Electroacupuncture ameliorates intestinal inflammation by activating α7nAChR-mediated JAK2/STAT3 signaling pathway in postoperative ileus

Abstract

Inflammatory cytokines produced by muscularis macrophages largely contribute to the pathological signs of postoperative ileus (POI). Electroacupuncture (EA) can suppress inflammation, mainly or partly via activation of vagal efferent. The goal of this study was to investigate the mechanisms by which EA stimulation at an hindlimb region ameliorates inflammation in POI. Methods: Intestinal motility and inflammation were examined after 24 h after intestinal manipulation (IM)-induced POI in mice. Local immune response in the intestinal muscularis, expression of macrophages, α7 nicotinic acetylcholine receptor (α7nAChR), Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) were determined by flow cytometry, Western Blot, qPCR and immunofluorescence. The effects of α7nAChR antagonists (methyllycaconitine and α-bungarotoxin) and JAK2/STAT3 inhibitors (AG490 and WP1066) were also administered in a subset of mice prior to EA. In the parasympathetic pathways, intestinal motility and inflammation were determined after cervical vagotomy and sub-diaphragmatic vagotomy. The expression of gamma absorptiometry aminobutyric acid (GABA_A) receptor in dorsal motor nucleus of vagal (DMV) cholinergic neurons was assessed by immunofluorescence and the response to DMV microinjection of bicuculine (antagonist of GABA_A receptor) or muscimol (agonist of GABA_A receptor) were assessed. Results: EA suppressed intestinal inflammation and promoted gastrointestinal motility. Mechanistically, EA activated the α7nAChR-mediated JAK2/STAT3 signaling pathway in macrophages which reduced the production of inflammatory cytokines. Furthermore, we also demonstrated that hindlimb region stimulation drove vagal efferent output by inhibiting the expression of GABA_A receptor in DMV to ameliorate inflammation. Conclusions: The present study revealed that EA of hindlimb regions inhibited the expression of GABA_A receptor in DMV neurons, whose excited vagal nerve, in turn suppressed IM-induced inflammation via activation of α7nAChR-mediated JAK2/STAT3 signaling pathway.

Keywords: GABAA receptor; JAK2/STAT3 signaling pathway; gastrointestinal motility; macrophages; α7nAChR.

Figure 1

EA attenuates IM-induced motility dysfunction and inflammatory in serum and intestinal muscularis. (A) Schematics showing the hindlimb ST36 acupoint and non-acupoint. (B) The mean gastrointestinal transit was calculated 24h after surgery. The expression of IL-6 (C) and TNF-α (D) in intestinal muscularis was analyzed by qPCR. The expression of serum IL-6 (E) and TNF-α (F) was analyzed by ELISA. ^&p < 0.05 versus Sham group, ^*p < 0.05 versus IM group, ^Δp < 0.05 versus IM + Non-acu group.

Figure 2

The anti-inflammatory effect of EA was medicated by α7nAChR. The frequencies of CD45⁺CD11b⁺MHCII⁺CD64⁺F4/80^+/- macrophages (A) and newly recruited macrophages (B) among CD45⁺ cells by flow cytometry. The expression of α7nAChR in intestinal muscularis was evaluated by Western blot (C). The frequencies α7nAChR⁺ macrophages (D.E) or newly recruited macrophages (F). The mean gastrointestinal transit (G) and the level of serum TNF-α (H) and IL-6 (I) was analyzed by ELISA. ^&p < 0.05 versus Sham group, ^*p < 0.05 versus IM group, ^Δp < 0.05 versus IM + Non-acu group, ^#p < 0.05 versus IM + Nacl + EA group.

Figure 3

EA stimulation activated the α7nAChR-mediated JAK2/STAT3 pathway in macrophages. The level of JAK2 and STAT3 in intestinal muscularis was evaluated by Western blot (A) or immunofluorescence staining (B, C). × 100 magnification. The frequencies JAK2⁺ macrophages or newly recruited macrophages (D, E). The expression of JAK2 was evaluated by Western blot with α7nAChR antagonist MLA and α-BGT (F). The mean gastrointestinal transit was evaluated in each group (G). The expression of serum IL-6 (H) and TNF-α (I) was analyzed by ELISA. ^&p < 0.05 versus Sham group, ^*p < 0.05 versus IM group, ^Δp < 0.05 versus IM + Non-acu group, ^#p < 0.05 versus IM + DMSO + EA group.

Figure 4

The protective function of EA was nearly abolished by vagotomy in POI. (A) Schematic showing the location of vagotomy. (B and G) The mean gastrointestinal transit was evaluated after LCV or SV. The expression of intestinal muscularis IL-6 (C, H) and TNF-α (D, I) was analyzed by qPCR. The expression of serum IL-6 (E, J) and TNF-α (F, K) was analyzed by ELISA. ^*p < 0.05 versus IM + LCV or IM + SV group, ^#p < 0.05 versus IM + EA + LCSV or IM + EA + SSV group. LCV, left cervical vagotomy; LCSV, left cervical sham vagotomy; SV, sub-diaphragmatic vagotomy; SSV, sub-diaphragmatic sham vagotomy.

Figure 5

Vagal nerve activity was mediated by GABA_A receptors in ChAT. The level of GABA_A was evaluated by immunofluorescence staining(A). × 400 magnification. (B) The gastrointestinal transit was measured in each brain microinjection group. The expression of IL-6 (C) and TNF-α (D) in intestinal muscularis was analyzed by qPCR. The expression of serum IL-6 (E) and TNF-α (F) was analyzed by ELISA. ^*p < 0.05 versus IM + EA + Nacl group, ^#p < 0.05 versus IM + BIM group. BIM, bicuculline.