The Negative Feedback Of CAMP/PKA Pathway Regulates The Effects Of Endoplasmic Reticulum Stress Induced NLRP3 Inflammasome Activationon Type Ⅱ Alveolar Epithelial Cell Pyroptosis-the New Mechanism Of Pulmonary Fibrosis

Background:Endoplasmic reticulum stress(ER stress)induced type II alveolar epithelial cell death plays an critical role in the pathogenesis of pulmonary fibrosis.It has been found that endoplasmic reticulum stress also promotes NLRP3 inflammasome activation and promotes pyroptosis.In addition,a number of important studies have proved that the upregulation of cAMP/PKA pathway inhibit the activation of NLRP3 inflammasome.Therefore,it is speculated that endoplasmic reticulum stress promotes NLRP3 inflammasome activation,and type II alveolar epithelial cell pyroptosis,which promotes the progression of lung fibrosis.Furthermore,cAMP/PKA pathway activation inhibits ER stress induced NLRP3 inflammasome activation,which relieves pulmonary fibrosis.This study explores the potential interaction between ER stress,NLRP3 inflammasome and cAMP/PKA pathway,whose potential impact on pulmonary fibrosis,and seeks targets for pulmonary fibrosis.Methods:In vivo,20 C57BL/6J mice were randomly divided into two groups,the Control group(saline instilled into the trachea)and the BLM group(bleomycin,5mg/kg instilled into the trachea).4 weeks later,the mice were sacrificed,the right lung tissue were embedded into wax.Histological changes of the lungs were evaluated by hematoxylin and eosin(H&E)and Masson’s staining.Immunohistochemical staining detected related protein expressions.And colocalizations between proteins were detected by immunofluorescence.In vitro,using type Ⅱ alveolar epithelial cell line A549,perform the following experiments:(1)Control,Tunicamycin(Tunicamycin,ER stress inducer),Tunicamycin+4PBA(4PBA,ER stress inhibitor)(2)Control,Tunicamycin,Tunicamycin+forskolin(Forskolin,ADCY inducer)(3)Control,Tunicamycin,Tunicamycin+KH7(KH7,adenylate cyclase inhibitor)(4)Control group,Tunicamycin,Tunicamycin+H89(H89,PKA inhibitor).LDH release assay and CCK8 assay were used to detect cell death.Western blotting and RT-qPCR detected separately related protein levels and mRNA levels.Colocalizations between proteins were detected by immunofluorescence.And Elisa detected intracellular cAMP levels.Results:In vivo,compared with the Control group,the BLM group has damaged alveolar structure,increased collagen,and inflammatory cell infiltration.The BLM group showed that the expression of chaperone protein Grp78 decreased but ER stress related protein Grp94 and CHOP increased in alveolar area,confirming that pulmonary fibrosis promotes endoplasmic reticulum stress due to the loss of chaperon protein Grp78,which is the same as the previous report[1].In addition,the expression of NLRP3 inflammasome-related proteins and PKA increased in alveolar area.Compared with the Control group,SPC/CHOP,SPC/NLRP3,CHOP/NLRP3,CHOP/IL1β,SPC/PKA,SPC/cAMP,and cAMP/NLRP3 colocalized in the BLM group.In vitro,compared with the Control group,in the Tunicamycin group showed that Grp94,CHOP,NLRP3,IL1β,GSDMD protein and mRNA levels increased,intracellular cAMP levels and PKA protein increased,cell death increased,and the colocalization of Grp94 and NLRP3 increased,while 4PBA could reverse the above-mentioned proteins and mRNA changes and cAMP changes,reduce pyroptosis and the co-localization of Grp94/NLRP3,CHOP/NLRP3,Grp94/cAMP,and NLRP3/cAMP.Forskolin inhibited the expressions of NLRP3 and ASC promoted by Tunicamycin and the increased pyroptosis promoted by Tunicamycin.Both KH7 and H89 could further aggravate pyroptosis promoted by Tunicamycin.Conclusion:In pulmonary fibrosis,ER stress induces NLRP3 inflammasome activation in type Ⅱalveolar epithelial cell and promotes cell pyroptosis.In addition,ER stress promotes activation of cAMP/PKA and the upregulation of cAMP/PKA pathway could inhibit NLRP3 inflammasome activation induced by ER stress in type Ⅱ alveolar epithelial cell.