GluN2A/ERK/CREB Signaling Pathway Involved in Electroacupuncture Regulating Hypothalamic-Pituitary-Adrenal Axis Hyperactivity

Abstract

The hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis caused by stress will inevitably disrupt the homeostasis of the neuroendocrine system and damage physiological functions. It has been demonstrated that electroacupuncture (EA) can modulate HPA axis hyperactivity during the perioperative period. As the initiating factor of the HPA axis, hypothalamic corticotrophin-releasing hormone (CRH) is the critical molecule affected by EA. However, the mechanism by which EA reduces CRH synthesis and secretion remains unclear. Activated N-methyl-D-aspartate receptor (NMDAR) has been linked to over-secretion of hypothalamic CRH induced by stress. To determine whether NMDAR is involved in EA regulating the over-expression of CRH, a surgical model of partial hepatectomy (HT) was established in our experiment. The effect of EA on hypothalamic NMDAR expression in HT mice was examined. Then, we investigated whether the extracellular regulated protein kinases (ERK)/cyclic adenosine monophosphate response element-binding protein (CREB) signaling pathway mediated by NMDAR was involved in EA regulating HPA axis hyperactivity. It was found that surgery enhanced the expression of hypothalamic CRH and caused HPA axis hyperactivity. Intriguingly, EA effectively suppressed the expression of CRH and decreased the activation of GluN2A (NMDAR subunit), ERK, and CREB in HT mice. GluN2A, ERK, and CREB antagonists had similar effects on normalizing the expression of CRH and HPA axis function compared with EA. Our findings suggested that surgery enhanced the activation of the hypothalamic GluN2A/ERK/CREB signaling pathway, thus promoting the synthesis and secretion of CRH. EA suppressed the phosphorylation of GluN2A, ERK, and CREB in mice that had undergone surgery, indicating that the GluN2A/ERK/CREB signaling pathway was involved in EA alleviating HPA axis hyperactivity.

Keywords: CRH; GluN2A/ERK/CREB signaling pathway; HPA axis; electroacupuncture; surgical trauma.

FIGURE 1

EA ameliorates the HPA axis hyperactivity induced by surgery. (A) Timeline of experimental protocol. (B) ACTH and (C) CORT in the peripheral serum of mice in the NC, HT, and HT + EA groups (n = 6 for each group). (D,E) CRH protein and (F) mRNA expression in the hypothalamus. The CRH protein bands were normalized to β-tubulin. (G,H) Representative immunofluorescence images and quantification for CRH-positive cells in the PVN (n = 4 for each group). Data are expressed as mean ± SEM. *vs. NC group (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001); #vs. HT group (^#p < 0.05, ^##p < 0.01, ^###p < 0.001). One-way analysis of variance (ANOVA). CRH, Corticotrophin-releasing hormone; ACTH, Adrenocorticotropic hormone; CORT, Corticosterone; PVN, Paraventricular nucleus.

FIGURE 2

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).

FIGURE 3

Hypothalamic CRH expression was downregulated after EA treatment in NMDA-administered mice. (A) Cannula implantation was performed and fluorescent dye (DiIC18) was injected into the hypothalamus through the cannula to determine the accuracy of drug injection (Administration location: AP 0.6 mm, ML ± 0.2 mm, DV 4.5 mm). For the GluN2A agonist, different doses of NMDA (0.08 nmol/μL, 0.4 nmol/μL, and 2 nmol/μL) were injected into the bilateral hypothalamus. (B) Quantification of hypothalamic CRH mRNA. (C,D) Representative WB images and quantification for CRH protein (n = 6 for each group) 24 h after NMDA injection. Quantification of CRH (E) mRNA and (F,G) protein in the NS, NMDA, NMDA + EA, and EA groups (n = 5 for each group). Data are expressed as mean ± SEM. *vs. NS group (*p < 0.05, **p < 0.01, ***p < 0.001); #vs. NMDA group (^#p < 0.05, ^##p < 0.01).

FIGURE 4

EA inhibits the phosphorylation of hypothalamic ERK and CREB. (A,B) Expression of hypothalamic phosphorylated ERK (pERK) and total ERK proteins in the NC, HT, and HT + EA groups (n = 6 for each group). (C,D) Immunofluorescence representative images and quantitative analysis of pERK-positive cells in the PVN (n = 4 for each group). (E,F) Expression of hypothalamus phosphorylated CREB and total CREB proteins (n = 5 for each group). The pERK and pCREB protein bands were normalized to ERK or CREB. 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). ERK, Extracellular regulated protein kinases; CREB, cAMP-response element binding protein.

FIGURE 5

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).