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

doi:10.12114/j.issn.1007-9572.2023.0326

中国全科医学 ›› 2024, Vol. 27 ›› Issue (02): 177-183.DOI: 10.12114/j.issn.1007-9572.2023.0326

所属专题：阿尔茨海默病最新文章合集；阿尔茨海默病最新文章合集

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

樊赟¹, 窦润鹏², 胡久略³, 侯紫君³, 周春祥¹^,^*()

¹210046　江苏省南京市，南京中医药大学
²473000　河南省南阳市中心医院
³473000　河南省南阳市，南阳理工学院张仲景国医国药学院　河南省张仲景方药与免疫调节重点实验室

收稿日期:2023-04-11 修回日期:2023-07-20 出版日期:2024-01-15 发布日期:2023-10-23
通讯作者: 周春祥
作者贡献：樊赟提出主要研究目标，负责研究的构思与设计，研究的实施，撰写论文；窦润鹏、胡久略进行数据收集与整理，统计学处理，图、表的绘制与展示；樊赟、侯紫君进行论文的修订；周春祥负责最终版本修订，对论文负责。
基金资助:
国家自然科学基金资助项目(81774021，82074504)

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

FAN Yun¹, DOU Runpeng², HU Jiulue³, HOU Zijun³, ZHOU Chunxiang¹^,^*()

¹Nanjing University of Traditional Chinese Medicine, Nanjing 210046, China
²Nanyang Central Hospital, Nanyang 473000, China
³Nanyang Institute of Technology, Zhang Zhongjing Traditional Chinese Medicine College/Zhang Zhongjing Key Laboratory of Prescriptions and Immunomodulation, Nanyang 473000, China

Received:2023-04-11 Revised:2023-07-20 Published:2024-01-15 Online:2023-10-23
Contact: ZHOU Chunxiang

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摘要/Abstract

摘要： 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。

关键词: 阿尔茨海默病, 茯苓酸, 核因子E2相关因子2/溶质载体家族7A11/GPX4信号通路, 铁死亡, 大鼠

Abstract:

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.

Key words: Alzheimer disease, Pachymic acid, Nrf2/SLC7A11/GPX4 signal pathway, Ferroptosis, Rats

樊赟,窦润鹏,胡久略,等. 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[J]. 中国全科医学, 2024, 27(02): 177-183. DOI: 10.12114/j.issn.1007-9572.2023.0326.
FAN Yun,DOU Runpeng,HU Jiulue, et al. Modulation of Iron Death by Poric Acid through Nrf2/SLC7A11/GPX4 Signal Pathway in the Improvement of Cognitive Impairment of Alzheimer's Disease Rats[J]. Chinese General Practice, 2024, 27(02): 177-183. DOI: 10.12114/j.issn.1007-9572.2023.0326.

图/表 7

表1 4组大鼠Morris水迷宫实验结果（±s）

Table 1 Results of Morris water maze experiment in the 4 groups of rats

分组	只数	逃避潜伏期（s）			平台停留时间（s）	穿越平台次数（次）
分组	只数	第2天	第4天	第6天	平台停留时间（s）	穿越平台次数（次）
Control组	15	42.42±6.25	25.38±4.67	13.32±2.45	48.34±4.23	15.20±3.42
Model组	15	76.31±7.13^a	57.55±6.26^a	45.63±7.41^a	23.65±4.14^a	8.40±2.35^a
PA组	15	57.39±6.23^b	36.47±5.83^b	27.39±4.69^b	34.42±3.82^b	12.33±3.54^b
PA+ML385组	15	68.42±7.57^c	48.35±5.81^c	34.52±5.88^c	28.34±4.23^c	9.27±2.23^c
F值		69.631	91.488	93.560	102.071	16.589
P值		<0.001	<0.001	<0.001	<0.001	<0.001

图1 4组大鼠神经元Nissl染色结果（×400）注：a为Control组，b为Model组，c为PA组，d为PA+ML385组；Control组=对照组，Model组=AD模型组，PA组=茯苓酸治疗组，PA+ML385组=PA+核因子E2相关因子2抑制剂组。

Figure 1 Results of Nissl staining of neurons in the 4 groups of rats

图2 4组大鼠海马组织普鲁士蓝染色结果（×400）注：a为Control组，b为Model组，c为PA组，d为PA+ML385组。

Figure 2 Results of Prussian blue staining in hippocampus of the 4 groups of rats

图3 4组大鼠免疫荧光染色结果（×200）注：NeuN=神经元核抗原，GPX4=谷胱甘肽过氧化物酶4，DAPI=4'，6-二脒基-2-苯基吲哚。

Figure 3 Results of immunofluorescence staining in the 4 groups of rats

表2 4组大鼠海马组织GSH、MDA、Fe2+含量比较（±s）

Table 2 Comparison of GSH，MDA and Fe2+ contents in hippocampus of the 4 groups of rats

组别	只数	GSH（μmol/g）	MDA（μmol/g）	Fe²⁺（μg/g）
Control组	5	19.85±3.43	13.75±2.69	26.21±3.76
Model组	5	5.76±0.97^a	38.24±5.73^a	53.41±5.64^a
PA组	5	14.34±2.78^b	20.25±3.61^b	25.26±4.23^b
PA+ML385组	5	9.42±1.65^c	27.34±4.52^c	38.55±3.81^c
F值		32.308	29.906	44.304
P值		<0.001	<0.001	<0.001

图4 4组大鼠海马组织Nrf2、SLC7A11、GPX4蛋白Western blotting分析图注：Nrf2=核因子E2相关因子2，SLC7A11=溶质载体家族7A11。

Figure 4 Western blotting analysis of Nrf2，SLC7A11 and GPX4 proteins in hippocampus of the 4 groups of rats

表3 4组大鼠海马组织Nrf2、SLC7A11、GPX4蛋白相对表达量（±s）

Table 3 Relative expression of Nrf2，SLC7A11 and GPX4 proteins in hippocampus of rats in the 4 groups of rats

组别	只数	Nrf2	SLC7A11	GPX4
Control组	5	1.21±0.14	1.05±0.12	1.13±0.11
Model组	5	0.43±0.05^a	0.23±0.05^a	0.19±0.03^a
PA组	5	0.97±0.10^b	0.85±0.08^b	0.57±0.06^b
PA+ML385组	5	0.72±0.08^c	0.43±0.05^c	0.35±0.04^c
F值		58.195	109.664	185.348
P值		<0.001	<0.001	<0.001

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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

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