目的 基于核因子E2相关因子2（nuclear factor erythroid 2-related factor 2，Nrf2）/谷胱甘肽过氧化物酶4（glutathione peroxidase 4，GPX4）通路探究补阳还五汤改善糖尿病认知障碍（diabetes cognitive impairment，DCI）的作用机制。方法 采用高脂高糖饲料喂养联合小剂量链脲佐菌素（streptozotocin，STZ）注射法建立DCI大鼠模型，将造模成功大鼠随机分为模型组及补阳还五汤低、高剂量（7.125、28.500 g/kg）组和二甲双胍（0.2 g/kg）组，另设置对照组。给药4周后，采用Morris水迷宫评价大鼠认知记忆能力；苏木素-伊红染色观察大鼠海马组织病理变化；生化法检测大鼠血清中超氧化物歧化酶（superoxide dismutase，SOD）、过氧化氢酶（catalase，CAT）活性及谷胱甘肽（glutathione，GSH）、丙二醛（malondialdehyde，MDA）和脑组织中亚铁离子（Fe²⁺）水平；免疫组化法检测大鼠海马CA1区Nrf2、GPX4蛋白表达； Western blotting检测大鼠海马组织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、转铁蛋白受体（transferrin receptor，TFR）、膜铁转运蛋白1（ferroportin 1，FPN1）、铁蛋白重链（ferritin heavy chain，FTH）蛋白表达。体外培养人神经母细胞瘤SHSY-5Y细胞并用160 mmol/L葡萄糖诱导建立损伤模型，设置对照组、模型组及补阳还五汤低、高剂量含药血清组和补阳还五汤高剂量含药血清＋Nrf2抑制剂ML385（10μmol/L）组，生化法检测细胞中SOD、CAT活性及GSH、MDA水平；JC-1荧光探针检测细胞线粒体膜电位变化；Western blotting检测细胞中Keap1、Nrf2、HO-1、SLC7A11和GPX4蛋白表达。结果 体内实验结果显示，补阳还五汤能够延缓DCI大鼠体质量下降（P＜0.05），降低空腹血糖（P＜0.05、0.01）；缩短逃避潜伏期（P＜0.05、0.01），增加目标象限移动距离、停留时间及穿越平台次数（P＜0.05、0.01）；减轻海马神经元细胞损伤；降低血清中MDA及脑组织Fe²⁺水平（P＜0.05、0.01），升高血清中SOD、CAT活性及GSH水平（P＜0.05、0.01）；下调海马组织中TFR、Keap1蛋白表达（P＜0.05、0.01），上调Nrf2、HO-1、SLC7A11、GPX4、FTH、FPN1蛋白表达（P＜0.05、0.01）。体外实验结果显示，补阳还五汤含药血清能够显著提高高糖诱导的SHSY-5Y细胞存活率（P＜0.01），降低MDA水平（P＜0.05），升高SOD、CAT活性及GSH水平（P＜0.05、0.01），升高线粒体膜电位，下调Keap1蛋白表达（P＜0.01），上调Nrf2、HO-1、SLC7A11、GPX4蛋白表达（P＜0.05、0.01）；与补阳还五汤高剂量含药血清组相比，给予ML385后显著逆转上述指标变化（P＜0.05、0.01）。结论 补阳还五汤能够改善DCI大鼠的认知功能障碍，减轻氧化应激损伤并改善铁代谢紊乱，其作用可能与激活Nrf2/GPX4信号通路、调控铁死亡相关过程有关。

Objective To investigate the mechanism of Buyang Huanwu Decoction (补阳还五汤) in ameliorating diabetes cognitive impairment (DCI) based on nuclear factor erythroid 2-related factor 2 (Nrf2)/glutathione peroxidase 4 (GPX4) signaling pathway. Methods A rat model of DCI was established by feeding a high-fat and high-sugar diet combined with low-dose streptozotocin (STZ) injection. The successfully modeled rats were randomly divided into model group, Buyang Huanwu Decoction low-, high-dose (7.125, 28.500 g/kg) groups and metformin (0.2 g/kg) group, control group was set up separately. After four weeks of administration, the cognitive and memory abilities of rats were evaluated by Morris water maze. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of hippocampal tissue in rats. The activities of superoxide dismutase (SOD), catalase (CAT) and levels of glutathione (GSH), malondialdehyde (MDA) in serum and ferrous ions (Fe2+) in brain tissue of rats were detected by biochemical methods. Immunohistochemistry was used to observe the expressions of Nrf2 and GPX4 proteins in CA1 region of hippocampus in rats. Western blotting was used to detect the expressions of Kelch-like ECH-associated protein 1 (Keap1), Nrf2, heme oxygenase-1 (HO-1), solute carrier family 7 member 11 (SLC7A11), GPX4, transferrin receptor (TFR), ferroportin 1 (FPN1) and ferritin heavy chain (FTH) proteins in hippocampal tissue of rats. Human neuroblastoma cells SHSY-5Y were cultured in vitro and a damage model was established by inducing with 160 mmol/L glucose. Control group, model group, Buyang Huanwu Decoction low-, high-dose drug containing serum groups and Buyang Huanwu Decoction high-dose drug containing serum + Nrf2 inhibitor ML385 (10 μmol/L) group were set up. Biochemical methods were used to detect the activities of SOD, CAT and levels of GSH, MDA in cells. JC-1 fluorescent probe was used to detect changes in mitochondrial membrane potential in cells. Western blotting was used to detect the expressions of Keap1, Nrf2, HO-1, SLC7A11 and GPX4 proteins in cells. Results The results of in vivo experiments showed that Buyang Huanwu Decoction could delay weight loss of DCI rats (P < 0.05), reduce fasting blood glucose (P < 0.05, 0.01), shorten the escape latency (P < 0.05, 0.01), increase the distance moved, dwell time and number of crossings in target quadrant (P < 0.05, 0.01), alleviate the damage of hippocampal neurons, reduce the levels of MDA in serum and Fe2+ in brain tissue (P < 0.05, 0.01), increase the activities of SOD, CAT and levels of GSH in serum (P < 0.05, 0.01), downregulate the expressions of TFR and Keap1 proteins in hippocampus (P < 0.05, 0.01), upregulate the expressions of Nrf2, HO-1, SLC7A11, GPX4, FTH and FPN1 proteins (P < 0.05, 0.01). The results of in vitro experiments showed that Buyang Huanwu Decoction drug-containing serum could significantly increase the survival rate of SHSY-5Y cells induced by high glucose (P < 0.01), reduce MDA level (P < 0.05), increase the activities of SOD, CAT and level of GSH (P < 0.05, 0.01), increase the mitochondrial membrane potential, downregulate the expression of Keap1 protein (P < 0.01), and upregulate the expressions of Nrf2, HO-1, SLC7A11 and GPX4 proteins (P < 0.05, 0.01). Compared with Buyang Huanwu Decoction high-dose drug containing serum group, the administration of ML385 significantly reversed the changes in the above indicators (P < 0.05, 0.01). Conclusion Buyang Huanwu Decoction could improve cognitive dysfunction, alleviate oxidative stress damage, and improve iron metabolism disorders in DCI rats. Its effects may be related to the activation of Nrf2/GPX4 signaling pathway and the regulation of ferroptosis processes.

R285.5

国家自然科学基金资助项目（82304504）；陕西省食品药品安全监测重点实验室开放课题资助项目（SXKLFDM202502）