OBJECTIVE: 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 AAl 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, suggesting 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.
OBJECTIVE: 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 AAl 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, suggesting 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.
Authors: Tao Chen; Lei Guo; Lu Zhang; Leming Shi; Hong Fang; Yongming Sun; James C Fuscoe; Nan Mei Journal: BMC Bioinformatics Date: 2006-09-06 Impact factor: 3.169