【作者】 谢晓燕；

【导师】 金发光； 张博；

【作者基本信息】 第四军医大学 ， 内科学， 2011， 硕士

【摘要】 研究背景:淹溺是全球最重要的公共健康问题之一,是意外伤害致死的第二大原因。海水淹溺不仅是海上生产作业、航海事故的主要死亡原因,更是军队在海上作战、训练等非战斗减员的重要原因。海水淹溺后,除因喉头、气管反射性痉挛引起患者急性窒息外,引起死亡的主要原因是海水淹溺性肺水肿（pulmonary edema of seawater drowning,PE-SWD）,若得不到及时有效的救治,则可能进展为海水型呼吸窘迫综合征（seawater respiratory distress syndrome,SW-RDS）,死亡率较高。因此有效治疗PE-SWD及其引起的损伤是成功救治淹溺患者的关键。有研究表明,肺泡上皮细胞钠-钾ATP酶（Na+/K+-adenosine triphosphatase,NKA）在促进肺泡内液体的吸收中发挥着重要作用。因此上调NKA活性对促进肺水肿液的吸收,改善PE-SWD的预后至关重要。丹参酮IIA磺酸钠（sodium tanshinone IIA sulphonate,STS）是丹参酮IIA的衍生物,在丹参中含量最多。STS具有多种药理作用,已广泛用于心脑血管疾病的治疗,还具有抗炎及抗氧化的作用。亦有文献报道STS在脂多糖引起的急性肺损伤中可减轻肺水肿及改善缺氧,但有关其在PE-SWD中的作用尚缺乏研究。本实验通过复制大鼠PE-SWD模型来观察STS对PE-SWD的治疗作用及对NKA的影响,并通过细胞实验进一步探讨STS发挥作用的可能机制,为临床应用提供实验依据。目的:通过建立大鼠PE-SWD模型及培养A549细胞,探讨STS在减轻PE-SWD中的作用,和其对NKA的影响,并通过细胞实验研究了ERK信号通路在STS调节NKA中的潜在机制。方法:动物实验1、实验分组及模型的制备:将48只大鼠随机分为正常组（normal group）、STS组（STS group）、海水组（SW group）、海水+STS组（SW+STS grouop）,每组12只。气管内滴入4ml/kg海水以建立大鼠PE-SWD模型,在海水灌注后立即腹腔内注射25mg/kgSTS。2、观察指标:1)PaO₂:通过血气分析检测各组海水灌注前、海水灌注后30 min、1h、2h、4h、6h的PaO₂。2)肺组织病理学:造模4小时后,观察各组肺组织病理学改变。3)肺水肿指数检测:海水灌注4小时后,测定肺湿/干重比值（W /D）和肺毛细血管通透性（PMVP）。4)NKA的表达及活性:利用试剂盒检测各组肺组织匀浆中NKA活性,采用Western blot检测NKAα1、β1亚基蛋白表达,并用RT-PCR方法检测NKAα1、β1mRNA水平。细胞实验1、实验分组及细胞的处理:将A549细胞随机分为正常组（normalgroup）、STS组（STS group）、海水组（SW group）、海水+STS组（SW+STS grouop）、海水+STS+ERK抑制剂（U0126）组（SW+STS+U0126 grouop）。海水（海水:培养液=1:4）刺激细胞4h,在加入海水后立即加入25ug/mlSTS, U0126在加入STS前30min作用于细胞。2、观察指标:1)NKA的表达及活性:利用试剂盒检测各组细胞的NKA活性,采用Western blot检测各组NKAα1、β1亚基。2)ERK1/2的表达:采用Western blot检测各组ERK1/2蛋白的表达。结果:动物实验1、PaO₂:PE-SWD模型建立后30min,PaO₂即降低到最低点,后缓慢升高,但低氧仍明显,STS治疗组从海水灌注后2h开始PaO₂显著高于海水组（P < 0.05）,到4h时PaO₂值增高达高峰（P < 0.01）,表明STS可改善海水灌注导致的缺氧。2、病理学改变:海水组的肺组织出血、水肿,肺泡间隔明显增厚、断裂及炎性细胞渗入肺泡腔,而STS治疗组肺损伤病理学结果较海水组明显减轻,表明STS可减轻海水灌注导致的肺组织损伤。3、肺水肿指标:海水吸入导致肺W/D、PMVP增加,而STS治疗组较海水组的W/D、PMVP明显下降（P < 0.05）,表明STS可减轻海水引起的肺水肿。4、肺组织中NKA表达及活性:海水吸入后肺组织的NKA活性及NKAα1、β1亚基蛋白表达及mRNA水平均明显下降,而STS治疗组的NKAα1、β1亚基蛋白表达及NKA活性均较海水组明显增加（P < 0.05）。而在mRNA水平上,STS组的NKAβ1亚基的mRNA显著增加（P < 0.05）,但NKAα1亚基的mRNA却与海水组无明显变化。细胞实验1、A549细胞的NKA表达及活性:海水浸泡A549细胞后,细胞的NKAα1、β1亚基蛋白表达、NKA活性均显著降低,而STS治疗组使上述指标均有所增高（P < 0.05）（这与大体实验的结果基本一致）,但加入了ERK抑制剂（U0126）后,NKAα1、β1亚基蛋白表达、NKA活性均较STS治疗组明显降低,表明STS对NKA的上调作用可部分被ERK抑制剂所阻断。2、A549细胞的ERK1/2表达:海水使细胞的ERK1/2蛋白表达降低,而STS治疗组增加了ERK1/2蛋白的表达,表明STS可刺激ERK1/2蛋白的表达。结论:1、在鼠PE-SWD中,STS可改善缺氧、减轻肺水肿及肺组织损伤。2、大体实验和细胞实验中,STS可通过上调NKA的表达及活性,从而促进肺泡内液体的吸收,以减轻肺水肿。3、在细胞实验中,STS可刺激ERK1/2蛋白的表达,而ERK1/2抑制剂可部分阻断STS对NKA活性的上调,表明STS可通过刺激ERK1/2信号通路来上调NKA的表达及活性。更多还原

【Abstract】 Background:Drowning is one of the most important public health problems , ranking the second death-causing factor in fatal accidents. Seawater drowning is not only the main death cause of offshore operation and maritme accident, but also an important reason for member depletion of army sea war, training and non-combat affairs. Besides the acute asphyxia caused by reflex laryngismus and tracheospasm, pulmonary edema of seawater drowning, PE-SWD is the major death cause drown by the sea. If the patient was not treated in time, seawater respiratory distress syndrome,SW-RDS will occur which enhances the death rate. Therefore, relieving pulmonary edema and injury caused by pulmonary edema is the key of a successful treatment to seawater drowning patients.It is known that basolateral Na,K-adenosine triphosphatase （Na-K-ATPase） located in the alveolar epithelial cells（AEC） is critical for clearance of edema fluid from the pulmonary alveoli. So up-regulating the activity of Na-K-ATPase accelerates clearance of edema fluid of alveolar and improves PE-SWD.Sodium tanshinone IIA sulphonate （STS）,a derivative of phenanthrenequinone, is a major compound extracted from Danshen. It exhibits multiple pharmacological actions and has been commonly used in traditional oriental herbal medicine to prevent or manage cardiovascular diseases and treat inflammatory diseases.Recent studies have reported that it can reduce lung edema and protect lung from LPS-induced acute injury.However, little is known about its effect and mechanism on seawater aspiration-induced acute pulmonary edema. In the present study, we explored the effect of STS on PE-SWD both in vivo and in vitro,and examine the effects of STS on Na-K-ATPase in PE-SWD and its underlying mechanisms.Aim of the study:To investigate whether STS treatment attenuates PE-SWD,and examine the effects of STS on Na-K-ATPase in vivo and in vitro and its underlying mechanisms by ERK signaling pathway.Methods:Animal experiment1.Group of experiments and model making:48 rats were randomly divided into four groups: normal group（NG）, STS group, seawater（SW） group, and seawater （SW）+STS group . The model of PE-SWD in rats were established by seawater （4 ml/kg body weight） instillation.STS（25mg/kg） was injected through peritoneal cavity at once after seawater aspiration.2. Index of experiments: 1).PaO₂: Arterial blood gas of rats were observed before seawater aspiration, and 30 min, 1 h, 2 h, 4 h and 6h after seawater aspiration. 2).Lung histology: At 4h after seawater aspiration, lung histology was observed. 3).Index of Pulmonary edema : At 4h after seawater aspiration, lung wet/dry （W/D） ratio and pulmonary microvascular permeability （PMVP ）were observed. 4).Measurement of Na-K-ATPase activity and expression: The activity of Na-K-ATPase was measured by a Na-K-ATPase activity assay kit. Western blot、RT-PCR were used to test the effects of STS on Na-K-ATPaseα1、β1 subunit plasma membrane protein expression and mRNA level in PE-SWD.Cell experiment1.Group and preparation of the cells: The A549 cells were randomly divided into five groups: normal group（NG）, STS group, seawater（SW） group, seawater（SW） +STS group, seawater（SW）+ STS+ ERK inhibitor（U0126） group. The A549 cells were incubated for 4 h with seawater. STS （25 mg/L） was added immediately after seawater treatment. The A549 cells were pretreated with U0126 as inhibitor of the ERK1/2 activity for 30 min before STS administration.2. Index of experiments: 1).Na-K-ATPase activity and expression : We detected Na-K-ATPase activity and Na-K-ATPaseα1、β1 subunit protein expression by western blot. 2).ERK1/2 expression: Western blot was used to test the protein expression of ERK1/2.Results:Animal experiment1.PaO₂:The oxygen partial pressure reached the lowest in 30 minutes after seawater aspiration,then increased slowly,but manifested obvious hypoxemia. Treatment with STS, PaO₂ was significantly increased compared with that of seawater group from 2 hours（P<0.05）,and increased to peak at 4h（P<0.01） post seawater instillation. It suggested that STS improved hypoxemia.2.Lung histopathology: After seawater aspiration, there were serious lung injuries such as hemorrhage, the markedly thickened alveolar wall, and the infiltration of many inflammatory cells in the alveolar spaces. However, treatment with STS the destruction of lung structure was reduced significantly.3.Index of Pulmonary edema: The lung W/D ratio and PMVP in rats were significantly increased following seawater aspiration. STS treatment reversed such effect in part （P<0.05）. It suggested that lung edema induced by seawater was attenuated by STS.4.The activity and expression of Na-K-ATPase in lung tissue: Seawater aspiration decreased the Na-K-ATPase activity,protein expression and mRNA level. STS reversed seawater-induced reduction of Na-K-ATPase activity and the western blot results showed that both Na-K-ATPaseα1 andβ1 subunit plasma membrane protein expressions following seawater aspiration were increased by STS treatment compared with seawater group（P<0.05）.However, administrated with STS, the Na-K-ATPaseβ1 mRNA level following seawater aspiration was increased compared with seawater group （P<0.05）. There was no significant difference in the Na-K-ATPaseα1 mRNA level between seawater group and seawater with STS treatment group.Cell experiment1.The activity and expression of Na-K-ATPase in A549 cells:The cell experiment indicted that the change of Na-K-ATPase protein expression and activity in A549 cells were similar to the lung tissues. Moreover, pretreated with ERK1/2 inhibitor U-0126,Na-K-ATPase protein expression and activity were decreased compared with STS treatment following seawater incubation （P<0.05）.2.The protein expression of ERK1/2: Seawater induced the decrease in ERK1/2 protein expression. However, STS treatment increased ERK1/2 phosphorylation following seawater incubation, which manifested STS can activat ERK1/2 in A549 cells.Conclusion1.Hypoxemia, acute pulmonary edema and lung injury induced by seawater aspiration were improved by STS administration.2.STS treatment could improve PE-SWD by up-regulating Na-K-ATPase activity and expression both in vivo and in vitro.3.STS activated ERK1/2 in A549 cells following seawater incubation. Moreover, the ERK1/2 inhibitor partially blocked the improved effect of STS on the Na-K-ATPase expression and activity,which indicated that activation of ERK1/2 mediated STS up-regulating the Na-K-ATPase protein expression and activity.更多还原