The Influence Of Airway Tight Junction Under Acidic Stress

Objective：To reveal the influence of airway acidic–circumstance on the airwayepithelial tight junctions (TJs) under clumps of airway chronicinflammatory diseases, as well as its molecular mechanism. To investigatethe variation of airway barrier function under acidic stress.Methods:（1）The influence of acidic stress on the airway epithelial barrierfunction16HBE cells, the immortalized human airway epithelial cell line, werecultured in vitro to achieve cellular conjugation. The synthesized TJs weredetected by Western Blot. The culture mediums were adjusted byhydrochloric acid to different concentrations. Followed by exposure todifferent acidic culture mediums, trans-epithelium electrical resistant (TER)of16HBE cells were measured and recorded. The permeability of16HBEcells was measured by a HRP flow measurement. Meanwhile, total cellularTJs proteins and their cellular distribution were analyzed by Western Blotor cell immunohistochemistry.（2）The possible molecular mechanisms of airway epithelialbarrier disfunction induced by acidic stress Transient receptor potential cation channel subfamily V member1(TRPV1) defected16HBE cells were obtained by transfection of TRPV1specific short hairpin RNA (shRNA). By pretreatment with TRPV1inhibitor capsazepine, intracellular chelating agent BAPTA-AM, PKCspecific inhibitor Safingol, or Calpain inhibitor MDL28170, we aimed toreveal the internal molecular mechanisms of depredated airway epithelialbarrier function induced by acidic airway circumstance.Results:（1）Acidic stress induced a time-and dose-dependent epithelialbarrier disfunction of16HBE cellsAcidic stress depredated the TER of16HBE cells. Despite slightly andnon-significant increase of TER was detected in the16HBE cells culturedin pH6.0mediums, significant decrease of TER was detected in16HBEcells cultured in the acidic mediums with pH<6.0. Generally, exposed to apH5.0culture medium for8h led to a decrease of TER at about61.0±6.6%in16HBE cell line. Exposed to a pH4.0culture medium for8h led to adecrease of TER at about52.9±6.7%. According to the HRP flowmeasurement, exposed to a pH5.0culture medium for8h led to a (4.1±0.4)-fold increase of HRP flow in16HBE cells. pH4.0culture mediumfor8h led to a (4.9±0.5)-fold increase of HRP flow in16HBE cells.（2）Acidic stress induced an degradation of Claudin-3andClaudin-4protein in the membranal protein extracts of16HBE cells Further study with Western Blot analysis indicated significantdecrease expression levels of Claudin-3and Claudin-4, but not Occludin、Claudin-1、 Claudin-5、 Claudin-7or ZO-1. Meanwhile, a significantdecrease of fluorescence intensity between16HBE cells indicated adecreased expression of Claudin-3or Claudin-4.（3）Acidic stress inducing the degradation of Claudin-3andClaudin-4protein as well as the epithelial barrier function of16HBEcells is probably depending on the activation of TRPV1-[Ca2+]i-PKCα-Calpain signaling cascadeTRPV1defect16HBE cells were acquired by transfected of TRPV1shRNA. With pretreatment of TRPV1inhibitor capsazepine, intracellularCa2+chelating agent BAPTA-AM, PKC specific inhibitor Safingol, orcalpain inhibitor MDL28170, the degradation of intercellular Claudin-3andClaudin-4as well as the degradation of TER in16HBE cells induced byacidic stress were partially attenuated.Conclusion:Acidic airway circumstance induces a depletion of airway epitheliumbarrier function meanly through the degradation of Claudin-3andClaudin-4. These effects probably depend on the activation ofTRPV1-[Ca2+]i-PKC-Calpain cascade.