Post-Treatment Sevoflurane Protects Against Hypoxic-Ischemic Brain Injury in Neonatal Rats by Downregulating Histone Methyltransferase G9a and Upregulating Nuclear Factor Erythroid 2-Related Factor 2 (NRF2)

Abstract

BACKGROUND Perinatal hypoxia and subsequent reduction of cerebral blood flow leads to neonatal hypoxic-ischemic brain injury (HIBI), resulting in severe disability and even death. Preconditioning or post-conditioning with sevoflurane protects against cerebral injury. This study investigated the mechanism of sevoflurane in HIBI. MATERIAL AND METHODS The HIBI model of neonatal rats was established and the model rats were post-treated with sevoflurane. The oxygen-glucose deprivation (OGD) cell model was established, and the OGD cells were transfected with NRF2-siRNA plasmid and post-treated with sevoflurane. The Morris water maze test was used to detect the motor activity, spatial learning, and memory ability of HIBI rats. Histological stainings were performed to observe the area of cerebral infarction, record the number of neurons in the hippocampus, and assess neuron apoptosis. The levels of inflammatory factors were detected by ELISA. The protein levels of histone methyltransferase G9a and histone H3 lysine 9 (H3K9me2) were detected by western blot assay. The apoptosis was detected by flow cytometry. RESULTS Sevoflurane post-treatment significantly shortened the escape latency of HIBI neonatal rats, increased the density of neurons, reduced the area of cerebral infarction, and decreased the levels of inflammatory factors and neuronal apoptosis. Sevoflurane post-treatment decreased G9a and H3K9me2 levels, and G9a level was negatively correlated with NRF2 level. NRF2 silencing reversed the alleviation of sevoflurane post-treatment on OGD-induced cell injury. CONCLUSIONS Sevoflurane post-treatment promotes NRF2 expression by inhibiting G9a and H3K9me2, thus alleviating HIBI in neonatal rats.

Figure 1

Sevoflurane post-treatment attenuates cognitive impairment in HIBI neonatal rats. (A) Escape latency of rats in each group assessed by Morris water maze test, N=18, ^# P<0.001, Sham group vs HI group; * P<0.001, HI group vs HI+S group. (B) The platform crossing times of rats in each group measured by spatial probe test, N=18. (C) The cerebral infarct size in rats of each group measured by TTC staining, N=6. The data are expressed as the mean±standard deviation. The data between groups were analyzed by one-way ANOVA, followed by Tukey’s multiple comparisons test. * P<0.05.

Figure 2

Sevoflurane post-treatment relieves inflammation and nerve cell apoptosis in HIBI neonatal rats. (A) Nissl staining was performed on the hippocampus of rat brain tissues in each group, and we compared the neuronal density ratios. (B) ELISA was used to measure the levels of inflammatory factors (TNF-α, IL-1β, IL-6, and IL-8) in rats of each group. (C) TUNEL staining was carried out to observe nerve cell apoptosis in brain tissues of rats in each group. N=6. The data are expressed as the mean±standard deviation. The data among groups were analyzed by one-way ANOVA, followed by Tukey’s multiple comparisons test. * P<0.05.

Figure 3

Sevoflurane post-treatment inhibits G9a and H3K9me2 expression in brain tissues of HIBI neonatal rats. The protein levels of G9a and H3K9me2 in rat brain tissues of each group were detected by western blot analysis. N=6. The data are expressed as the mean±standard deviation. The data among groups were analyzed by one-way ANOVA, followed by Tukey’s multiple comparisons test. * P<0.05.

Figure 4

Sevoflurane post-treatment reduces the levels of G9a and H3K9me2 in OGD-induced PC12 cells to alleviate OGD-induced cell damage. (A) The protein levels of G9a and H3K9me2 in cells of each group were detected by western blot analysis. (B) ELISA was utilized to measure the levels of inflammatory factors (TNF-α, IL-1β, IL-6, and IL-8) in cells of each group. (C) Flow cytometry was performed to detect the apoptosis rate of cells in each group. The data are expressed as the mean±standard deviation. The data among groups were analyzed by one-way ANOVA, followed by Tukey’s multiple comparisons test. * P<0.05.

Figure 5

Silencing NRF2 reverses the alleviating effect of sevoflurane post-treatment on HIBI neonatal rats. (A) NRF2 protein level in cells of each group was determined by western blot assay. (B) ELISA was utilized to measure the levels of inflammatory factors (TNF-α, IL-1β, IL-6, and IL-8) in cells of each group. (C) Flow cytometry was carried out to observe apoptosis in cells of each group. The data are expressed as the mean±standard deviation. The data among groups were analyzed by one-way ANOVA, followed by Tukey’s multiple comparisons test. * P<0.05.