Cytotoxic effect and mechanism of bakuchiol and bakuchiol combined with psoralen on HK-2 cell.

OBJECTIVE To study the nephrotoxicity induced by bakuchiol alone and bakuchiol combined with psoralen and to explore its mechanism.METHODS The cytotoxicities of bakuchiol and bakuchiol combined with psoralen were investigated using human renal tubular epithelial cell lines(HK-2),in presence or absence of hepatic S9 mixture.The HK-2 cells were exposed to culture medium alone(blank control),0.5% DMSO(vehicle control),aristolochic acid Ⅰ(AAⅠ;positive control),psoralen 5 μmol·L-1 group,bakuchiol 5,10,20,30 and 40 μmol·L-1 groups,and bakuchiol+psoralen(20+5),(30+5) and(40+5)μmol·L-1 groups,respectively.The cell viabilities were examined by MTT assay;cell membrane injuries were examined by detecting lactate dehydrogenase(LDH) release rate;and the morphological changes in HK-2 cells were observed with contrast microscope.The rate of cell apoptosis was detected by AnnexinⅤ/PI staining,and cell cycle was detected by PI staining with flow cytometry.RESULTS No cytotoxicity was found in psoralen 5 μmol·L-1 group.The HK-2 cell viabilities were significantly reduced after 4,24,48 and 72 h of exposure to either bakuchiol 20,30 and 40 μmol·L-1groups or bakuchiol+psoralen(20+5),(30+5) and(40+5)μmol·L-1 groups in a time-and concentration-dependent manner.The IC_50 values of bakuchiol were(26.4±4.8),(21.8±0.6) and(24.1±0.8)μmol·L-1 for 24,48 and 72 h exposure,respectively.The cytotoxicity of bakuchiol was significantly decreased in presence of hepatic S9 mixture.The LDH release rate of HK-2 cell increased significantly after 24 h of exposure to bakuchiol 20,30 and 40 μmol·L-1 or bakuchiol+psoralen groups.With the concentration and time increasing,the HK-2 cells became more and more contracted and rounded.In bakuchiol 40 μmol·L-1 or bakuchiol+psoralen(20+5),(30+5) and(40+5)μmol·L-1 groups,HK-2 cells showed apoptotic characters.In bakuchiol or bakuchiol+psoralen groups,apoptotic cells significantly increased and cells in G_2 phase markedly decreased.CONCLUSION Bakuchiol has a significant cytotoxicity in HK-2 cells,and combined with psoralen can not decrease its toxicity.The cytotoxicity of bakuchiol is significantly reduced in the presence of hepatic S9 mixture.The possible mechanisms of the renal cytorotoxicity of bakuchiol are as follows:① direct damage to the cell membrane;② inducing cell apoptosis;③ inhibiting intracellular DNA synthesis and block cell mitosis and proliferation.