Magnesium Protects Against Sepsts By Relieving Extracellular Histone-induced Macrophage Damage

Background and objectiveThe immune response of macrophages is involved in the pathogenesis of sepsis.Extracellular histones have been identified as important mediators of sepsis that can cause cytotoxicity and further affect the innate immune system.Therefore,it is crucial to find effective drugs with few side effects that counteract histone-mediated immune abnormalities in sepsis.Mg2+ is one of the essential nutrients involved in the regulation of normal immune function of the body.We aim to explore the mechanism of extracellular histone-induced macrophage damage and to elucidate the possible protective role of Mg2+.We hope to provide an important experimental basis for the pathogenesis and clinical treatment of sepsis.Content and methods1.Investigation the role of extracellular histones in inducing macrophage damage(1)Bioinformatic analysis indicated that the apoptotic signalling pathway may be involved in the pathogenic mechanism of extracellular histones in sepsis.(2)Elaboration the role of extracellular histones on macrophage damage by cell activity assay,apoptosis assay,western blotting and mitochondrial membrane potential assay.2.Investigation the protective effect of Mg2+ on macrophage damage under extracellular histone stimulationDemonstration the protective role of Mg2+ on extracellular histone-induced macrophage damage by cell activity assay,apoptosis assay,western blotting and mitochondrial membrane potential assay.3.Elaboration the cellular signal transduction mechanism of Mg2+ inhibition of extracellular histone-induced macrophage damage(1)Bioinformatic analysis indicated that the calcium signalling pathway may be important in the pharmacological mechanism of Mg2+action.(2)Verification the role of extracellular histones on intracellular Ca2+signaling alteration by measuring the calcium fluorescence probe(fluo-3 AM),and elucidation the effect of Mg2+ on intracellular calcium signaling.(3)Demonstration which types of calcium channels are involved in extracellular histone-induced cellular damage through various calcium channel inhibitors.4.Elucidation the association of Mg2+and the fate of immune macrophages in sepsis state in vivo(1)An intravenous histone injection model was constructed to assess the effect of Mg2+on mouse survival and apoptosis of peritoneal macrophages.And a CLP model was constructed to assess the effect of Mg2+ on the apoptosis of murine peritoneal macrophages.(2)The data of septic patients in ICU were obtained through the MIMIC-Ⅳdatabase to assess the relationship between serum magnesium levels and monocyte counts in these patients.Result1.Extracellular histones mediated macrophage apoptosis via the mitochondrial pathway.2.Mg2+inhibited extracellular histone-induced apoptosis in macrophages through the mitochondrial pathway.Mg2+ was very important for cell survival under stress.3.Elevated intracellular Ca2+levels were an important event in extracellular histone-induced macrophage damage.Altered calcium signalling associated with PLC/IP3R/STIM might be involved in the protective mechanism of Mg2+.4.Mg2+ attenuated extracellular histone-mediated mouse death and reduced extracellular histone or ClP-induced apoptosis of peritoneal macrophages in mice.In addition,clinical data showed that low magnesium levels in sepsis patients were associated with low monocyte counts.ConclusionsMg2+ may inhibit extracellular histone-induced mitochondria-related apoptosis in macrophages via the PLC/IP3R/STIM-mediated calcium signalling pathway,suggesting that Mg2+might protect against the immunosuppressive state of sepsis induced by extracellular histones.Furthermore,Mg2+ treatment significantly reduced the susceptibility of mice to extracellular histone-mediated death.Magnesium supplementation may be a promising treatment for septic patients,especially those with elevated histone levels.