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Hypoxic Preconditioning Attenuates Neuroinflammation via Inhibiting NF-κB/NLRP3 Axis Mediated by p-MLKL after Transient Global Cerebral Ischemia

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Abstract

Hypoxic preconditioning (HPC) has been reported to alleviate neuronal damage and microglial activation in hippocampal CA1 after transient global cerebral ischemia (tGCI). However, the molecular mechanism is unclear. Recent studies identified that nuclear factor-kappa-B (NF-κB)/oligomerization domain-like receptors protein (NLRP) 3 inflammasome pathway is mainly involved in the activation of microglia and that phosphorylated (p)-mixed lineage kinase domain-like (MLKL) is related to the regulation of NF-κB/NLRP3 axis. Hence, in this study, we set out to investigate whether HPC attenuates neuronal damage and microglial activation through inhibiting NF-κB/NLRP3 axis mediated by p-MLKL after tGCI in CA1 of male rats. We found that HPC decreased NLRP3 inflammasome in microglia and inhibited M1 polarization of microglia in CA1 after tGCI. Mechanistically, HPC inhibited the activation of NF-κB signaling pathway and reduced the mRNA and protein levels of NLRP3 inflammasome after tGCI. Additionally, the knockdown of p-MLKL by short hairpin RNA (shRNA) administration inhibited the activation of the NF-κB signaling pathway and reduced the formation of NLRP3 inflammasome, thus attenuating M1 polarization of microglia and decreasing the release of interleukin 1 beta (IL-1β) and necrosis factor alpha (TNF-α) in CA1 post ischemia. We consider that p-MLKL in microglia may be derived from necroptotic neurons after tGCI. In conclusion, the new finding in this study is that HPC-induced neuroprotection against tGCI through inhibiting NF-κB/NLRP3 pathway mediated by p-MLKL.

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Data Availability

The data supporting the conclusions of this article are included within the article. Original slides, photographs, and blot images are retained. All regents used in this study are available from scientific supply companies. The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

AAV :

Adeno-associated virus

ALIX :

Also known as programmed cell death 6-interacting protein

Arg-1 :

Arginase-1

ASC :

Apoptosis-associated speck-like protein containing a CARD

Caspase-1 :

Cysteinyl aspartate–specific proteinase-1

CD68 :

Cluster of differentiation 68

CNS :

Central nervous system

EVs :

Extracerllular vesicles

GAPDH :

Glyceraldehyde-phosphate dehydrogenase

GFAP :

Glial fibrillary acidic protein

GFP :

Green fluorescent protein

HPC :

Hypoxic preconditioning

Iba-1 :

Ionized calcium binding adaptor molecule-1

IκBα :

Nuclear factor-kappa-B inhibitor α

IL-10 :

Interleukin 10

IL-1β :

Interleukin 1β

MLKL :

Mixed lineage kinase domain-like

NeuN :

Neuronal nuclei

NF-κB :

Nuclear factor kappa-B

NLRP3 :

Nucleotide binding and oligomerization domain-like receptors protein 3

PDTC :

Pyrrolidine dithiocarbamate

P-MLKL :

Phosphorylated MLKL

RIP3 :

Receptor interacting protein 3

shRNA :

Short hairpin RNA

tGCI :

Transient global cerebral ischemia

TNF-α :

Tumor necrosis factor-α

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Acknowledgements

Our sincere thanks go to Haixia Wen and Kongping Li (Institute of Neurosciences and Department of Neurology of the Second Affiliated Hospital of Guangzhou Medical University) for the modification of the figures.

Funding

This work is supported by the Natural Science Foundation of Guangdong Province, China (No. 2021A1515011269), and the National Natural Science Foundation of China (No. 82071281 and 82271330).

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Authors and Affiliations

  1. Department of Neurology, Institute of Neuroscience, Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, China

    Xiaomei Lu, Lixuan Zhan, Guorong Chai, Meiyan Chen, Weiwen Sun & En Xu

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  1. Xiaomei Lu
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  2. Lixuan Zhan
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  3. Guorong Chai
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  4. Meiyan Chen
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Contributions

E. Xu, X. Lu, and L. Zhan conceived the study, designed and assembled all the figures. X. Lu performed the experiments with the help of G. Chai, M. Chen, and W. Sun. X. Lu performed data analysis. X. Lu prepared the figures with the help of G. Chai and M. Chen. E. Xu and X. Lu wrote the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to En Xu.

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All surgical procedures and animal experiments were performed according to the Animal Research: Reporting In Vivo Experiments guidelines and were approved and monitored by the Animal Care and Use Committee of Guangzhou Medical University.

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Supplementary Fig. S1

(A) Immunohistochemistry of Iba-1 in different regions of brain. Representative images were showed: (a) Sham group; (h) 168 h after tGCI group. The regions displayed in the right panel were cortex (b, i), CA1 (c, j), CA2 (d, k), CA3 (e, l), DG (f, m) and thalamus (g, n). (B) Immunohistochemistry of ASC in different regions of brain. Representative images were showed, (a) at 168 h after tGCI. The regions displayed in the right panel were cortex (b), CA1 (c), CA2 (d), CA3 (e), DG (f). (C) Representative photomicrographs with fluorescent staining of NLRP3 (red), Iba-1 (green) and DAPI (blue) in CA1. Representative images were showed in Sham group (a-d), 72 (e-h) and 168 (i-l) h after tGCI groups, respectively. (PNG 7590 kb)

High resolution image (TIF 9589 kb)

Supplementary Fig. S2

(A) Representative photomicrographs with fluorescent staining of NLRP3 (red), NeuN/Iba-1/GFAP (green) and DAPI (blue) in CA1. (B) Representative photomicrographs with fluorescent staining of RIP3 (red), NeuN/Iba-1/GFAP (green) and DAPI (blue) in CA1. (PNG 7292 kb)

High resolution image (TIF 9848 kb)

Supplementary Fig. S3

(A) Representative images of western blotting showing the expression of NLRP3 in CA1. The histogram presents the quantitative analysis of NLRP3 in CA1 (n = 4 independent experiments). Each bar represents the mean±S.D. *p < 0.05 vs. Sham+Vehicle group. (B) Representative images of western blotting showing the expression of p-p65 in CA1. The histogram presents the quantitative analysis of p-p65 in CA1 (n = 4 independent experiments). Each bar represents the mean±S.D. *p < 0.05 vs. Sham+Vehicle group. (C) Representative photomicrographs with fluorescent staining of p-p65 (red), Iba-1 (green) and DAPI (blue) in CA1. (D) (a-e) Representative photomicrographs show the co-localization of GFP (green), p-MLKL (gray), NeuN (red) and and DAPI (blue) in CA1 from Sham animals with AAV-hSyn-MLKL injection. (f-j) Representative photomicrographs show the co-localization of GFP (green), p-MLKL (gray), GFAP (red) and and DAPI (blue) in CA1 from Sham animals with AAV-hSyn-MLKL injection. (E) (a-e) Representative photomicrographs show the co-localization of GFP (green), p-MLKL (gray), Iba-1 (red) and and DAPI (blue) in CA1 from Sham animals with AAV-hSyn-MLKL injection. (f-j) Representative photomicrographs show the co-localization of GFP (green), p-MLKL (gray), GFAP (red) and and DAPI (blue) in CA1 from Sham animals with AAV-hSyn-MLKL injection. (PNG 5486 kb)

High resolution image (TIF 7421 kb)

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Lu, X., Zhan, L., Chai, G. et al. Hypoxic Preconditioning Attenuates Neuroinflammation via Inhibiting NF-κB/NLRP3 Axis Mediated by p-MLKL after Transient Global Cerebral Ischemia. Mol Neurobiol 61, 1080–1099 (2024). https://doi.org/10.1007/s12035-023-03628-w

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