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目的探讨原花青素B2(PC-B2)与黄曲霉毒素B_1(AFB_1)对人胚胎肝细胞L-02细胞色素P450(CYP)亚酶1A2、3A4活力及其基因表达的影响。方法 L-02细胞体外培养后分空白对照、溶剂对照、AFB_1染毒、PC-B2处理和PC-B2干预共5组,其中PC-B2处理分为3个亚组,分别采用3、10、30μg/ml PC-B2干预,PC-B2干预分为3各亚组,分别采用3、10、30μg/ml PC-B2预处理12 h后,再加不同浓度PC-B2与AFB_1干预24 h。测定各组细胞的CYP1A2、CYP3A4酶活力以及CYP1A2、CYP3A4基因的mRNA表达水平;测定细胞存活率,倒置荧光显微镜观察细胞形态。结果与空白对照组相比,AFB_1染毒组细胞存活率降低(P<0.05),胞膜结构不清,漂浮细胞增多,CYP1A2、CYP3A4酶活力及其mRNA表达水平明显升高(P<0.05)。与空白对照组相比,3、10μg/ml PC-B2处理组细胞相关指标无明显变化,但30μg/ml PC-B2处理组的细胞存活率升高,CYP1A2、CYP3A4酶活力下降(P<0.05)。与AFB_1染毒比较,30μg/ml PC-B2干预组的细胞存活率升高(P<0.05),各浓度PC-B2干预组的细胞CYP1A2、CYP3A4酶活力及基因表达均降低(P<0.05),且干预浓度越高,CYP1A2、CYP3A4酶活力及基因表达越低(P<0.05)。结论 AFB_1能明显诱导肝细胞CYP1A2、CYP3A4酶活力及其mRNA表达,促使肝细胞凋亡,PC-B2干预可促进肝细胞生长并抑制上述CYP二个亚酶的活力及其基因表达,提示PC-B2可能通过抑制I相代谢酶CYP对AFB_1的活化而对肝细胞有良好保护作用。
Objective To investigate the effects of proanthocyanidin B2 (PC-B2) and aflatoxin B_1 (AFB_1) on the activity and gene expression of cytochrome P450 (CYP) 1A1 and 3A4 in human embryonic hepatocytes L-02. Methods L-02 cells were divided into blank control group, solvent control group, AFB 1 group, PC-B2 group and PC-B2 group. The PC-B2 group was divided into 3 subgroups, The PC-B2 intervention group was divided into three subgroups: 30μg / ml PC-B2 group and 3,10,30μg / ml PC-B2 group for 12 hours respectively. The activity of CYP1A2 and CYP3A4 and the mRNA expression of CYP1A2 and CYP3A4 in each group were determined. The cell viability was measured and the cell morphology was observed by inverted fluorescence microscope. Results Compared with the blank control group, the survival rate of AFB_1 cells was decreased (P <0.05), the membrane structure was unclear and the number of floating cells increased. The activities of CYP1A2 and CYP3A4 and their mRNA expressions were significantly increased (P <0.05) . Compared with the blank control group, there was no significant change in the relevant parameters of the cells treated with 3 and 10μg / ml PC-B2, but the cell viability increased and the activities of CYP1A2 and CYP3A4 decreased (P <0.05 ). Compared with AFB_1 group, the cell viability in 30μg / ml PC-B2 group was significantly increased (P <0.05). The activity and gene expression of CYP1A2 and CYP3A4 in cells treated with PC-B2 at various concentrations were decreased (P <0.05) , And the higher the intervention concentration, the lower the CYP1A2, CYP3A4 activity and gene expression (P <0.05). Conclusion AFB-1 can obviously induce the activity and mRNA expression of CYP1A2 and CYP3A4 in hepatocytes, and induce the apoptosis of hepatocytes. PC-B2 can promote the growth of hepatocytes and inhibit the activity and expression of CYP2A2 and CYP2A1, B2 may have a good protective effect on hepatocytes by inhibiting the activation of AFB_1 by the phase I metabolizing enzyme CYP.