钙拮抗剂对马兜铃酸Ⅰ所致LLC-K1细胞凋亡及细胞内钙离子浓度的作用

VARIATION OF INTRACELLULAR FREE CALCIUM CONCENTRATION IN ARISTOLOCHIC ACID I INDUCED APOPTOSIS IN LLC PK1 CELLS AND INHIBITION OF APOPTOTIC DAMAGE BY LACIDIPINE

目的：研究马兜铃酸Ｉ（ＡＡＩ） 所致ＬＬＣＰＫ１ 细胞凋亡时细胞内游离钙离子浓度（［ Ｃａ２＋ ］ｉ） 的变化和钙拮抗剂拉西地平对细胞凋亡的作用。 方法：应用已建立的ＡＡＩ 所致ＬＬＣＰＫ１ 细胞凋亡模型，使用光镜、ＡｎｎｅｘｉｎＶＦｌｕｏｓ 凋亡检测试剂盒和流式细胞仪检测细胞凋亡，经Ｆｌｕｏ３／ＡＭ 染色，激光扫描共聚焦显微镜测定平均［Ｃａ２＋］ｉ 。 结果：０－０４ ｇ／Ｌ 的ＡＡＩ 作用２４ｈ 可致ＬＬＣＰＫ１ 细胞出现明显细胞凋亡和平均［Ｃａ２＋］ｉ 显著升高；拉西地平（１００ ｎｇ／Ｌ，１ μｇ／Ｌ） 可显著抑制ＡＡＩ 所致的［Ｃａ２ ＋］ｉ 升高并能降低凋亡细胞比例。 结论：在体外条件下ＡＡＩ 所致ＬＬＣＰＫ１ 细胞凋亡的发生可能与［Ｃａ２＋］ｉ 升高有关；拉西地平可能通过抑制［ Ｃａ２＋］ｉ 升高而抑制ＡＡＩ 所致的凋亡。

To examine the changes of intracellular free calcium concentration([Ca 2+ ] i)in aristolochic acid I(AAI) induced apoptosis in LLC PK1 cells,and the effects of Lacidipine(a calcium antagonist)on the changes of [Ca 2+ ] i and apoptosis induced by AAI. METHODOLOGY LLC PK1 cells were treated in different groups:(a)normal,without treatment;(b)with AAI alone(0 04 g/L);(c)with Lacidipine alone(10,10 2,10 3 ng/L);(d)with AAI(0 04 g/L)plus Lacidipine(10 or 10 2 or 10 3 ng/L).Mean[Ca 2+ ] i was measured by laser confocus microscopy using Fluo 3/AM staining.Light microscopy,Annexin V Flous apoptosis detection kit and flow cytometry using propidium iodiode staining were used to identify or quantify the apoptosis of LLC PK1 cell. RESULTS AAI induced apoptosis of LLC PK1 cells was found to be dose dependent.Mean[Ca 2+ ] i was significantly higher in cells treated with AAI than that in normal cells (58 01±18 89 vs 22 66±4 78, P <0 001).In group treated with AAI plus Lacidipine (10 2 or 10 3ng/L),mean[Ca 2+ ] i was significantly lower than that treated with AAI alone (35 47±10 16 ,28 55±12 85 vs 58 01±18 89, P <0 001).And the apoptotic cells ratios in group treated with AAI plus Lacidipine (10 2,10 3 ng/L)were also significantly lower than that treated with AAI alone(19 0%,27 8% vs 34 7%, P <0 001). CONCLUSION The increase of [Ca 2+ ] i in AAI treated LLC PK1 cells may be related to AAI induced apoptosis.Lacidpine may reduce AAI induced apoptosis by inhibiting increase of [Ca 2+ ] i in LLC PK1 cell.