泽泻醇提物对大鼠肾毒性及其分子机制的探究实验

Study on nephrotoxicity of ethanol extract from Alismatis Rhizoma in rats and its molecular mechanism

泽泻醇提物长期喂养大鼠,探究其对大鼠的肾毒性作用,并研究其肾毒性的可能的分子机制。60只雌性大鼠随机分为空白组,雷公藤对照组,庆大霉素对照组,泽泻高、中、低剂量组,连续给药6个月。利用高效液相色谱仪(HPLC)对泽泻提取物成分进行分析,同时采用HE染色检测肾脏病理变化,通过Western blot法、免疫组织化学和q-PCR法检测Kim-1,clusterin,LCN2,osteopontin,ceruloplasmin和TIMP1蛋白和m RNA水平。HPLC解析出泽泻醇提物中的8个成分(环氧泽泻烯,泽泻醇F,泽泻醇A,24-乙酰泽泻醇F,泽泻醇A-24-醋酸酯,泽泻醇B,23-乙酰泽泻醇B和23-乙酰泽泻醇C)。HE染色结果显示,高、中剂量的大鼠肾脏间质炎性细胞浸润,肾小管上皮细胞水肿且形态发生变化。与空白组相比,雷公藤和庆大霉素对照组大鼠肾脏Kim-1,clusterin,LCN2,osteopontin和TIMP1蛋白和m RNA水平显著地提高,而ceruloplasmin蛋白和m RNA水平显著地降低;给予泽泻高、中剂量6个月后,大鼠肾脏Kim-1,clusterin,LCN2,osteopontin和TIMP1蛋白和m RNA水平也有显著地提高,而ceruloplasmin蛋白和m RNA水平显著地降低。长期大剂量服用泽泻醇提物可导致雌性大鼠产生肾毒性,其分子机制可能是通过调节Kim-1,ceruloplasmin,clusterin,LCN2,osteopontin和TIMP1等蛋白的表达来实现。

Female rats were fed with ethanol extraction of Alismatis Rhizonm for 6 months to study its nephrotoxicity and molecular mechanism. HPLC was used to determine the components in ethanol extraction of Alismatis Rhizoma. An assessment of renal pathology was determined hy HE staining. Meanwhile Western blot, immunohistochemical assay and q-PCR were used to assess the protein ex- pression and mRNA level of Kim-1, clusterin, LCN2, osteopontin, ceruloplasmin and TIMP1 in rat kidney. Eight components were identified in ethanol extraction of Alismatis Rhizoma. Tubule-interstitial inflammation, renal tubular epithelial cell exfoliation and mor- phological changes of cell were observed in rat kidney. Comparing with control blank group, the protein expression of clusterin, Kim-1, LCN2, osteopontin and TIMP1 in rat kidney was significantly increased while the protein expression of ceruloplasmin was significantly decreased. The mRNA level of Kim-1, TIMP1, osteopontin, clusterin and LCN2 was significantly increased while the mRNA level of eeruloplasmin was significantly decreased. In this study, it was inferred that there is chronic toxicity in kidney by using high dosage of ethanol extraction of Alismatis Rhizoma for a long time. And the underlying molecular mechanism was related to regulate the protein ex- pression of ceruloplasmin, clnsterin, Kim-1, LCN2, osteopontin and TIMP1.