To examine the effect of different concentrations of aristolochic acid I (AAI) in inducing apoptosis of cultured porcine renal cell line LLC PK1 and to investigate the relationship between intracellular free calcium ...To examine the effect of different concentrations of aristolochic acid I (AAI) in inducing apoptosis of cultured porcine renal cell line LLC PK1 and to investigate the relationship between intracellular free calcium concentration ([Ca ++ ]i) and LLC PK1 apoptosis induced by AAI and the influence of a calcium antagonist, lacidipine on apoptosis and [Ca ++ ]i Methods LLC PK1 cells were treated in different groups: a the normal group without treatment; b the group with AAI alone (0 01?g·L 1 , 0 02?g·L 1 , 0 04 ?g·L 1 , 0 08?g·L 1 ); c the group with lacidipine alone (10?ng·L 1 , 10 2 ?ng·L 1 , 10 3?ng·L 1 ); d the group with AAI (0 04?g·L 1 ) plus lacidipine (10?ng·L 1 , 10 2? ng·L 1 , 10 3?ng·L 1 ) Light microscopy, agarose gel electrophoresis, Annexin V Flous apoptosis detection kit and flow cytometry using propidium iodide staining to identify or quantify the apoptosis of LLC PK1 cells Mean [Ca ++ ]i was measured by laser confocus microscopy using Fluo 3/AM staining Results A series of morphologic changes that were characteristic of apoptosis, Annexin V Flous staining positive apoptotic cells and “DNA ladder' were identified in AAI (0 02?g·L 1 -0 08?g·L 1 ) treated LLC PK1 cells Quantitative analysis of apoptotic cells showed that the percentage of apoptotic cells in AAI (0 02?g·L 1 , 0 04?g·L 1 or 0 08?g·L 1 ) group was significantly higher than that in normal group (5 3%, 48 5%, 78 7% vs 2 6%, P <0 001) Mean [Ca ++ ]i was significantly higher in cells treated with AAI (0 04?g·L 1 ) than that in normal cells (58 01±18 89 vs 22 66±4 78, P <0 001) In group treated with AAI plus lacidipine (102?ng·L 1 , 103?ng·L 1 ), mean [Ca ++ ]i was significantly lower than that treated with AAI alone (35 47±12 85, 28 55±10 16 vs 58 01±18 89, P <0 001) And the percentage of apoptotic cells in group treated with AAI plus lacidipine (10 2?ng·L 1 , 10 3?ng·L 1 ) was also significantly lower than that treated with AAI alone (19 0%, 27 8% vs 34 7%, P <0 001) Conclusions High concentrations of AAI may induce apoptosis of LLC PK1 cells The mean [Ca ++ ]i in AAI treated LLC PK1 cells was increased significantly, sugviping that the increase of [Ca ++ ]i may be related to apoptosis in LLC PK1 cells Lacidipine may decrease the raised mean [Ca ++ ]i levels caused by AAI and the percentage of apoptotic cells, and lacidipine may ameliorate AAI induced apoptotic damage by inhibiting the increase of [Ca ++ ]i in LLC PK1 cells展开更多
文摘To examine the effect of different concentrations of aristolochic acid I (AAI) in inducing apoptosis of cultured porcine renal cell line LLC PK1 and to investigate the relationship between intracellular free calcium concentration ([Ca ++ ]i) and LLC PK1 apoptosis induced by AAI and the influence of a calcium antagonist, lacidipine on apoptosis and [Ca ++ ]i Methods LLC PK1 cells were treated in different groups: a the normal group without treatment; b the group with AAI alone (0 01?g·L 1 , 0 02?g·L 1 , 0 04 ?g·L 1 , 0 08?g·L 1 ); c the group with lacidipine alone (10?ng·L 1 , 10 2 ?ng·L 1 , 10 3?ng·L 1 ); d the group with AAI (0 04?g·L 1 ) plus lacidipine (10?ng·L 1 , 10 2? ng·L 1 , 10 3?ng·L 1 ) Light microscopy, agarose gel electrophoresis, Annexin V Flous apoptosis detection kit and flow cytometry using propidium iodide staining to identify or quantify the apoptosis of LLC PK1 cells Mean [Ca ++ ]i was measured by laser confocus microscopy using Fluo 3/AM staining Results A series of morphologic changes that were characteristic of apoptosis, Annexin V Flous staining positive apoptotic cells and “DNA ladder' were identified in AAI (0 02?g·L 1 -0 08?g·L 1 ) treated LLC PK1 cells Quantitative analysis of apoptotic cells showed that the percentage of apoptotic cells in AAI (0 02?g·L 1 , 0 04?g·L 1 or 0 08?g·L 1 ) group was significantly higher than that in normal group (5 3%, 48 5%, 78 7% vs 2 6%, P <0 001) Mean [Ca ++ ]i was significantly higher in cells treated with AAI (0 04?g·L 1 ) than that in normal cells (58 01±18 89 vs 22 66±4 78, P <0 001) In group treated with AAI plus lacidipine (102?ng·L 1 , 103?ng·L 1 ), mean [Ca ++ ]i was significantly lower than that treated with AAI alone (35 47±12 85, 28 55±10 16 vs 58 01±18 89, P <0 001) And the percentage of apoptotic cells in group treated with AAI plus lacidipine (10 2?ng·L 1 , 10 3?ng·L 1 ) was also significantly lower than that treated with AAI alone (19 0%, 27 8% vs 34 7%, P <0 001) Conclusions High concentrations of AAI may induce apoptosis of LLC PK1 cells The mean [Ca ++ ]i in AAI treated LLC PK1 cells was increased significantly, sugviping that the increase of [Ca ++ ]i may be related to apoptosis in LLC PK1 cells Lacidipine may decrease the raised mean [Ca ++ ]i levels caused by AAI and the percentage of apoptotic cells, and lacidipine may ameliorate AAI induced apoptotic damage by inhibiting the increase of [Ca ++ ]i in LLC PK1 cells
