目的 探究枸杞多糖（Lycium barbarum polysaccharides，LBP）调控核因子E2相关因子2（nuclear factor E2 related factor 2，Nrf2）/谷胱甘肽过氧化物酶4（glutathione peroxidase 4，GPX4）通路对缺氧/复氧（hypoxia/reoxygenation，H/R）诱导的心肌细胞铁死亡的影响。方法 设置对照组、模型组及LBP低、中、高剂量（25、50、100 mg/L）组和LBP（100 mg/L）＋Nrf2抑制剂ML385（5 μmol/L）组。构建H9c2细胞H/R模型，并给予相应药物处理。采用CCK-8检测细胞活力；采用TUNEL染色检测细胞凋亡；采用试剂盒检测细胞Fe²⁺、谷胱甘肽（glutathione，GSH）、丙二醛（malondialdehyde，MDA）水平及乳酸脱氢酶（lactate dehydrogenase，LDH）、超氧化物歧化酶（superoxide dismutase，SOD）、过氧化氢酶（catalase，CAT）活性；采用二氢乙锭（dihydroethidium，DHE）荧光探针检测细胞活性氧（reactive oxygen species，ROS）水平；采用透射电镜观察细胞线粒体形态；采用Western blotting检测转铁蛋白受体1（transferrin receptor 1，TfR1）、二价金属离子转运蛋白1（divalent metal transporter 1，DMT1）、铁蛋白重链1（ferritin heavy chain 1，FTH1）、酰基辅酶A合成酶长链家族成员4（acyl-CoA synthetase long-chain family member 4，ACSL4）、Kelch样ECH相关蛋白1（Kelch like ECH associated protein 1，Keap1）、Nrf2、血红素氧合酶-1（heme oxygenase-1，HO-1）、溶质载体家族7成员11（solute carrier family 7 member 11，SLC7A11）、GPX4蛋白表达。结果 与对照组比较，模型组细胞活力显著降低（P＜0.001），Fe²⁺、MDA、ROS水平和LDH活性显著升高（P＜0.001），GSH水平和SOD、CAT活性显著降低（P＜0.001），线粒体缩短、膜密度增高且嵴数量减少，TfR1、Nrf2、HO-1、SLC7A11、GPX4蛋白表达水平显著降低（P＜0.001），DMT1、FTH1、ACSL4、Keap1蛋白表达水平显著升高（P＜0.001）。与模型组比较，LBP组细胞活力显著升高（P＜0.01），Fe²⁺、MDA、ROS水平和LDH活性显著降低（P＜0.05、0.01、0.001），GSH水平和SOD、CAT活性显著升高（P＜0.05、0.01、0.001），线粒体形态较为规则，TfR1、Nrf2、HO-1、SLC7A11、GPX4蛋白表达水平显著升高（P＜0.01、0.001），DMT1、FTH1、ACSL4、Keap1蛋白表达水平显著降低（P＜0.05、0.01、0.001）；而ML385能够抑制枸杞多糖对细胞铁死亡的改善作用（P＜0.05、0.01、0.001）。结论 枸杞多糖可能通过激活Nrf2/GPX4通路改善H/R诱导的心肌细胞铁死亡。

Objective To investigate the effect of Lycium barbarum polysaccharides (LBP) on ferroptosis induced by hypoxia/reoxygenation (H/R) in cardiomyocytes by regulating nuclear factor E2 related factor 2 (Nrf2)/glutathione peroxidase 4 (GPX4) pathway. Methods Control group, model group, LBP low-, medium-, high-dose (25, 50, 100 mg/L) groups and LBP (100 mg/L) + Nrf2 inhibitor ML385 (5 μmol/L) group were set up. H9c2 cells H/R model was constructed and treated with corresponding drugs. CCK-8 was used to detect cell viability; TUNEL staining was used to detect cell apoptosis; The reagent kits were used to detect levels of Fe²⁺, glutathione (GSH), malondialdehyde (MDA), as well as activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and catalase (CAT) in cells; Dihydroethidium (DHE) fluorescence probe was used to detect cellular reactive oxygen species (ROS) level; Transmission electron microscopy was used to observe the mitochondrial morphology of cells; Western blotting was used to detect the expressions of transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), ferritin heavy chain 1 (FTH1), acyl-CoA synthetase long-chain family member 4 (ACSL4), Kelch like ECH associated protein 1 (Keap1), Nrf2, heme oxygenase-1 (HO-1), solute carrier family 7 member 11 (SLC7A11) and GPX4 protein. Results Compared with control group, cell viability in model group was significantly reduced (P < 0.001), levels of Fe²⁺, MDA, ROS and LDH activity were significantly increased (P < 0.001). GSH level and activities of SOD and CAT were significantly reduced (P < 0.001), mitochondria was shortened, membrane density was increased, and the number of cristae was decreased, the protein expression levels of TfR1, Nrf2, HO-1, SLC7A11 and GPX4 were significantly reduced (P < 0.001), while the protein expression levels of DMT1, FTH1, ACSL4 and Keap1 were significantly increased (P < 0.001). Compared with model group, cell viability in LBP group was significantly increased (P < 0.01), Fe²⁺, MDA, ROS levels and LDH activity were significantly decreased (P < 0.05, 0.01, 0.001), GSH level and SOD, CAT activities were significantly increased (P < 0.05, 0.01, 0.001), the morphology of mitochondria were relatively regular, protein expression levels of TfR1, Nrf2, HO-1, SLC7A11 and GPX4 were significantly increased (P < 0.01, 0.001), protein expression levels of DMT1, FTH1, ACSL4 and Keap1 were significantly decreased (P < 0.05, 0.01, 0.001); ML385 could inhibit the improvement effect of LBP on cell ferroptosis (P < 0.05, 0.01, 0.001). Conclusion LBP may improve H/R-induced ferroptosis in cardiomyocytes by activating the Nrf2/GPX4 pathway.

R285.5

安徽省宿州市卫生健康科研项目（SZWJ2023a040）