大黄中游离蒽醌对HK-2细胞系的毒性作用研究

Cytotoxicity of free anthraquinone from Radix et Rhizoma Rhei to HK-2 Cells

目的:观察大黄中大黄素等游离蒽醌对人近曲小管上皮细胞(HK-2)的细胞毒性作用及毒性作用机制。方法:体外培养HK-2细胞,利用MTT法评价大黄素等对HK-2细胞的增殖抑制作用,观察不同浓度药物对细胞形态和LDH释放率的影响,利用TUNEL染色检测大黄素等对HK-2细胞凋亡的影响,细胞线粒体膜电位和活性氧(ROS)生成的检测采用流式细胞技术,分别以DCFH-DA和罗丹明-123为荧光探针。结果:大黄素、大黄酸和大黄素甲醚作用HK-2细胞48 h后,明显抑制细胞增殖,其IC50值分别为130.65,82.97和76.02μmol.L-1,芦荟大黄素轻微抑制HK-2细胞增殖,大黄酚作用不明显。大黄素、大黄酸和大黄素甲醚能够导致细胞皱缩和空泡化,LDH漏出率增加以及促使细胞凋亡,3种蒽醌均使细胞的线粒体膜电位降低,而ROS生成减少,80μmol.L-1大黄素预处理细胞能够明显降低H2O2引起的ROS升高。结论:大黄素、大黄酸和大黄素甲醚可能是大黄中主要的肾毒性物质,能够引起HK-2细胞的凋亡,其机制可能涉及线粒体膜电位途径,但是不包括ROS生成途径。

Objective：To assess the cytotoxic effect of free anthraquinones from Radix et Rhizoma Rhei on human proximahubular epithelial cell line （HK-2）. Methods： MTT assay was used to evaluate the IC50 of anthraquinone on HK-2 cells. The changes of cell morphology and LDH leakage were observed. The apoptosis effect was detected by TUNEL assay. 2＇, 7＇-dichlorofluorescein diacetate and rhodamine 123 were used as fluoresent probes to measure the cytosolic reactive oxygen species （ROS） content and mitochondrial membrane potential （MMP） , respectively, by flow cytometer. Results：Emodin, rhein and physcion significantly inhibited the proliferation of HK-2 cells after 48 hours cultures, showing IC50 values of 130.65,82.97 and 76.02umol· L^-1 , respectively. Aloe-emodin had mild inhibitory effects and chrysophanol showed no effects on the proliferation of HK-2 cells. Emodin-, rhein- and physciontreated HK-2 cells manifested cellular shrinkage and vacuolation and increased LDH leakage and apoptosis at 40umol·L^-1. All of the three anthraquinones reduced MMP, leading to decreases of cytosolic ROS level. The emodin-treated HK-2 cells significantly decreased the H2O2 -induced intracellular ROS level. Conclusion： Emodin, rhein and physcion as major nephrotoxic components from Radix et Rhizoma Rhei induced apoptosis of HK-2 cells by modulating MMP rather than the ROS production.