Fig 2: The GluN2A, ERK, and CREB antagonist reverses HPA axis hyperactivity induced by surgery. The GluN2A antagonist PEAQX (1 µg/µL, 0.25 µL/side) was administered to both sides of the hypothalamus. Hypothalamus was dissected 24 h after the PEAQX administration. (A–D) Representative bands and quantification of hypothalamic pERK, pCREB, and CRH protein among the NS, HT + NS, HT + PEAQX, and HT + EA groups (n = 4 for each group). The ERK inhibitor MEK1/2 Inhibitor IV (0.1 nmol/µL, 0.5 µL/side) was administered stereotaxically into the hypothalamus. (E–G) Representative bands and quantification of pCREB and CRH protein in the NS, HT + NS, HT + MEK, and HT + EA groups (n = 5 for each group). 666-15 (an antagonist of CREB, 0.1 nmol/µL, 0.5 µL/side) was administrated to investigate the relationship between CREB and HPA axis. (H,I) Western Blot analysis of hypothalamic CRH protein in the NS, HT + NS, HT + 666-15, and HT + EA groups (n = 5 for each group). (J) Relative hypothalamic CRH mRNA expression after surgical trauma and drugs administration (n = 5 for each group). (K,L) ACTH and CORT levels in the peripheral serum of mice (n = 5 for each group). Data are expressed as mean ± SEM. *vs. NS group (*p < 0.05, **p < 0.01, ***p < 0.001); #vs. HT group (#p < 0.05, ##p < 0.01, ###p < 0.001).

Fig 3: EA reduces hypothalamic GluN2A expression after surgery. Quantification of hypothalamic (A) GluN1, (B) GluN2A, and (C) GluN2B mRNA levels in the NC, HT, and HT + EA groups (n = 6 for each group). (D–F) Expression of phosphorylated GluN2A (pGluN2A) and total GluN2A protein in the hypothalamus was analyzed by western blot (n = 6 for each group). (G,H) Representative images and quantification for Co-labeled CRH and GluN2A-positive cells in the PVN (n = 4 for each group). Data are expressed as mean ± SEM. *vs. NC group (**p < 0.01, ***p < 0.001); #vs. HT group (#p < 0.05, ##p < 0.01, ###p < 0.001). One-way analysis of variance (ANOVA).