| Background and ObjectiveSeawater inhalation can cause acute lung injury (SW-ALI) or acute respiratory distress syndrome (SW-ARDS), which was characterized by progressive dyspnea, refractory hypoxemia and high mortality. In the past few years, studies of inflammatory mediators and cytokines in molecular level demonstrated the crucial role of inflammation in the ALI. So when we study ALI, we turn attention to the regulation of inflammation. Protease-activated receptor 2 (PAR2) belongs to a family of G protein-coupled receptors. It can be activated by serine proteases and then induce inflammation. But there is still a dispute on the role of PAR2 in pulmonary inflammation, no information concerning the role of PAR2 on ALI induced by seawater, and the downstream signal transduction of PAR2 remain unclear. FUT-175 is a synthetic inhibitor of serine proteases, which can block PAR2 activation and then reduce the release of inflammatory mediators and cytokines, but it's protective effects was not mentioned on ALI. Therefor, in this study we will detect the influence of seawater on PAR2 mRNA and protein expression of A549 cells, investigate the influence of FUT-175 and PAR2-antibody on seawater-activated PAR2 signal transduction in vitro, and observe the effects of FUT-175 on pulmonary inflammation and pulmonary microvascular permeability in the a model of seawater inhalation-induced ALI in vivo. The purposes of this study are to elucidate the role and the mechanism of PAR2 activation on pathogenesis of SW-ALI, confirm the protective effects of FUT-175 on SW-ALI.Methods1. A549 cells were randomly divided into 5 groups: control group, 2 h group, 4 h group, 8 h group and 16 h group, which were treated with seawater for 2 h, 4 h, 8 h and 16 h respectively. After seawater treated, cells were collected for Real-time quantitative polymerase chain reaction (PCR) and Western blot analysis to determine the expression of PAR2 mRNA and protein. Supernatant were collected for IL-8 and TNF-αdetection. 2. A549 cells were randomly divided into 7 groups: control group, seawater group, PAR2-antibody group, FUT-175 group, SB203580 group, PD98059 group and SP600125 group. Control group remained untreated as blank control; seawater group was incubated with seawater; PAR2-antibody group was treated with 2mg/L PAR2-antibody for 1 h before incubated with seawater; FUT-175 group was treated with 10μM FUT-175 for 1 h before incubated with seawater; SB203580 group was treated with 10μM SB203580 for 1 h before incubated with seawater; PD98059 group was treated with 10μM PD98059 for 1 h before incubated with seawater; SP600125 group was treated with 10μM SP600125 for 1 h before incubated with seawater. After 8 h, cells of control group, seawater group, PAR2-antibody group and FUT-175 group were collected for Western blot analysis and electrophoretic mobility shift assays to determine the expression of phosphorylated p38MAPK, phosphorylated ERK, phosphorylated JNK protein and NF-κB binding activity. Supernatant of all groups were collected for IL-8 and TNF-αdetection.3. ALI was induced by inhaling seawater (4ml/kg). Forty-eight Wistar rats were randomly divided into four groups: control group, 2 h group, 4 h group and 8 h group, which were 2 h, 4 h and 8 h after seawater inhalation respectively. Lung tissues were collected for real-time quantitative PCR, western blot analysis and immunohistochemistry to determine the expression of PAR2 mRNA and protein in lung tissue of rats.4. ALI was induced by inhaling seawater (4ml/kg) and LPS (4mg/kg). Ninety-six Wistar rats were randomly divided into four groups: control group, seawater group, LPS group and FUT-175 group. In seawater group, 2ml normal saline (NS) was administered through jugular vein at 20min after seawater inhalation; in FUT-175 group, instead of 2ml of NS, 10mg/kg body weight of FUT-175 in 2ml of NS was administered through jugular vein at 20min after seawater inhalation. During experiment, arterial blood was collected for blood gas analysis at 0.5 h, 1 h, 2 h, 4 h and 8 h time point after model established. After 8h of experiment, serum was collected for IL-8 and TNF-αdetection. Pulmonary microvascular permeability (PMVP), wet/dry ratio (W/D) of lung tissue and myeloperoxidase activity (MPO) of lung tissue were detected by biochemical methods. The histopathologic changes of lung tissue were observed under optical microscope.Results1. The expression of mRNA for PAR2 on A549 cells increased after seawater treated, increasing significantly after 4 h(P<0.05), peaking at 8h(1.8-fold) post-seawater treated and lasted for 16 h(P<0.01).And the expression of protein for PAR2 on A549 cells increased too after seawater treated, increasing significantly after 4 h(P<0.05), peaking at 8h(2.2-fold) post-seawater treated and lasted for 16 h(P<0.01).The level of IL-8 and TNF-αin supernatant increased significantly after seawater treated, peaking at 2 h post-seawater treated and then followed a little decrease which still higher than those of control group significantly(P<0.01).2. The level of p-p38 MAPK, p-ERK, p-JNK and NF-κB binding activity on A549 cells all increased significantly after seawater treated(3.1-fold, 1.8-fold, 3.2-fold and 6.7-fold respectively)(P<0.01-0.05). The level of p-p38 MAPK, p-ERK, p-JNK and NF-κB binding activity on A549 cells all decreased significantly pre-treated with FUT-175 and PAR2-antibody compared with seawater group(P<0.01-0.05). The level of IL-8 and TNF-αin supernatant increased significantly after seawater treated(P<0.01), and decreased significantly pre-treated with FUT-175, PAR2-antibody, SB203580, PD98059 and SP600125(P<0.01).3. The expression of PAR2 mRNA on lung tissue of rats increased after seawater inhalation, increasing significantly after 2 h(P < 0.05), peaking at 4h (2.8-fold) post-seawater inhalation and lasted for 8 h(P<0.01). The expression of PAR2 protein on lung tissue of rats increased too after seawater inhalation, increasing significantly after 2 h(P<0.05), peaking at 4h (4.2-fold) post-seawater inhalation and lasted for 8 h(P<0.01). PAR2 immunoexpression located in alveoli, bronchial epithelium and pulmonary macrophage cells of rats in control group showed mild immunostaining. At 2h after seawater inhalation, stronger positive staining was not only observed in the alveolar and bronchial epithelium cells, but also observed in the pulmonary microvascular endothelium and airways smooth muscle.4. The level of IL-8 and TNF-αin plasma, MPO activity, PMVP, W/D and pathomorphological score of lung tissue increased significantly after seawater and LPS inhalation, which is followed by a significant reduction in arterial PaO2 (P<0.01). PaO2 in seawater group was significant lower than that of LPS group, while MPO, W/D and PVMP were significant higher than those of LPS group(P<0.01). The above parameters of seawater group all ameliorated after FUT-175 administering(P<0.01-0.05). Conclusion1. Seawater up-regulate the expression of PAR2 on A549 cells, induce IL-8 and TNF-αrelease, and change the structure of A549 Cells.2. Seawater activate PAR2 of A549 cells, induce IL-8 and TNF-αrelease through MAPK pathway and NF-κB pathway.3. Serine proteases FUT-175 and PAR2-antibody have protective effects on A549 cells through blocking PAR2 activation, reducing IL-8 and TNF-αrelease.4. Seawater inhalation up-regulate the expression of PAR2 on lung tissue of rats, which has crucial role in the seawater inhalation-induced ALI.5. Serine proteases FUT-175 have protective effects on the seawater inhalation-induced ALI in rats through blocking PAR2 activation and downstream inflammatory reaction, ameliorating lung injury, pulmonary microvascular permeability and pulmonary edema.
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