Elsevier

Brain Research Bulletin

Volume 159, June 2020, Pages 25-31
Brain Research Bulletin

Hypothermia protects neurons against ischemia/reperfusion-induced pyroptosis via m6A-mediated activation of PTEN and the PI3K/Akt/GSK-3β signaling pathway

https://doi.org/10.1016/j.brainresbull.2020.03.011Get rights and content

Highlights

  • Hypothermia protected hippocampal neurons against from H/R induced pyroptosis.

  • The PTEN and PI3K/Akt/GSK-3β signaling participated in activating inflammasome and promoting pyroptosis.

  • Methylated level of PTEN mRNA was elevated in hippocampal neurons upon H/R.

Abstract

Cerebral ischemia/reperfusion (I/R) injury often leads to irreversible neuronal injury and even death, and hypothermia is the only therapeutic method that has been proven to be effective. However, the molecular mechanisms underlying the effect of hypothermia treatment on I/R injury have not been fully elucidated. In the present study, we aimed to evaluate the neuroprotective effects and mechanisms of hypothermia against hypoxia/reoxygenation (H/R)-induced neuronal damage. Primary hippocampal neurons were exposed to H/R and were then treated with hypothermia. We observed that hypothermia significantly increased cellular viability, downregulated the expression of pyroptosis-related proteins—including NLR pyrin domain containing 3 (NLRP3), apoptotic speck-like protein containing CARD (ASC), cleaved Caspase-1, and Gasdermin-D (GsdmD) p30—and reduced secretion of the pro-inflammatory cytokines, IL-1β and IL-18. Additionally, pretreatment with MCC950, a specific small-molecule inhibitor of the NLRP3 inflammasome, yielded a protective effect on cellular viability that was comparable to that of hypothermia treatment. Furthermore, hypothermia also significantly elevated the expression level of phosphatase and tensin homologous protein (PTEN) and activated the phosphorylation levels of protein kinase B (Akt) and glycogen synthase kinase-3β (GSK-3β). These protective effects of hypothermia on pyroptosis-related proteins and pro-inflammatory cytokines were partially reversed by the specific PI3K/Akt inhibitor, LY294002. Moreover, the methylated level of PTEN mRNA was elevated in hippocampal neurons upon H/R, whereas this level remained stable in the hypothermia group. Therefore, our findings suggest that hypothermia protects neurons against neuronal H/R-induced pyroptosis, and that m6A-mediated activation of PTEN and the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt/GSK-3β signaling pathway may play crucial roles during this process.

Introduction

Advances in clinical resuscitation in recent years have increased survival rates following cardiac arrest (CA) (Mallikethi-Reddy et al., 2017; Yamaguchi et al., 2017). However, cerebral injury remains the major cause of morbidity and death among CA victims (Lemiale et al., 2013). Therefore, alleviating cerebral injury after cardiopulmonary resuscitation (CPR) is key to improving patient neurological functions and survival rates. However, aside from target temperature management (TTM), which has been recommended by the latest CPR guidelines (2015) for coma patients after successful CPR by maintaining their body temperature at 32–36 °C (Callaway et al., 2015; Donnino et al., 2015), few other therapeutic methods are clinically available worldwide.

Previous studies have found that cerebral injury after CPR following CA histologically manifests as necrosis, apoptosis, and autophagy (Lalaoui et al., 2015). In recent years, mounting evidence has suggested that neuronal pyroptosis also has a critical impact on the pathogenesis of cerebral ischemia/reperfusion (I/R) injury (Guo et al., 2016) and may represent a potentially therapeutic target. Pyroptosis is a highly inflammatory form of Caspase-1-dependent programmed cell death and is distinct from apoptosis (Gaidt and Hornung, 2016). The NLR pyrin domain containing 3 (NLRP3) inflammasome is one of the best-characterized inflammasomes and consists of NLRP3, apoptotic speck-like protein containing CARD (ASC), and Caspase-1 (Guo et al., 2016). Recent research has demonstrated that the NLRP3 inflammasome plays a vital role in the development of I/R-induced cerebral injury (Qiu et al., 2016; Wang et al., 2015). In addition, NLRP3 inflammasome inhibitors (e.g., MCC950) have been shown to alleviate neurological I/R injury (Wang et al., 2019a,b,c). Thus, controlling NLRP3 inflammasome activation may provide a beneficial therapeutic strategy for CA. Furthermore, hypothermia has been shown to improve cerebral function by inhibiting the NLRP3 inflammasome and its downstream inflammatory factors in vitro (Tu et al., 2019) and in vivo (Qian et al., 2019). However, the underlying mechanisms of hypothermia-mediated neuroprotection by inhibiting pyroptosis remain largely unknown.

The phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B (PI3K/Akt) signaling pathway is one of the major survival pathways in cells and has been increasingly implicated in neuronal survival after I/R injury. Phosphorylation levels of Akt levels have been found to increase rapidly with reperfusion after cerebral ischemia (Zhao et al., 2005). Furthermore, activation of the PI3K/Akt signaling pathway alleviates but does not eliminate cerebral injury (Endo et al., 2006), which suggests that some PI3K/Akt-independent mechanisms may also be responsible for cerebral I/R injury. Phosphatase and tensin homologous protein (PTEN) negatively regulates the PI3K/Akt pathway; inhibition of PTEN has been shown to protect against oxygen-glucose deprivation/reperfusion (OGD/R)-induced neuronal death (Farajdokht et al., 2018) and cardiac arrest induces neurological injury (Li et al., 2015) via the PI3K/AKT signaling pathway. However, the underlying mechanisms by which PTEN is regulated remain largely unknown.

Methylation of the adenosine at the N6 position that leads to the formation of N6-methyladenosine (m6A) is the most abundant and reversible methylation modification, accounting for >80 % of all mRNA-base methylations in the adult brain (Meyer et al., 2012). Recent studies have found that both stroke (Chokkalla et al., 2019) and traumatic brain injury (Wang et al., 2019a,b,c) alter cerebral m6A modifications, the latter of which may represent a therapeutic target for cerebral I/R injury. However, whether hypothermia attenuates cerebral I/R injury by m6A-mediated activation of PTEN and the PI3K/Akt/GSK-3β signaling pathway has not yet been investigated.

Accordingly, the purpose of the present study was to determine the roles and mechanisms of hypothermia in primary hippocampal neurons in protecting against hypoxia/reoxygenation (H/R)-induced pyroptosis.

Section snippets

Cell culture

Primary hippocampal neurons isolated from newborn rats (1–3 days) were used in the present study. After disinfection, rat cerebral tissues were quickly removed and placed into a culture dish containing phosphate-buffered saline (PBS). Under an operating microscope, hippocampal tissues were separated and transferred into a centrifuge tube containing Dulbecco’s modified eagle’s medium (DMEM)/F12 medium (Thermo Fisher, Carlsbad, CA, USA). The mixture was subsequently centrifuged at 1000 rpm for

Hypothermia protects hippocampal neurons against H/R-induced pyroptosis

To explore the protective effect of hypothermia on H/R-injured hippocampal neurons, the CCK-8 assay was performed to assess cellular viability. As shown in Fig. 1A, cellular viability dramatically decreased in the H/R group compared with that of the normoxic group. Conversely, hypothermia significantly preserved cellular viability compared with that of the normal H/R group. Moreover, H/R significantly induced pyroptosis, which is a kind of programmed cell death indicated by the levels of

Discussion

The present study demonstrated that hypothermia significantly protected primary hippocampal neurons against H/R-induced cellular death. The protective effects of hypothermia were achieved via PTEN inhibition and enhanced PI3K/Akt signaling, which further inhibited pyroptosis. In addition, hypothermia significantly reduced inflammatory injury in hippocampal neurons by depressing the expression levels of the NLRP3 inflammasome and its related cytokines. In contrast, inhibition of PI3K/Akt pathway

CRediT authorship contribution statement

Meng-Yuan Diao: Conceptualization, Methodology, Investigation, Writing - original draft. Ying Zhu: Investigation, Validation. Jing Yang: Investigation, Writing - original draft. Shao-Song Xi: Methodology, Investigation. Xin Wen: Investigation. Qiao Gu: Formal analysis, Visualization. Wei Hu: Conceptualization, Validation, Writing - review & editing, Supervision.

Declaration of Competing Interest

The authors declare no conflicts of interest, financial or otherwise.

Acknowledgments

We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

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