Regulatory effect of hemichannels protein Pannexin-1 on P2X7 receptor activity in the lungs of mice with lung injury / 中华危重病急救医学

ABSTRACT

Objective To investigate the regulative effect of hemichannels protein Pannexin-1 on P2X7 receptor activation and caspase-1 mediated inflammatory response in the lungs of mice with lung injury. Methods Sixty male C57BL/6 mice were randomly divided into five groups with 12 mice in each group sham operation group (Sham group), mechanical ventilation (MV) group, MV + low dose lipopolysaccharide (LPS) group (MLL group), MV + medium and high dose LPS group (MML group) and MV + high dose LPS group (MHL group). A "two-hit" lung injury model was reproduced by MV with high tidal volume combined with LPS injection in airway. All the mice underwent tracheotomy and intubation. After operation, the mice in Sham group were maintained spontaneous breathing, and those in other four groups were put on small animal ventilators to give MV with a large tidal volume of 28 mL/kg. After stable respiration in mice, those in the Sham group and MV group were injected 8 mL/kg of normal saline (NS) into the airway, and those in MLL, MML and MHL groups were given 2, 5 and 8 mg/kg of LPS respectively (diluted with NS into 8 mL/kg). After 4 hours on MV, the mice were sacrificed, and bronchoalveolar lavage fluid (BALF) was extracted to determine intracellular and extracellular ATP concentration. Lung tissue was harvested and water containing ratio of lungs was measured. The degree of lung pathological damage was observed after hematoxylin-eosin (HE) staining, and lung injury score was calculated. The expression of Pannexin-1 protein in lung tissue was calculated with immunohistochemistry. Western Blot and fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) were used to detect the protein and mRNA expressions of Pannexin-1, P2X7 receptor, caspase-1 and interleukin-1β (IL-1β). Results There was no obvious pathological change in lung tissue in Sham group, intracellular ATP concentration was higher than extracellular ATP concentration, water content in lung tissue was lower, Pannexin-1 expression was low in lung tissue by immunohistochemical staining, and Pannexin-1, P2X7 receptor, caspase-1 and IL-1β were only expressed in micro-protein and mRNA in lung tissue. Compared with the Sham group, the alveolar lesions and hemorrhages in the MV group were not obvious, and lung injury score was slightly increased. There was no significant fluctuation between intracellular ATP concentration and extracellular ATP concentration. The water content in lung tissue was increased significantly, while the expressions of Pannexin-1, P2X7 receptor, caspase-1 and IL-1β in lung tissue were increased slightly. After LPS intervention, progressively increased lung exudation, ruptured alveoli, dilated capillaries, and inflammatory cells were found, and lung injury score was increased without significant difference among the three LPS doses groups. With the increase in LPS dosage, the concentration of extracellular ATP in BALF was increased, the concentration of intracellular ATP was decreased, the water containing ratio of lung tissue was increased gradually, and the protein and mRNA expressions of Pannexin-1, P2X7 receptor, caspase-1 and IL-1β in lung tissue were increased gradually in a dose-dependent manner. The parameters in MHL group showed significant differences as compared with those in MV group [lung injury score 8.25±0.45 vs. 3.50±0.52; intracellular ATP concentration (μmol/L) 198.76±150.77 vs. 896.69±281.11, extracellular ATP concentration (μmol/L) 336.57±90.28 vs. 141.52±42.22; lung water containing rate (6.37±0.11)% vs. (5.05±0.14)%; Pannexin-1 protein (gray value) 3.20±0.70 vs. 1.54±0.76, Pannexin-1 mRNA (2-&Delta;&Delta;CT) 7.86±0.86 vs. 2.47±0.92; P2X7 receptor protein (gray value) 3.18±0.88 vs. 1.80±0.72, P2X7 receptor mRNA (2-&Delta;&Delta;CT) 7.17±0.96 vs. 2.31±0.45; caspase-1 protein (gray value) 3.00±0.45 vs. 0.93±0.51, caspase-1 mRNA (2-&Delta;&Delta;CT) 4.39±0.91 vs. 2.74±0.41; IL-1β protein (gray value) 2.54±1.08 vs. 1.16±0.53, IL-1β mRNA (2-&Delta;&Delta;CT) 132.34±41.48 vs. 19.67±8.67; all P < 0.05]. Conclusion Pannexin-1 may be involved in LPS and MV induced lung injury, which may be regulated by intracellular release of ATP to the extracellular site and binding to P2X7 receptor on the cell surface, thereby regulating active caspase-1 production and release, involving in the production of IL-1β and other inflammatory factors eventually which leads to the occurrence and development of lung injury.