The Effects Of Atp Over-Release On The High Externalization Of Mucin In Airway Epithelial Cells By Mechanical Stretching

Objective:To explore the effects of ATP over-release on mucin over-externalization in airway epithelial cells by mechanical stretching.Methods:(1) The airway epithelial cells line HBE16cultured in vitro were stimulated by rhythmically tilting the culture dish.0,10,15,30,45s after tilting the culture dish in30°(with one third of the cells had no culture liquid covered and exposed to the air), the cell activity was assessed by methylthiazolyl tetrazolium method (MTT), and the optimization tilting time was determined;(2) The shear stress and compressive stress were provided by liquid surface tension, atmospheric pressure and liquid gravity with tilting the culture dishes. And we injected medical oxygen into closed boxes to increase tension to simulate mechanical ventilation.(3) The airway epithelial cells were divided into8groups:①control group,②tilt group,③tilt+intra-cellular Ca2+-chelator BAPTA-AM group,④tilt+BAPTA-AM+extracellular Ca2+-chelator EGTA group,⑤tilt+air pressure group,⑥tilt+air pressure+P2Y receptor blocking pharmacon: reactive blue-2(RB-2) group,⑦tilt+air pressur+BAPTA-AM group,⑧tilt+air pressure+BAPTA-AM+EGTA group.(4) The levels of MUC5AC protein in culture medium were detected with enzyme-linked immunosorbent assay; The concentration changes of ATP were detected with high performance liquid chromatography; The levels of MUC5AC mRNA in culture cells were detected with RT-PCR; The concentration changes of Ca2+were detected with inverted fluorescence microscope.Results:(1) The expression levels of MUC5AC mRNA in culture cells, its protein and ATP in culture medium of tilt+air pressure group were (11.8±0.01),(7.41±0.45) μmol/g and (0.77±0.26) respectively, which were increased significantly (all P<0.05) as compared with those in tilt group[(6.6±0.01),(2.76±0.47)μmol/g,(0.25±0.01)] and control group [(3.4±0.01),(0.00±0.01)μmol/g,(0.02±0.01)].(2) Pre-treated with RB-2, EGTA+BAPTA-AM in tilt+air pressure group, the mRNA expression in the HBE16cells [(10.5±0.03) and (11.9±0.11),] was not significantly decreased as compared with tilt+air pressure group(11.80±0.01)(all P＞0.05),but increased significantly as compared with control group (3.40±0.01)(P<0.05).(3) After the HBE16cells were pre-treated with RB-2, EGTA+BAPTA-AM in tilt+air pressure group,the concentration of MUC5AC protein in supernatant [(0.07±0.05),(0.08±0.05)] was significantly decreased as compared with tilt+air pressure group (0.74±0.27)(all P＜0.05). Compared with control group (0.02±0.01), the concentration of MUC5AC protein in supernatant was significantly increased in tilt+air pressure group (P<0.05).(4) After the HBE16cells were pre-treated with RB-2, EGTA+BAPTA-AM in tilt+air pressure group,the concentration of ATP in supernatant[(0.05±0.02) μmol/g,(0.08±0.02) μmol/g] was significantly decreased as compared with tilt+air pressure group [(7.41±0.45) μmol/g] (all P<0.05). Compared with control group [(0.00±0.01) μmol/g], the concentration of ATP in supernatant was significantly increased in tilt+air pressure group (P＜0.05).(5) The bigger the angle of tilt, the larger the concentration of intra-cellular Ca2+. The maximum concentration of intra-cellular Ca2+in tilt group was significantly higher than that in control group (P<0.05); The maximum concentration of intra-cellular Ca2+in tilt+air pressure group was significantly higher than that in tilt group (P＜0.05); After the cells were pre-treated with RB-2, BAPTA-AM in tilt+air pressure group,the maximum concentration of intra-cellular Ca2+was significantly decreased as compared with tilt+air pressure group (all P＜0.05); Pre-treated with EGTA in tilt+air pressure group,the maximum concentration of intra-cellular Ca2+was not significantly decreased(P<0.05).Conclusion:(1) Mechanical ventilation could increase the secretion and expression of MUC5AC in airway epithelial cells.(2) Ca2+-dependent ATP over-release from the airway epithelial cells may be closely related with MUC5AC over-externalization in airway epithelial cells by mechanical stretching, but has no correlation with the expression level of MUC5AC mRNA.(3) In this mechanism, almost all needed Ca2+was provided by intra-cellular Ca2+...