The Role Of Inflammatory Factors Induced By CLCA In Airway Epithelial Cells During The Development Of ARDS

Background:Acute respiratory distress syndrome(ARDS)is an acute,diffuse,inflammatory lung injury.The incidence of ARDS is increasing and the hospital mortality is as high as 38%.Pulmonary acute respiratory distress syndrome(ARDSp)is the most common clinical type of ARDS.Its pathogenic factors mainly include pulmonary infection,lung trauma,drowning and toxic gas inhalation,among which pulmonary infection is the most common cause.Studies have confirmed that there were significant differences between ARDSp and ARDS.These differences may have great affects on clinical research,basic research,clinical treatment and prognosis of ARDS.The pathogenesis of ARDSp involves activation of lung macrophages,infects relevant activation factors,neutrophils and inflammatory cells accumulation and infiltration,inflammatory factors over release.Neutrophil percentage and tumor necrosis factor α(TNF-α)content in bronchoalveolar lavage fluid(BALF)are monitoring indicators commonly used to reflect the degree of inflammation.However,the main reason why ARDSp mortality remains high is that the initiation mechanism of inflammation has not been fully clarified.Airway epithelial cells are located at the first barrier of the respiratory system.Goblet cells in the airway epithelium can secrete mucin,inflammatory factors and other types of proteins,and their role in pulmonary inflammation has received increasing attention.Calcium-activated chloride channel(CLCA),a protein secreted by airway goblet cells under the stimulation of some external factors,has gradually attracted attention.At first,CLCA was considered to be a transmembrane ion channel protein anchored to the cell membrane,and subsequent studies have shown that it is a protein secreted to the extracellular space.Researchers found that CLCA is not only involved in the occurrence and development of chronic inflammatory diseases of the airway,but also in the formation of pulmonary infectious diseases.Recent studies have revealed that human CLCA1(hCLCA1)can directly act on airway macrophages and induce their expression of pro-inflammatory factors such as IL-1β,IL-6,TNF-α and IL-8.mCLCA3 is a heterologous mouse genotype of hCLCA1.In the mouse model of Staphylococcus aureus pneumonia,the deletion of mCLCA3 can reduce the expression of chemokines CXCL1 and IL-17,and reduce the infiltration of neutrophils in the lung.These studies provide direct evidence for hCLCA1/mCLCA3 as a signaling molecule in the regulation of inflammation.However,up to now,the role of airway epithelial goblet cells and hCLCA1 in the pathogenesis of ARDSp remains unclear.Our group has proposed the hypothesis that “Airway goblet cell paracrine CLCA can promote epithelial cells to produce proinflammatory factors CXCL and related inflammatory factors,thus initiating and aggravating neutrophil-dominated ARDS inflammation”.To clarify that CLCA acts on airway epithelial cells to initiate and aggravate the occurrence of ARDS inflammation is expected to provide new ideas and intervention targets for the clinical prevention and treatment of ARDS.Objective:1.To investigate the relationship between the expression of hCLCA1 in airway epithelium and the level of pulmonary inflammation and clinical prognosis in patients with endogenous acute respiratory distress syndrome(ARDSp).2.The expression of Cl CA in mice ARDS model was observed.3.To explore the mechanism of Cl CA induced lung inflammation in mice.4.In vitro cell experiments,the mechanism of Cl CA involved in ARDS was further studied,and the possible signal pathways were explored.Methods:1.General data of each group was collected and analyzed.Lavage fluid was collected based on lavage target according to the imaging exudative lesions in the ARDSp and CAP group,and normal lung lobes in the Normal group.ARDSp patients were divided into two subgroups: survival group and death group based on whether they died within 28 days.BALF was collected at the time of admission and 7 days of treatment.Cells in BALF were counted by light microscope.The supernatant was collected after BALF solution was centrifuged.Enzyme-linked immunosorbent assay(ELISA)kits were usd to detecte the level of TNF-α and hCLCA1.2.The ARDSp model of mice was established by intratracheal instillation of LPS with different concentrations.The lung injury score and W/D value were calculated.BALF was collected,the total protein content,the expression level of TNF-α and IL-6,and the neutrophil count of BALF were measured to determine the optimal concentration of LPS.The lung histopathology,BALF neutrophils,Cl CA and CXCL1/CXCL2 were observed in the ARDSp mice and the control group.3.N-h Cl CA1,an N-terminal fragment of hCLCA1,was infused into the airway of normal mice to calculate the lung injury score,observe the HE staining changes of lung tissue and detect the expression of BALF cytokineschanges of the morphological,inflammatory factors were observed.4.The N-hCLCA1 fragment was used to stimulate human airway epithelial cell line 16 HBE cells.The expression level of cytokines was detected by RT-PCR and ELISA in the collected cells and culture supernatant.The biological function of n-hclca1 fragment was determined.The effect of N-hCLCA1 on the activity of MAPK kinase family was detected.The results were verified by the use of corresponding kinase inhibitors.Results:1.Twenty-six patients were included in the ARDSp group,including 15 males and 11 females,aged 35 to 65 years.ARDSp group was divided into two subgroups based on whether they died within 28 days,of which 14 were in the survival group,with an average age of 57.2 ± 7.8 years,and the male-to-female ratio was 8: 6;12were in the death group,with an average age of 61.2 ± 3.2 years,and the male-to-female ratio was 7: 5.The baseline clinical indicators for admission in the survival and death groups were Pa O2 / Fi O2(142.5 ± 20.1,133.6 ± 15.5),APACHEⅡ scores(17.3 ± 3.1,18.4 ± 7.2),and SOFA scores(6.5 ± 1.4,7.2 ± 2.5).There was no significant difference between them.Twenty-six patients were enrolled in the CAP group,with an average age of 53.84 ± 14.60 years.The ratio of male to female was 8: 5.Twenty-eight patients were enrolled in the Normal group,with an average age of 50.03 ± 15.3 years,and the male to female ratio was 4: 3.There was no significant difference in gender and age between the three groups(P> 0.05).2.Compared with the normal control group,the percentage of hCLCA1,TNF-αand neutrophils in BALF of ARDSp group and CAP group increased significantly(P<0.01).The percentage of hCLCA1,TNF-α and neutrophils in BALF of ARDSp group was higher than that of CAP group(P<0.05 or P<0.01).3.Comparison of hCLCA1,TNF-α and neutrophil percentage in BALF of patients with different prognosis before and after treatment.Before treatment,hCLCA1 and TNF-α in BALF of the dead group were higher than those of the living group(P=0.035 and <0.01 respectively).There was no significant difference in the percentage of neutrophils in BALF between the two groups.Compared with before treatment,the percentage of hCLCA1,TNF-α and neutrophils in BALF of the dead group of ARDSp had no significant difference after 7 days of treatment.The percentage of hCLCA1,TNF-α and neutrophils in BALF of the survival group of ARDSp decreased after 7 days of treatment(P <0.05).4.Correlation analysis of BALF indexes in patients with ARDSp.Linear correlation analysis showed that hCLCA1 was positively correlated with TNF-α in BALF(r=0.823,P<0.05).The percentage of BALF neutrophils was positively correlated with TNF-α(r=0.417,P <0.05)and hCLCA1(r=0.43,P<0.05).5.The ARDSp mouse model was established successfully.The results showed that LPS intervention at the concentration of 10 mg / kg led to high mortality in mice.In the control of LPS(2.5mg / kg)and LPS(5mg / kg),it was found that when the concentration of LPS was 5mg / kg,there were more differences in lung injury score,W/ D,BALF protein content and cytokines content in BALF,which were all within a reasonable range.6.Time curve of mCLCA3 concentration in BALF of LPS-induced ARDS mice:Within 24 hours,the protein level of mCLCA3 significantly increased,and the difference was statistically significant compared with the control group(P <0.05).The mCLCA3 level reached a peak at 6h.After 36 hours,the level of mCLCA3 decreased close to the PBS group.7.N-hCLCA1,the N-segment form of hCLCA1,can induce pathological lung injury and alveolar edema in mice.The content of protein in BALF increased,and the content of TNF-α,IL-6,CXCL1 and CXCL2 increased significantly(P < 0.05).8.The recombinant protein of N-hCLCA1 interferes with the 16 HBE cell line of human airway epithelial cells.The Boiled N-hCLCA1 has no significant effect on the cell line,while N-hCLCA1 can significantly increase the mRNA content of TNF-α,IL-6,CXCL1 and CXCL2 in the cell culture supernatant,and increase the content of CXCL1 / CXCL2 in the cell culture supernatant,but has no significant effect on the content of TNF-α and IL-6.When N-hCLCA1 intervened in 16 HBE cell line,the phosphorylation of p38 increased.After sb2035800,the increasing trend of CXCL1 and CXCL2 in cell culture supernatant was inhibited,but it was still higher than that of the control group.Conclusions:1.The hCLCA1 content in BALF of ARDSp patients is notably higher than those in patients with CAP and normal lung,that in death group is higher than that in survival group,and is positively correlated with TNF-α and neutrophil percentage in BALF.It is speculated that hCLCA1 may be correlated with the pulmonary inflammation level and prognosis of ARDSp patients.However,as time goes on,the reason for the progressive decline of Cl CA level is not clear.2.The mouse ARDS model was successfully established and the optimal concentration of LPS was determined.CLCA was increased at an early stage,but decreased progressively over time.It may be due to the proliferation and metaplasia of goblet cells,so that the secretion of Cl CA is also increased.3.When N-hCLCA1 was infused into the airway of normal mice,edema of lung tissue,aggravation of lung injury,increase of protein content in BALF,and increase of TNF-α,IL-6 and CXCL1 / CXCL2 were observed.Although it is not as strong as LPS intervention in airway level,it has significant statistical significance compared with the control group.The results suggest that Cl CA may play a role in the early occurrence and development of ARDS.4.N-hCLCA1 can significantly increase the mRNA content of TNF-α,IL-6,CXCL1 and CXCL2 in 16 HBE cells,and increase the content of CXCL1 and CXCL2 in the cell culture supernatant.It is clear that p38 is one of the signal pathways of hCLCA1 to stimulate the secretion of CXCL1 and CXCL2 in 16 HBE cells.It is speculated that the highly secreted Cl CA can induce the production of chemokines in epithelial cells,which is the mechanism of promoting neutrophil dominated ARDS inflammation one.