基于HK-2细胞的泽泻萜类组分体外肾毒性评价及其诱导细胞凋亡作用的探究

Evaluation of nephrotoxicity induced by total terpenoids from Alismatis Rhizoma on HK-2 cells in vitro and its induction of apoptosis

用人肾小管上皮细胞(HK-2)评价泽泻萜类组分的肾毒性作用,同时探究其诱导HK-2细胞凋亡的作用,为存在争议的泽泻肾毒性研究提供参考。采用肾小管上皮细胞株,运用MTT比色法测定细胞活力,流式细胞术分析细胞凋亡,同时用Western blot检测HK-2细胞caspase-3,Bcl-2,Bcl-xl,Kim-1,clusterin,TFF-3蛋白表达水平,最后用q-PCR检测caspase-3,Bcl-2,Bcl-xl,Kim-1,clusterin,TFF-3 mRNA的表达。流式细胞仪检测结果表明,泽泻萜类组分(6.25×10-5,3.125×10-5,1.562 5×10-5g·m L-1)给药组细胞凋亡率分别为(37.48±1.76)%,(26.91±1.91)%,(25.61±2.05)%。同时,Western blot结果说明泽泻萜类组分不同剂量给药组中Bcl-2和Bcl-xl蛋白水平都有显著地降低,而caspase-3蛋白水平显著地提高。q-PCR结果与Western blot结果一致,泽泻萜类组分不同剂量给药组中Bcl-2和Bcl-xl的mRNA水平都有显著的降低,而caspase-3 mRNA水平有显著的提高。Western blot和q-PCR结果说明,泽泻萜类组分不同剂量给药组中细胞的clustrein,Kim-1和TFF-3蛋白表达和mRNA水平均有显著地提高。HK-2细胞系体外评价结果表明,泽泻萜类组分具有肾毒性作用,但是还需要进一步的研究证明,同时可诱导HK-2细胞凋亡,为泽泻肾毒性研究以及临床用药安全提供依据。

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- 5g·m L- 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- 5g·m L- 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- 5g·m L- 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- 5g· m L- 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.