IL-17A deletion reduces sevoflurane-induced neurocognitive impairment in neonatal mice by inhibiting NF-κB signaling pathway

Abstract

We investigated the role of IL-17A in sevoflurane-inducedneurocognitive impairment in neonatal mice. Seventy-two wild-type (WT) and 42 IL-17A knockout (KO) neonatal mice were randomly divided into WT (n = 36), IL-17A^-/- (n = 6), sevoflurane (Sev, n = 36), and IL-17A^-/- + sevoflurane (IL-17A^-/- + Sev, n = 36) groups. The latter two groups were given 3% sevoflurane for 2 h per day on postnatal days (P) 6-8. Behavioral experiments were performed on P30-36. At P37, RNA sequencing and qRT-PCR of the hippocampus was performed, neurons were detected by Nissl staining, and neuropathological changes were evaluated by HE staining. NF-κB pathway-related proteins were evaluated by western blot and immunofluorescence analyses, IL-1β and IL-6 levels were assessed by ELISA. RNA sequencing identified 131 differentially expressed genes, highlighting several enriched biological processes (chemokine activity, immune response, extracellular region, extracellular space, inflammatory response) and signaling pathways (IL-17 signaling pathway, chemokine signaling pathway, cytokine-cytokine receptor interaction, ECM-receptor interaction and influenza A). Repeated sevoflurane exposures induced long-term cognitive impairment in WT mice. The cognitive impairment was comparatively less severe in IL-17A KO mice. In addition, the increased levels of NF-κB p65, iNOS, COX-2, IL-17A, IL-6 and IL-1β, reduced neuronal numbers and neuropathological changes were ameliorated in neonatal mice in the IL-17A^-/- + Sev group compared with neonatal mice in Sev group. IL-17A deletion protects against long-term cognitive impairment induced by repeated sevoflurane exposure in neonatal mice. The underlying mechanism may relate to inhibiting NF-κB signaling pathway as well as the reducing neuroinflammation.

Keywords: IL-17A; NF-κB signaling pathway; cognitive dysfunction; neuroinflammation; sevoflurane.

Graphical abstract

Figure 1.

Experimental flow chart. Mice received anesthesia on postnatal days 6, 7 and 8 (P6–8) with 3% sevoflurane supplemented with 60% oxygen for 2 h daily. RNA-seq and RT-PCR were performed at P30, the Morris water maze test was conducted on P30 to P36. At P36, after the Morris water maze test, Nissl staining, HE staining, ELISA, western blot and immunofluorescence assay were performed.

Figure 2.

DEG identification and GO analysis of DEGs. (a) Representative heat map of differentially expressed genes in WT and WT + sevoflurane group. (b) Representative heat map of differentially expressed genes in IL-17A^−/− and IL-17A^−/− + Sev group. (c) Differential gene volcano map in WT and WT + sevoflurane group. (d) Differential gene volcano map in IL-17A^−/− and IL-17A^−/− + Sev group. (e-f) GO enrichment analysis upregulated pathways in WT and WT + sevoflurane group. (g-h) GO enrichment analysis downregulated pathways in WT and WT + sevoflurane group.

Figure 3.

KEGG pathway analysis of DEGs, PPI network construction and hub gene identification (a) KEGG pathway enrichment scatter diagram in WT and WT + sevoflurane group. (b) KEGG pathway enrichment scatter diagram in IL-17A^−/− and IL-17A^−/− + Sev group. (c) The protein–protein interaction network of DEGs in WT and WT + sevoflurane group. (d)Ten hub genes with the highest degree in WT and WT + sevoflurane group(Red square indicates a higher degree, and yellow square indicates a lower degree). (e) GSEA gene enrichment analysis diagram. (f) Differential expression of IL-17 was confirmed by RT-PCR. Data are expressed as mean ± SD. Compared with WT group, *P < 0.05.

Figure 4.

IL-17A deletion ameliorates learning and memory impairment induced by repeated sevoflurane exposure in neonatal mice. (a) Swimming trajectory in spatial exploration experiment. (b) Average escape latency. (c) Times of crossings of the platform area. (d) Average swimming speed in the MWM test. Data are expressed as mean ± SD (n = 30 for each group). Compared with the WT group, *P < 0.05; compared with the Sev group, ^#P < 0.05.

Figure 5.

IL-17A deletion ameliorates neuronal injury after repeated sevoflurane exposure in neonatal mice. (a) Representative photomicrographs of Nissl staining in the hippocampal CA1 region; scale bar = 10 μm. (b) Quantification of the number of Nissl bodies using Nissl staining in the hippocampal CA1 region. (c) Representative images of histopathological changes in the hippocampal CA1 of neonatal mice. Data are expressed as mean ± SD (n = 5 for each group). Compared with the WT group, *P < 0.05; compared with the Sev group, ^#P < 0.05.

Figure 6.

IL-17A deletion diminishes the increase in inflammatory factor levels induced by repeated exposure to sevoflurane in neonatal mice. The levels of hippocampal IL-6 (a) and IL-1β (b). Data are presented as mean ± SD (n = 5 for each group). Compared with the WT group, *P < 0.05; compared with the Sev group, ^#P < 0.05.

Figure 7.

IL-17A deletion ameliorates activation of repeated sevoflurane exposure-induced NF-κB signaling pathway in neonatal mice. (a) Representative western blot of IL-17A, NF-κB p65, iNOS and COX-2. (b) Representative histogram of the relative expression of IL-17A, NF-κB p65, iNOS and COX-2. Data are expressed as mean ± SD (n = 10 for each group). Compared with the WT group, *P < 0.05; compared with the Sev group, ^#P < 0.05.

Figure 8.

IL-17A deletion reduces the upregulation of NF-κB p65 in repeated sevoflurane exposure-induced hippocampal neuronal cells in neonatal mice. (a) Representative photomicrographs of NF-κB p65/NeuN/DAPI staining (NF-κB p65: red; NeuN: green; DAPI: blue); scale bar = 10 μm. (b) Percentages of NF-κB p65/NeuN/DAPI positive cells. Data are expressed as mean ± SD. Compared with the WT group, *P < 0.05; compared with the Sev group, ^#P < 0.05.