Effect Of Hemin In Exacerbating Lung Injury Of Sepsis Mice Via Up-regulating STAT3 Phosphorylation

BackgroundThe problem of red blood cells(RBCs)storage lesion has been concerned.A large number of clinical studies have shown that transfusion of stored packed RBCs is associated with patient poor outcomes.As the degradation product of hemoglobin,heme,which is oxidized to hemin immediately when it does not bind to proteins,is the main damage associated molecular patterns(DAMPs)of hematolysis,and also is the main toxic substance released from stored RBCs.The concentration of heme in the supernatant of stored RBCs is increased with the prolongation of storage time,and excessive heme can cause inflammatory response through varieties of mechanisms.Acute respiratory distress syndrome(ARDS)is a common complication of organ dysfunction in sepsis patients,and causes extremely high mortality.It has been shown that transfusion of stored RBCs can increase the incidence of ARDS in sepsis patients.However,the effect of heme and its related mechanism on lung injury induced by sepsis are unclear.Signal transducers and activators of transcription 3(STAT3)is an important transcription factor,and plays a key role in the regulation of inflammatory response of ARDS.Heme can activate STAT3 to regulate inflammatory damage caused by hemolytic diseases such as malaria.However,whether the role of heme in the inflammatory injury of sepsis-induced ARDS is linked to STAT3 activity remains unknown.ObjectivesTo investigate the effect and the underlying mechanism of hemin on lung injury of lipopolysaccharide(LPS)-induced sepsis mice and LPS induced murine RAW264.7 cells.Materials and methodsC57BL/6 mice were intraperitoneally injected with LPS(20 mg/kg)to reproduce sepsis mouse model in vivo,hemin was intraperitoneally injected with LPS at the same time.To observe the effects of different doses of hemin(5,10,30,50 mg/kg)on 48 hours survival rate of sepsis mice,mice were divided into hemin group,LPS group and LPS+hemin group.To investigate the effects of hemin(10 mg/kg)on lung injury of LPS-induced sepsis mice,mice were divided into control group,hemin group,LPS group and LPS+hemin group.To investigate the role of STAT3 phosphorylation in vivo,the LPS+hemin+vehicle group and LPS+hemin+stattic group were added,and mice were pre-treated 45 min with stattic(20 mg/kg),an inhibitor of STAT3 phosphorylation.Blood and lung tissues were collected at 4 h,6 h and 12 h after administration.The changes of pulmonary morphological were evaluated by using hematoxylin-eosin staining.The levels of pro-inflammatory cytokines TNF-α and IL-6 in murine serum and lung tissues were detected by using ELISA.The levels of STAT3 phosphorylation in murine lung tissues were detected by using western blot.Murine RAW264.7 cells were treated with LPS(100 ng/ml)to reproduce sepsis cell model in vitro.The cells were divided into control group,hemin group,LPS group and LPS+hemin group,and cells were stimulated with different concentrations of hemin(10,30,50 μmol/L)at the same time.To investigate the effect of hemin(50 μmol/L)on STAT3 phosphorylation of sepsis cells,the LPS+hemin+vehicle group and LPS+hemin+stattic group were added.To investigate the changes of pro-inflammatory cytokines in sepsis cells after inhibiting STAT3 phosphorylation,the hemin+stattic group,LPS+stattic group and LPS+hemin+stattic group were added,and cells were pre-treated 45 min with stattic(10 μmol/L).Cells and culture medium supernatant were collected after 4 h for detecting the changes on the mRNA and protein expression of TNF-α and IL-6 by using qPCR and ELISA.Cells were collected after 2 h for analyzing the level of STAT3 phosphorylation by using western blot.Results1.Comparing with LPS group,the survival time of sepsis mice in LPS+hemin group was significantly decreased,and the mortality was increased in a dose-dependent manner of hemin(P<0.05),whereas there was no death of mice in hemin group.The sensitivity of mice to LPS could be enhanced by hemin,and the LPS dose of inducing half-death of mice was reduced with the treatment of hemin(P<0.05).2.The histopathology of lung tissue and injury score showed that hemin exacerbated lung injury of LPS-induced sepsis mice(P<0.05).3.Hemin increased the levels of pro-inflammatory cytokines TNF-α and IL-6 in peripheral blood and lung tissue of LPS-induced sepsis mice(P<0.05).4.Hemin increased TNF-α and IL-6 mRNA and protein expression on LPS-treated RAW264.7 cells in a dose-dependent manner(P<0.05).5.Hemin up-regulated the phosphorylated levels of STAT3 in lung tissues of LPS induced sepsis mice and LPS-treated RAW264.7 cells(P<0.05).While LPS-induced sepsis mice and LPS-treated RAW264.7 cells were treated with stattic,such hemin-mediated STAT3 phosphorylation could be inhibited,the levels of pro-inflammatory cytokines in mice and RAW264.7 cells were also decreased(P<0.05),and the severity of lung injury was attenuated and the survival time of mice was prolonged(P<0.05).ConclusionsHemin may amplify the levels of pro-inflammatory cytokines,exacerbate lung injury and increase mortality in LPS-induced sepsis mice by enhancing the phosphorylation of STAT3.