1.桂林医学院;2.湖南医药学院
:湖南省自然科学基金(2021JJ30484,2022JJ30426)；广西自然科学基金(2023JJA140042)
1.Guilin Medical College;2.Hunan Medical University
Natural Science Foundation of Hunan Province or Guangxi (2021J30484; 2022J30426; 2023JJA140042)
目的 探讨香菇多糖（LNT）对亚砷酸钠（SA）染毒小鼠肝脏铁死亡的干预效应与机制。方法 以C57BL/6雄性小鼠为对象，实验分为对照组、SA低剂量组、SA高剂量组和LNT干预+SA高剂量组。生化法和苏木精-伊红（HE）染色评价肝组织病理损伤；酶联免疫吸附（Elisa）法检测肿瘤坏死因子α（TNFα）、白细胞介素6（IL-6）、微管相关蛋白1轻链3B（MAP1LC3B）、谷胱甘肽过氧化酶4（GPX4）和铁离子（Fe）水平；免疫印迹法（WB）或免疫共沉淀法（IP）检测铁蛋白重链（FTH1）、LC3B/A表达或FTH1与LC3或泛素（Ub）共表达；分子对接软件分析线粒体铁蛋白（FTMT）与LC3A/B或Ub互作。结果 与对照组相比，SA染毒组肝组织显示病理损伤与AST、ALT、TNFα和IL-6水平升高，铁自噬标志物LC3B、FTH1和Fe含量升高，铁死亡标志物GPX4水平下调（P<0.05）；与SA染毒组相比，LNT干预组肝组织病理损伤程度明显减轻，AST、ALT、TNFα、IL-6、LC3B、FTH1和Fe水平下调，而GPX4水平上调（P<0.05）；WB或IP实验显示，相比对照组，SA染毒组FTH1、LC3B/A水平上调，FTH1与LC3B或Ub共表达水平升高。相比SA染毒组，LNT干预组FTH1、LC3B/A水平或FTH1与LC3B/Ub共表达水平降低（P<0.05）。分子对接模拟显示FTMT与LC3A/B或Ub蛋白发生较稳定的氢键结合。 结论 香菇多糖拮抗亚砷酸钠染毒小鼠肝组织病理损伤和铁死亡，可能与TNFα-铁自噬信号抑制有关。
Objective: To explore the intervention effect and mechanism of Lentinan (LNT) on liver ferroptosis in mice exposed to sodium arsenite (SA). Methods: C57BL/6 male mice as experimental subjects, The experiment was divided into control group, SA low-dose group, SA high-dose group, and LNT intervention+SA high-dose group. Biochemical methods and Hematoxylin eosin (HE) staining of liver tissue were applied to evaluate hepatic pathological damage; Enzyme linked immunosorbent assay (Elisa) was used to detect the levels of tumor necrosis factorα (TNFα), microtubule associated protein 1 light chain 3B (MAP1LC3B), Interleukin-6 (IL-6), glutathione peroxidase 4 (GPX4), and iron ions (Fe); Immunoblotting (WB) or immunoprecipitation (IP) methods were used to detect the levels of ferritin heavy chain (FTH1), ratio of LC3B to LC3A, or the co-expressions of FTH1 and LC3, or FTH1 and ubiquitin (Ub); Molecular docking software was applied to analyze the interactions between mitochondrial ferritin (FTMT) and LC3A, LC3B, or Ub. Results: Compared with control group, SA exposure group exhibited hepatic pathological damage and the elevated levels of AST, ALT, TNFα, and IL-6, and the elevated ferritinophagy markers LC3B, FTH1, and Fe, while the levels of ferroptosis biomarkers GPX4 decreased (P<0.05); Compared with SA exposure group, LNT intervention showed a significant reduction in hepatic pathological damage, and showed the downregulations of AST, ALT, TNFα, IL-6, LC3B, FTH1, and Fe, while the level of GPX4 upregulated (P<0.05); WB or IP experiment showed that SA exposure induced the upregulated levels of FTH1 and LC3B/A, and the higher co-expressions of FTH1 and LC3B or Ub protein compared to control group, while LNT intervention showed the downregulated levels of FTH1 and LC3B/A, and the decreased co-expressions of FTH1 and LC3B or Ub protein compared to SA exposure group (P<0.05). Molecular docking simulations showed that FTMT binds stably to LC3A, LC3B or Ub by hydrogen bond. Conclusion: Lentinan antagonizes against sodium arsenite exposure mice hepatic injury and ferroptosis, which may be associated with the inhibition or TNFα-ferritinophagy signaling.
