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

Figures & data

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.

Table 1. Top 10 up-regulated and down-regulated DEGs.

Gene ID	Gene name	Transcript ID	Log2FC	P value
Up-regulated gene
ENSRNOG00000015992	Ccl20	ENSRNOT00000021730	13.39	3.84072981942949E-10
ENSRNOG00000002843	Cxcl6	ENSRNOT00000003823	3.71	3.39523089132731E-07
ENSRNOG00000050760	LOC103690035	ENSRNOT00000073192	12.71	0.0000688090643603203
ENSRNOG00000012660	Postn	ENSRNOT00000084527; ENSRNOT00000017453	1.45	0.000815097780700335
ENSRNOG00000002548	Tnn	ENSRNOT00000003452; ENSRNOT00000076616	3.44	0.00200342823930197
ENSRNOG00000016164	Fcrl2	ENSRNOT00000021938	1.26	0.00223843358506834
ENSRNOG00000020951	Slc4a1	ENSRNOT00000092102; ENSRNOT00000028445	1.98	0.00348500772958391
ENSRNOG00000000716	Htr1f	ENSRNOT00000000907	1.29	0.00411615717014051
ENSRNOG00000005960	RGD1311744	ENSRNOT00000036054	1.11	0.00607683506368763
ENSRNOG00000001915	Chodl	ENSRNOT00000031889; ENSRNOT00000090878	2.33	0.00609892431996525
Down-regulated gene
ENSRNOG00000008816	Gpnmb	ENSRNOT00000011945	−1.70	1.06602538530999E-10
ENSRNOG00000047350	Gns	ENSRNOT00000064349	−14.85	8.78140095997793E-08
ENSRNOG00000047657	C4a	ENSRNOT00000083833; ENSRNOT00000072377; ENSRNOT00000078871	−2.09	1.73016156759426E-07
ENSRNOG00000023340	Tmem72	ENSRNOT00000037397	−5.09	1.52166007039475E-06
ENSRNOG00000018735	Cd74	ENSRNOT00000025344; ENSRNOT00000025354	−1.03	2.26256481219666E-06
ENSRNOG00000039107	Mfrp	ENSRNOT00000059813; ENSRNOT00000085578	−5.27	3.25447958914502E-06
ENSRNOG00000010378	Slc4a5	ENSRNOT00000082336; ENSRNOT00000014249	−4.89	3.45959002077057E-06
ENSRNOG00000016275	Ttr	ENSRNOT00000022113	−5.03	4.79364146850667E-06
ENSRNOG00000047471	LOC103689994	ENSRNOT00000064063	−12.55	7.34042831884681E-06
ENSRNOG00000048322	LOC100911492	ENSRNOT00000074488	−13.33	0.00609892431996525

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.

Table 2. Degree of top 10 hub genes.

Rank	Gene ID	Gene name	Description	Degree
1	ENSRNOG00000014288	Fn1	fibronectin 1	32
2	ENSRNOG00000016611	Hras	HRas proto-oncogene, GTPase	30
2	ENSRNOG00000000513	Mapk14	mitogen activated protein kinase 14	30
4	ENSRNOG00000005659	Aurkb	aurora kinase B	24
4	ENSRNOG00000014183	Gnaq	G protein subunit alpha q	24
4	ENSRNOG00000013536	Cdc42	cell division cycle 42	24
7	ENSRNOG00000020311	Cxcl5	C-X-C motif chemokine ligand 5	22
7	ENSRNOT00000065721	Sgol1	shugoshin 1	22
7	ENSRNOT00000031074	Cenpc1	Centromere protein C 1	22
7	ENSRNOG00000001068	Rac1	Rac family small GTPase 1	22

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.

Data availability statement

The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.