NNMT调控铁死亡在非酒精性脂肪肝细胞模型中的作用研究

The Role of NNMT in Regulating Ferroptosis in Non-alcoholic Fatty Liver Disease

目的探讨尼克酰胺-N-甲基转移酶(NNMT)在非酒精性脂肪肝(NAFL)中的作用,阐明NNMT调控铁死亡的分子机制。方法用油红O染色和尼罗红染色检测不同浓度游离脂肪酸混合物FFA(油酸钠∶棕榈酸钠=2∶1)处理的HepG2细胞脂滴的积聚,探针法检测脂质过氧化水平和Fe^(2+)的水平,并用Western blot检测FFA、铁死亡抑制剂(Fer-1)及NNMT抑制剂(NNMTi)处理下HepG2细胞的NNMT和GPX4、SLC7A11等铁死亡相关蛋白的表达变化。结果FFA处理HepG2细胞后,细胞脂滴增加,NNMT蛋白表达增加;FFA降低铁死亡负调控蛋白GPX4和SLC7A11的表达,提高细胞脂质过氧化水平和Fe^(2+)含量,诱导细胞铁死亡,而铁死亡抑制剂能阻止FFA所致的细胞铁死亡;且NNMTi促进GPX4和SLC7A11的表达,降低细胞脂质过氧化水平和Fe^(2+)含量,缓解铁死亡。结论FFA诱导下,高表达的NNMT介导肝细胞铁死亡,为NAFL病的发生发展提供新机制。

Objective To explore the role of nicotinamide N-methyltransferase(NNMT)in non-alcoholic fatty liver(NAFL)and to elucidate the molecular mechanism of NNMT regulating ferroptosis.Methods The accumulation of lipid droplets in HepG2 cells treated with different concentrations of free fatty acid mixture FFA(sodium oleate∶sodium palmitate=2∶1)was detected using Oil Red O staining and Nile Red staining.The levels of lipid peroxidation and Fe^(2+)were detected using the probe method.Western blot was used to detect the expression changes of iron death related proteins such as NNMT,GPX4 and SLC7A11 in HepG2 cells treated with FFA,iron death inhibitor(Fer-1)and NNMT inhibitor(NNMTi).Results After HepG2 cells were treated with FFA,the number of cellular lipid droplets increased and the expression of NNMT protein increased.FFA reduced the expression of iron death negative regulatory proteins GPX4 and SLC7A11,increased cell lipid peroxidation levels and Fe^(2+)content,and induced cell iron death,whileiron death inhibitors could prevent FFA induced cell iron death.Moreover,NNMTi promoted the expression of GPX4 and SLC7A11,reduced cellular lipid peroxidation levels and Fe^(2+)content,and alleviated ferroptosis.Conclusion Under FFA induction,highly expressed NNMT mediates ferroptosis in liver cells,providing a new mechanism for the occurrence and development of NAFL disease.