| Introduction Vanherweghen reported that a cluster of patients with interstitial renal fibrosis rapidly progressing to end-stage renal disease after having followed a slimming regimen containing powdered extracts of Chinese herbs in Brussels, Belgium in 1993. The herbal product was found to contain Aistoltchia fangchi. The nephropathy induced by Chinese medicinal herbs that contain aristolochic acid (AA) has been concerned throughout the world. The term "Chinese Herb Nephropathy (CHN)"has been widely used in scientific nephrology publications. However, the researchers in China thought that "Aristolochic Acid Nephropathy (AAN)"shoud be used. There were some related reports about AAN in China, Japan and England. In the 1980s, studies had found that aristolochic acid(AA) is a nephrotoxic and carcinogenic extract from aristolochia herbs. IARC had ranked herbs containing AA as carcinogens to human in 2002. Studies in mechanisms of AAN showed that AA induced renal tubular necrosis or apoptosis, tubular epithelial-myofibroblast transdifferentiation, AA-DNA adducts formation and so on. But it is unknown whether there was DNA oxidative damage in the kidney tissue that injured by AA. In this study, LLC-PK1 cells and rats were used as experimental system and had investigated the mechanisms of AA-induced nephrotoxicity from the aspects of oxidative stress and DNA damage. It would provide help to reveal the mechanism of its nephrotoxicity and carcinogenicity. Methords In vitro test, LLC-PK1 cells were used as experimental system. LLC-PK1 cells were seeded in culture plate and incubated at 37℃for at the least 24h prior to use. Then serial amounts of AA were added to the cells. Cytotoxicity was determined by MTT color assay. ROS in cells was measured by fluorescence method. DNA strand-breaks were determined by single cell gel electrophoresis (SCGE) assay. In vivo, prepared Manshurian Duthmanspipe Stem(MDS) decoction with traditional method and determined its contents of AA. 36 male adult Sprague-Dawley rats were randomly divided into 6 groups. Rats were treated with MDS (50g/kg body weight) orally twice a day for different days: MDS for 3 days group, MDS for 5 days group, MDS for 7 days group and corresponding three control groups. On Day 4, Day 6 and Day 8 , the rats were sacrificed. The following indices were observed and determined: body weight, kidney index and histological examination, contents of blood urea nitrogen (BUN). Changes in malondialdehyde (MDA) and DNA strand-breaks in kidney. Results MTT test showed there were significant difference in IC50 values in LLC-PK1 cells treated with AA for different time, i.e. IC50 for 12h, 24h and 48h was 37.7μg/mL, 14.3μg/mL and 5.5μg/mL , respectively. ROS was increased significantly after different concentrations of AA treated LLC-PK1 for 1 h. SCGE assay showed AA at concentrations of above 20μg/mL can induce DNA strand-breaks. In vivo test, MDS , 50g/kg per day orally, the dose equivalent to AA 97mg/kg, for 3 consecutive days caused kidney damage in rats, for 5 and 7 consecutive days caused noticeable kidney damage and characterized by body weight loss, increase of kidney index and contents of BUN and MDA, which have significance compared with the control groups. SCGE assay observed there were DNAstrand-breaks in all treated groups. Histologically, edema, denaturation and necrosis in renal tubular epithelium were observed. The 8-OH-dG positive cells obviously increased in treated groups. Conclusion AA-induced cytotoxicity and increased genetation of ROS in LLC-PK1 cells. AA induced DNA strand-breaks in vitro and in vivo test. MDS, 50g/kg per day orally, the dose equivalent to AA 97mg/kg, for 3 consecutive days caused kidney damage in rats, in this study, the kidney damage was the most serious on Day 7. There were 8-OH-dG expression in kidney from treated groups. These results suggest that AA-induced increase of generation of ROS, and subsequent oxidative damage for lipid and DNA may contribute to nephrotoxicity and carcinogenicity induced by AA. |
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