| BackgroundSepsis is inflammatory response disorder in vivo,and leading to life-threatening organ dysfunction.Currently,sepsis is a major cause of death in intensive care units across the world.Sepsis-induced myocardial dysfunction is a complication of severe sepsis that indicates poor prognosis and and the incidence is as high as 50%.The inflammatory cells of innate immune response include macrophages,dendritic cells(DC)and nk cells and adaptive immune cells T/B lymphocytes in Sepsis.They was activated,then apoptosis or autophagy,which destroys the body's normal immune response and exacerbates sepsis.In addition,the sympathetic nervous system was activated,catecholamine was released in large quantities and cardiac function was decreased.Heart rate rapid increases myocardial load and oxygen consumption,leads to inadequate ventricular diastole and insufficient coronary artery perfusion,exacerbates myocardial injury,and plays an important role in the development of cardiac dysfunction in the end.ObjectiveTo observe the role of Dendritic cell(DC)in the myocardial dysfunction induced by lipopolysaccharide(LPS),the interaction between DC and sympathetic nervous system in sepsis and mechanism of myocardial injury and cardiac dysfunction in sepsis.This study will provide theoretical basis for finding new therapeutic targets in clinic.Methods1.Effect of LPS on bone marrow-derived dendritic cellsMale C57B6/L mice(n=5)were housed the specific pathogen free(SPF)facilities.Culture of femur bone marrow cells from mice was prepared:Cell phenotype was analysis and identificated by flow Cytometry.The DC was divided into two groups.One group co-cultured with 25?g/ml 1ps and the other group without lps as control group for 24 hour.The expression of IL-6,IL-12 and TNF-a were detected by qPCR and Western Blot.2.Role of dendritic cells in myocardial injury induced by sepsisMale C57B6/L mice(n=80)were housed the specific pathogen free(SPF)facilities and randomly divided into four groups,the control group of wild type(WT-Sham),the inhibitor control group(WT-LPS-VAG539),wild type test group the test group(WT-LPS)and the inhibitor test group(WT-LPS-VAG539).DC cells inhibitors VSG539 to establish DC cells inhibited mice model(30 mg/kg,WT+VAG539 and WT-LPS-VAG539)2 times every day for 2days,then establishment of mouse cardiac dysfunction model by intraperitoneal injection of LPS(10mg/kg,WT-LPS and WT-LPS-VAG539).To observe four groups of animals mortality at 14 days,and through the small animal tail cuff method to measure mean arterial pressure(MBP),small animal cardiac function was evaluated by echocardiography:evaluation indexes including heart rate,left ventricular ejection fraction(EF%)and shortening(FS%),flow cytometry analysis and ELISA were adopted to detect the myocardium DC maturation and tissue inflammatory cytokines in mice:the expression of tumor necrosis factor alpha(TNF alpha),interleukin 12(IL-12),and interleukin 6(IL-6),immunohistochemical detection of cardiac myocyte apoptosis markers bax and bcl-2.3.Effects of sympathetic excitation and dendritic cell activation on myocardial injury in sepsisMale SD rats(n=40)were housed in the specific pathogen free(SPF)facilities and randomly divided into four groups,the control group of wild type(WT-Sham),wild type test group the test group(WT-LPS)and the inhibitor test group(WT-LPS-VAG539 and WT-LPS-Ate).DC cells inhibitors VSG539 to establish DC cells inhibited mice model(30 mg/kg,WT-LPS-VAG539)2 times every day for 2days,then establishment of mouse cardiac dysfunction model by intraperitoneal injection of LPS(10mg/kg,WT-LPS,WT-LPS-VAG539 and WT-LPS-Ate)and after 24 hour,the WT-LPS-Ate group was injected atenolol(5 mg/kg).The sympathetic discharges and hemodynamic indexes were recorded by powerlab system.The contents of norepinephrine in plasma were measured by HPLC and TNF-a and dendritic cells in myocardium were measured by immunohistochemistry respectively.Results1.Effect of LPS on bone marrow-derived dendritic cellsLps could significantly up-regulate the expression of cd40,cd80 and cd86(P<0.05).Although it could increased the expression of MHCII,there was no significant difference.The results of qPCR and Western blotting showed that the expression levels of IL-6.IL-12 and TNF-a were increased(P<0.05).2.Role of dendritic cells in myocardial injury induced by sepsisAt 24 h of LPS intraperitoneal injection,the mortality rate of the WT-LPS-sham group was 50%in 48 hours,80%in 96 hours and 85%in 14 days.The mortality rate of the WT-LPS-sham group was 15%in 48 hours,35%in 96 hours and 45%in 14 days.Compared with WT-LPS-sham group,the mortality rate of the WT-LPS-VAG539 group was significantly reduced(P<0.05).After LPS treatment,the mice in group WT+VAG539 HR was decreased,mean blood pressure(MBP),ejection fraction(EF%)and fractional shortening(FS%)was significantly improved when compared with WT mice(P<0.05).Flow cytometry showed that the number of DC s in myocardium increased after LPS treatment,increased from 1.8%to 6.5%in WT group,and increased from 1.6%to 3.7%in WT+VAG539 group.The expression level of inflammatory factors in mice serum was detected by ELISA method.The level of TNF-?,IL-12 and IL-6 was higher after LPS treatment,and in WT-VAG539 group was significantly lower than that in the WT group after LPS treatment(p<0.05).The expression levels of Bax was higher and Bcl-2 was lower after LPS treatment(p<0.05).The unbalanced state of increasing bax and decreasing Bcl-2 has been improved in WT-LPS-VAG539 group when compare with WT-LPS-sham(p<0.05).3.Effects of sympathetic excitation and dendritic cell activation on myocardialinjury in sepsisAt 24 h of LPS intraperitoneal injection,LVEDP and HR was significantly increased,andądp/dtmax was reduced when compared with WT-sham group(P<0.05).When rats treatment with atenolol or DC inhibitor,these symptoms had improved compare with WT-LPS-sham group(P<0.05).WT-sham group rats had higher levels of NE than other groups(P<0.05).Treatment with atenolol or DC inhibitor,the NE of the plasma reduced compare with WT-LPS-sham group(P<0.05)Myocardium levels of TNF-? was higher in model group induced by LPS when compared with WT-sham group.Treatment with atenolol or DC inhibitor,levels of TNF-a was reduced when compare with WT-LPS-sham group.Model group rats induced by LPS exhibited higher sympathetic nerve activity(%of max)whe-n compared with WT-sham rats.Treatment with atenolol or VAG539 inhibited sympathetic nerve activity.ConclusionLPS promote the maturation and aggregation of DC in LPS-induced sepsis.The expression of inflammatory factors is increased by activating multiple immune cells,which lead to myocardial injury,dysfunction and cardiomyocyte apoptosis.Excessive activation of sympathetic nervous system promotes DC-induced inflammatory response,and the activation of DCs also upregulated sympathetic nervous system excitation,which contributes to the impairment of cardiac function in sepsis. |
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