Differences In Expression And Function Of Airway Epithelial CLCA Between Direct And Indirect ARDS

Background:Nowadays,Science and technology are highly developed.Even so,Acute respiratory distress syndrome is still an awkward medical problem.Its clinical treatment is difficult,the mortality is relatively high and the pathogenesis is still not entirely clear.What’s especially frustrating is that clinical researches on new therapeutic strategies for ARDS have failed repeatedly.Clinicians are aware of the importance of typing ARDS,such a highly heterogeneous group of clinical syndromes.Only accurate clinical classification,enabling individualized and precise clinical treatment programs,can break the ice of the current treatment of ARDS.Depending on the cause we type ARDS into direct and indirect forms,the pathogenesis of both forms is different.Previous studies focused mainly on neutrophils,macrophages,alveolar epithelial cells,endothelial cells and other inflammatory mediators secreting cells.Airway epithelial cells as an important barrier airway cells,secreting a variety of inflammatory cytokines,play an important role in inflammatory lung diseases.Wherein the airway goblet cell under external stimuli can secrete a specific calcium-activated chloride channel protein(CLCA),thereby promoting the synthesis and release of mucin,to help reach the airway defense function.CLCA,which is highly active in asthma and other chronic inflammatory diseases,promote the formation of mucus hypersecretion symptom by increasing the expression of mucin.It is not clear how CLCA play a role in the pathogenesis of ARDS.Based on the above,our group intends to compare clinical differences in direct and indirect appearance of ARDS,and discuss what is the role of CLCA in the two types of ARDS respectively,in order to provide new ideas and new molecular targets for diagnosis and treatment of ARDS.Objective:We intend to compare the clinical features and differences in prognosis of direct and indirect ARDS patients.We want to establish lipopolysaccharides(LPS)induced mouse models of direct and indirect ARDS respectively,in order to compare the lung injury levels and differences in the expression of inflammation-related factors.Furthermore,differential expressions of m RNA and protein levels of CLCA and mucin(Mucin 5AC,MUC5AC)are compared in both models.We need to intervene CLCA reversely to observe the change of lung injury and inflammation levels,in order to investigate the role of CLCA played in two types of ARDS.Methods:1.Diagnosed with ARDS patients in the ICU of our hospital were analyzed retrospectively.156 cases meet the diagnostic criteria of The Berlin definition.After exclusion criteria,patients included a total of 100 cases in the analysis of cohort data.51 cases of patients with ARDS were direct,49 cases were indirect ARDS patients.Clinical characteristics between two groups of patients were compared and independent prognostic risk factor were analyzed using SPSS 25.2.C57 mice with lipopolysaccharide airway instillation established direct ARDS model,while mice injected intraperitoneally with LPS established indirect ARDS model.HE staining demonstrated pathological changes in mouse lung,and lung injury score was calculated.Wet lung tissue/dry weight ratio(W/D value)was recorded.The change in total protein content of bronchoalveolar lavage fluid(BALF)of mice was tested by BCA assay.We set up control groups,using of the above three indicators,to compare lung damage among the groups.3.We use ELISA assay to detect the content of TNF-α,IL-β,IL-6,KC,SP-D,Ang-2of BALF and serum.Comparison of inflammatory cytokines expression were analyzed between groups.4.Using PCR and Western Blot assay,we detect m RNA and protein expression of m CLCA3 and MUC5 AC in mouse lung tissue,in order to compare different expression among groups.5.Immunohistochemistry was uesd to detect the expression level of m CLCA3 in mouse lung tissue,in order to further verify whether m CLCA3 expressed differently in each group.6.We intervened m CLCA3 reversely by the use of non-specific blocker niflumic acid(NFA)and CLCA specific antibody(ab-m CLCA3)respectively,to observe changes of lung injury levels and inflammatory factors.Results:1.Compared to indirect ARDS patients,direct ARDS patients’ age(65 vs.55,p<0.01),complicated with chronic respiratory disease cases(18 vs.1,p < 0.01),baseline oxygenation index(219.28 vs.153.66,p <0.01)was high;the SOFA score(6 vs.9,p <0.01),shock(24 vs.34,p<0.05),the case of organ failure(organ failure numbers≥3)(15 vs.24,p<0.05),the total number of hospitalizations days(12 vs.14,p<0.05)were low.Hospital mortality between the two groups was of no significant difference(39.2% vs.36.7%).On the whole,patients with ARDS,lung injury score,SOFA score,organ failure after treatment,as well as increased mortality oxygenation index was an independent risk factor.In subgroup analysis,the lung injury score increased direct ARDS independent risk factor for mortality(OR 10.187,p<0.05),SOFA score was increased indirect ARDS appearance independent risk factor for mortality(OR 1.932,p<0.05).2.HE staining: compared to the control groups of mice,both the direct group(IT group)and the indirect group(IP group)has shown a significant destruction,a large number of inflammatory cells within the alveolar accompanied by protein deposition.Lung injury score:(1)Compared to direct control group(ITC group),IT group lung injury scores were significantly increased(p<0.05);compared to indirect control group(IPC group)IP group increased significantly(p<0.05).(2)6 hours after the LPS challenge,no significant difference(p>0.05)was observed between IT and IP groups;24 hours after the LPS challenge,no significant difference(p>0.05)was observed between IT and IP groups.(3)Among the IT group,lung injury scores significantly increased 6 hours after the intervention of LPS than 24 hours(p<0.05);Among the IP group,lung injury scores significantly increased 6 hours after the intervention of LPS than 24 hours(p<0.05).W/D value:(1)Compared with the control groups,both IT and IP groups were significantly increased(p<0.05).(2)6 hours after the intervention of LPS,we found no difference between the IT and IP groups.The IT and IP groups demonstrated no significant difference after 24 hours(p>0.05).(3)In both IT and IP groups,compared to 6 hours,W/D value decreased after 24 hours(p<0.05).BALF total protein content:(1)Compared with the control groups,The IT and IP groups were significantly higher(p<0.05).(2)During the same period of time,we found no difference between the IT and IP groups.(3)In IT and IP groups respectively,the total protein content 24 hours after intervention decreased than 6hours(p<0.05).In summary,the IT group and IP group had no significant difference in the degree of lung injury,while the level of lung injury gradually weakened during the first 24 hours.3.The expression of inflammatory cytokines in BALF: In the IT group,TNF-α,IL-β,IL-6,KC,SP-D were higher than that of the IP group(p<0.05);Ang-2 of The IP group intrapulmonary was higher than the IT group(p<0.05).TNF-α,IL-β,IL-6,KC gradually decreased(p<0.05),SP-D in the IT group was maintained at a high level at both time points,while Ang-2 was high in the IP group(p>0.05).Expression of inflammatory cytokines in serum: TNF-α,IL-β expressed higher in the IT than the IP group(p<0.05).6hours IL-6,KC were higher in the IP group than the IT(p<0.05);there was no significant difference of IL-6,KC at 24 hours(p>0.05).SP-D was higher in the IT group(p<0.05),whereas Ang-2 expression in the IP group was higher(p <0.05).4.PCR and Western Blot results: m CLCA3,Muc5 ac expression in the lungs were significantly higher in the IT than in the IP group(p<0.05);no significant difference was observed between the IP and IPC groups(p>0.05).5.Immunohistochemistry results: m CLCA3 located mainly in airway epithelial cells,There was higher expressed m CLCA3 in the IT group than in other groups(p<0.05);no significant difference was observed between the IP and IPC groups(p>0.05).6.using of niflumic acid(NFA)and m CLCA3 specific antibody(ab-m CLCA3)to intervene mice lungs reversely,lung injury levels of the correlation results:(1)lung injury score: there was no significant difference between the NFA+LPS treated group and the group with LPS(p>0.05);Whereas the lung injury score was lower in the ab-m CLCA3+LPS treated group(p <0.05).(2)W/D value: there was no significant difference between the NFA+LPS and LPS treated group(p>0.05);ab-m CLCA3+LPS treated group compared with LPS group(p<0.05).(3)BALF Total protein concentration:NFA+LPS treated group,the ab-m CLCA3+LPS group and the LPS group had no significant difference(p>0.05).Reverse intervention of the IP group lung injury levels of the correlation results:(1)lung injury score: NFA+LPS treated group,ab-m CLCA3+LPS group and LPS group had no significant difference(p>0.05);(2)W/D value: NFA+LPS treated group,the ab-m CLCA3 + LPS treated group and LPS group were not significantly different(p>0.05);(3)BALF total protein concentration: NFA+LPS treated group,ab-m CLCA3+LPS treated group and LPS group were not significantly different(p> 0.05).7.Application m CLCA3 specific antibody(ab-m CLCA3)Reverse intervention lungs of mice,inflammatory cytokine correlation results show: TNF-α,CXCL-1,CXCL-2 was significantly decreased(p<0.05),whereas IL-1β,IL-6 did not change significantly(p>0.05).Reverse intervention pulmonary mice: inflammatory cytokines levels did not change significantly(p> 0.05).Conclusions:1.There was no significant difference of in-hospital mortality in patients with both direct and indirect ARDS;lung injury score and SOFA score were independent risk factors of death in patients with both direct and indirect ARDS respectively.2.inflammatory cytokines and chemokines expressed significant differences in the direct and indirect mouse models of ARDS;Alveolar epithelial cells injured heavier in indirect ARDS,while direct ARDS demonstrate a harder pulmonary endothelial damage.3.m CLCA3 expressed significantly higher in the lungs of direct ARDS mice,while there was no significant difference in indirect ARDS models.4.With reverse intervention of m CLCA3 function of direct ARDS mice,the extent of lung damage and inflammation reduced;With reverse intervention lungs of m CLCA3 of indirect ARDS mice,inflammatory and lung injury levels had no significant change.CLCA played a Proinflammatory effect during the form of lung inflammation of direct ARDS mice.