目的 探讨丁酸钠（sodium butyrate,NaB）是否可通过诱导铁自噬发挥其抗结直肠癌作用。方法 以MTT、平板克隆和PI染色观察NaB对结直肠癌HCT-116细胞增殖和死亡的影响;自噬检测试剂盒（MDC法）、亚铁离子荧光探针FerroOrange、Western blot检测p62、LC3、FTH1、NCOA4表达探究NaB是否诱导结直肠癌HCT-116细胞发生铁自噬。结果 MTT和平板克隆实验结果显示,2 mmol/L NaB即可显著抑制HCT-116细胞活力（P<0.001）和克隆球形成（P<0.01）;PI染色结果显示,NaB可使死亡细胞数量增加;铁离子螯合剂去铁胺（DFO）可逆转NaB对HCT-116细胞增殖活性的抑制和促死亡作用（P<0.05）;自噬抑制剂氯喹（CQ）可进一步增强NaB对HCT-116细胞增殖活性的抑制作用（P<0.001）;MDC法和FerroOrange检测结果显示,NaB可增加HCT-116细胞自噬体形成和细胞内Fe2+水平（P<0.001）,DFO和CQ均可逆转NaB对HCT-116细胞内Fe2+水平的上调作用（P<0.05）;Western blot结果显示,NaB可下调HCT-116细胞p62、FTH1蛋白表达水平（P<0.001）,上调LC3-II、NCOA4蛋白表达水平（P<0.001,P<0.05）,CQ可逆转NaB对HCT-116细胞FTH1蛋白表达水平的下调作用（P<0.05）。结论 NaB可通过诱导铁自噬发挥抗结直肠癌作用。
Abstract
Objective To investigate whether sodium butyrate (NaB) can exert its anti-colorectal cancer action by inducing ferritinophagy. Methods The effects of NaB on the proliferation and death of colorectal cancer HCT-116 cells were investigated using MTT assay, plate cloning assay, and PI staining. The induction of ferritinophagy in HCT-116 cells by NaB was explored through the use of autophagy detection kit (MDC method), ferrous ion fluorescent probe FerroOrange. Western blot analysis was performed to detect the expression of p62, FTH1, LC3 and NCOA4 molecules. Results The results of the MTT and colony formation experiments showed that 2 mmol/L NaB significantly inhibited the viability (P<0.001) and clone formation (P<0.01) of HCT-116 cells. The PI staining results showed that NaB increased the number of dead cells, and the iron chelator deferoxamine (DFO) reversed the inhibitory and pro-apoptotic effects of NaB on HCT-116 cells (P<0.05). The autophagy inhibitor chloroquine (CQ) further enhanced the inhibitory effect of NaB on HCT-116 cells (P<0.001). The results of MDC method and FerroOrange detection showed that NaB increased the formation of autophagic vacuoles and intracellular Fe2+ levels in HCT-116 cells (P<0.001). The upregulation of intracellular Fe2+ levels by NaB was reversed by DFO and CQ (P<0.05). Western blot analysis revealed that NaB downregulated the protein expression levels of p62 and FTH1 (P<0.001) and upregulated the protein expression levels of LC3-II, NCOA4 in HCT-116 cells (P<0.001, P<0.05). The downregulation of FTH1 protein expression level by NaB was reversed by CQ (P<0.05). Conclusion NaB can exert an anti-colorectal cancer action by inducing ferritinophagy in HCT-116 cells.
关键词
丁酸钠 /
结直肠癌 /
铁自噬
Key words
sodium butyrate /
colorectal cancer /
ferritinophagy
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基金
国家自然科学基金（No.81773429）; 广东省自然科学基金（No.2022A1515011631）
