| BackgroundAcute lung injury（ALI）is a pulmonary manifestation of an acute systemic inflammation.Due to the impairment of the pulmonary epithelial-vascular endothelial barrier caused by various pathogenic factors,ALI has many prominent features,such as diffuse alveolar damage,pulmonary inflammatory cell infiltration,and surfactant dysfunction.Severe ALI can further develop into acute respiratory distress syndrome（ARDS）,which is an important cause of death from lung diseases,with a mortality rate as high as 45%.When ALI occurs,the main feature of the lungs is the damage of the alveolar barrier,followed by increased infiltration of neutrophils,the release of inflammatory cytokines,and the promotion of inflammatory responses leading to pulmonary edema and increased protein content.Alveolar epithelial cells（AECs）are a layer of epithelial cells covering the surface of the alveoli and participate in the formation of the alveolar barrier.As a result,they become main target cells that are damaged during the development of ALI/ARDS.After injury,they release inflammatory mediators,which further increases the permeability of the alveolar barrier and exacerbates the inflammatory response.Therefore,it is of great significance to investigate the role of AEC in the occurrence and development of ALI/ARDS for understanding the pathogenesis and treatment of ALI/ARDS.Heat shock protein 70（HSP70）is the most common member of the heat shock protein family with a molecular weight of about 70 k D.The expression level of HSP70 in normal cells is low,and its production is increased under stress conditions such as high temperature and microbial infection.Some scholars have found that the expression of HSP70 is elevated in the lung tissue of patients with chronic obstructive pulmonary disease,and when HSP70 is secreted outside the cell,it can trigger an inflammatory response in airway epithelial cells.Chase’s study showed that HSP70 stimulates primary airway epithelial cells to release inflammatory factors and trigger an inflammatory response.Wheeler’s group showed that HSP70 can be released from virus-infected airway epithelial cells,leading to the recruitment and activation of neutrophils.Since HSP70 is involved in the recruitment,activation and inflammatory response of neutrophils,HSP70 may be involved in the pathogenesis of ALI.Objective: To explore the role and related mechanism of HSP70 in the pathogenesis of acute lung injury in mice.Method:（1）To construct ALI mouse models,the ICR strain mice were randomly divided into two groups: the control group（instillation of PBS through the airway）,ALI model group（instillation of LPS through the airway）,after 24 h,Western Blot was used to tested the expression of HSP70 protein in lung tissue,and the content of HSP70 in BALF was detected by ELISA.（2）The ICR strain mice were randomly divided into four groups: Control group,ALI model group（follow method 1）,anti-HSP70+ALI group（Mice were administered antiHSP70（0.25 mg/kg）for 2 h before and 6 h after the nasal instillation of LPS）,Ig G+ALI group（the treatment control group with Ig G instead of anti-HSP70）.Lung tissues of mice were taken out for HE staining and observed pathological changes.The wet-dry weight ratio of lung tissue was weighed and calculated.The secretion of proinflammatory factors and total protein concentration were respectively tested by ELISA and BCA assay.Neutrophil counts were performed after smearing and Wright staining of cells in BALF.（3）AEC was stimulated with different concentrations of HSP70（0 ng/ml,10 ng/ml,50 ng/ml,100 ng/ml,200 ng/ml,500 ng/ml）for 24 h,then cocultured with FITC-labeled apoptotic cells for 4 h,and the effect of HSP70 on AEC phagocytosis was detected by flow cytometry and fluorescence microscopy.Primary alveolar epithelial cells（PAEC）were sorted by magnetic bead sorting technology.HSP70（200 ng/ml）stimulated PAEC for 24 h,then cocultured with FITC-labeled apoptotic cells for 4 h,and flow cytometry tested the effect of PAEC phagocytosis of apoptotic cells.（4）HSP70（200 ng/ml）stimulated AEC for 12 h to detect expression of p Akt at protein level by Western blot.AEC was pretreated with Wortmannin（1 μM）which is PI3K/Akt signaling pathway inhibitor or NF-κB blocker BAY11-7082（1 μM）or IRF3 blocker MRT67307（1 μM）for 2 h,then treated with HSP70（200 ng/ml）for 24 h,cocultured with FITC-labeled apoptotic cells for 4 h,and the effect of AEC phagocytosis of apoptotic cells was tested by flow cytometry.（5）After AEC was stimulated by HSP70（200 ng/ml）,the expressions of CD36,CD204 and CD366 were detected by flow cytometry.CD36 blocking antibody（CD36-Ab,1 μg/ml）stimulated AEC for 6 h,and the fluorescence intensity of CD36 on the surface of AEC was tested by flow cytometry.AEC was pre-stimulated with CD36-Ab（1 μg/ml）or Iso-Ab（1 μg/ml）for 6 h,then stimulated with HSP70（200 ng/ml）for 24 h,and cocultured with apoptotic cells for 4 h,then flow cytometry was used to observe changes of AEC phagocytosis of apoptotic cells.（6）Western blot and ELISA respectively detected the production of TNF-α in lung tissue and BALF of ALI mice.Western blot and ELISA respectively detected the effect of HSP70（200 ng/ml）on the production of TNF-α in AEC.HSP70（200 ng/ml）stimulated AEC for 12 h was to detect expression of p-NF-κB p65 and p-IRF3（ser396）at protein level by Western blot.Following TLR4-Ab（1 μg/ml）,BAY11-7082（1 μM）or MRT67307（1 μM）stimulation,AEC was stimulated with HSP70（200 ng/ml）for 24 hours,and the production and secretion of TNF-α in AEC were tested by Western blot and ELISA.Result:（1）Compared with the mice in the control group,the protein expression of HSP70 in the lung tissue and the secretion of HSP70 in the BALF were increased in the ALI group（all P < 0.01）.（2）Compared with mice in ALI group,the infiltration of inflammatory cells in lung tissue were reduced,and wet-dry specific gravity of the lung was decreased（P < 0.05）.The secretion of pro-inflammatory factors,the number of neutrophils and the protein content in BALF were reduced in anti-HSP70+ALI group（all P < 0.05）.（3）Compared with the control group,the percentage of AEC phagocytosed apoptotic cells in HSP70（100 ng/m L,200 ng/m L,500 ng/ml）stimulation group were reduced,and HSP70（200 ng/m L）stimulation group significantly reduced（P < 0.01）.The percentage of apoptotic cells phagocytosed by PAEC also decreased after HSP70（200 ng/ml）stimulation（P < 0.01）.（4）HSP70（200 ng/m L）enhanced expression of p Akt protein in AEC（P < 0.05）.Compared with HSP70 stimulation group,the phagocytosis rate of AEC in Wortmannin+HSP70 group was increased（P < 0.05）,and did not change significantly in BAY11-7082+HSP70 group and MRT67307+HSP70 group.（5）Compared with the control group,the expression of CD36 receptor in AEC was decreased after HSP70 stimulation（P < 0.05）,while there was no significant change in CD204 and CD366.The fluorescence intensity of CD36 receptor on the surface of AEC was decreased after CD36-Ab blockade.The phagocytosis rate of AEC in CD36-Ab group was reduced compared with control group,and CD36-Ab+HSP70 group was reduced compared with HSP70 group（all P < 0.05）.（6）Compared with control group,the production of TNF-α in AEC was increased after HSP70 stimulation（P < 0.05）.HSP70 enhanced expressions of p-NF-κB p65 and pIRF3（ser396）protein in AEC（all P < 0.05）.Compared with HSP70 single stimulation group,the production of TNF-α by AEC was decreased in TLR4-Ab+HSP70 group,BAY11-7082+HSP70 group and MRT67307+HSP70 group（all P < 0.05）.Conclusion:（1）HSP70 promotes lung inflammation in ALI mice.（2）HSP70 inhibits AEC phagocytosis of apoptotic cells through PI3K/Akt signaling.（3）HSP70 reduces the function of phagocytosing apoptotic cells by inducing the decrease of CD36 receptor on the surface of AEC.（4）HSP70 promotes TNF-α production from AEC via TLR4-NF-κB/IRF3 signaling. |
