Influence Of Heat Shock Protein A12B On Pulmonary Vascular Endothelial Injury And Its Mechanisms In Septic Mice

Sepsis is associated with a systemic inflammatory responsethat affectsseveral organs.Severe sepsis can lead to septic shock,even threaten the life.The respiratory system is the most frequently susceptible organ system,and diffuse inflammationof lung parenchyma and severe lung dysfunction arethe first steps in the development of multiple organ failure andone of the leading causes of death in sepsis.Nearly 50% ofpatients with severe sepsis will develop acute lung injury(ALI)and its more severe form,acute respiratory distresssyndrome(ARDS).ALIinvolved with several key pathological processes,includingloss of vascular integrity,neutrophil infiltration,and accumulationof protein-rich fluid in the airspaces of the lung.The potential pathological mechanism of sepsis is developing,but not completely clear.Thus,it is a challenge to prevent the patients from sepsis-induced acute lung injury.In recent years,lots of attention is paid to the heat shock protein A12B(HSPA12B)—a new member of heat shock protein 70 family.HSPA12 B are mainly expressed on endothelial cells and also widely distributed in various tissues in mammals.Many studies have found that HSPA12 B protects endothelial cells against a variety of harmful factors,such as cerebral ischemia/reperfusion injury and cardiac dysfunction.Moreover,HSPA12 B can reduce LPS-induced endothelial injury in human umbilical vein endothelial cells.In addition,wepreviously found that plasma HSPA12 B was elevated in both septic mice and patients and it might be a good predictor for prognosis in patients with sepsis.However,the protection of HSPA12 B in the sepsis-induced pulmonary endothelial function and its potential mechanisms has not beenclear.Our research discussed the effect of HSPA12 B on the pulmonary vascular endothelial injury and its possible mechanisms by in vitro and in vivo experiments.Firstly,we verified the efficiency of inhibiting the expression of HSPA12 B by the nasal inhalation of HSPA12 B siRNA.Secondly,we investigated the role of HSPA12 B in the sepsis-induced acute injury by survival rate,pathological damage and inflammatory response.Finally,we further explored the potential mechanisms of HSPA12 B in the sepsis-induced pulmonary vascular endothelial dysfunction.HSPA12B in mice lung by the nasal inhalation siRNA Part 1 Theinterference efficiency of inhibiting the expression ofObjective Verify the interference efficiency of inhibiting the expression of HSPA12 B in mice lung treated by nasal inhalation siRNA.Methods C57BL/6 mice were randomly divided into two groups.Mice were treated by nasal inhalation of 2-Ome modified HSPA12 B siRNA or Negative Control siRNA(NC siRNA).After transfecting 24 h,mice were sacrificed and removed the heart,lung,liver and kidney tissues.The m RNA and protein levels of HSPA12 B were measuredby RT-q PCR and Western blot.Results 1.The m RNA expression level of HSPA12 B in lung tissue with nasal inhalation was specifically inhibited in mice lung,but no significant difference in other tissues(p<0.05).2.The protein expression level of HSPA12 B in lung with nasal inhalation were obviously down regulated,compared with the NC siRNA(p<0.05)?ConclusionNasal inhaling HSPA12 B siRNA can down regulate the expression of HSPA12 B in mice lung tissues,and specifically reduced the expression in lung.It provided the reliable basis for the subsequent experiments.Part 2 The effect of HSPA12 B on the survival rate in sepsis-induced acute lung injury in vivoObjective Research the survival rate in CLP-induced lung injury mice by knockdown HSPA12B.Methods C57BL/6 mice were randomly divided into four groups(n=15): sham operation group,CLP group,HSP12 B siRNA-CLP group,NC siRNA-CLP group.Mice were treated by nasal inhalation of 2-Ome modified HSPA12 B siRNA or Negative Control siRNA(NC siRNA).After transfecting 24 h,mice were performed the CLP surgery.A mortality study was conducted and the survival of CLP mice was monitored every 24 h for a total of seven days.Results The seven-day survival rate of different group were observed and recorded.HSPA12 B siRNA-CLP group was 0,significantly lower than NC siRNA-CLP group(40%),CLP group(33.33%)and Sham group(100%)(p<0.05).Conclusion Knockdown of HSPA12 Breduced the survival rate of mice when challenged with CLP surgery.Weconcluded that HSPA12 B provided survival benefits in sepsis-induced ALI.Part 3 The influence of HSPA12 B on the pathological damage and inflammatory response in sepsis-induced acute lung injury in vivoObjectiveStudy the influence of HSPA12 B on the pathological damage and inflammatory response in sepsis-induced acute lung injury in vivoMethods C57BL/6 mice were randomly divided into four groups(n=8): sham operation group,CLP group,HSP12 B siRNA-CLP group,NC siRNA-CLP group.Mice were treated by nasal inhalation of 2-Ome modified HSPA12 B siRNA or Negative Control siRNA(NC siRNA).After transfecting 24 h,mice were performed the CLP surgery.1.HE staining and pathological scores were used to assess the pathological changes.2.The levels of IL-1?,TNF-?,IL-6 in bronchoalveolar lavage were quantified by ELISA.3.The MPO activity was measured by ELISA and the number of neutrophil and alveolar macrophagecount were analyzed by flow cytometry.4.The wet/dry ratio of lung tissues was detected.Results 1.HE staining showed that the pathological changes in HSPA12 B siRNA-CLP group was obviously increased,even the damage to 24 h after CLP surgery were more serious than 48 h after CLP surgery.The pathological score was significantly higher than other treatment groups(p<0.05).2.The levels of IL-1?,TNF-?,IL-6 in bronchoalveolar lavage were higher in the HSPA12 B siRNA-CLP group(p<0.05).3.MPO activity and neutrophil count in the HSPA12 B siRNA-CLP group were significantly increased(p<0.05).4.The W/D ratio of lung tissue in HSPA12 B siRNA-CLPgroup wassignificantly elevated compared with othertreatment groups(p<0.05).Conclusion Knockdown of HSPA12 Bamplified the pathological injury and inflammation response in the sepsis-induced lung injury,suggested HSPA12 B play an important role in sepsis-inducedpulmonary endothelial injury.Part 4 The potential mechanisms of HSPA12 B in pulmonary endothelial protectionObjective Explore thepotential mechanisms of HSPA12 B in pulmonary endothelial protection in vivo and in vitroMethods 1.C57BL/6 mice were randomly divided into four groups(n=8): sham operation group,CLP group,HSP12 B siRNA-CLP group,NC siRNA-CLP group.Mice were treated by nasal inhalation of 2-Ome modified HSPA12 B siRNA or Negative Control siRNA(NC siRNA).After transfecting 24 h,mice were performed the CLP surgery.Mice were sacrificed after CLP surgery 24 h,removed the lung tissues.Endothelial apoptosis was detected by TUNEL.The expression of MAPK family moleculeswere measured by Western Blot.2.HUVECs were cultured in vitro,and transfected transiently with HSPA12 B plasmid and siRNA.With induction of LPS,the expression of MAPK family molecules were measured by Western Blot.Results 1.TUNEL staining showed that pulmonary endothelial cell apoptosis in HSPA12 B siRNA-CLP group was obviously increased,compared with other treatment groups.The index of apoptosis was significantly higher(p<0.05).2.Western Blot suggested that HSAP12 B inhibit ERK phosphorylation.Conclusion HSPA12 B preveted endothelial from inflammation response by repressing ERK/MAPK signaling pathways and inhibited endothelium apoptosis,thus protected endothelial function in sepsis-induced acute lung injury.