Riboflavin (Vitamin B2) Deficiency Induces Apoptosis Mediated by Endoplasmic Reticulum Stress and the CHOP Pathway in HepG2 Cells.
Riboflavin (Vitamin B2) Deficiency Induces Apoptosis Mediated by Endoplasmic Reticulum Stress and the CHOP Pathway in HepG2 Cells.
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核黄素是维持细胞内环境稳定的必需微量营养素，是黄素单核苷酸和黄素腺嘌呤二核苷酸的前体。核黄素缺乏（RD）诱导细胞凋亡。内质网应激（endoplasmic reticulum，ER）可诱导细胞凋亡，而C/EBP同源蛋白（C/EBP homologous protein，CHOP）是参与这一过程的关键途径。然而，RD诱导的细胞凋亡是否由ER应激和CHOP通路介导仍不清楚，需要进一步研究。因此，目前的研究显示RD对人肝癌细胞系（HepG 2）中ER应激和凋亡的影响。首先，在RD培养基（4.55 nM核黄素）和对照（CON）培养基（1005 nM核黄素）中培养细胞。我们进行了细胞显微结构表征和确定凋亡的观察。随后，4-苯基丁酸（4-PBA），一个ER应激抑制剂，在HepG 2细胞中，研究ER应激在RD诱导的细胞凋亡中的作用。最后，将CHOP siRNA转染到HepG 2细胞中，以验证RD是否通过CHOP途径触发ER应激介导的细胞凋亡。结果表明，RD可抑制细胞增殖，引起内质网应激，并增加内质网应激标志物（CHOP，78 kDa葡萄糖调节蛋白，转录激活因子6）的表达（p < 0.05）。此外，RD还可增加细胞凋亡率，增加促凋亡标志物（B细胞淋巴瘤2相关X，Caspase 3）的表达，降低抗凋亡标志物（B细胞淋巴瘤2）的表达（p < 0.05）。4-PBA处理和CHOP敲低显著减轻RD诱导的细胞凋亡。这些结果表明RD通过触发ER应激和CHOP途径诱导细胞凋亡。
Riboflavin is an essential micronutrient and a precursor of flavin mononucleotide and flavin adenine dinucleotide for maintaining cell homeostasis. Riboflavin deficiency (RD) induces cell apoptosis. Endoplasmic reticulum (ER) stress is considered to induce apoptosis, and C/EBP homologous protein (CHOP) is a key pathway involved in this process. However, whether RD-induced apoptosis is mediated by ER stress and the CHOP pathway remains unclear and needs further investigation. Therefore, the current study presents the effect of RD on ER stress and apoptosis in the human hepatoma cell line (HepG2). Firstly, cells were cultured in a RD medium (4.55 nM riboflavin) and a control (CON) medium (1005 nM riboflavin). We conducted an observation of cell microstructure characterization and determining apoptosis. Subsequently, 4-phenyl butyric acid (4-PBA), an ER stress inhibitor, was used in HepG2 cells to investigate the role of ER stress in RD-induced apoptosis. Finally, CHOP siRNA was transfected into HepG2 cells to validate whether RD triggered ER stress-mediated apoptosis by the CHOP pathway. The results show that RD inhibited cell proliferation and caused ER stress, as well as increased the expression of ER stress markers (CHOP, 78 kDa glucose-regulated protein, activating transcription factor 6) (p < 0.05). Furthermore, RD increased the cell apoptosis rate, enhanced the expression of proapoptotic markers (B-cell lymphoma 2-associated X, Caspase 3), and decreased the expression of the antiapoptotic marker (B-cell lymphoma 2) (p < 0.05). The 4-PBA treatment and CHOP knockdown markedly alleviated RD-induced cell apoptosis. These results demonstrate that RD induces cell apoptosis by triggering ER stress and the CHOP pathway.