蛋白酶激活受体-2在海水吸入大鼠急性肺损伤中的作用及机制研究
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摘要
背景与目的:
海水淹溺可引起海水吸入型急性肺损伤(SW-ALI),进一步发展可导致海水型急性呼吸窘迫综合征(SW-ARDS),病情凶险,死亡率高。由于炎症反应是ALI发病的关键环节,人们将对ALI的研究转向炎症调控机制的研究,以期控制炎症反应,减轻损伤,从而降低ALI的发病率和死亡率。蛋白酶激活受体-2(PAR2)属G蛋白偶联受体家族,激活后可参与炎症反应,但在肺部炎症反应中的致病作用还存在争议,在SW-AIL中的作用尚未提及,且PAR2激活后下游信号通路仍不清楚,有待进一步探讨。FUT-175作为一种丝氨酸蛋白酶抑制剂,可抑制PAR2的激活,减少炎症介质和细胞因子的释放,但其抗炎作用在ALI的研究中尚未提及,有待进一步研究。为此,本课题拟从细胞水平检测海水对A549细胞PAR2表达的影响,从分子水平探讨PAR2激活后的下游信号转导通路以及FUT-175和PAR2抗体对其下游信号通路的干预作用,从器官水平观察FUT-175对SW-ALI大鼠肺部炎症反应和肺微血管通透性的影响,旨在明确PAR2在SW-ALI中的作用及机制,从抗炎的角度揭示FUT-175对SW-ALI大鼠的保护作用,为临床应用FUT-175治疗SW-ALI提供更坚实的理论基础和实验依据。
方法:
1.将培养的A549细胞接种于6孔板中,随机分为对照组(control)、海水干预2、4、8、16h组,各组细胞干预相应的时间后进行指标观测。收集上清检测TNF-α和IL-8浓度,收集细胞用实时荧光定量PCR法和Western blot法检测PAR2 mRNA和蛋白表达水平。
2.将培养的A549细胞接种于6孔板中,随机分为对照组(control组)、海水干预组(seawater组)、海水+PAR2抗体组(PAR2-antibody组)、海水+FUT-175组(FUT-175组)、海水+SB203580组(SB203580组)、海水+PD98059组(PD98059组)、海水+SP600125组(SP600125组)。对照组加入无血清培养液,海水组加入海水培养8h;PAR2-antibody组用PAR2单克隆抗体2mg/L预处理1h后,加入海水继续培养8h;FUT-175组用FUT-175 10μM预处理1h后,加入海水继续培养8h;SB203580组用SB203580 10μM预处理1h后,加入海水继续培养8h;PD98059组用PD98059 10μM预处理1h后,加入海水继续培养8h;SP600125组用SP600125 0μM预处理1h后,加入海水继续培养8h。control组、seawater组、PAR2-antibody组和FUT-175组收集细胞用Western印迹法和EMSA法分别检测磷酸化p38MAPK蛋白表达量、磷酸化ERK蛋白表达量、磷酸化JNK蛋白表达量和NF-κB活化水平;各组细胞收集上清检测TNF-α和IL-8浓度。
3.采用海水吸入法复制大鼠ALI模型,吸入海水(4ml/kg)后成功建立ALI模型。将48只Wistar大鼠随机分为对照组、海水吸入2、4、8h组,取肺组织用实时荧光定量PCR法检测PAR2 mRNA表达水平,用Western blot法和免疫组化法检测PAR2蛋白表达情况。
4.采用海水吸入法和LPS吸入法复制大鼠ALI模型,96只Wistar大鼠随机分为对照组(control)、海水组(seawater组)、LPS组(LPS组)和FUT-175治疗海水吸入组(FUT-175组)。海水组和LPS组分别吸入海水(4ml/kg)和LPS(4mg/kg)建立ALI模型。seawater组:模型复制成功后20min,经尾静脉注射生理盐水2ml/kg。FUT-175组:海水型ALI模型复制成功后20min,经尾静脉注射FUT-175 10mg/kg。4组大鼠观察8小时,分别于模型建立及干预后0.5、1、2、4、8h进行动脉血气分析,最后检测肺微血管通透性(PMVP)、肺湿/干重比(W/D)、肺组织髓过氧化物酶(MPO)、血浆IL-8和TNF-α水平,并观察病理学变化。
结果:
1.与正常对照组比较,海水处理后,A549细胞PAR2 mRNA在4h时显著升高(P<0.05),随着时间延长,在8h点达最高峰(为对照组的1.8倍),此后一直显著高于对照组(P<0.01)。A549细胞PAR2蛋白表达量也在4h时显著升高(P<0.05),在8h点达最高峰(为对照组的2.2倍),此后一直显著高于对照组(P<0.01)。A549细胞经海水处理后,上清液中的IL-8和TNF-α迅速升高,2 h达最高峰,显著高于对照组(P<0.01)。此后有所下降,但在所有时间点均显著高于对照组(P<0.01)。
2.与正常对照组比较,海水处理后,A549细胞p38MAPK磷酸化水平、ERK磷酸化水平和JNK磷酸化水平显著升高(分别为对照组的3.1倍、1.8倍和3.2倍)(P<0.01-0.05),NF-κB活化水平显著升高(为对照组的6.7倍)(P<0.01),而FUT-175和PAR2抗体预处理组同海水处理组比较,A549细胞p38MAPK磷酸化水平、JNK磷酸化水平、ERK磷酸化水平和NF-κB活化水平显著降低(P<0.01-0.05)。海水处理后,A549细胞培养上清液中IL-8和TNF-α显著升高(P<0.01),而FUT-175、PAR2抗体、SB203580、PD98059和SP600125预处理组同海水处理组比较,上清液中IL-8和TNF-α则显著降低(P<0.01)。
3.与正常对照组比较,海水吸入后,大鼠肺组织PAR2 mRNA和蛋白表达量在2h时即显著升高(P<0.05),随着时间延长,在4h点达最高峰(为对照组的2.8倍),此后均显著高于对照组(P<0.01)。同时大鼠肺组织PAR2蛋白表达量在2h时即显著升高(P<0.05),随着时间延长,在4h点达最高峰(为对照组的4.2倍),此后均显著高于对照组(P<0.01)。PAR2免疫组化显示,在正常对照组,PAR2微弱表达于肺泡、支气管上皮和肺泡巨噬细胞,海水吸入2h后,阳性反应明显增强,不仅见于肺泡和支气管上皮细胞,还见于肺微血管内皮细胞和气道平滑肌细胞。
4.与正常对照组比较,海水和LPS吸入致伤组大鼠PaO2下降、W/D比值增大、肺微血管通透性增高、肺组织MPO活性增高、血浆IL-8和TNF-α升高、肺组织病理形态学积分增加(P<0.01);海水组PaO2明显低于LPS组,而MPO、W/D和PVMP明显高于LPS组(P<0.01)。FUT-175治疗海水吸入组上述指标均显著改善(P<0.01-0.05)。
结论:
1.海水处理A549细胞,可上调A549细胞PAR2的表达,导致IL-8和TNF-α释放的增加,同时可导致A549细胞结构的改变。
2.海水激活A549细胞PAR2后,通过MAPK信号通路和NF-B信号通路,导致IL-8和TNF-α释放增加,是海水处理A549细胞导致细胞损伤的可能机制。
3.丝氨酸蛋白酶抑制剂FUT-175和PAR2抗体可通过抑制PAR2的激活,对海水处理的A549具有保护作用。
4.海水吸入后大鼠肺组织PAR2表达上调,在海水所致急性肺损伤中起重要作用。
5.丝氨酸蛋白酶抑制剂FUT-175对海水吸入所致急性肺损伤大鼠具有保护作用,其机制可能是通过抑制肺组织PAR2的激活,阻断其下游的炎症反应,减轻肺损伤,降低肺血管通透性,减轻肺水肿。
Background and Objective
Seawater 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.
Methods
1. 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.
Results
1. 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).
Conclusion
1. 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.
海水淹溺可引起海水吸入型急性肺损伤(SW-ALI),进一步发展可导致海水型急性呼吸窘迫综合征(SW-ARDS),病情凶险,死亡率高。由于炎症反应是ALI发病的关键环节,人们将对ALI的研究转向炎症调控机制的研究,以期控制炎症反应,减轻损伤,从而降低ALI的发病率和死亡率。蛋白酶激活受体-2(PAR2)属G蛋白偶联受体家族,激活后可参与炎症反应,但在肺部炎症反应中的致病作用还存在争议,在SW-AIL中的作用尚未提及,且PAR2激活后下游信号通路仍不清楚,有待进一步探讨。FUT-175作为一种丝氨酸蛋白酶抑制剂,可抑制PAR2的激活,减少炎症介质和细胞因子的释放,但其抗炎作用在ALI的研究中尚未提及,有待进一步研究。为此,本课题拟从细胞水平检测海水对A549细胞PAR2表达的影响,从分子水平探讨PAR2激活后的下游信号转导通路以及FUT-175和PAR2抗体对其下游信号通路的干预作用,从器官水平观察FUT-175对SW-ALI大鼠肺部炎症反应和肺微血管通透性的影响,旨在明确PAR2在SW-ALI中的作用及机制,从抗炎的角度揭示FUT-175对SW-ALI大鼠的保护作用,为临床应用FUT-175治疗SW-ALI提供更坚实的理论基础和实验依据。
方法:
1.将培养的A549细胞接种于6孔板中,随机分为对照组(control)、海水干预2、4、8、16h组,各组细胞干预相应的时间后进行指标观测。收集上清检测TNF-α和IL-8浓度,收集细胞用实时荧光定量PCR法和Western blot法检测PAR2 mRNA和蛋白表达水平。
2.将培养的A549细胞接种于6孔板中,随机分为对照组(control组)、海水干预组(seawater组)、海水+PAR2抗体组(PAR2-antibody组)、海水+FUT-175组(FUT-175组)、海水+SB203580组(SB203580组)、海水+PD98059组(PD98059组)、海水+SP600125组(SP600125组)。对照组加入无血清培养液,海水组加入海水培养8h;PAR2-antibody组用PAR2单克隆抗体2mg/L预处理1h后,加入海水继续培养8h;FUT-175组用FUT-175 10μM预处理1h后,加入海水继续培养8h;SB203580组用SB203580 10μM预处理1h后,加入海水继续培养8h;PD98059组用PD98059 10μM预处理1h后,加入海水继续培养8h;SP600125组用SP600125 0μM预处理1h后,加入海水继续培养8h。control组、seawater组、PAR2-antibody组和FUT-175组收集细胞用Western印迹法和EMSA法分别检测磷酸化p38MAPK蛋白表达量、磷酸化ERK蛋白表达量、磷酸化JNK蛋白表达量和NF-κB活化水平;各组细胞收集上清检测TNF-α和IL-8浓度。
3.采用海水吸入法复制大鼠ALI模型,吸入海水(4ml/kg)后成功建立ALI模型。将48只Wistar大鼠随机分为对照组、海水吸入2、4、8h组,取肺组织用实时荧光定量PCR法检测PAR2 mRNA表达水平,用Western blot法和免疫组化法检测PAR2蛋白表达情况。
4.采用海水吸入法和LPS吸入法复制大鼠ALI模型,96只Wistar大鼠随机分为对照组(control)、海水组(seawater组)、LPS组(LPS组)和FUT-175治疗海水吸入组(FUT-175组)。海水组和LPS组分别吸入海水(4ml/kg)和LPS(4mg/kg)建立ALI模型。seawater组:模型复制成功后20min,经尾静脉注射生理盐水2ml/kg。FUT-175组:海水型ALI模型复制成功后20min,经尾静脉注射FUT-175 10mg/kg。4组大鼠观察8小时,分别于模型建立及干预后0.5、1、2、4、8h进行动脉血气分析,最后检测肺微血管通透性(PMVP)、肺湿/干重比(W/D)、肺组织髓过氧化物酶(MPO)、血浆IL-8和TNF-α水平,并观察病理学变化。
结果:
1.与正常对照组比较,海水处理后,A549细胞PAR2 mRNA在4h时显著升高(P<0.05),随着时间延长,在8h点达最高峰(为对照组的1.8倍),此后一直显著高于对照组(P<0.01)。A549细胞PAR2蛋白表达量也在4h时显著升高(P<0.05),在8h点达最高峰(为对照组的2.2倍),此后一直显著高于对照组(P<0.01)。A549细胞经海水处理后,上清液中的IL-8和TNF-α迅速升高,2 h达最高峰,显著高于对照组(P<0.01)。此后有所下降,但在所有时间点均显著高于对照组(P<0.01)。
2.与正常对照组比较,海水处理后,A549细胞p38MAPK磷酸化水平、ERK磷酸化水平和JNK磷酸化水平显著升高(分别为对照组的3.1倍、1.8倍和3.2倍)(P<0.01-0.05),NF-κB活化水平显著升高(为对照组的6.7倍)(P<0.01),而FUT-175和PAR2抗体预处理组同海水处理组比较,A549细胞p38MAPK磷酸化水平、JNK磷酸化水平、ERK磷酸化水平和NF-κB活化水平显著降低(P<0.01-0.05)。海水处理后,A549细胞培养上清液中IL-8和TNF-α显著升高(P<0.01),而FUT-175、PAR2抗体、SB203580、PD98059和SP600125预处理组同海水处理组比较,上清液中IL-8和TNF-α则显著降低(P<0.01)。
3.与正常对照组比较,海水吸入后,大鼠肺组织PAR2 mRNA和蛋白表达量在2h时即显著升高(P<0.05),随着时间延长,在4h点达最高峰(为对照组的2.8倍),此后均显著高于对照组(P<0.01)。同时大鼠肺组织PAR2蛋白表达量在2h时即显著升高(P<0.05),随着时间延长,在4h点达最高峰(为对照组的4.2倍),此后均显著高于对照组(P<0.01)。PAR2免疫组化显示,在正常对照组,PAR2微弱表达于肺泡、支气管上皮和肺泡巨噬细胞,海水吸入2h后,阳性反应明显增强,不仅见于肺泡和支气管上皮细胞,还见于肺微血管内皮细胞和气道平滑肌细胞。
4.与正常对照组比较,海水和LPS吸入致伤组大鼠PaO2下降、W/D比值增大、肺微血管通透性增高、肺组织MPO活性增高、血浆IL-8和TNF-α升高、肺组织病理形态学积分增加(P<0.01);海水组PaO2明显低于LPS组,而MPO、W/D和PVMP明显高于LPS组(P<0.01)。FUT-175治疗海水吸入组上述指标均显著改善(P<0.01-0.05)。
结论:
1.海水处理A549细胞,可上调A549细胞PAR2的表达,导致IL-8和TNF-α释放的增加,同时可导致A549细胞结构的改变。
2.海水激活A549细胞PAR2后,通过MAPK信号通路和NF-B信号通路,导致IL-8和TNF-α释放增加,是海水处理A549细胞导致细胞损伤的可能机制。
3.丝氨酸蛋白酶抑制剂FUT-175和PAR2抗体可通过抑制PAR2的激活,对海水处理的A549具有保护作用。
4.海水吸入后大鼠肺组织PAR2表达上调,在海水所致急性肺损伤中起重要作用。
5.丝氨酸蛋白酶抑制剂FUT-175对海水吸入所致急性肺损伤大鼠具有保护作用,其机制可能是通过抑制肺组织PAR2的激活,阻断其下游的炎症反应,减轻肺损伤,降低肺血管通透性,减轻肺水肿。
Background and Objective
Seawater 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.
Methods
1. 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.
Results
1. 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).
Conclusion
1. 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.
引文
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2. Hirota Y, Osuga Y, Hirata T, et al. Activation of protease-activated receptor 2 stimulates proliferation and interleukin (IL)-6 and IL-8 secretion of endometriotic stromal cells[J] . Human Reproduction, 2005;20(12):3547-3553.
3. Yagi Y; Otani H; Ando S, et al. Involvement of Rho signaling in PAR2-mediated regulation of neutrophil adhesion to lung epithelial cells[J]. Euro J Pharma, 2006; 536 (1-2) : 19 - 27.
4. Ossovskaya VS, Bunnet NW. Protease-activated receptors: contribution to physiology and disease[J]. Physiol Rev, 2004;84(2): 579-621.
5. Asokananthan N, Graham PT, Fink J, et al. Activation of protease-activated receptor (PAR)-1, PAR2, and PAR-4 stimulates IL-6, IL-8, and prostaglandin E2 release from human respiratory epithelial cells [J]. Immunol,2002a;168: 3577–3585.
6. Henry PJ. The protease-activated receptor2 (PAR2)-prostaglandin E2-prostanoid EP receptor axis: a potential bronchoprotective unit in the respiratory tract? [J] Eur J Pharmacol, 2006; 533(1-3): 156-170.
7. Kawabata A, Kanke T, Yonezawa D, et al.2004a. Potent and metabolically stable agonists for protease-activated receptor-2: evaluation of activity in multiple assay systems in vitro and in vivo[J]. Pharmacol Exp Ther,2004a; 309: 1098–1107.
8. Uehara A, Muramoto K, Takada H, et al. Neutrophil serine proteinases activate human nonepithelial cells to produce inflammatory cytokines through proteinase-activated receptor 2 [J]. J Immunology, 2003; 170(11): 5690-5696.
9. Morello S, Vellecco V, Roviezzo F, et al. A protective role for proteinase activated receptor 2 in airways of lipopolysaccharide-treated rats. Biochem Pharmacol, 2005; 71(1-2): 223-230.
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16.王海燕,何韶衡,林珏龙。蛋白酶激活受体-2介导A549细胞胞浆游离钙的释放。汕头大学医学院学报,2005;18(2): 81-85.
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22. Jesmin S, Gando S, Zaedi S,et al. Chronological expression of PAR isoforms in acuteliver injury and its amelioration by PAR2 blockade in a rat model of sepsis. Thrombosis and Haemostasis, 2006; 96(6):830-838.
23. Kazerani HR, Plevin R, Kawagoe J, et al. Lack of effect of proteinase-activated receptor-2 (PAR2) deletion on the pathophysiological changes produced by lipopolysaccharide in the mouse: comparison with dexamethasone. J Pharm Pharmacol, 2004; 56:1015–1020.