| Traditional Chinese medicine and compound preparations containing AA are variety, and there still exist clinical application, it is necessary to carry out the safety evaluation of renal toxicity. Aristolochia kwangsiensis Chun et How extract component is one important part of the Tongdi capsule.High Content Screening (HCS) can dynamic screens toxic substances at a cellular level, HCS can not only illustrate the interaction relationship between test drug and targets, but also can predict the test drug toxicity by observed the change of morphology of cell. HCS can be used to predict toxicity early, rapid and high throughput. Therefore in this study, the renal toxicity of Aristolochia kwangsiensis was researched. Based on HK-2 cells by MTT assay, LDH release assay and High Content Analysis of renal toxicity experiments carried out on different components of Aristolochia kwangsiensis. Compare the acute toxicity and renal toxicity of different compenents of Aristolochia kwangsiensis to provide experimental date for safe use in clinal, including 95% alcohol extract components,75% alcohol extract components, and water extract components.The study was divided into three parts:The first part, establish a high content screening method in vitro for kidney injury based HK-2 cells;The second part, different extraction components of Aristolochia kwangsiensis in vitro study of renal toxicity. The third part, renal toxicity study including acute toxicity in mice andacute renal failure in rats.The First Part:To establish a method for predicting toxicity based HK-2 cells by High Content Screening analysis technologyThe results show:DDP and AA could be detected clearly HK-2 cells toxicity. To DDP, the earliest indicator of changes was to reduce the permeability of cell membrane. To AA, the earliest indicators of changes were the lowering of the mitochondrial membrane potential and the raising of the cell membrane permeability. Rosiglitazone even at a concentration of maximus plasma concentration of 1000 times the situation still couldn’t detect significant HK-2 renal cell toxicity. The results were in keeping with the actual situation of the clinical consensus, the above results suggested that HK-2 cells could be used for renal cell toxicity screening and provide a more efficient method of cytotoxicity in vitro.The Second Part:The nephrotoxicity study in vitro based on HK-2 cells of different extraction components.First:The influence of different components to cell viability by MTT assayThe results show:MTT assay was used to examine cell proliferation under the different polar parts treated 48h. The IC50 value of AA was 52.58ug·mL-1, and the IC50 value of 95% and 75% alcohol extraction components were respectively 59.43 ug·mL-1 and 122.60 ug·mL-1. The ability of cell proliferation was AA> 95% alcohol extraction components>75% alcohol extraction components> water extraction.Second:Influence study of different components to cell membranes by LDH releasing assay.The results show:AA could increase the cell LDH release rate gradually at a concentration within 7.81～62.5mg·mL-1.95% alcohol extraction components could increase the cell LDH release rate at the concentration of 125 mg·mL-1, and no significant increase of LDH was found in the other concentrations. The 75% alcohol extraction components and water extraction components couldn’t increase the rate of cellular release of LDH in the concentration range.Third:Cytotoxicity analysisof different components based on HK-2 cells by HCSThe results show:Cytotoxicity of 95% alcohol extraction components can be detected at a lower concentration, and its toxicity was stronger than that of the 75% alcohol extraction group. Early toxicity of 95% and 75% alcohol extraction group showed the injury of cell membrane integrity, with the concentration would lead to increased apoptosis and cell proliferation was significantly inhibited. It is suggested that the toxic components of 95% alcohol extraction and 75% alcohol extraction may be the same, but the content is different. Cytotoxicity of water extraction components was not obvious, and the most sensitive change is mitochondrial membrane potential. This is different with alcohol extraction components,It was suggested that the toxic components of the water extraction group were less than alcohol extraction components, and the toxic components were different from them. The HCS results were consistent with the experimental results of MTT assay, LDH assay and the acute toxicity in ICR mice, but compared with MTT and LDH test HCS was more sensitive and intuitive, and compared with animals experiments HCS was more simple and efficient. The use of HCS technology could be achieved on different extraction components of traditional Chinese medicine for the analysis of cytotoxicity study in vitro. HCS could not only to be used to compare the toxicity of different components, but also explore the mechanisms of toxicity based on the indicators change.The Third Part:Aristolochia kwangsiensis renal toxicity studyFirst:Comparative study on acute toxicity of different components of Aristolochia kwangsiensis in mice.The results show:The deaths mainly occurred in 4-7 d. The LD50 of 95% alcohol extraction components and 75% alcohol extraction components in Aristolochia kwangsiensis were 47.97g·kg-1 and 59.98g·kg-1. The all-components MTD results calculated in accordance with crude drug content was 320g·kg-1. The acute toxicity of different components in Aristolochia kwangsiensis were:95% alcohol extraction components> 75% alcohol extraction components> all components> water extraction components, and the sequence of components and content of AA keep consistent.Second:The 95% alcohol extraction components of Aristolochia kwangsiensisinduce acute renal failure in ratsThe results show:In the 95% alcohol extraction components group, the body weight and renal coefficient of rats were higher compared with the control group. Blood biochemical indexes such as urea nitrogen (Urea), creatinine (CR),alkaline phosphatase (ALP) and y-glutamyl transpeptidase (y-GT) changed as compared with the control group, and with dose-effect relationship.Urine NAG content changed as compared with the control group. There was obviously tubular injury showed in the histopathologic examination. Conclusion: The short-term administration of 95% alcohol extraction components to rats could induce obvious nephrotoxicity injury, and with dose-effect relationship. NAG enzyme sensitivity was much higher than other indicators. |
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