Effect Of Lidocaine On Glycolysis In LPS-stimulated Macrophages And Its Potential Mechanism

BackgroundSepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection.But the molecular mechanism wasn`t illustrated clearly and its effective treatment also wasn`t found.Inflammatory cytokines were mainly released by macrophages in which glycolysis played an important role.There is increasing emphasis on the important role of macrophage glycolysis in the treatment of sepsis.Pathogen-associated molecular mode receptors(such as LPS)act on the surface of macrophages to cause changes,such as upregulation of HIF-1?.HIF-1? is a central link in regulating glycolysis,and the high expression of HIF-1? also leads to the high expression of glycolysis molecules,including glucose transporter 1(GLUT1)and hexokinase 2(HK2).This paper investigated the effects of lidocaine on LPS-stimulated inflammatory cytokines and the mechanism of glycolysis in mouse peritoneal macrophages.Methods1.The mouse peritoneal macrophages(PMs)were isolated and cultured in vitro.The cells were stimulated with lidocaineand LPS simultaneously.After 6h,the supernatant was collected to measure the level of TNF-? and IL-6 by ELISA.PMs were pre-incubated with various concentrations of lidocaine for 6h.PMs were stimulated with various concentrations of lidocaine for 6 hours.CCK-8 reagent was added for measuring cell viability after 2 hours.2.The effect of lidocaine on glycolysis were evaluated.PMs were stimulated with lidocaine and LPS simultaneously.After 1h,the mRNA expression of GLUT1,HK2 were detected in PMs.3.Granulocyte macrophage colony stimulating factor(GM-CSF)enhances macrophages glycolytic activity.The effect of lidocaine on PMs were studied by pre-incubating of GM-CSF.PMs were activated with or without GM-CSF for 2.5h before treatment of lidocaine,followed by LPS exposure for 6h.The level of TNF-? and IL-6 were measured by ELISA.The mRNA expression of GLUT1,HK2 were detected after 1h.4.PMs were stimulated with lidocaine and LPS simultaneously.The mRNA expression of HIF-1? was measured after 6h.Pretreatment of DMOG can markedly increase expression level of HIF-1?.Whether DMOG can reverse the effect of lidocaine was observed.PMs were incubated with or without DMOG for 2h before treatment with lidocaine,followed by LPS for 6h.TNF-? and IL-6 were measured by ELISA.And the mRNA expression of GLUT1,HK2 were determined by PCR after LPS stimulation for 1h.Results1.Lidocaine can significantly inhibit the production of TNF-? and IL-6 from LPS-stimulated PMs.In addition,lidocaine dose-dependently inhibited TNF-? and IL-6 production.Lidocaine,at concentration of 1–5 ?M did not significantly influence the viability of PMs.2.Lidocaine can significantly inhibit mRNA expression of GLUT1,HK2 in LPS-stimulated PMs.3.After pretreatment with GM-CSF,lidocaine partially reduced the increase of TNF-? and IL-6 in LPS-stimulated PMs,and reversed the high mRNA expression of GLUT1 and HK2 in PMs.4.After pretreatment with DMOG,lidocaine partially reduced the increase of TNF-? and IL-6 in LPS-stimulated PMs,and reversed the high mRNA expression of HIF-1?,GLUT1 and HK2 in PMs.ConclusionBy inhibiting the HIF-1?-mediated glycolytic pathway,lidocaine can significantly inhibit LPS-stimulated inflammatory cytokine in mouse peritoneal macrophages.