补骨脂素在2D/3D肝细胞模型上的毒性比较及对线粒体融合分裂的影响

Toxicity comparison of psoralen in 2D /3D hepatocyte models andits effect on mitochondrial fusion and fission

目的分别使用二维(2D)和三维(3D)培养的人肝癌细胞(human hepatocellular carcinomas,HepG2)模型评价补骨脂素的毒性并比较其差异。方法采用低吸附U形底多孔板法构建了HepG2细胞的3D细胞模型。补骨脂素处理24 h后,CCK-8法检测细胞存活率,试剂盒检测LDH漏出量,TMRM染色检测线粒体膜电位。Q-PCR检测补骨脂素对线粒体融合分裂蛋白DRP1、Mfn-2及OPA1的mRNA的影响。结果3D模型可以长时间保持高水平白蛋白和尿素氮分泌,较2D模型具有更高的CYP1A2、CYP2E1、CYP3A4和UGT1A1表达水平。在3D模型中,补骨脂素较低浓度出现细胞存活率的明显降低,LDH漏出量明显升高以及线粒体膜电位的下降。Q-PCR结果显示补骨脂素诱导线粒体分裂蛋白DRP1表达明显增加,而线粒体融合蛋白OPA1明显下降。结论成功构建了3D HepG2细胞模型并应用于对补骨脂素肝毒性的评价;3D细胞培养模型对补骨脂素毒性反应更为敏感,线粒体在补骨脂素诱导细胞损伤中可能起关键性作用。

Aim To evaluate and compare the toxicity of psoralen on two-dimensional(2D)and three-dimensional(3D)cultured human hepatocyte HepG2 models.Methods The 3D cell model of HepG2 cells was constructed by low adsorption U-shaped bottom porous plate method.After treated with psoralen for 24 hours,the cell viabilities of 2D and 3D HepG2 cells were detected by CCK-8 assay,LDH leakage was detected by kit,and the mitochondrial membrane potential was detected by TMRM staining.The effect of psoralen on the mRNA of mitochondrial fusion-fission proteins DRP1,Mfn-2 and OPA1 was detected by Q-PCR.Results 3D model maintained a high level of albumin and urea secretion for a long time.And the expression levels of CYP1A2,CYP2E1,CYP3A4 and UGT1A1 in 3D model were higher than those in 2D model.In 3D model lower concentrations of psoralen showed a significant decrease in cell viability,a significant increase in LDH leakage,and a decrease in mitochondrial membrane potential.Q-PCR results showed that psoralen induced a marked increase in the expression of mitochondrial fission protein DRP1,while a significant decrease in mitochondrial fusion protein OPA1.Conclusions A 3D HepG2 cell model is successfully constructed and applied to the evaluation of psoralen hepatotoxicity;the 3D cell culture model is more sensitive to psoralen toxicity,and mitochondria may play a key role in psoralen-induced cell damage.