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GW24-e2104 Epinephrine enhanced LPS-induced pro-inflammatory cytokines release by BMMCs: a cross-talking between catecholamine, circadian rhythm and inflammation
  1. Huang Xiao,
  2. Liang Chun,
  3. Zhao Xinna,
  4. He Zhiqing,
  5. Wu Zonggui
  1. Changzheng Hospital, Shanghai, China

Abstract

Objectives Occurring of acute coronary syndromes (ACSs) displayed circadian rhythms as well as the levels of catecholamines and pro-inflammatory cytokines in vivo. Such circadian rhythms were proved to exist also in mast cells which secreted plenty of pro-inflammatory cytokines and accumulated in the shoulder region of human coronary atherosclerosis plaques. Rev-erb α, a circadian rhythm modulating molecular, has been shown to modulate the release of pro-inflammatory cytokines in vitro and in vivo. Interestingly, an enhancing effect of epinephrine on pro-inflammatory cytokine release has been observed recently in human gingival fibroblasts. We hypothesised that there might be an cross-talking between catecholamine and circadian rhythm which could modulate production of pro-inflammatory cytokines in mast cells. We investigated whether epinephrine affects pro-inflammatory cytokines released from mast cells and whether rev-erb α modulates pro-inflammatory cytokines production in mast cells.

Methods Cultured murine bone marrow derived mast cells (BMMCs) were stimulated by lipopolysaccharide (LPS) with or without epinephrine at different concentrations from 10-6 M to 10-4 M for 2 hours. Then, SB203580(p38MAPK inhibitor), PD98059 (ERK1/2 MAPK inhibitor), PDTC (NF-κB inhibitor), SR8278 and GSK 4112 (rev-erb α antagonist and agonist) were added into cultures according to the protocol, respectively. Rev-erb α siRNA was transfected in to BMMCs by an nucleofector II device to confirm the role of rev-erb α in modulating pro-inflammatory cytokines production in BMMCs. IL-6, TNF-α and MCP-1 levels in medium were determined by ELISA. And, NF-κB p65, MAPK p38 and ERK1/2 phosphorylation and rev-erb α protein were assessed by western blot. BMMCs viability was assessed by WST-1 kit.

Results The production of IL-6, TNF-α and MCP-1 from LPS challenged BMMCs was significantly enhanced at the epinephrine concentration of 10-4M. Epinephrine increased phosphorylated p38 MAPK and phosphorylated p65 NF-κB expression, and LPS plus epinephrine could further increase the expression of phosphorylated p38, ERK1/2 and p65. SB203580, PD98059 and PDTC inhibited such enhancing effect of epinephrine on IL-6, TNF-α and MCP-1 from LPS challenged BMMCs. LPS increased rev-erb α protein expression in BMMCs, and epinephrine could exert further increasing effect on rev-erb α protein expression in LPS challenged BMMCs. Both GSK4112 and SR8278 significantly inhibited IL-6 production in LPS-challenged BMMCs. But a decreased cell viability was detected in GSK4112 activated BMMCs by WST-1 kit. And epinephrine had no significant enhancing effect on LPS-induced IL-6 release from SR8278 cultured BMMCs. Suppressive siRNA inhibited rev-erb α mRNA expression efficiently, and a decreased IL-6 releasing was observed in siRNA transfected BMMCs, compared to scramble control siRNA transfected BMMCs. No significant enhancing effect of epinephrine on IL-6 production was observed in siRNA transfected BMMCs.

Conclusions Epinephrine could further increase the production of pro-inflammatory cytokines from LPS-challenged BMMCs. MAPK and NF-κB pathway activations were involved in such effects of epinephrine on pro-inflammatory cytokines production. Rev-erb α activation negatively regulated LPS-induced pro-inflammatory cytokines release from BMMCs. The enhancing effect of epinephrine on pro-inflammatory cytokines was rev-erb α dependent.

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