The Role Of Receptor For Advanced Glycation End Products In Vascular Endothelial Cells In Acute Lung Injury Induced By Heatstroke

Objective:1 To study the role of RAGE in acute lung injury induced by heatstroke in vivo.2 To study the role of RAGE in the endothelial barrier dysfunction induced by heat stress and further explore the signal pathway by which RAGE involved in heat-induced endothelia response in cell levels,and find a novel target for the clinical treatment of ALI induced by heatstroke.Methods1 Established the animal model of heatstroke by using RAGE-/-mice,and observed the role of RAGE in acute lung injury induced by heatstroke in mice by evaluating the leukocytes,neutrophils and protein concentration in BALF,lung wet/dry ratio,histopathological changes and morphological ultrastructure of lung tissue and arterial blood gas analysis.2 The role of RAGE in the endothelial barrier dysfunction induced by heat stress and the signal pathway by which RAGE involved in heat-induced endothelia response.2.1 The role of RAGE in the endothelial barrier dysfunction induced by heat stress2.1.1 Established heat stress model of HUVEC and study the influence of heat stress on HUVEC monolayer permeability for different temperature,heat stress time or rewarming time by detecting the transendothelial electrical resistance.2.1.2 Detected the influence of heat stress on RAGE protein expression for different temperature,heat stress time or rewarming time by western blot.2.1.3 Explore the influence of suppression of RAGE expression by transfecting RAGE siRNA on heat-induced endothelial barrier dysfunction.2.2 The role of MAPK families in the heat-induced endothelia barrier dysfunction in which RAGE involved.2.2.1 Explore the influence of heat stress on HUVEC JNK,ERK and P38 phosphorylation and total protein expression by western blot.2.2.2 A series of experiments were accomplished in cells by transfecting RAGE siRNA,ad-RAGE or pre-incubated with RAGE blocking antibody,and the effect of RAGE involved in heat-induced JNK,ERK and P38 phosphorylation was explored by western blot.2.2.3 HUVECs were pre-treated with JNK,ERK and P38 inhibitor and the effect of MAPK signal pathway in heat-induced endothelia barrier dysfunction was assayed by detecting TEER.2.3 The role of HSF1 and c-Jun involved in heat-induced RAGE expression changes2.3.1 Analyzed the promoter region of RAGE gene to locate transcription factor-binding sites using the bioinformatics program MatInspector.2.3.2 Detected the influence of heat stress on HSF1 and c-Jun phosphorylation and total protein expression.2.3.3 HUVECs were pre-infected with HSF1 siRNA or ad-HSF1,and the effect of HSF1 involved in heat-induced RAGE protein expression changes was monitored by western blot.2.3.4 HUVECs were pre-treated with AP-1 inhibitor to suppress c-Jun expression,and the effect of c-Jun involved in heat-induced RAGE expression changes was explored by western blot.2.3.5 Explore the interaction between transcription factor c-Jun and RAGE gene promoter region by chromatin immunoprecipitation assay.Results1 Knockout of RAGE gene played a protective role in acute lung injury induced by heatstroke in mice.1.1 Knockout of RAGE gene significantly decreased the leukocytes,neutrophils and protein concentration in BALF of heatstroke mice.1.2 Knockout of RAGE gene significantly decreased lung wet/dry ratio and water content of heatstroke mice.1.3 Knockout of RAGE gene significantly attenuated histopathological changes and morphological ultrastructure injury in lung tissue of heatstroke mice.1.4 Knockout of RAGE gene significantly improved arterial blood gas parameters of heatstroke mice.2 The role of RAGE in the endothelial barrier dysfunction induced by heat stress and the signal pathway by which RAGE involved in heat-induced endothelia response.2.1 The role of RAGE in the endothelial barrier dysfunction induced by heat stress2.1.1 Heat stress increased HUVEC monolayer permeability in a time-and temperature-dependent manner.2.1.2 Heat stress increased RAGE protein expression in a time-and temperature-dependent manner.2.1.3 The expression of RAGE gene increased immediately after heat stress.2.1.4 The increasing of heat-induced HUVEC monolayer permeability was prevented by inhibiting RAGE expression.2.2 The role of MAPK families in the heat-induced endothelia barrier dysfunction in which RAGE involved.2.2.1 Heat stress increased phosphorylation levels of JNK,ERK and P38.Knockdown of RAGE using siRNA suppressed the phosphorylation of ERK and P38,but had no similar effect on JNK phosphorylation.2.2.2 Overexpress of RAGE using adenovirus vector significantly increased the phosphorylation levels of ERK and P38 after heat stress,but had no similar effect on JNK phosphorylation.2.2.3 Pre-treatment with RAGE blocking antibody suppressed the phosphorylation of ERK and P38 after heat stress,but had no similar effect on JNK phosphorylation.2.2.4 Pre-treatment with ERK and P38 inhibitor prevented endothelial barrier dysfunction after heat stress.2.3 The role of HSF1 and c-Jun involved in heat-induced RAGE expression changes2.3.1 The result of bioinformatics analysis indicated transcription factor HSF1 and c-Jun may locate to the promoter region of RAGE gene.2.3.2 Heat stress increased phosphorylation levels of HSF1 and c-Jun.2.3.3 Knockdown of HSF1 using siRNA significantly increased the expression of RAGE protein after heat stress.While Overexpress of RAGE using adenovirus vector suppressed the expression of RAGE protein after heat stress.2.3.4 Pre-incubation with AP-1 inhibitor prevented the increasing of RAGE protein expression after heat stress.2.3.5 The result of ChIP assay indicated that transcription factor c-Jun located to the promoter region of RAGE gene after heat stress,but similar change had not detected in normal cells.Conclusion1 Heat stress increased HUVEC monolayer permeability in a time-andtemperature-dependent manner.2 RAGE was essential for heat-induced increase in HUVEC monolayerpermeability.3 ERK and P38 MAPK involved in the signal pathway by which RAGE involved in heat-induced endothelia barrier dysfunction.4 C-Jun located to the promoter region of RAGE gene and increased the RAGE protein expression after heat stress.HSF1 suppressed RAGE protein expression after subjecting to heat stress.The two transcription factors co-regulated the change of HUVEC monolayer permeability after heat stress.5 Knockout of RAGE gene played a protective role in acute lung injury induced by heatstroke in mice.