once a day for 14 days. The control group was treated with distilled water. Urinary protein, 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) was chosen to recognize the specific antigens, which deposited in injury sites. All animals given AA developed elevation of urinary protein, NAG, BUN, blood glucose. The histological examination was observed the typical AAN: tubular atrophy, interstitial infiltration and fibrosis. In the immunofluorescence stain assay, macrophage, TGF-β, MMP-9 were localized in the renal tissue. After discontinuation of AA, renal function was not improved. The amount of urinary protein, NAG, BUN, blood sugar were decreased in the BR 300 mg/kg, saikosaponin A, D, P, BR combine with P-treated groups compared with the control group. The histological examination was observed the alleviation in all treatment groups. The staining areas of macrophage, MMP-9 in the interstitium were significantly increased, and TGF-β was decreased among all groups. Our result demonstrate the therapeutics effect of the treatment as following: BR combine with P have more prior effect than P, BR 300 mg/kg, saikosaponin D, saikosaponin A. This present study offers a new therapeutic trend of utilizing the crude drug preparation with medicine.

並列關鍵字

Aristolochic acid nephropathy (AAN) ； Bupleuri Radix ； Macrophage ； TGF-β (transforming growth factor-β) ； MMP-9 (matrix metalloproteinase-9)

參考文獻

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