摘要
海水淹溺不仅是航海事故、海上生产作业、旅游的死亡原因之一,而且是军队海上训练、作战、抢滩登陆战斗和非战斗减员的重要原因。水运交通事故,潜水等水上运动引起的淹溺在沿海地区更为常见。海水吸入肺内,可以直接损伤肺泡I、II型上皮细胞和肺毛细血管内皮细胞,也可以激发肺部急性炎症反应,引起肺水肿,急性肺损伤(ALI)/急性呼吸窘迫综合症(ARDS)。
水通道蛋白(aquaporins,AQPs)主要介导自由水跨生物膜转运,对保持细胞内外环境的稳态平衡起重要作用,也参与执行机体的一些重要生理功能。水通道蛋白(AQPs)在肺泡毛细血管间的水跨膜转运中发挥重要作用,在调节肺泡上皮细胞的液体清除中已受到高度重视。AQPs与肺组织液体转运密切相关,在肺水平衡调节上有重要作用。但是水通道蛋白在海水淹溺性肺损伤中的作用仍不甚清楚,在海水引起的肺损伤中,AQP1,5有什么变化,起什么作用,这方面的研究也较少。为此,在本研究中,我们以海水浸泡A549细胞和海水淹溺性肺损伤大鼠动物模型为研究对象,观察AQP1,5在A549细胞内和肺组织的表达情况,以及地塞米松干预后的变化规律,同时观察肺组织的炎症反应的变化规律,旨在探讨海水型急性肺损伤(SW-ALI)/海水型呼吸窘迫综合征(SW- RDS)状态下,肺组织内AQP1,5的动态变化规律及其在肺水肿中的作用。
研究内容:
1.应用MTT法、光镜、免疫荧光和PT-PCR法检测海水对肺腺癌系细胞A549生长曲线、生长抑制率、细胞形态的影响及AQP1和AQP5在mRNA和蛋白水平的表达情况。
2. 130只健康雄性Wistar大鼠随机分为3组,即海水淹溺组(n=60)、地塞米松处理组(n=10)和对照组(n=60)。海水淹溺组大鼠行气管切开、插管、吸入海水法建立;对照组大鼠只行气管切开,气管插管;地塞米松处理组大鼠,吸入海水同时给予腹腔内注射地塞米松(1mg/kg)。各组大鼠分别于致伤后0.5 h、1 h、2h、4h、8h时间点,取大鼠颈动脉血做血气分析观察动脉血氧分压(PaO2)的变化;光镜下观察肺组织病理变化;ELISA法测定外周血中肿瘤坏死因子-α(TNF-α)和白介素-6(IL-6)的含量;伊文思兰法测定肺组织通透性变化。
3.分别采用免疫组织化学法和RT-PCR方法检测肺组织中AQP1,5表达与蛋白分布。
结果:
1.肺腺癌细胞A549经海水浸泡15分钟后光镜下可见到细胞膜皱缩,细胞变小,但未见到细胞器及胞膜破裂。海水浸泡(1,2,4,8,16min)后,24小时内A549细胞生长受到抑制,但24小时后两组间无显著差异(P>0.05)。
2.海水浸泡组A549细胞AQP1在mRNA和蛋白水平表达(0.78±0.07,27.3±2.7)明显高于对照组(0.41±0.04,18.6±1.5) (P <0.01;P<0.05)。
3. A549细胞AQP5mRNA和蛋白的表达海水浸泡组(0.53±0.06,36.9±4.4)明显高于对照组(1.07±0.13,51.3±6.2) (P <0.01;P<0.05)。
4.海水吸入后,大鼠动脉血氧分压(PaO2)和PaO2 /FiO2迅速下降, 0.5小时时下降最为明显(42.4±6.4,202.2±30.5),以后逐步上升,各时相点(0.5, 1, 2, 4, 8h)海水吸入组明显低于对照组(P<0.01);PaCO2仅在0.5h时有轻度增高(43.1±4.2);HCO3-下降迅速,在2h时达到最低点(8.3±4.6);pH在1h时下降到最低点(7.13±0.03)。
5.光镜下可见海水淹溺组大鼠各时点肺组织有毛细血管充血、肺间隔增宽、肺间质、肺泡水肿和灶性出血,肺泡腔可见大量红细胞,8小时组最为明显,对照组未见明显病理变化。
6.大鼠外周血TNF-α,在吸入海水后迅速增加,1小时即达到328.23±34.68pg/ml,2小时340.24±30.25pg/ml,之后逐步下降,海水吸入组各时相点明显高于对照组(P<0.01)。
7.大鼠外周血IL-6,在吸入海水后逐步增加,在8小时达最大(1106.4±180.6pg/ml)。海水吸入组各时相点明显高于对照组(P<0.01)。
8.大鼠肺毛细血管通透性海水吸入后明显增大(30.7±2.9μg/ml),显著高于对照组(18.4±1.7μg/ml)(P<0.01)。
9.大鼠肺湿/干比值(W/D)吸入海水后明显增大,在实验时间内逐步增加,8小时时最高(5.86±0.43),与相应时相对照组(4.46±0.37)比较有显著差异(P<0.01)。
10.海水淹溺组及对照组大鼠肺间质的毛细血管内皮AQP1均呈阳性表达;但海水淹溺组表达更强,光密度积分值显著高于对照组的表达(P<0.05);海水淹溺组大鼠肺组织AQP1mRNA表达也显著高于对照组(P<0.01)。
11.海水淹溺组大鼠肺实质AQP5的阳性表达,积分光密度值显著高于对照组的表达(P<0.05);海水淹溺组大鼠肺组织AQP5mRNA表达也显著高于对照组(P<0.01)。
12.地塞米松处理组大鼠,炎症介质TNF-α、IL-6的释放、肺泡和肺间质水肿、W/D、明显低于海水淹溺组,仍然高于对照组;PaO2及PaO2 /FiO2明显高于海水淹溺组;肺组织AQP1,5的表达积分光密度值显著高于海水淹溺组(P<0.05);肺组织AQP1,5mRNA表达也显著高于海水淹溺组(P<0.01)。
结论:
1.海水浸泡对A549细胞有损伤作用,但短时间内(16min)无致死作用。
2.海水浸泡可使A549细胞细胞变小,胞膜皱缩,细胞内水外渗,同时并存AQP1,5表达上调,表明AQP1,5参与细胞内外的水平衡调节。
3.海水吸入肺内可引起大鼠炎症介质TNF-α、IL-6的释放增多,肺泡、肺间质水肿,PaO2和PaO2 /FiO2下降,表明海水淹溺可引起急性肺损伤。
4.海水淹溺所致大鼠肺损伤时,随肺水肿加重,AQP1和AQP5 mRNA及蛋白表达显著上调,表明AQP1和AQP5可能与肺水肿的形成过程有关。
5.地塞米松可以改善海水淹溺ALI大鼠肺的毛细血管通透性、肺湿干比值、炎性因子的释放,上调AQP1和AQP5 mRNA及蛋白表达。这表明,AQP1和AQP5的上调表达可能是地塞米松保护海水淹溺大鼠ALI的重要机制之一。
Background and Objective
Seawater drowning is a major factor that cause disaster in the progress of voyage, work and travel on the sea. Boating accidents, and drownings related to aquatic sports such as diving are also more common in coastal regions. Sea water aspiration can lead to direct the alveolar epithelial and pulmonary capillary endothelial cells injury. Sea water aspiration also can induce acute lung injury (ALI) and its more severe form, the acute respiratory distress syndrome (ARDS), which are characterized by an acute inflammatory process in airspaces and lung parenchyma. These syndromes are manifestations of the loss of barrier function of the alveolar epithelial and pulmonary capillary endothelial cells.
Aquaporins constitute a family of water channels that facilitate membrane water permeability in various tissues of animals. Aquaporins can clear up excess water in the lung tissue, which take an important role in the barrier function of the alveolar epithelial and pulmonary capillary endothelial cells. The effect of hyperosmolar fluid aspiration (seawater) on lung fluid balance and aquaporins has not been well studied. Very few studies have been addressed on the expression of gene and protein of aquaporin1, 5 or the changes of fluid transport function in lung during ALI/ARDS induced by sea water. It is unclear whether aquaporin1, 5 is related to the development of pulmonary edema. Whether do aquaporins reduce or increase after sea water drowning? The study aims to observe the aquaporin1 and aquaporin5 expression at mRNA and protein level in A549 cell line soaked respectively, and the aquaporin1 and aquaporin5 expression in rats with acute lung injury induced by seawater aspiration.
Methods
1. Microculture tetrazolium assay (MTT),microscope, immunofluorescence and RT-PCR were applied to detect growth curve, inhibition rate, cell morphological changes and the expression of aquaporin1 and aquaporin5 at the mRNA and the protein level in A549 cells treated or untreated with sea water.
2. A total of 130 Wistar healthy male rats were randomly assigned into 3 groups: sea water aspiration group (n=60) which was reproduced by tracheotomy, intubatton and seawater aspiration (4ml/kg body weight), dexamethason intervention group (n=10)which was given intraperitoneal dexamethasone injection (1mg/kg) after sea water aspiration, control group (n=60) which underwent tracheotomy and intubatton only. All the indexes about ALI/ARDS were observed at 0.5h, 1h, 2h, 4h, 8h after treatment. Changes in partial pressure of oxygen in artery (PaO2 ) were determined by blood gas analysis. Pathological changes in pulmonary-tissue were observed by microscopy. Tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) levels in serum were determined using enzyme linked immunosorbent assay (ELISA). Pulmonary capillary permeability was measured with the Evan’s blue intravenously method.
3. RT-PCR and immunohischemistry were used to detect aquaporin1 and aquaporin5 expression at the mRNA and the protein level, respectively.
Results
1. After A549 cells were soaked for 15 minutes, cellular membrane crenulation was observed, while cellular organ injury and plasmatorrhexis was not found. The cell proliferation was suppressed after A549 cells soaked for 1,2,4,8 and 16min. However, there was no significant difference in cell proliferation rate between cells treated or untreated with sea water for 24 h.
2. The expression of AQP1 at the mRNA and the protein level in the sea water group(0.78±0.07,27.3±2.7) was significantly higher than that in control group (0.41±0.04,18.6±1.5) after treatment at 0.5 h (P <0.01and P<0.05).
3. The expression of AQP5 at the mRNA and the protein level in the sea water group (0.53±0.06,36.9±4.4)was significantly higher than that in control group(1.07±0.13,51.3±6.2)after treatment at 0.5 h (P <0.01and P<0.05).
4. The PaO_2 and PaO_2 /FiO_2 of the sea water aspiration group decreased quikly after sea water aspiration and reached the lowest point at the 0.5h( 42.4±6.4,202.2±30.5). The PaO_2 and PaO_2 /FiO_2 of the sea water aspiration group was significantly lower than that of control group at 0.5h, 1h, 2h, 4h and 8h after injury (P<0.01).
5. It was observed that there was hyperaemia in the capillary vessels of sea water aspiration group. Meanwhile, alveolar edema was also observed. No pathological changes were found in the control group.
6. After sea water aspiration, the level of TNF-αin the serum increased quickly and reached 328.23±34.68 pg/ml at 1h, 340.24±30.25 pg/ml at 2h, then dropped slowly, but was still higher than that in the control group at each time point (P <0.01).
7. After sea water aspiration, the level of IL-6 in the serum increased quickly and reached the highest point 1106.4±180.6 pg/ml at 8 h, and was higher than that in the control group at each time point (P <0.01).
8. Pulmonary capillary permeability rised significantly (30.7±2.9μg/ml) after sea water aspiration, higher than that in the control group (18.4±1.7μg/ml) (P <0.01).
9.The lung W/D increased and reached the highest point at 8h (5.86±0.43), and was significantly higher than that in the control group (P <0.01).
10. The expression of AQP1 at the mRNA and the protein level in the sea water group was significantly higher than that in control group after treatment (P <0.01and P<0.05).
11. The expression of AQP5 at the mRNA and the protein level in the sea water group was significantly higher than that in control group after treatment correspondingly(P <0.01and P<0.05).
12.In the dexamethason intervention goup, TNF-α,IL-6 levels in plasma,alveolar and interstitia edema and W/D were lower than those in the sea water group, PaO_2 and PaO_2 /FiO_2 were higher than those in the sea water goup, and the expression of AQP1,5 at the mRNA and the protein level were higher than that in the sea water aspiration group(P<0.05).
Conclusions
1. Sea water was harmful, while not lethal to A549 cells in a short time.
2. Cellular membrane crenulation was observed and the expression of AQP1, 5 both at the mRNA and the protein level were up-regulated after A549 was soaked, which suggestted that up-regulation of AQP1, 5 may contribute to the water exosmosing in A549 cells.
3. Sea water aspiration resulted in releasing of inflammation mediators (TNF-α,IL-6), pulmonary interstitial edema, PaO2 and PaO2 /FiO2 degression and ALI/ARDS.
4. The expression of AQP1,5 at the mRNA and the protein in pulmonary-tissue of rats with acute lung injury induced by sea water increased, suggesting that AQP1,5 may play an important role in fluid transportation in ALI/ARDS.
5. Dexamethasone could improve biological indexes, lung permeability, wet-to-dry weight ratio and up-regulate AQP1, 5, suggesting dexamethasone has an effect on protecting acute lung injury induced by sea water. The up-regulation of AQP1 and AQP5 might be one of the mechanisms of lung protection by the dexamethasone.
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