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. 2021 Jul 6:9:e11609.
doi: 10.7717/peerj.11609. eCollection 2021.

NLRP3 inflammasome activation mediates sleep deprivation-induced pyroptosis in mice

Kun Fan #  1 , Jiajun Yang #  2 , Wen-Yi Gong  3 , Yong-Chao Pan  2 , Peibing Zheng  2 , Xiao-Fang Yue  2
Affiliations

NLRP3 inflammasome activation mediates sleep deprivation-induced pyroptosis in mice

Kun Fan et al. PeerJ. .

Abstract

Background: Sleep deprivation (SD) has many deleterious health effects, including cognitive decline, work ability decline, inadequate alertness, etc. Neuroinflammation plays an important role in sleep deprivation. However, the underlying mechanism is still unclear.

Methods: In the present study, we detected the activation of microglia and apoptosis of nerve cells in sleep deprivation (SD) mice model using IHC, HE staining and TUNEL apoptosis assay. RT-PCR array data were used to detect the expression of inflammatory bodies in hippocampal CA1 region after sleep deprivation, to explore how NLRP3 inflammasome regulates neuronal apoptosis and how specific signaling pathways are involved in SD-induced activation of NLRP3/pyrosis axis.

Results: We found the number of microglia significantly increased in SD mice, while this effect was blocked by sleep recovery. RT-PCR array data suggested that NLRP3 inflammasome, but not other inflammasomes, was obviously increased in hippocampus CA1 region after sleep deprivation. Mechanistically, we found that NLRP3 mediated the pyroptosis of neurocyte through GSDMD-dependent way , and P38 and ERK-MAPK signaling pathway is involved in SD-induced activation of NLRP3/pyroptosis axis. All these results suggested that MAPK/NLRP3 axis mediated SD-induced pyroptosis.

Conclusion: NLRP3 plays an important role in SD-induced neuroinflammation. Thus, NLRP3 inflammasome is expected to be a potential therapeutic target for SD-induced neuroinflammation.

Keywords: MAPK; NLRP3; Pyroptosis; Sleep deprivation.

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Conflict of interest statement

The authors declare there are no competing interests.

Figures

Figure 1
Figure 1. Sleep deprivation induced the increase of microglia and neuronal damage.
(A and B) IHC for IBA-1 level in hippocampus CA1 region of control group (n = 10), SD group (n = 10) and SD+ recover group (n = 10). Scale bars =50 µm for IHC. (C and D) H&E staining for hippocampus CA1 region in control group (n = 10), SD group (n = 10) and SD+ recover group (n = 10). Scale bars =100 µm for H&E. (E and F) Cell apoptosis were detected in hippocampus CA1 region in control group (n = 10), SD group (n = 10) and SD+ recover group (n = 10) by Tunel staining. Scale bars =25 µm for Tunel staining. Results represent the mean ofthreeindependentexperiments * p < 0.05. IHC, immunohistochemical; IBA-1, Ionized calcium binding adaptor molecule-1; SD, sleep deprivation; H&E staining, hematoxylin-eosin staining.
Figure 2
Figure 2. Sleep deprivation promoted the activation of NLRP3/caspase 1 pathway.
(A) RT-PCR array for the expression levels of genes (IL-1 β, IL-18, Caspase-1, NLRP3, Caspase-4, ASC, GSDMD, NLRP1, NLRP2 and AIM2) in control group, SD group and SD+ recover group (n = 5). (B and C) Western blot analysis for Caspase-1, ASC and NLRP3 protein level in hippocampus CA1 region of control group, SD group and SD+ recover group (n = 5). (D) IHC for NLRP3 level in hippocampus CA1 region of control group, SD group and SD+ recover group. Scale bars = 50 µm for IHC (n = 5). Results represent the mean of three independent experiments *p < 0.05, **p < 0.01. RT-PCR, Quantitative real-time PCR; IHC, immunohistochemical; SD, sleep deprivation; ASC, -associated speck-like protein containing a CARD.
Figure 3
Figure 3. Sleep deprivation induced the pyroptosis of neuronal cell.
qPCR (A) and ELISA (B) analysis of IL-1β, IL-18 and TNF-α expression in hippocampus CA1 region of control group, SD group and SD+ recover group and qPCR analysis of IL-1 β, IL-18 and TNF-α expression in hippocampus of control group, SD group and SD+ recover group (n = 5). (C and D) Western blot analysis for GSDMD and bax protein level in mouse hippocampus CA1 region of control group, SD group and SD+ recover group (n = 5). (E) IHC for GSDMD level in mouse hippocampus CA1 region of control group, SD group and SD+ recover group (n = 5). Scale bars = 50 µm for IHC. (F) Fluorescence co-localization for GSDMD and NeuN in mouse hippocampus CA1 region of control group, SD group and SD+ recover group (n = 5). Scale bars = 20 µm. Results represent the mean of three independent experiments *p < 0.05, **p < 0.01. qPCR, Quantitative real-time PCR; IHC, immunohistochemical; SD, sleep deprivation.
Figure 4
Figure 4. Sleep deprivation induces the activation of P38 and ERK MAPKs pathway.
(A and B) Western blot analysis for p-P38 and P38 protein level in mouse hippocampus CA1 region of control group, SD group and SD+ recover group (n = 5). (C and D) Western blot analysis for p-ERK and ERK protein level in mouse hippocampus CA1 region of control group, SD group and SD+ recover group (n = 5). (E and F) Western blot analysis for p-AKT and AKT protein level in mouse hippocampus CA1 region of control group, SD group and SD+ recover group (n = 5). (G and H) Western blot analysis for NLRP3 and GSDMD protein level in mouse hippocampus CA1 region of control group, SD group, SD+ SB203580, and SD+ U0126 group (n = 5) Results represent the mean of three independent experiments * p < 0.05. SD, sleep deprivation.
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Grants and funding

This work was supported by the National Natural Science Foundation of China (grant number 31400953). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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