甘草酸二铵对马兜铃酸致肾小管上皮细胞损害保护作用的实验研究

Protective effects of diammonium glycyrrhizinate on the renal tubular epithelial cells injuried by aristolochic acid

目的 探讨甘草酸对马兜铃酸 (aristolochic acid,AA)肾损害的保护作用 ,观察甘草酸是否可以减轻 AA对培养的肾小管上皮细胞的损害 ,并初步探讨其可能机制。方法 5 % FCS RPMI- 16 4 0培养肾小管上皮细胞株 (L L C- PK1) ,采用结晶紫染色法观察细胞增殖 ;荧光染色观察活细胞数目 ;酶动力学检查 L DH活性 ,比色法检测 NAG水平 ;透射电镜观察细胞超微细胞结构。结果 1AA4 0、80、16 0μg/ ml可明显抑制细胞增殖 ,结晶紫 OD值显著减少 ,与无 AA对照组比较 P<0 .0 1。 2甘草酸二铵 (diammoniumglycyrrhizinate,DG)在 5、10、2 5 μg/ ml时结晶紫 OD值与无甘草酸对照组比较 P>0 .0 5 ,甘草酸在 5 0 μg/ ml时 P<0 .0 5 ,10 0 μg/ ml、2 0 0μg/ ml时 P<0 .0 1,提示大剂量甘草酸可改善 AA抑制细胞增殖的作用。 3在 AA4 0μg/ ml作用下随着时间延长 ,无甘草酸组的细胞存活数目逐渐减少 ,而甘草酸 (10 0μg/ ml、2 0 0μg/ ml)作用组 ,活细胞数目明显增多 ,提示高浓度甘草酸对细胞损伤有一定的保护作用。4甘草酸在 5、10、2 5 μg/ ml时 ,L DH释放率、NAG酶水平与无甘草酸对照组比较 P>0 .0 5 ,甘草酸在 5 0 μg/ ml时 P<0 .0 5 ,甘草酸在 10 0μg/ ml、2 0 0μg/ ml时 L DH释放率、NAG酶明显减少 。

Objective To investigate protective effects of diammonium glycyrrhizinate(DG) on the renal tubular epithelial cells injuried by aristolochic acid(AA), and its mechanism.Methods Renal tubular epithelial cell strain (LLC-PK1) was cultured in RPMI-1640/5% FCS with different concentration of DG and AA (40μg/ml). The cells proliferation were measured by the absorb value of crystal purple. The cytotoxicity was determined by the fluorescence staining, the lactate dehydrogenase (LDH) release rate, and the NAG level of the cultured medium were determined. The ultrastructural changes of the cells were studied by transmission electron microscopy. Results ①AA in the concentration of 40,80,160μg/ml can significantly inhibited the proliferation of LLC-PK1, while the absorb value of crystal purple decreased notably, as compared with the no AA group (P<0.01). ②When the cells were cultured in AA (40μg/ml), and DG 5,10,25μg/ml, the absorb value of crystal purple was no different statistically from the no DG control groups (P> 0.05 ), but when the DG 50μg/ml, P<0.05, while 100μg/ml and 200μg/ml, P<0.01. These suggested that increased DG concentration (100μg/ml, 200μg/ml) could meliorate AA's inhibiting proliferation effects. ③In AA 40μg/ml with effective time went on, the number of alive cells were increased distinctively in the groups of high DG concentration. ④Among DG 5,10,25μg/ml and no DG control group, the LDH release rates and NAG level were no different statistically (P> 0.05 ), DG in 100μg/ml and 200μg/ml, the LDH and NAG level were reduced markedly than no DG control group (P<0.01). These suggested that DG may protect cells from AA's cytotoxicity. ⑤The electron microscopy showed the ultra-structural changed dramatically after cells treated with AA(40μg/ml) for 24 hours, especially nuclear changes were notable, and swelling as well as malformed mitochondria etc. to manifest severe cell injuried changes. The severity were gentle in the group of DG (100μg/ml, 200μg/ml). Conclusions DG (100μg/ml, 200μg/ml) exerts certainly protective effects on the renal tubular epithelial cells injuried by AA, to lighten notably the effects on AA inhibiting cell proliferation, to elevate number of alive cells, to alleviate AA's cytotoxicity and the ultra-structural changes.