补骨脂酚及其与补骨脂素合用对HK-2细胞的毒性及其机制

目的研究补骨脂酚(bakuchiol)及补骨脂酚与补骨脂素(psoralen)合用的肾细胞毒性,并初步探讨其毒性机制。方法采用人肾近曲小管上皮细胞(HK-2),研究补骨脂酚、补骨脂酚与补骨脂素合用以及在大鼠肝匀浆S9作用下,对HK-2细胞的毒性作用。实验分为空白对照、溶剂对照、阳性对照马兜铃酸Ⅰ(AAⅠ)、补骨脂素5μmol.L-1、补骨脂酚5,10,20,30和40μmol.L-1,补骨脂酚与补骨脂素合用(20+5),(30+5),(40+5)μmo.lL-1组。MTT法检测细胞存活率;乳酸脱氢酶(LDH)释放实验检测细胞膜损伤;倒置显微镜观察细胞形态改变;AnnexinⅤ/PI染色法检测细胞凋亡;PI染色法检测细胞周期。结果补骨脂素5μmol.L-1作用于HK-2细胞,未观测到毒性作用;补骨脂酚、补骨脂酚与补骨脂素合用分别与HK-2细胞作用4,24,48和72h,在较高浓度下(20,30和40μmol.L-1)细胞存活被明显抑制(P<0.01),补骨脂酚24,48和72h的IC50值分别为(26.4±4.8),(21.8±0.6)和(24.1±0.8)μmol.L-1;在大鼠肝匀浆S9作用下,补骨脂酚的细胞毒性明显降低(P<0.01)。较高浓度(30和40μmol.L-1)的补骨脂酚、补骨脂酚与补骨脂素合用分别与HK-2细胞作用24h,LDH的释放率明显增高(P<0.01),呈浓度依赖性;倒置显微镜下观察细胞形态变化,HK-2细胞随着浓度的增高,作用时间的延长,细胞收缩、变小变圆和破裂脱落现象越明显。补骨脂酚40μmol.L-1、补骨脂酚与补骨脂素合用(20+5),(30+5)和(40+5)μmol.L-1可诱导HK-2细胞发生不同程度的凋亡,并且引起明显的细胞死亡。补骨脂酚10,20,30和40μmol.L-1、补骨脂酚与补骨脂素合用均影响细胞周期,主要表现为G2期减少,G1和S期增加或减少。结论补骨脂酚对HK-2细胞有明显的毒性,与补骨脂素合用不能降低其毒性,经大鼠肝匀浆S9作用后,补骨脂酚的细胞毒性明显降低。补骨脂酚对HK-2细胞毒性作用机制可能为:①直接对细胞膜造成损伤;②引发细胞凋亡;③抑制细胞内DNA合成,阻滞细胞有丝分裂,抑制细胞增殖。

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~(-1)groups 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 G2 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.