Extracellular Histones Induce Macrophage Apoptosis By Activating The Mitochondrial Pathway Via Ca²⁺Influx

BackgroundSepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection.It is a common complication of infection,trauma,shock,burns and is also an important cause of multiple organ dysfunction syndrome(MODS).According to epidemiological statistics,about 751,000 people in the United States are diagnosed with sepsis every year,and 215,000 died although accept active treatment.Sepsis is an urgent,rapid-developing critical disease with complex pathologic mechanisms,which is an important cause of death in critically ill patients.Immune suppression is prevalent in sepsis patients and multiple mechanisms are involved in the immunosuppression.Immune cell apoptosis plays an important role in immunosuppression.Macrophages are important innate immune cells,as well as important antigen presenting cells,which play an important role in the coordination of innate immunity and acquired immunity.Recent studies have shown that a lot of T cells apoptosis during the development of sepsis,macrophages also have a large of apoptosis.In animal experiments,there was also a large number of apoptosis of macrophages in the early stage of sepsis.In addition,a large number of inflammatory mediators such as TNF-a in the early phase and high-mobility group box-1 protein(HMGB1)in the late phase were released which active the related signaling pathways leading to apoptosis.Yao groups found that HMGB1 could significantly increase the activity of caspase-3 and induce apoptosis of macrophages.Academics have made clear that macrophages play an important role in maintaining the body’s immune response,a large number of immune cell apoptosis further induces immune system disorders which lead to sepsis shock and multiple organ dysfunction syndrome.Histone is an important part of the chromosome and is difficult to be detected in normal physiological conditions.While in clinical work,a large amount of histone was detected in patients with sepsis.Extracellular histone can not only induce the pro-inflammatory factor relwasing,play injury effects,but also can lead to mitochondrial dysfunction,calcium overload,and induce cell apoptosis.The body suffers immunosuppression or immune paralysis after large number of immune cells apoptosis which led to the collapse of the body’s immune system and fail to fight against remaining pathogens and then double infection,shock,multiple organ dysfunction or death are difficult to be.avoided.Therefore,extracellular histone plays an important role in the development of sepsis and is considered to be a key mediator to the death of patients with sepsis.Apoptosis is a programmed death by gene regulation.In the process of cell apoptosis,apoptotic genes and anti-apoptosis genes regulate apoptosis by influencing each other and apoptosis was mediated through the exogenous apoptosis signaling pathways,mitochondria and endoplasmic reticulum signal pathways in the cell.Intracellular Ca2+ overload,mitochondrial damage,Caspase and reactive oxygen species(ROS)are also involved in cell apoptosis.Calcium ions as a common signal factor which is involved in cell apoptosis at different stages.Therefore,Calcium ions are another important substance that induces apoptosis.Now,it is clear that there are lots of macrophages apoptosis in the development of sepsis,but it is not clear whether extracellular histones mediated the apoptosis of macrophages and the specific apoptosis mechanism.According to previous reports,we hypothesized that extracellular histones induce macrophage apoptosis by activating the mitochondrial pathway via Ca2+ influx.In this study,we use the Raw264.7 cells to explore the mechanism of histone in apoptosis.Further clarify the relationship between histone and intracellular Ca2+,mitochondrial,apoptosis.Objective1.To confirm whether the extracellular histones can cause Ca2+ influx.2.To explore whether extracellular histones can induce mitochondria damage through Ca2+ influx.3.To investigate whether extracellular histones mediated the apoptosis of Raw264.7 cells through mitochondrial damage.4.To clear the signaling pathway of apoptosis in Raw264.7 cell by histone-induced.Methods1.The concentration of Ca2+ in Raw264.7 cells was detected by Fluo3-AMfluorescence staining flow cytometry.2.The flow cytometry also was used to examine the mitochondrial membranepotential(Δψm,MMP)of Raw264.7 cells after Rhodamine 123.3.The apoptosis of Raw264.7cells was detected by flow cytometry withAnnexinV-FITC/PI staining.4.The expression of Bcl-2,cytoplasmic cytochrome C(Cyt-C)and CleavedCaspase-3 were detected by Western blot.Statistical AnalysisAll experimental data were analyzed using SPSS software version 19.0(SPSS Inc.,Chicago,IL,USA)and are presented as means ± SD.The differences between each group were assessed by a two-tailed unpaired t test.P<0.05 was considered significant.Results1.Histones influence Ca2+ influx in Raw264.7 cells significantly and have time-dependent and concentration-dependent,but the effect was inhibited by MgSO4,a blocker Ca2+ influx.2.Histones can induce mitochondrial damage by Ca2+ influx.3.Histones induce Raw264.7 cells apoptosis.4.Histones mediate Raw264.7 cells apoptosis though mitochondrial damage.ConclusionThe results show that extracellular histones mediate mitochondrial damage through Ca2+ influx which results the change in mitochondrial membrane permeability,then the Cyt-C was released to the cytoplasm and caspase-3 was activated,finally Raw264.7 cells apoptosis was triggered.