Wang Chun-Fei, Ma Liang, Feng Liang, Yin Mei-Rong, Gu Jun-Fei, Jia Xiao-Bin
School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230031, China.
Key Laboratory of New Drug Delivery System of Chinese Meteria Medica, Jiangsu Provincial Academy of Chinese Medicine, Nanjing 210028, China.
Zhongguo Zhong Yao Za Zhi. 2016 Feb;41(3):490-497. doi: 10.4268/cjcmm20160322.
To evaluate the nephrotoxicity of total terpenoids from Alismatis Rhizoma on human kidney proximal tubular cells (HK-2), explore the iraction in inducing apoptosis of HK-2 cells, and provide reference for the research of controversial nephrotoxicity of total terpenoids from Alismatis Rhizoma, HK-2 cells were used and cells viability was measured by MTT colorimetric method. An assessment of cells apoptosis was also conducted by using flow cytometry. Meanwhile western blot assay was used to detect the protein expressions of caspase-3, Bcl-2, Bcl-xl, Kim-1, clusterin and TFF-3. At last, q-PCR was used to detect the mRNA expressions of caspase-3, Bcl-2, Bcl-xl, Kim-1, clusterin and TFF-3. The flow cytometry results showed that cells apoptosis rate was (37.48±1.76)%, (26.91±1.91)% and (25.61±2.05)% respectively after treating with total terpenoids (6.25×10-5, 3.125×10-5, 1.562 5×10-5 g•mL⁻¹). Western blot results showed that Bcl-2 and Bcl-xl protein levels were significantly decreased after treating with total terpenoids (6.25×10-5, 3.125×10-5, 1.562 5×10-5 g•mL⁻¹), while the protein expression of caspase-3 was significantly increased. q-PCR results were the same with western blot results, that mRNA expressions of Bcl-2 and Bcl-xl were significantly decreased while mRNA expression of caspase-3 was significantly increased after treating with total terpenoids (6.25×10-5, 3.125×10-5, 1.562 5×10-5 g•mL⁻¹). Western blot results and q-PCR results showed that both mRNA and protein expressions of Kim-1, clusterin and TFF-3 were significantly increased after treating with total terpenoids from Alismatis Rhizoma (6.25×10-5, 3.125×10-5, 1.562 5×10-5 g•mL⁻¹). HK-2 cells in vitro evaluation results showed that, total terpenoids from Alismatis Rhizoma may have nephrotoxicity effect, but further study is still needed for verification; meanwhile, they could induce HK-2 cells apoptosis, providing basis for nephrotoxicity study and safe application of Alismatis Rhizoma.
为评价泽泻总萜对人肾近端小管上皮细胞（HK-2）的肾毒性，探讨其诱导HK-2细胞凋亡的作用，为泽泻总萜存在争议的肾毒性研究提供参考，采用HK-2细胞，运用MTT比色法检测细胞活力。同时采用流式细胞术评估细胞凋亡情况。此外，运用蛋白质免疫印迹法检测半胱天冬酶-3、Bcl-2、Bcl-xl、Kim-1、簇集素和三叶因子3的蛋白表达。最后，采用实时荧光定量聚合酶链反应检测半胱天冬酶-3、Bcl-2、Bcl-xl、Kim-1、簇集素和三叶因子3的mRNA表达。流式细胞术结果显示，经泽泻总萜（6.25×10⁻⁵、3.125×10⁻⁵、1.562 5×10⁻⁵ g•mL⁻¹）处理后，细胞凋亡率分别为（37.48±1.76）%、（26.91±1.91）%和（25.61±2.05）%。蛋白质免疫印迹结果显示，经泽泻总萜（6.25×10⁻⁵、3.125×10⁻⁵、1.562 5×10⁻⁵ g•mL⁻¹）处理后，Bcl-2和Bcl-xl蛋白水平显著降低，而半胱天冬酶-3的蛋白表达显著增加。实时荧光定量聚合酶链反应结果与蛋白质免疫印迹结果一致，即经泽泻总萜（6.25×10⁻⁵、3.125×10⁻⁵、1.562 5×10⁻⁵ g•mL⁻¹）处理后，Bcl-2和Bcl-xl的mRNA表达显著降低，而半胱天冬酶-3的mRNA表达显著增加。蛋白质免疫印迹结果和实时荧光定量聚合酶链反应结果显示，经泽泻总萜（6.25×10⁻⁵、3.12⁵×10⁻⁵、1.562 5×10⁻⁵ g•mL⁻¹）处理后，Kim-1、簇集素和三叶因子3的mRNA和蛋白表达均显著增加。体外HK-2细胞评价结果表明，泽泻总萜可能具有肾毒性作用，但仍需进一步研究验证；同时，其可诱导HK-2细胞凋亡，为泽泻肾毒性研究及安全应用提供依据。
