Forsythoside A Mitigates Alzheimer's Disease By Regulating Ferroptosis-mediated Neuroinflammation Via Nrf2/GPX4

Alzheimer's disease(AD)is a neurodegenerative disease characterized by progressive loss of cognitive abilities.The elderly group aged over 65 is the high-risk group.AD has become one of the most expensive,deadly and high-burden diseases with the aging of the world population.The pathological mechanism of AD is complex and has yet been elucidated.Researchers have proposed many hypotheses about AD pathogenesis and developed several AD treatment drugs.However,these drugs cannot delay AD progression and are associated with severe side effects.Therefore,there is an urgent need to develop drugs that can more effectively treat AD.In recent years,traditional medicines and active natural products derived from herbal medicines have attracted great attention to the scientific community due to their diverse pharmacological activities and no obvious toxic side effects.Forsythiaside A(FA),the active ingredient of Forsythia suspensa(Thunb.)Vahl.,is an active phenylethanoid glycoside.It has been proved to possess numerous pharmacological properties,among which anti-inflammation,antioxidation and neuroprotection are related to the etiology of AD,indicating that FA may play an effective role in the treatment of AD.However,there is still a lack of systematic research on the mechanism of FA on AD treatment.In this study,A?_1-42-induced N2a cells,lipopolysaccharide(LPS)-induced BV2 cells and APP/PS1 double transgenic mice were used to clarify the effect and mechanism of FA on AD.Label-free quantitative proteomics technology was used to dig deeper into the mechanism,and systematically verified the mechanism in the APP/PS1 double-transgenic mouse model and erastin-induced HT22 cells.Specific research contents are as follows:1.Study on the neuroprotective effect of FA on A?_1-42-induced N2a cells and LPS-stimulated BV2 cells.In the A?_1-42-induced N2a cells,FA increased the viability of N2a cells,but had no significant effect on healthy ones.FA ameliorated the imbalance of mitochondrial membrane potential by JC-1 fluorescence staining.In addition,FA significantly inhibited the production of intracellular reactive oxygen species(ROS),protected neurons from the attack of oxygen free radicals,and further reduced the production of malondialdehyde(MDA).Next,in the LPS-stimulated BV2cells,FA inhibited the production of nitric oxide(NO),interleukin(IL)-1?and IL-6.Above studies confirmed that FA significantly inhibit A?_1-42-induced oxidative damage and LPS-induced inflammatory response.2.Effects of FA on behavior and related pathology in APP/PS1 mice.First,the safety of FA in mice was evaluated by body weight,organ index analysis and H&E staining.The results showed that FA did not affect body weight and spleen index in mice,while reducing liver index and kidney index;FA did not cause obvious pathological changes in the liver,spleen,kidney and brain of mice,indicating its safety.Next,FA improved the learning and memory ability via the Y maze test and Morris water maze test.Finally,the results of pathological analysis of mice showed that FA administration could partially inhibit the deposition of A?,reduce the expression of p-tau,and restrain neuronal apoptosis in the brain of mice,indicating that FA significantly alleviated the memory impairment and pathological characteristics of APP/PS1 mice.3.The mechanism of FA in the treatment of AD based on proteomics.First,it was preliminarily determined that FA may further regulate the inflammatory response by activating the downstream cascade signaling of dopamine,improve the pathological conditions of AD mice,and exert neuroprotective effects via label-free quantitative proteomic analysis.Subsequently,dopaminergic and neuroinflammatory signaling pathways in the brains of APP/PS1 mice were detected by immunohistochemistry,immunofluorescence,enzyme-linked immunosorbent assay and western blot.The results showed that,after 6-week treatment,FA upregulated the expression of c AMP in the brain and activated the downstream c AMP response element binding protein(CREB)cascade signaling pathway.In addition,FA inhibited the expression levels of microglia activation marker ionized calcium-binding adaptor 1(Iba1)and astrocyte activation marker glial fibrillary acidic protein(GFAP)in the hippocampus and cortex of mouse brain,inactivated the I?B kinase(IKK)/inhibitor of nuclear factor-?B(I?B)/nuclear factor-?B(NF-?B)signaling pathway,reduced pro-inflammatory factors released by glial cells,such as monocyte chemoattractant protein 1,IL-1?and tumor necrosis factor alpha(TNF-?),and increased the secreted levels of anti-inflammatory factors,such as transforming growth factor-?,IL-4 and IL-10,indicating that FA significantly inhibited the inflammatory response in the brains of APP/PS1 mice.4.Effects of FA on ferroptosis in APP/PS1 mice and erastin-induced HT22cells.It has been confirmed that ferroptosis plays a vital role in AD pathogenesis,and is intertwined with neuroinflammation bidirectionally in the brain.To explore the regulatory effect of FA on ferroptosis,related indicators in APP/PS1 mice and erastin-induced HT22 cell models were examined.Further combine with molecular docking to find the target of FA.The results showed that,FA activated nuclear factor erythroid 2-related factor 2(Nrf2)signaling,increased expression levels of antioxidative proteins,such as heme oxygenase 1(HO-1),NAD(P)H quinone dehydrogenase 1(NQO1)and glutathione peroxidase 4(GPX4).FA also improved iron metabolism in the brain by regulating the expression of transferrin receptor(TFRC),divalent metal-ion transporter-1(DMT1),ferritin heavy chain(FTH)and ferritin light chain(FTL).In the erastin-induced HT22 cells,ferroptosis-related indicators were detected.The results showed that,FA inhibited erastin-induced cell death,reduced the expression levels of intracellular ROS and MDA,and effectively protected HT22 cells from lipid peroxidation.TEM results showed that FA alleviated the reduction in mitochondrial volume,increases in double membrane density,mitochondrial cristae and other hallmarks of ferroptosis induced by erastin.Western blot analysis showed that,FA upregulated the expression levels of Nrf2 and downstream antioxidants in HT22 cells,and improved iron metabolism in cells.The experimental results were consistent with the APP/PS1 mouse model.Molecular docking results showed that FA had a strong affinity for GPX4,and the optimal binding energy was-6.35 kcal/mol.Above studies indicate that FA inhibit ferroptosis in APP/PS1 mice and erastin-induced HT22 cells.5.Combined with si RNA transfection technology,the target of FA was explored in the erastin-induced HT22 cells.In order to investigate whether the inhibitory effect of FA on the occurrence and development of neuroinflammation in AD is based on its regulation of ferroptosis,the inflammation-related mediators in erastin-induced HT22 cells were detected,and the results showed that,FA suppressed the IKK/I?B/NF-?B pathway,decreased the secretion of pro-inflammatory cytokines,such as IL-1?and TNF-?,and boosted the release of anti-inflammatory cytokines IL-4and IL-10.The results of si RNA transfection experiments showed that,after GPX4 or Nrf2 were silenced,the regulation ability of FA on ferroptosis and inflammation-related proteins in the erastin-induced HT22 cell model was significantly inhibited,indicating that FA treatment exerted anti-AD properties via modulation of ferroptosis-mediated neuroinflammation by targeting the activation of the Nrf2/GPX4 axis.To summarize,in this study,A?_1-42-induced N2a cells,LPS-induced BV2 cells,erastin-induced HT22 cells and APP/PS1 double transgenic mice were used to investigate the protective effects of FA in vivo and in vitro.A systematic and in-depth excavation of the mechanism was carried out,and it was clear that FA could inhibit ferroptosis and neuroinflammation by activating the Nrf2/GPX4 signaling axis,thereby improving AD.It provides a theoretical foundation for further research and new drug development in the treatment of AD.