Abstract：
BACKGROUND: Moschus, first described in the Shennong\'s Classic of the Materia medicine, is a scarce and precious animal medicine. Modern pharmacological researches have suggested that Moschus has neuroprotective actions, and its mechanism is related to anti-inflammatory, antioxidant, and anti-apoptosis effects. Ferroptosis is one of the major pathologies of Alzheimer\'s disease (AD) and is widely implicated in the pathogenesis and progression of AD. Although previous studies have suggested that Moschus possesses neuroprotective effect, whether Moschus could mitigate neuronal damages by inhibiting the onset of ferroptosis is unknown in model cells of AD.
OBJECTIVE: The aim of study was to explore the water extract of Moschus (WEM) on ferroptosis caused by erastin and the potential mechanism.
METHODS: Erastin was used to stimulate HT22 cells to form ferroptosis model to evaluate the anti-ferroptosis effect of WEM by cell counting kit-8 and lactic dehydrogenase (LDH) tests. The malondialdehyde (MDA) and glutathione (GSH) kits are used for detection of MDA and GSH levels, and 2\',7\'-dichlorofluorescein diacetate and C11 BODIPY 581/591 fluorescence probe are used for evaluation of reactive oxygen species (ROS) and lipid peroxide (LOOH) levels. And Western blot was used to test nuclear factor erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), heme oxygenase-1 (HO-1), and ferroptosis associated proteins including glutathione peroxidase 4 (GPX4), cystine/glutamate antiporter subunit (SLC7A11), ferritin heavy chain 1 (FTH1), ferroportin1 (FPN1), transferrin receptor (TFRC). In addition, the Nrf2 inhibitor ML385 was applied to verify whether WEM prevents erastin-induced ferroptosis by activating the Keap1/Nrf2 pathway.
RESULTS: After WEM treatment, erastin-induced HT22 cell survival was significantly elevated, the accumulation of intracellular MDA, ROS, and LOOH were significantly reduced, the level of GSH and expressions of ferroptosis inhibitors GPX4 and SLC7A11 were significantly increased, and iron metabolism-related proteins TFRC, FPN1, and FTH1 were regulated. These effects of WEM are implemented by activating the Keap1/Nrf2 pathway.
CONCLUSIONS: This study demonstrated that WEM could perform neuroprotective effects by alleviating ferroptosis, verified that WEM treatment of AD can be mediated by the Keap1/Nrf2 pathway, and provided theoretical support for the application of WEM in the treatment of AD.
OBJECTIVE: The aim of study was to explore the water extract of Moschus (WEM) on ferroptosis caused by erastin and the potential mechanism.
METHODS: Erastin was used to stimulate HT22 cells to form ferroptosis model to evaluate the anti-ferroptosis effect of WEM by cell counting kit-8 and lactic dehydrogenase (LDH) tests. The malondialdehyde (MDA) and glutathione (GSH) kits are used for detection of MDA and GSH levels, and 2\',7\'-dichlorofluorescein diacetate and C11 BODIPY 581/591 fluorescence probe are used for evaluation of reactive oxygen species (ROS) and lipid peroxide (LOOH) levels. And Western blot was used to test nuclear factor erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), heme oxygenase-1 (HO-1), and ferroptosis associated proteins including glutathione peroxidase 4 (GPX4), cystine/glutamate antiporter subunit (SLC7A11), ferritin heavy chain 1 (FTH1), ferroportin1 (FPN1), transferrin receptor (TFRC). In addition, the Nrf2 inhibitor ML385 was applied to verify whether WEM prevents erastin-induced ferroptosis by activating the Keap1/Nrf2 pathway.
RESULTS: After WEM treatment, erastin-induced HT22 cell survival was significantly elevated, the accumulation of intracellular MDA, ROS, and LOOH were significantly reduced, the level of GSH and expressions of ferroptosis inhibitors GPX4 and SLC7A11 were significantly increased, and iron metabolism-related proteins TFRC, FPN1, and FTH1 were regulated. These effects of WEM are implemented by activating the Keap1/Nrf2 pathway.
CONCLUSIONS: This study demonstrated that WEM could perform neuroprotective effects by alleviating ferroptosis, verified that WEM treatment of AD can be mediated by the Keap1/Nrf2 pathway, and provided theoretical support for the application of WEM in the treatment of AD.
摘要：
背景：Moschus，在神农医学经典中首次描述，是一种稀缺而珍贵的动物药物。现代药理研究表明，Moschus具有神经保护作用，其机制与抗炎有关，抗氧化剂，和抗凋亡作用。铁凋亡是阿尔茨海默病(AD)的主要病理类型之一,与AD的发病机制和进展密切相关。尽管先前的研究表明Moschus具有神经保护作用，在AD模型细胞中，Moschus是否可以通过抑制铁凋亡的发生来减轻神经元损伤是未知的。
目的：本研究的目的是探讨麝香水提物（WEM）对艾司汀引起的铁凋亡的影响及其可能的机制。
方法：Erastin用于刺激HT22细胞形成铁凋亡模型，以通过细胞计数试剂盒-8和乳酸脱氢酶测试评估WEM的抗铁凋亡作用。丙二醛（MDA）和谷胱甘肽（GSH）试剂盒用于检测MDA和GSH水平，和2\'，7'-二氯荧光素二乙酸酯和C11BODIPY581/591荧光探针用于评估活性氧（ROS）和脂质过氧化物（LOOH）水平。用Westernblot检测核因子红系2相关因子2(Nrf2),Kelch样ECH相关蛋白1（Keap1），血红素加氧酶-1（HO-1），和铁凋亡相关蛋白，包括谷胱甘肽过氧化物酶4（GPX4），胱氨酸/谷氨酸反转运亚基（SLC7A11），铁蛋白重链1（FTH1），ferroportin1(FPN1),转铁蛋白受体（TFRC）。此外,Nrf2抑制剂ML385用于验证WEM是否通过激活Keap1/Nrf2通路来防止擦除素诱导的铁凋亡.
结果：WEM治疗后，erastin诱导的HT22细胞存活率显著升高,细胞内MDA的积累，ROS,LOOH显著减少，GSH水平和铁凋亡抑制剂GPX4和SLC7A11的表达显著增加，和铁代谢相关蛋白TFRC，FPN1和FTH1受调控。WEM的这些作用是通过激活Keap1/Nrf2途径实现的。
结论：这项研究表明，WEM可以通过减轻铁性凋亡来发挥神经保护作用，证实WEM治疗AD可通过Keap1/Nrf2通路介导,为WEM在AD治疗中的应用提供理论支持。
目的：本研究的目的是探讨麝香水提物（WEM）对艾司汀引起的铁凋亡的影响及其可能的机制。
方法：Erastin用于刺激HT22细胞形成铁凋亡模型，以通过细胞计数试剂盒-8和乳酸脱氢酶测试评估WEM的抗铁凋亡作用。丙二醛（MDA）和谷胱甘肽（GSH）试剂盒用于检测MDA和GSH水平，和2\'，7'-二氯荧光素二乙酸酯和C11BODIPY581/591荧光探针用于评估活性氧（ROS）和脂质过氧化物（LOOH）水平。用Westernblot检测核因子红系2相关因子2(Nrf2),Kelch样ECH相关蛋白1（Keap1），血红素加氧酶-1（HO-1），和铁凋亡相关蛋白，包括谷胱甘肽过氧化物酶4（GPX4），胱氨酸/谷氨酸反转运亚基（SLC7A11），铁蛋白重链1（FTH1），ferroportin1(FPN1),转铁蛋白受体（TFRC）。此外,Nrf2抑制剂ML385用于验证WEM是否通过激活Keap1/Nrf2通路来防止擦除素诱导的铁凋亡.
结果：WEM治疗后，erastin诱导的HT22细胞存活率显著升高,细胞内MDA的积累，ROS,LOOH显著减少，GSH水平和铁凋亡抑制剂GPX4和SLC7A11的表达显著增加，和铁代谢相关蛋白TFRC，FPN1和FTH1受调控。WEM的这些作用是通过激活Keap1/Nrf2途径实现的。
结论：这项研究表明，WEM可以通过减轻铁性凋亡来发挥神经保护作用，证实WEM治疗AD可通过Keap1/Nrf2通路介导,为WEM在AD治疗中的应用提供理论支持。
