Sevoflurane post-conditioning alleviates neonatal rat hypoxic-ischemic cerebral injury via Ezh2-regulated autophagy

Abstract

Background:

When neonatal rats suffer hypoxic-ischemic brain injury (HIBI), autophagy is over-activated in the hippocampus, and inhibition of autophagy provides neuroprotection. The aim of this study was to investigate the possible roles of autophagy and Ezh2-regulated Pten/Akt/mTOR pathway in sevoflurane post-conditioning (SPC)-mediated neuroprotection against HIBI in neonatal rats.

Methods:

Seven-day-old Sprague–Dawley rats underwent left common artery ligation followed by 2 h hypoxia as described in the Rice–Vannucci model. The roles of autophagy and the Ezh2-regulated Pten/Akt/mTOR signaling pathway in the neuroprotection conferred by SPC were examined by left-side intracerebroventricular injection with the autophagy activator rapamycin and the Ezh2 inhibitor GSK126.

Results:

SPC was neuroprotective against HIBI through the inhibition of over-activated autophagy in the hippocampus as characterized by the rapamycin-induced reversal of neuronal density, neuronal morphology, cerebral morphology, and the expression of the autophagy markers, LC3B-II and Beclin1. SPC significantly increased the expression of Ezh2, H3K27me3, pAkt, and mTOR and decreased the expression of Pten induced by HI. The Ezh2 inhibitor, GSK126, significantly reversed the SPC-induced changes in expression of H3K27me3, Pten, pAkt, mTOR, LC3B-II, and Beclin1. Ezh2 inhibition also reversed SPC-mediated attenuation of neuronal loss and behavioral improvement in the Morris water maze.

Conclusion:

These results indicate that SPC inhibits excessive autophagy via the regulation of Pten/Akt/mTOR signaling by Ezh2 to confer neuroprotection against HIBI in neonatal rats.

Keywords:

• sevoflurane post-conditioning
• hypoxic-ischemic brain injury
• neonatal rat
• autophagy
• Ezh2
• Pten/Akt/mTOR

Acknowledgments

This study was supported by the following grants: the National Natural Science Foundation of China (No. 81671311), the Outstanding Scientific Fund of Shengjing Hospital (No. 201708), and the Colleges and Universities Basic Research Project Fund of Liaoning Province (No. LQNK201709).

Abbreviations list

HIBI, hypoxic-ischemic brain injury; SPC, sevoflurane post-conditioning; CA, cornu ammonis; MWM, morris water maze; DG, dentate gyrus; CNS, central nervous system; MAC, minimum alveolar concentration.

Highlights

1. As high as 2.5% sevoflurane post-conditioning for 30 mins provided neuroprotection of the hippocampus in neonatal rats after hypoxic ischemia.

2. Sevoflurane post-conditioning conferred neuroprotection through the inhibition of over-activated autophagy in the hippocampus.

3. The Pten/Akt/mTOR pathway was involved in sevoflurane post-conditioning conferred neuroprotection.

4. Sevoflurane post-conditioning mediated neuroprotection through Ezh2, which functioned as an epigenetic regulator and down-regulated Pten expression by catalyzing trimethylated histone3 at lys 27.

Disclosure

The authors report no conflicts of interest in this work.

Supplementary materials

Figure S1 Autophagy markers expression levels in sham and sham + R groups. Representative western blots (A) and quantification of (B) Beclin1 and (C) LC3B-II, values are presented as mean±SD (n=6/group). (D) Representative immunofluorescent staining of LC3B (green) with NeuN (red) in hippocampus, scale bar =100 μm.

Figure S2 Expression levels of Ezh2 regulated Pten/Akt/mTOR pathway in sham and sham + G groups. Representative western blots (A) and quantification of (B) Ezh2 (C) H3K27me3/H3 (D) Pten, (E) pAkt/Akt, and (F) mTOR, values are presented as mean±SD (n=6/group).

Figure S3 Autophagy markers expression levels in sham and sham + G groups. Representative western blots (A) and quantification of (B) Beclin1 and (C) LC3B-II, values are presented as mean±SD (n=6/group). (D) Representative immunofluorescent staining of LC3B (green) with NeuN (red) in hippocampus, scale bar =100 μm.