茯苓酸通过Nrf2/SLC7A11/GPX4信号通路调控铁死亡改善阿尔茨海默病大鼠认知障碍的研究

Modulation of Iron Death by Poric Acid through Nrf2/SLC7A11/GPX4 Signal Pathway in the Improvement of Cognitive Impairment of Alzheimer's Disease Rats

背景阿尔茨海默病(AD)是一种常见且不可逆转的神经退行性脑疾病,严重影响患者的生活品质和生存质量,目前仍缺乏有效的治疗方法延缓或阻止疾病进展。中药及其活性成分在防治AD方面具有重要潜力。目的探讨茯苓酸(PA)对AD大鼠认知障碍及核因子E2相关因子2(Nrf2)/溶质载体家族7A11(SLC7A11)/谷胱甘肽过氧化物酶4(GPX4)信号通路的影响。方法2022年1—9月将75只6~8周龄雄性SPF级SD大鼠依据随机数字表法分为对照组(Control组)、AD模型组(Model组)、PA治疗组(PA组),PA+Nrf2抑制剂组(PA+ML385组),除Control组外制备AD大鼠模型。造模成功后,PA组腹腔注射50 mg/kg PA,PA+ML385组腹腔注射50 mg/kg PA+30 mg/kg Nrf2抑制剂ML385,Control组与Model组腹腔注射等量0.9%氯化钠溶液。末次给药24 h后进行Morris水迷宫实验,第2、4、6天进行定位航行实验,记录大鼠到达平台的时间(逃避潜伏期),第7天移走平台,记录大鼠在120 s内滞留平台时间和穿越平台次数。Nissl染色后观察各组大鼠海马神经元病理变化,普鲁士蓝染色检测海马组织铁沉积,免疫荧光染色检测海马神经元GPX4表达,检测海马组织谷胱甘肽(GSH)、丙二醛(MDA)、Fe2+含量,蛋白质印迹法(Western blotting)检测大鼠海马组织Nrf2、SLC7A11、GPX4蛋白表达。结果末次给药第2、4、6天Model组逃避潜伏期高于Control组、PA组,PA+ML385组高于PA组,差异有统计学意义(P<0.05);Model组平台停留时间、穿越平台次数低于Control组、PA组,PA+ML385组低于PA组,差异有统计学意义(P<0.05)。Nissl染色结果示Model组神经元坏死严重,细胞核皱缩,尼氏体数目减少;PA组神经元坏死减少,排列紧密且尼氏体增多;PA+ML385组神经元损伤明显加重,尼氏体数目减少。普鲁士蓝染色结果示Model组铁沉积较Control组增加,与Model组比较,PA组铁沉积减少,PA+ML385组较PA组铁沉积增多。免疫荧光染色结果示Model组绿色荧光减弱,GPX4阳性细胞减少,PA组细胞绿色荧光较Model组增强,GPX4阳性细胞增多,PA+ML385组较PA组GPX4阳性细胞减少。Model组GSH低于Control组、PA组,PA+ML385组低于PA组,差异有统计学意义(P<0.05);Model组MDA、Fe2+高于Control组、PA组,PA+ML385组高于PA组,差异均有统计学意义(P<0.05)。Model组Nrf2、SLC7A11、GPX4蛋白相对表达量低于Control组、PA组,PA+ML385组低于PA组,差异有统计学意义(P<0.05)。结论PA可改善AD大鼠认知障碍,其机制可能与通过激活Nrf2/SLC7A11/GPX4信号通路抑制铁死亡有关。

Background Alzheimer's disease(AD)is a common and irreversible neurodegenerative brain disease that severely affects the quality of life and survival of patients,while there is still a lack of effective treatments to delay or stop disease progression.Traditional Chinese medicine(TCM)and its active ingredients have important potential in the prevention and treatment of AD.Objective To investigate the effects of poric acid(PA)on cognitive impairment and nuclear factor E2-related factor 2(Nrf2)/solute carrier family 7A11(SLC7A11)/glutathione peroxidase 4(GPX4)signaling pathway in AD rats.Methods Seventy-five male SPF grade SD rats aged 6 to 8 weeks were divided into the control group(Control group),AD Model group(Model group),PA treatment group(PA group)and PA+Nrf2 inhibitor group(PA+ML385 group)by random number table method to prepare AD rat model from January to September,2022.After successful modeling 50 mg/kg PA was intraperitoneally injected into the PA group,50 mg/kg PA and 30 mg/kg ML385 was intraperitoneally injected into the PA+ML385 group,and 0.9%sodium chloride solution was intraperitoneally injected into the Control group and Model group.The Morris water maze experiment was performed 24 h after the last dose,and the positioning navigation experiment was carried out on days 2,4 and 6 to record the time when the rats arrived at the platform(escape latency).The platform was removed on day 7,and the duration of the rats staying on the platform and the number of times they crossed the platform within 120 s were recorded.The pathological changes of hippocampal neurons in each group were observed after Nissl staining.Iron deposition was detected by Prussian blue staining,GPX4 expression and GSH,MDA and Fe2+contents were detected by immunofluorescence staining.The protein expression levels of Nrf2,SLC7A11 and GPX4 in rat hippocampus were detected by Western blotting.Results The escape latency of the Model group was higher than that of the Control group and PA group,and escape latency of the PA+ML385 group was higher than that of the PA group at 2,4 and 6 days after the last administration.The platform residence time and platform crossing times in the Model group were lower than those in the Control group and PA group,and those in the PA+ML385 group were lower than those in PA group,and the difference was statistically significant(P<0.05).The results of Nissl staining showed severe neuronal necrosis,nucleus shrinkage and decreased number of Nissl bodies in the Model group,decreased neuronal necrosis with tight arrangement and increased number of Nissl bodies in the PA group,significantly increased neuronal damage and decreased the number of Nissl bodies in the PA+ML385 group.The Prussian blue staining results showed that iron deposition in the Model group was higher than that in the Control group,iron deposition in the PA group was lower than that in the Model group,and iron deposition in the PA+ML385 was higher than that in the PA group.The results of immunofluorescence staining showed that green fluorescence was weakened and GPX4 positive cells were reduced in the Model group,green fluorescence was enhanced and GPX4 positive cells were increased in the PA group compared with the Model group,and GPX4 positive cells were decreased in the PA+ML385 group compared with the PA group.GSH in the Model group was lower than that in the Control group and PA group,GSH in the PA+ML385 group was lower than that in the PA group.MDA and Fe2+in the Model group were higher than those in the Control group and PA group,and those in the PA+ML385 group were higher that the PA group,and the differences were statistically significant(P<0.05).The relative expression levels of Nrf2,SLC7A11 and GPX4 in the Model group were lower than those in the Control group and PA group,and those in the PA+ML385 group were lower than those in the PA group,and the differences were statistically significant(P<0.05).Conclusion PA can improve the cognitive impairment of AD rats,and its mechanism may be related to the inhibition of iron death by activating Nrf2/SLC7A11/GPX4 signal pathway.