Icariin enhances PINK1-associated mitophagy to attenuate NLRP3/GSDMD-related microglial pyroptosis and cognitive impairment in Alzheimer’s disease

i

This article has been updated. A Correction to this article was published on .

i

The Original Article was published on

i

The Original Article was published on

i

This article has been updated

i

This article was retracted on

i

This article has been updated

Abstract

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder in which microglia-mediated neuroinflammation contributes to disease progression. Icariin (ICA), a major bioactive flavonoid from Epimedium, has neuroprotective activity, but how it regulates mitochondrial quality control and inflammatory injury in microglia remains unclear. In the present study, APP/PS1 transgenic mice and Aβ_1-42-stimulated BV-2 microglia were used to examine the effects of ICA on mitophagy, mitochondrial stress, NLRP3/GSDMD-related pyroptotic changes, and neuroinflammation. Behavioral testing, histological analysis, transcriptomic profiling, immunofluorescence, Western blotting, ELISA, molecular docking, molecular dynamics simulation, cellular thermal shift assay, Mdivi-1 co-treatment, and siRNA-mediated Pink1 knockdown were performed. ICA improved cognitive deficits in APP/PS1 mice and reduced microglial inflammatory activation. Transcriptomic and experimental analyses showed that ICA enhanced mitophagy-related responses, restored mitochondrial membrane potential, and reduced excessive mitochondrial reactive oxygen species accumulation. Molecular docking, molecular dynamics simulation, and cellular thermal shift assay supported potential engagement of ICA with PINK1. ICA also reduced NLRP3 inflammasome-associated inflammatory injury, GSDMD-related pyroptotic changes, and IL-1β and IL-18 release. In APP/PS1 mice, Mdivi-1 co-treatment weakened the anti-inflammatory effects of ICA. In Aβ_1-42-stimulated BV-2 microglia, Pink1 knockdown reduced ICA-induced LC3/TOM20 colocalization and partially reversed the inhibitory effects of ICA on NLRP3 inflammasome-associated inflammatory markers. Overall, ICA enhances PINK1-associated mitophagy, alleviates mitochondrial stress, and attenuates NLRP3/GSDMD-related microglial pyroptotic injury in AD-related models. These findings suggest that PINK1-associated mitochondrial quality control contributes to the neuroprotective effects of ICA.

Keywords

Alzheimer’s disease / Icariin / mitophagy / NLRP3 inflammasome / Microglial pyroptosis

CHANGE HISTORY

A Correction to this paper has been published:
https://doi.org

The Retraction Note was updated to reflect reception of statements of disagreement from all authors.

This article has been retracted.Please see the Retraction Notice for more detail:
https://doi.org

References

[1] Graff-Radford J, Yong KXX, Apostolova LG, et al. New insights into atypical Alzheimer’s disease in the era of biomarkers. Lancet Neurol. 2021; 20(3):222-234. https://doi.org/10.1016/S1474-4422(20)30440-3.

[2] 2024 Alzheimer’s disease facts and figures. Alzheimers Dement. 2024; 20(5):3708-3821. https://doi.org/10.1002/alz.13809.

[3] Ren RL, Qi JL, Lin SH, et al. The China Alzheimer report 2022. Gen Psychiatr. 2022; 35(1):e100751. https://doi.org/10.1136/gpsych-2022-100751.

[4] Mangialasche F, Solomon A, Winblad B, et al. Alzheimer’s disease: clinical trials and drug development. Lancet Neurol. 2010; 9(7):702-716. https://doi.org/10.1016/S1474-4422(10)70119-8.

[5] Calsolaro V, Edison P . Neuroinflammation in Alzheimer’s disease: current evidence and future directions. Alzheimers Dement. 2016; 12(6):719-732. https://doi.org/10.1016/j.jalz.2016.02.010.

[6] Singh D. Astrocytic and microglial cells as the modulators of neuroinflammation in Alzheimer’s disease. J Neuroinflamm. 2022; 19:206. https://doi.org/10.1186/s12974-022-02565-0.

[7] Deczkowska A, Amit I, Schwartz M. Microglial immune checkpoint mechanisms. Nat Neurosci. 2018; 21(6):6. https://doi.org/10.1038/s41593-018-0145-x.

[8] Fu W, Vukojevic V, Patel A, et al. Role of microglial amylin receptors in mediating beta amyloid (Aβ)-induced inflammation. J Neuroinflamm. 2017; 14(1):199. https://doi.org/10.1186/s12974-017-0972-9.

[9] Chen Y, Song SH, Parhizkar S, et al. APOE3ch alters microglial response and suppresses Aβ-induced tau seeding and spread. Cell. 2023; 187(2):428-445.e20. https://doi.org/10.1016/j.cell.2023.11.029.

[10] Feng YS, Tan ZX, Wu LY, et al. The involvement of NLRP3 inflammasome in the treatment of Alzheimer’s disease. Ageing Res Rev. 2020; 64:101192. https://doi.org/10.1016/j.arr.2020.101192.

[11] Heneka MT, Kummer MP, Stutz A, et al. NLRP3 is activated in Alzheimer´s disease and contributes to pathology in APP/PS1 mice. Nature. 2013; 493(7434):674-678. https://doi.org/10.1038/nature11729.

[12] Ising C, Venegas C, Zhang SS, et al. NLRP3 inflammasome activation drives tau pathology. Nature. 2019; 575(7784):669-673. https://doi.org/10.1038/s41586-019-1769-z.

[13] Yao J, Wang Z, Song WH, et al. Targeting NLRP3 inflammasome for neurodegenerative disorders. Mol Psychiatry. 2023; 28(11):4512-4527. https://doi.org/10.1038/s41380-023-02239-0.

[14] Bergsbaken T, Fink SL, Cookson BT . Pyroptosis: host cell death and inflammation. Nat Rev Microbiol. 2009; 7(2):99-109. https://doi.org/10.1038/nrmicro2070.

[15] Zhong Z, Liang S, Sanchez-Lopez E, et al. New mitochondrial DNA synthesis enables NLRP3 inflammasome activation. Nature. 2018; 560(7717):198-203. https://doi.org/10.1038/s41586-018-0372-z.

[16] Shimada K, Crother TR, Karlin J, et al. Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis. Immunity. 2012; 36(3):401-414. https://doi.org/10.1016/j.immuni.2012.01.009.

[17] Wang YF, Shi PL, Chen Q, et al. Mitochondrial ROS promote macrophage pyroptosis by inducing GSDMD oxidation. J Mol Cell Biol. 2019; 11(12):1069-1082. https://doi.org/10.1093/jmcb/mjz020.

[18] Klemmensen MM, Borrowman SH, Pearce C, et al. Mitochondrial dysfunction in neurodegenerative disorders. Neurotherapeutics. 2023; 21(1):e00292. https://doi.org/10.1016/j.neurot.2023.10.002.

[19] Luo ZY, Dong JC, Wu JF . Impact of icariin and its derivatives on inflammatory diseases and relevant signaling pathways. Int Immunopharmacol. 2022; 108:108861. https://doi.org/10.1016/j.intimp.2022.108861.

[20] Wu Y, Wang XQ, Wu JY, et al. A tri-compound formula comprising ginsenoside Rg1, tetrandrine and icariin alleviates atopic dermatitis symptoms in a mouse model. Phytomedicine. 2025; 141:156737. https://doi.org/10.1016/j.phymed.2025.156737.

[21] Liu YQ, Li HL, Wang XW, et al. Anti-Alzheimers molecular mechanism of icariin: insights from gut microbiota, metabolomics, and network pharmacology. J Transl Med. 2023; 21:277. https://doi.org/10.1186/s12967-023-04137-z.

[22] Wang J, Liu YY, Wu YX, et al. Anti-inflammatory effects of icariin in the acute and chronic phases of the mouse pilocarpine model of epilepsy. Eur J Pharmacol. 2023; 960:176141. https://doi.org/10.1016/j.ejphar.2023.176141.

[23] Zhang TC, Lin YC, Sun NN, et al. Icariin, astragaloside a and puerarin mixture attenuates cognitive impairment in APP/PS1 mice via inhibition of ferroptosis-lipid peroxidation. Neurochem Int. 2024; 175:105705. https://doi.org/10.1016/j.neuint.2024.105705.

[24] Zhao H, Zhang SS, Gong Z, et al. Icariin mitigates depressive-like behavior in rats by regulating mitochondrial function. J Ethnopharmacol. 2025; 350:120036. https://doi.org/10.1016/j.jep.2025.120036.

[25] Min-Kaung-Wint-Mon, Kida H, Kanehisa I, et al. Adverse effects of Aβ_1-42 oligomers: impaired contextual memory and altered intrinsic properties of CA1 pyramidal neurons. Biomolecules. 2024; 14(11):1425. https://doi.org/10.3390/biom14111425.

[26] Li HL, Xiao Q, Zhu LM, et al. Targeting ceramide-induced microglial pyroptosis: icariin is a promising therapy for Alzheimer’s disease. J Pharm Anal. 2025; 15(4):101106. https://doi.org/10.1016/j.jpha.2024.101106.

[27] Deacon RM . Assessing nest building in mice. Nat Protoc. 2006; 1(3):1117-1119. https://doi.org/10.1038/nprot.2006.170.

[28] Denninger JK, Smith BM, Kirby ED . Novel object recognition and object location behavioral testing in mice on a budget. JoVE. 2018(141):58593. https://doi.org/10.3791/58593.

[29] Seibenhener ML, Wooten MC . Use of the open field maze to measure locomotor and anxiety-like behavior in mice. J Visualized Exp. 2015(96):52434. https://doi.org/10.3791/52434-v.

[30] Kraeuter AK, Guest PC, Sarnyai Z . The Y-Maze for Assessment of Spatial Working and Reference Memory in Mice. In: Guest PC, ed. Pre-Clinical Models . Vol 1916. Methods in Molecular Biology. Springer New York; 2019:105-111. https://doi.org/10.1007/978-1-4939-8994-2_10.

[31] Bespalov A, Steckler T . Pharmacology of anxiety or pharmacology of elevated plus maze? Biol Psychiat. 2021; 89(12):e73. https://doi.org/10.1016/j.biopsych.2020.11.026.

[32] Vorhees CV, Williams MT . Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc. 2006; 1(2):848-858. https://doi.org/10.1038/nprot.2006.116.

[33] Hickman S, Izzy S, Sen P, et al. Microglia in neurodegeneration. Nat Neurosci. 2018; 21(10):1359-1369. https://doi.org/10.1038/s41593-018-0242-x.

[34] Kovacs SB, Miao EA . Gasdermins: effectors of pyroptosis. Trends Cell Biol. 2017; 27(9):673-684. https://doi.org/10.1016/j.tcb.2017.05.005.

[35] Chen WF, Wu L, Du ZR, et al. Neuroprotective properties of icariin in MPTP-induced mouse model of Parkinson’s disease: involvement of PI3K/Akt and MEK/ERK signaling pathways. Phytomedicine. 2017; 25:93-99. https://doi.org/10.1016/j.phymed.2016.12.017.

[36] Cai YW, Chai YH, Fu YX, et al. Salidroside ameliorates Alzheimer’s disease by targeting NLRP3 inflammasome-mediated pyroptosis. Front Aging Neurosci. 2022; 13:809433. https://doi.org/10.3389/fnagi.2021.809433.

[37] Moonen S, Koper MJ, Van Schoor E, et al. Pyroptosis in Alzheimer’s disease: cell type-specific activation in microglia, astrocytes and neurons. Acta Neuropathol. 2023; 145(2):175-195. https://doi.org/10.1007/s00401-022-02528-y.

[38] Fang EF, Hou Y, Palikaras K, et al. Mitophagy inhibits amyloid-β and tau pathology and reverses cognitive deficits in models of Alzheimer’s disease. Nat Neurosci. 2019; 22(3):401-412. https://doi.org/10.1038/s41593-018-0332-9.

[39] Gu YY, Zhao XR, Zhang N, et al. Mitochondrial dysfunction as a therapeutic strategy for neurodegenerative diseases: current insights and future directions. Ageing Res Rev. 2024; 102:102577. https://doi.org/10.1016/j.arr.2024.102577.

[40] Kim J, Lee HJ, Park SK, et al. Donepezil regulates LPS and Aβ-stimulated neuroinflammation through MAPK/NLRP3 inflammasome/STAT3 signaling. Int J Mol Sci. 2021; 22(19):10637. https://doi.org/10.3390/ijms221910637.

[41] Zheng LY, Wu SC, Jin HC, et al. Molecular mechanisms and therapeutic potential of icariin in the treatment of Alzheimer’s disease. Phytomedicine. 2023; 116:154890. https://doi.org/10.1016/j.phymed.2023.154890.

[42] Wang SG, Ma JR, Zeng YQ, et al. Icariin, an up-and-coming bioactive compound against neurological diseases: network pharmacology-based study and literature review. Drug Des Devel Ther. 2021; 15:3619-3641. https://doi.org/10.2147/DDDT.S310686.

[43] Wang YL, Shang C, Zhang Y, et al. Regulatory mechanism of icariin in cardiovascular and neurological diseases. Biomed Pharmacother. 2023; 158:114156. https://doi.org/10.1016/j.biopha.2022.114156.

[44] Xu WJ, Ding J, Kuang SD, et al. Icariin-Curcumol promotes docetaxel sensitivity in prostate cancer through modulation of the PI3K-Akt signaling pathway and the Warburg effect. Cancer Cell Int. 2023; 23(1):190. https://doi.org/10.1186/s12935-023-03042-1.

[45] El-Shitany NA, Eid BG . Icariin modulates carrageenan-induced acute inflammation through HO-1/Nrf2 and NF-κB signaling pathways. Biomed Pharmacother. 2019; 120:109567. https://doi.org/10.1016/j.biopha.2019.109567.

[46] Mangan MSJ, Olhava EJ, Roush WR, et al. Targeting the NLRP3 inflammasome in inflammatory diseases. Nat Rev Drug Discov. 2018; 17(8):588-606. https://doi.org/10.1038/nrd.2018.97.