The Mediated Effect Of Tensition-aensitive Cation Channel In Airway Basic Mucus Secretion

Objective To investigate the regulation of the secretion of basictension sensitivity cation of airway mucin (MUC), to further enrich thetheory of the airway of airway mucus secretion mechanism of airwaymucustreatment of secretory disorders provide a theoretical basis.Methods Gas/liquid interface and cultured16HBE airway epithelialcells, to enable two devices simulate normal breathing rhythm of pressurewaves in cultured cells, testing reflects the important indicator of airwayepithelial mucus layer steady-state. Experimental groups:(1) Static controlgroup: no rhythmic pressure wave, the culture medium16HBE cells;(2)rhythmic pressure wave: Start of the pressure wave generated by the abovedevice to simulate normal breathing in16HBE cells;(3) rhythmic pressurewaves+tension sensitivity of cation channel blocker ruthenium red (RR)group: in the16HBE cells with ruthenium red pretreatment, and thensimulate the pressure wave generated by the normal breathing for16HBEcells;(4) rhythmic pressure wave+verapamil: first HBE16cells in thecalcium channel blockers verapamil, and then simulate the pressure wavegenerated by the normal breathing for16HBE cells; protein kinase Cinhibition (5) rhythmic pressure waves+agent (GF109203x): pre-AddGF109203x pretreatment, and then simulate the rhythmic pressure waves incultured cells. Measured by MTT assay groups16HBE cell activity;grouping cultured for24h to extract total protein within the cells in eachgroup, the use of ELISA for detection of each group of MUC protein level (2,5AC and5B); immunocytochemistry confocal laser scanning microscopyto observe the MUC2protein expression of the group; RT-PCR detection ofTRPV4and MUC5AC mRNA levels; Western blot detection of MARCKSand SNAP-23protein expression levels of; observed by confocal Ca2+concentration changes.Results (1)16HBE cells, respectively, the survival rate (93.9+3.1)%，(99.2+4.3)%、(94.6+7.3)%、(96.6+2.3)%and (96.5+5.0)%. Control groupthan in the rhythmic pressure waves stimulate cell survival of groups toreduce the static.(2) rhythmic pressure wave of MUC(2,5AC and5B)protein content was significantly higher (P<0.01); of MUC after RRtreatment group and verapamil, the PKC inhibitor (2,5AC and5B) contentthan the rhythmic pressure waves was significantly decreased (P<0.01).(3)MARCKS, pMARCKS and SNAP-23, pSNAP-23protein expression in thepressure wave are1.08±0.48、0.84±0.40、1.15±0.35、0.34±0.56weresignificantly higher (P<0.01); give the RR, verapamil, the PKC inhibitorpretreatment, MARCKS and pSNAP-23protein expression wassignificantly decreased (P<0.01).(4) immunocytochemistry laser confocalimaging to show the normal basis of16HBE cells MUC2protein expressionwas mainly localized in the cytoplasm; rhythmic pressure waves comparedwith the control group, expression of MUC2protein expression levels weresignificantly increased.(5) The rhythmic pressure wave group TRPV4mRNA (0.63±0.03) was significantly higher than the static control group(0.28±0.05)(P<0.01); giving RR pretreatment, TRPV4mRNA expressionwas significantly lower (0.46±0.08), but still higher than those in the controlgroup (P<0.01).Conclusion1.rhythmic pressure wave generated by the normalbreathing of the airway involved in to maintain the balance of basic MUCsecretion in the airway microenvironment. 2. rhythmic pressure wave airway sticky protein secretion through theactivation of TRPV4protein channels, the main mechanism by Ca2+/PKC/MARCKS signaling pathway...