基于MDCKⅡ细胞的中药成分体外肾毒性评价

Evaluation of nephrotoxicity induced by Chinese herbal ingredients with MDCKⅡ cells in vitro

目的用狗肾近端小管上皮（MDCKⅡ）细胞系研究中药成分的肾毒性，探讨其作为评价中药成分肾毒性体内动物实验替代方法的可能性。方法以氯化汞（HgCl2）为阳性对照，研究已知有肾毒性的马兜铃酸Ⅰ（AAⅠ）和马兜铃酸Ⅱ（AAⅡ），以及未见肾毒性报道的苦参碱、氧化苦参碱和肉豆蔻醚对MDCKⅡ细胞的毒性作用。用MTT法检测细胞存活；倒置显微镜观察细胞形态改变；乳酸脱氢酶（LDH）释放实验检测细胞膜损伤；流式细胞术分析细胞周期及细胞凋亡，用Hoechst33258染色法观察凋亡细胞形态变化。结果AAⅠ和AAⅡ分别与MDCKⅡ细胞作用24，48和72h细胞存活均被明显抑制，AAⅠ的IC50值分别为（63．4±6．6），（44．8±6．0）和（37．3±4．6）μmol·L^-1，AAⅡ的IC50值分另4为（125．7±6．2），（106．7±20．4）和（94．2±9．7）μmol·L^-1；在5—300μmol·L^-1时AAⅠ和AAⅡ分别与MDCKⅡ 细胞作用24h，LDH的释放率明显增高；AAⅠ（75μmol·L^-1）和AAⅡ（150μmol·L^-1）分别与MDCKⅡ细胞作用24h，倒置显微镜下可见细胞收缩变圆，部分细胞破裂脱落；用流式细胞仪和Ho-echst33258染色法观察亦发现，AAⅠ和AAⅡ均可使细胞周期S期阻滞，诱导MDCKⅡ细胞发生凋亡。苦参碱、氧化苦参碱和肉豆蔻醚在2～10mmol·L^-1浓度范围内分别与MDCKⅡ细胞作用24h，对上述指标均无明显影响。结论 MDCKⅡ细胞系对有肾毒性的AAⅠ和AAⅡ和未见肾毒性报道的苦参碱、氧化苦参碱和肉豆蔻醚的反应不同，可用于中药成分体外肾毒性评价。

AIM To evaluate the nephrotoxicity induced by Chinese herbal ingredients with Madin Darby canine kidney type Ⅱ （ MDCK Ⅱ） cells and explore the possibility of using MDCK Ⅱ cells to early assess the nephrotoxicity of Chinese herbal ingredients in vitro. METHODS The cytotoxicities of aristolochic acid Ⅰ （ AA Ⅰ ）, aristolochic acid Ⅱ （ AA Ⅱ ）, matrine, oxymatrine and myristicin were investigated. The cell survival inhibition rate of MDCKⅡ cells after treatment of these drugs were studied by MTr assay, and the morphological changes of MDCK Ⅱ cells were examined with contrast microscopy. Cell membrane injury was observed by detecting lactate dehydrogenase （LDH） release rate. Cell cycle and apoptosis rate were determined by using flow cytometry. The morphological changes of apoptosis cells were examined with Hoechst 33258 staining. RESULTS MDCK Ⅱ cell survival was inhibited after exposed to AA Ⅰ and AA Ⅱ , respectively. The IC50 values of AA Ⅰ for 24, 48 and 72 hwere （63.4±6.6）, （44.8±6.0） and （37.3 ± 4.6 ）μmol· L^-1, respectively. The IC50 values of AA Ⅱ for 24, 48 and 72 h were （125.7±6.2）, （106.7±20.4） and （94.2±9.7 ） μmol· L^- 1, respectively. The LDH release rate of MDCK Ⅱ cells significantly increased after treated with AA Ⅰ or AA Ⅱ for 24 h. MDCK Ⅱcells were contracted and rounded after treated with AA Ⅰ （75 μmol·L - 1 ） or AA Ⅱ （ 150 μmol· L^- 1 ） for 24 h. The further research also indicated that AA Ⅰ and AA Ⅱ affected cell cycle and induced cell apoptosis. After treated with matrine, oxymatrine and myristicin （ 2- 10 mmol·L^-1） for 24 h, all above the parameters of MDCK Ⅱ cells had no obvious changes. CONCLUSION MDCKⅡ cells have different responses to AAⅠ and AAⅡ with nephrotoxicity, and matrine, oxymatrine and myristicin without reports of nephrotoxicity. MDCK Ⅱ cells may be useful for assessment of nephrotoxicity of Chinese herbal ingredients in vitro.