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Inhibition of the NLRP3 Inflammasome Activation/Assembly through the Activation of the PI3K Pathway by Naloxone Protects Neural Stem Cells from Ischemic Condition

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Abstract

Naloxone is a well-known opioid antagonist and has been suggested to have neuroprotective effects in cerebral ischemia. We investigated whether naloxone exhibits anti-inflammatory and neuroprotective effects in neural stem cells (NSCs) injured by oxygen-glucose deprivation (OGD), whether it affects the NOD-like receptor protein 3 (NLRP3) inflammasome activation/assembly, and whether the role of the phosphatidylinositol 3-kinase (PI3K) pathway is important in the control of NLRP3 inflammasome activation/assembly by naloxone. Primary cultured NSCs were subjected to OGD and treated with different concentrations of naloxone. Cell viability, proliferation, and the intracellular signaling proteins associated with the PI3K pathway and NLRP3 inflammasome activation/assembly were evaluated in OGD-injured NSCs. OGD significantly reduced survival, proliferation, and migration and increased apoptosis of NSCs. However, treatment with naloxone significantly restored survival, proliferation, and migration and decreased apoptosis of NSCs. Moreover, OGD markedly increased NLRP3 inflammasome activation/assembly and cleaved caspase-1 and interleukin-1β levels in NSCs, but naloxone significantly attenuated these effects. These neuroprotective and anti-inflammatory effects of naloxone were eliminated when cells were treated with PI3K inhibitors. Our results suggest that NLRP3 inflammasome is a potential therapeutic target and that naloxone reduces ischemic injury in NSCs by inhibiting NLRP3 inflammasome activation/assembly mediated by the activation of the PI3K signaling pathway.

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Data Availability

The datasets used and/or analyzed in the current study are available from the corresponding authors on reasonable request.

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Acknowledgements

Not applicable.

Funding

This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI20C0253, HU21C0007, and HU21C0113) and the Medical Research Center (2017R1A5A2015395), and was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2022R1F1A1072546, 2022R1A2C1092835).

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  1. Ji Young Kim, Mina Hwang and Na-Young Choi contributed equally to this study.

Authors and Affiliations

  1. Department of Nuclear Medicine, Hanyang University College of Medicine, Hanyang University Guri Hospital, 153, Gyeongchun-ro, Guri-si, Gyeonggi-do, 11923, Republic of Korea

    Ji Young Kim

  2. Department of Neurology, Hanyang University College of Medicine, 153, Gyeongchun-ro, Guri-si, Gyeonggi-do, 11923, Republic of Korea

    Mina Hwang, Na-Young Choi & Seong-Ho Koh

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Contributions

J.Y.K., M.H., N.Y.C., and S.H.K. designed the study; M.H. and N.Y.C. performed experiments; J.Y.K. and S.H.K. analyzed and interpreted data; M.H. and N.Y.C. generated figures; J.Y.K. and M.H. wrote original draft; S.H.K. revised manuscript; All authors provided critical insight and review of the manuscript. All authors have read and agreed to published version of the manuscript.

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Correspondence to Seong-Ho Koh.

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All animal procedures were conducted in accordance with Hanyang University’s guidelines for the care and use of laboratory animals, and approved by the Institutional Animal Care and Use Committee (IACUC) of Hanyang University (2019-0162 A and 2021-0039 A). All efforts were made to minimize the number of animals used and animal suffering.

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Supplementary Fig. 1

: Effects of naloxone on the viability and cytotoxicity of hNSCs damaged by OGD. (A) hNSCs viability was decreased after 16 h of OGD but significantly increased after naloxone treatment. (B) Cytotoxicity of hNSCs also increased after OGD but was significantly reduced by naloxone. All data are represented as mean (% of control) ± standard deviation of a minimum of three independent experiments and were analyzed using Tukey’s test after a one-way analysis of variance (n ≥ 3). *p < 0.05, **p < 0.01 (vs. the control group); and #p < 0.05, ##p < 0.01 (vs. the group subjected to OGD alone). hNSCs, human neural stem cells; OGD, oxygen-glucose deprivation.

Supplementary Fig. 2

: Effects of OGD on the expression of NLRP3 inflammasome related factors at various time points. Alterations of NLRP3 (A) and IL-1β (B) were compared in OGD-treated NSCs over time via western blotting. Their expression was the highest at 6 h after exposure to OGD. All data are shown as the mean (% of control) ± standard deviation of a minimum of three independent experiments and were analyzed using Student’s t-test (n ≥ 3). *p < 0.05, **p < 0.01 (vs. the control group). OGD, oxygen-glucose deprivation; NLRP3, NOD-like receptor protein 3.

Supplementary Fig. 3

: Original western blotting images to demonstrate that the antibodies are specific. Uncut membrane images showing molecular weight of protein bands (A–K) and Ponceau S staining images of the western blotting membrane (L). (M) Western blotting involving only secondary antibody labeling, without primary antibody, to confirm the specificity of antibodies.

Supplementary Fig. 4

: Western blotting images containing protein marker. (A, B) A protein marker was used to identify the molecular weight of each protein band, but markers were not detected in the chemical reaction.

Supplementary Fig. 5

: The effect of PI3K pathway and naloxone on the OGD-injured NSCs through Aim2 inflammasome. (A-B) Aim2 inflammasome protein expression levels through western blotting. Data are represented as the mean (% of control) ± standard deviation of the minimum of three independent experiments and were analyzed using Tukey’s test after one-way analysis of variance (n ≥ 3). *p < 0.05, **p < 0.01 (vs. the control group); #p < 0.05, ##p < 0.01 (vs. the group subjected to OGD alone); and †p < 0.05, ††p < 0.01 (vs. the group subjected to OGD and 1 µM naloxone). PI3K, phosphatidylinositol 3-kinase; OGD, oxygen-glucose deprivation; NSCs, neural stem cells.

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Kim, J.Y., Hwang, M., Choi, NY. et al. Inhibition of the NLRP3 Inflammasome Activation/Assembly through the Activation of the PI3K Pathway by Naloxone Protects Neural Stem Cells from Ischemic Condition. Mol Neurobiol 60, 5330–5342 (2023). https://doi.org/10.1007/s12035-023-03418-4

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