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凉膈散对内毒素型急性肺损伤肺组织水液转运的影响及机制研究

The Effect and Mechanism of Lianggesan on Fluid TransPortation in Endotoxin Acute Lung Injury Tissue

【作者】 王国全

【导师】 余林中;

【作者基本信息】 南方医科大学 , 中药学, 2014, 博士

【摘要】 目的与意义:急性肺损伤(Acute lung injury, ALI)是指由严重感染、创伤、休克等多种因素引起肺组织结构发生特征性的病理改变而出现的一种临床综合症,是急救医学中发病率和死亡率都很高的一种临床重症。诊断上以弥漫性肺泡毛细血管膜损伤导致肺水肿和肺不张为主要病理特征。临床表现为呼吸窘迫,伴有顽固性低氧血症、肺顺应性下降和扩散性炎症浸润等症状,其病死率高达45%-50%。感染性败血症是其发生的常见原因,革兰氏阴性菌是主要致病菌,起损伤作用的是内毒素(endotoxin)。迄今为止ALI确切的发病机理尚不清楚,因此深入研究ALI的发病机理对急症医学领域显得尤为重要。凉膈散出自《太平惠民和剂局方》,由大黄、芒硝、甘草、栀子、薄荷、黄芩、连翘、竹叶、蜂蜜组成,具有清上凉膈,泻火通便功效,主治上中二焦火热证,是中医治疗温热病的经典名方。课题组前期试验发现该方对内毒素导致的急性肺损伤大鼠有明显的保护作用,它可以明显对抗内毒素,控制炎症发展,但是凉膈散是否通过改善作用于钠通道及其相关水液通路的功能而发挥保护作用,这方面研究还未见报道。本实验就是通过LPS型ALI模型大鼠研究凉膈散对ALI的防治作用,如行动脉血气分析、测定肺组织湿/干质量比值(W/D)、肺脏水通透性(LPI)等指标检测,并且在明确其改善肺组织水液代谢障碍作用的基础上,继续观测该方对ALI模型大鼠肺组织α,β,γ-rENaC,Na+-K+-ATase,AQP-3及其mRNA表达水平的影响,探讨凉膈散改善ALI大鼠肺水肿的作用机制和作用靶点,从而为该方的临床应用提供理论和实验依据。实验方法:1.动物分组及模型取健康成年Wistar雄性大鼠300只,体重180-220g,分为正常对照组(ig生理盐水)、ALI模型组(ig生理盐水,气管滴注LPS2mg/kg),凉膈散高剂量+钠通道抑制剂Bemzamil组(ig30g生寥/kg,iv Bemzamil50mmol/L,气管滴注LPS2mg/kg),凉膈散高剂量组(ig30g生药/kg,气管滴注LPS2mg/kg),凉膈散低剂量组(ig7.5g生药/kg,气管滴注LPS2mg/kg),地塞米松组(ig DEX0.27mg/kg,气管滴注LPS2mg/kg),每组10只。钠通道抑制剂Bemzamil组从预防给药开始每天尾静脉注射Bemzamil (50mmol/L)0.2ml,直至ALI造模当天。各组大鼠在给予生理盐水或LPS后1、2、4、8、16h不同时相处死、取材。2.大鼠LPS致急性肺损伤模型的筛选及建立3.凉膈散对LPS诱发ALI模型大鼠肺水转运障碍的治疗作用研究3.1大鼠的一般体征观察3.2肺水含量(W/D)的变化3.3呼吸功能测定(呼吸频率,呼吸周期)3.4血气分析(Pa02和PaCO2变化)3.5肺泡通透指数(LPI)测定3.6血管外肺水(EVLW)含量的测定3.7病理形态学检查和评分4.凉膈散对LPS诱发ALI模型大鼠肺组织水液转运障碍的保护作用机制研究4.1免疫组化(SP)法检测LPS诱发ALI模型大鼠肺组织α-rENaC,β-rENaC, γ-rENaC,Na+-K+-ATase蛋白的分布及表达4.2免疫印迹分析(WesternBlotting)法检测LPS致发ALI模型大鼠肺组织α-rENaC,β-rENaC,β-rENaC,APQ-3和Na+-K+-ATase蛋白表达4.3荧光反转录聚合酶链式反应(qRT-PCR)检测LPS致ALI模型大鼠肺组织α-rENaC、β-rENaC、γ-rENaC,Na+-K+-ATase基因的表达5.统计学分析采用SPSS17.0统计软件,计量资料均用均数±标准差(χ±s)表示,单独效应分析,各实验组间均数间多重比较均被采用,使用单因素方差分析(One-Way ANOVA),方差齐时各组间两两比较采用LSD法,方差不齐时使用Dunnett’s T3法,所有统计结果均以P<0.05表示差异具有统计学意义,P<0.01表示有显著性差异。实验结果:1.LPS型ALI大鼠模型的筛选及建立通过采用两种不同内毒素和三种给药方式,筛选和建立合适的ALI大鼠模型。结果发现大鼠气管滴注浓度为2mg/kg LPS0111:B4所导致的ALI模型损伤程度适中可控,符合实验要求,即定为实验目标内毒素、给药途径和实验浓度。2.大鼠的一般体征的观察各组支气管滴加LPS诱发大鼠ALI后,大鼠均出现心跳加快、呼吸急促、精神不振、烦躁不安、行动迟缓、恶寒、蜷缩成团状等症状,地塞米松组和凉膈散高低剂量组大鼠症状均好于模型组和Benzamil+凉膈散高剂量组。3.肺水含量(W/D)的变化LPS可以诱导大鼠肺组织湿质量/干质量值(W/D)升高。其中2、4、8、16h时间点,W/D在ALI模型组较对照组显著增加(P<0.01),凉膈散各剂量组、地塞米松组较ALI模型组显著降低(P<0.05,P<0.05,P<0.01)。4.呼吸频率的变化LPS可以导致大鼠肺组织呼吸频率升高,其中ALI组在2、4、8、16h时间点呼吸频率较正常对照组显著加快(P<0.01),凉膈散各剂量组、地塞米松组较ALI模型组显著降低(P<0.01,P<0.01,P<0.01)。5.呼吸周期的变化LPS可以导致大鼠肺呼吸周期缩短,其中ALI组在2、4、8、16h时间点呼吸周期较正常对照组显著缩短(P<0.01),凉膈散各剂量组、地塞米松组较ALI模型组显著延长(P<0.01,P<0.01,P<0.05)。6.血气分析PaO2检测ALI模型大鼠肺PaO2显著降低,其中ALI组在2、4、8、16h时间点PaO2较正常对照组显著降低(P<0.01),凉膈散各剂量组、地塞米松组较ALI模型组显著升高(P<0.05,P<0.05,P<0.01)。7.血气分析PaCO2检测ALI模型大鼠肺PaCO2升高,其中ALI组在2、4、8、16h时间点PaCO2较正常对照组显著升高(P<0.01),凉膈散各剂量组、地塞米松组较LPS组显著降低(P<0.01,P<0.05,P<0.01)。8.肺泡通透指数(LPI)测定LPS诱导的ALI模型大鼠肺组织LPI显著升高,其中2、4、8、16h点LPI在ALI组较正常对照组显著增加(P<0.01),凉膈散大小剂量组、地塞米松组较ALI模型组显著降低(P<0.05,P<0.01,P<0.01)。9.血管外肺水(EVLW)含量的测定LPS可以诱导大鼠肺组织血管外肺水(EVLW)升高,其中2、4、8、16h时间点EVLW在ALI模型组较正常对照组显著增加(P<0.01),凉膈散高低剂量组、地塞米松组较ALI模型组显著降低(P<0.01,P<0.01,P<0.01)。10.病理形态学检查和评分肺大体观察,大鼠LPS致损后2h,双肺体积增大,明显肺水肿。随着时间的延长,肺水肿程度越来越明显,表面广泛分布斑点状红色出血灶。光镜下可见肺泡隔增厚,肺间质及肺泡水肿,出血,并有大量炎性细胞的浸润,部分肺不张,肺泡结构破坏。凉膈散各剂量组及地塞米松组可不同程度的减少肺泡间隔和炎性细胞的浸润。正常对照组肺泡结构完整、清晰,无渗出。11.凉膈散对LPS诱发大鼠ALI肺组织a-rENaC、β-rENaC、γ-rENaC,Na+-K+-ATase和AQP-3蛋白表达的影响11.1α、β、γ-rENaC免疫组化检测结果α、β、γ-rENaC蛋白为膜蛋白,其主要分布在细胞膜和细胞质中,在各组肺组织中均有表达,各组α、β、γ-rENaC蛋白相比较都有显著的统计学差异(F=110.376,P<0.01; F=132.29,P<0.01; F=826.509,P<0.05)。ALI模型组和Benzamil+凉膈散低剂量组α、β、γ-rENaC蛋白表达显著低于正常对照组(P<0.01),ALI模型组和Benzamil+凉膈散低剂量组α-rENaC蛋白相比较无统计学差异(P>0.05)。地塞米松组和凉膈散组高低剂量组的表达高于ALI模型组(P<0.05、P<0.01、P<0.05)。说明凉膈散能有效提高LPS致ALI模型大鼠肺组织α、β、γ-rENaC蛋白低表达。11.2α-Na+-K+-ATPase免疫组化检测结果α-Na+-K+-ATPase蛋白广泛分布于细胞当中,在各组肺组织中均有表达,各组α-Na+-K+-ATPase蛋白相比较都有显著的统计学差异(F=568.495,P<0.05)。ALI模型组和Benzamil+凉膈散高剂量组α-Na+-K+-ATPase蛋白表达显著低于正常对照组(P<0.01),地塞米松组、凉膈散高低剂量组的表达高于ALI模型组(P<0.05)。说明凉膈散可促进LPS致ALI模型大鼠肺组织α-Na+-K+-ATPase蛋白的表达。11.3α、β、γ-rENaC免疫印迹检测结果各组α、β、β-rENaC表达比较有显著的统计学差异(F=493.841,P<0.01;F=370.742, P<0.01;F=6.526,P<0.01), ALI模型组大鼠肺组织α、β、γ-rENaC表达与正常对照组相对比明显降低,统计学有显著性差异(P<0.01),凉膈散高低剂量组与ALI模型组比较升高,有显著性差异(P<0.01),α、β、γ-rENaC表达与凉膈散给药剂量高低有关,地塞米松组与ALI模型组比较,升高作用较明显,有统计学差异(P<0.01、P<0.05、P<0.05),表明凉膈散可提高ALI模型大鼠肺组织α、β、γ表达水平。11.4AQP-3免疫印迹检测结果各组AQP-3表达比较有显著的统计学差异(F=73.925,P<0.05), ALI模型组肺组织AQP-3表达与正常对照组相对比明显降低,统计学有显著性差异(P<0.05),凉膈散高低剂量组与ALI模型组比较升高,有显著性差异(P<0.01,P<0.05), AQP-3表达与凉膈散给药剂量高低有关,地塞米松组与ALI模型组比较,升高作用较明显,有统计学差异(P<0.01)。Benzamil+凉膈散高剂量组与ALI模型组比较,Benzamil+凉膈散高剂量组的AQP-3蛋白表达明显提高(P<0.05),这说明当Benzamil抑制钠通道功能时,凉膈散还能通过调节AQP-3蛋白表达来改善ALI模型大鼠肺水转运障碍。11.5α-Na+-K+-ATPase免疫印迹检测结果各组α-Na+-K+-ATPase表达比较有显著的统计学差异(F=86.189,P<0.05),ALI模型组肺组织α-Na+-K+-ATPase表达与正常对照组相对比明显降低,统计学有显著性差异(P<0.05),凉膈散高低剂量组与ALI模型组比较升高,有显著性差异(P<0.01),α-Na+-K+-ATPase表达与凉膈散给药剂量高低有关,地塞米松组ALI模型组比较,升高作用较明显,有统计学差异(P<0.05),而且我们发现当Benzamil抑制钠通道功能时,Benzamil+凉膈散高剂量组与ALI模型组比较,Benzamil+凉膈散高剂量组的α-Na+-K+-ATPase蛋白表达明显提高,表明凉膈散减轻ALI模型大鼠肺水代谢障碍可能通过提高肺组织α-Na+-K+-ATPase表达水平来实现。当钠通道被抑制时,他还可以提高α-Na+-K+-ATPase和水通道蛋白来改善水液代谢。12.反转录聚合酶链式反应(qRT-PCR)检测凉膈散对内毒素诱发大鼠ALI肺组织α-rENaC、β-rENaC、γ-rENaC,Na+-K+-Atase基因表达的影响12.1qRT-PCR检测凉膈散对内毒素诱发大鼠ALI肺组织α、β、γ-rENaC基因表达的影响正常对照组α、β、γ-rENaC基因表达明显高于ALI模型组,有显著性差异(P<0.01,P<0.01,P<0.05);地塞米松阳性对照组的α、β、γ-rENaC基因表达高于ALI模型组,具有统计学差异(P<0.01,P<0.01,P<0.05);凉膈散各剂量组α、β、γ-rENaC基因表达高于与ALI模型组,有显著性差异(P<0.01,P<0.01,P<0.05)。12.2qRT-PCR检测凉膈散对内毒素诱发大鼠ALI肺组织α-Na+-K+-ATPase基因表达的影响ALI模型组的α-Na+-K+-ATPase基因表达低于正常对照组(P<0.01),说明造模后大鼠体内α-Na+-K+-ATPase基因表达受到了影响。凉膈散各剂量组和地塞米松组α-Na+-K+-ATPase基因表达均高于与ALI模型组,有显著性差异(P<0.01,P<0.05,P<0.05)。实验结论:1.LPS支气管滴注诱发大鼠急性肺损伤后,大鼠肺组织肺水含量(W/D)、肺泡通透指数(LPI)、血管外肺水(EVLW)、病理形态学检查评分以及呼吸频率显著升高,而呼吸周期和Pa02显著下降,提示造模成功。凉膈散对大鼠急性肺损伤肺水肿有保护作用,能明显提高ALI模型大鼠呼吸周期和PaO2指数,降低肺组织肺水含量(W/D)、肺泡通透指数(LPI)和血管外肺水(EVLW)等指标。2.免疫组化、免疫印迹以及反转录聚合酶链式反应分析法检测结果提示,LPS诱发大鼠ALI后,与对照组比较,ALI模型大鼠肺组织内α,β,γ-rENaC. α-Na+-K+-ATPase和AQP-3蛋白表达明显减少。凉膈散可通过上调ALI模型大鼠肺组织内α,β,γ-rENaC,α-Na+-K+-ATPase和AQP-3蛋白表达水平和mRNA的表达水平,改善肺组织液体的异常转运,减轻ALI模型大鼠肺水肿症状。

【Abstract】 Objectives and significance:Acute lung injury (ALI) is a syndrome characterized by hypoxemia, noncardiogenic pulmonary edema, low lung compliance and widespread capillary leakage. When accompanied by more severe hypoxemia, it is called acute respiratory distress syndrome (ARDS). ALI is characterized by acute hyPoxemic respiratory failure with several potential causes, including trauma, shock, viruses, and bacterial endotoxins. ALI can lead to injury of alveolar epithelial cells and capillary endothelial cells and diffuse Pulmonary interstitial and alveolar edema. When lung tissue is affected by a virus or bacterial endotoxin, G Protein-coupled receptors (membrane Proteins) in lung cells are activated. Development of ALI/ARDS has been associated with short and long term morbidity, prolonged hospitalization, and high health-care costs. Given the clinical consequences attributable to ALI/ARDS, identifying risk factors for prevention of ALI/ARDS is of great importance and is a priority in intensive care unit. Inhibit or reduce the Pulmonary edema may be one of the feasible methods to Prevent or treat ALI.Lianggesan (contained Song"He Ji Ju Fang") is the common famous complex Prescription of warm disease. In Previous studies, it was found that Lianggesan can reduce lung tissue damage induced by LPS. Combining organ Picture theory of traditional chinese medicine and the knowledge of modern medicine on the ALI, according to the ALI pulmonary pathology, starting from the perspective of transmembrane transport of water, the influence of Lianggesan on pulmonary edema and changes of expression of AQP-1-.5in the endotoxin-induced acute lung injury in rat were studied in order to exPlore the Protective effect of Lianggesan on endotoxin-induced ALI and the mechanism of Lianggesan in the treatment of ALI, Provide exPerimental evidence for Lianggesan in the treatment of ALI, and ProPose new ideas for the treatment of ALI through integrative medicine.Methods:1.180Wistar male rats which could weigh between180-220g in SPF leve were selected and randomized into6groups, including the control group(ig NS, iv NS), the ALI model group(ig NS, Bronchial instillation LPS2mg/kg), LPS+DEX treatment group (ig DEX0.27mg/kg, Bronchial instillation LPS2mg/kg), Bemzamil+LPS+Lianggesan (ig DEX0.27mg/kg, Bronchial instillation LPS2mg/kg, iv Bemzamil50mmol/kg),LPS+Lianggesan two different treatment grouPs (ig7.5,30g/kg, Bronchial instillation LPS2mg/kg), which were treated with corresponding therapies. The rats were killed in batches at lth,2th,4th,8th,8th hour and the lung tissues were reselected for further examinations.2. The screening and establishment of acute lung injury induced by LPS in rats3. The Study of Lianggesan’s Influence to male rats with ALI and the protective Effect on Lung3.1The observation Common signs of in the rat3.2The change of lung water content (W/D)3.3The change of the ResPiratory function(The resPiratory cycle and The respiratory frequency)3.4The change of Blood gas analysis (PaO2、PaCO2)3.5The change of Alveolar transparent index3.6The change of extravascular lung water3.7The Lung tissue histoPathological examination and pathological evaluation 4. The study of Lianggesan on the protective Effect in the LPS-induced acute lung injury in rat4.1The changes of α-rENaC、β-rENaC、γ-rENaC and Na+-K+-ATase exPression in the lungs of rats were analyzed by immunohistochemistry (SP)4.2The changes ofα-rENaC、β-rENaC、γ-rENaC、AQP-3and Na+-K+-ATaseexpression in the lungs of rats were analyzed by western blot4.3The changes ofα-rENaC、β-rENaC、γ-rENaC、AQP-3and Na+-K+-ATase expression in the lungs of rats were analyzed by qRT-PCR.5. Statistical analysis.Results are presented as mean±SD of n experiments by SPSS16.0, unless otherwise indicated. The statistical significance of differences between the means of multiple groups or the means of an individual group at multiple time points was determined by one-way ANOVA followed by Newman-Keuls multiple intergroup comparisons probability,0.05was considered statistically significant.RESULTS:1. The establishment of three rat models of ALI induced by different injection of LPS.These models are good experimental models for clinical ARDS induced by the similar common causes. With these the appropriate model we would be able to study the pathogenesis. Rat trachea drip concentration of2mg/kg LPS:0111B4ALI model with is conform to the requirements of the experiments,2. The observation Common signs of in the ratWe found that the rat of ALI group is heartbeat, shortness of breath, depressed, irritable, sluggish, fur bent handstand, lacklustre, evil cold, curled uP to make, the symPtom such as blood in the urine. But DEX and Lianggesan group was n’t.3. The change of lung water content (W/D)The LPS can lead to highter W/D in rats. ComPared with the control group, the LPS group showed significantly higher W/D in the1h,2h,4h,8h and was on the rise(P<0.01); W/D was lower in the high dose and low dose Lianggesan groups, and in the DEX group.(P<0.05, P<0.05, P<0.01).4. The change of the resPiratory frequencyThe LPS can lead to breathing rate increases in rats. Compared with the control group, the LPS group showed significantly higher the respiratory frequency in the1h,2h,4h,8h and was on the rise(P<0.01);The respiratory frequency was lower in the high dose and low dose Lianggesan groups, and in the DEX group.(P<0.01, P<0.01, P<0.01).[1] The change of the respiratory cycleThe LPS can lead to shorter the resPiratory cycle in rats. Compared with the control group, the LPS group showed significantly shorter the respiratory cycle in the,2h,4h,8h,16h and was gradually on the short (P<0.01); the respiratory cycle was longer in the high dose and low dose Lianggesan groups, and in the DEX group.(P<0.01, P<0.05, P<0.01).6. The change of PaO2The LPS can lead to lower PaO2in rats. Compared with the control group, the LPS grouP showed significantly lower PaO2and was gradually on the short in the2h,4h,8h,16h (P<0.01); PaO2was rised in the high dose and low dose Lianggesan grouPs, and in the DEX grouP.(P<0.01, P<0.05, P<0.01).7. The change of PaCO2The LPS can lead to higher PaO2in rats. Compared with the control group, the LPS group showed significantly lower higher and was gradually on the rise in the2h,4h,8h,16h (P<0.01); PaC>2was lower in the high dose and low dose Lianggesan grouPs, and in the DEX grouP.(P<0.01, P<0.01, P<0.01).8. The change of LPIThe LPS can lead to higher LPI in rats. ComPared with the control group, the LPS group showed significantly lower higher and was gradually on the rise in the2h,4h,8h,16h (P<0.01); LPI was lower in the high dose and low dose Lianggesan groups, and in the DEX grouP.(P<0.05, P<0.05, P<0.01). 9. The change of EVLWThe LPS can lead to higher EVLW in rats. Compared with the control group, the LPS group showed significantly lower higher and was gradually on the rise in the4h,8h,16h (P<0.05); EVLW was lower in the high dose and low dose Lianggesan grouPs, and in the DEX grouP.(P<0.05, P<0.01, P<0.01).10. The change of thePathology and Pathology scoresWhole lung observation in LPS group:the lungs in the two sides enlarged with obvious pulmonary edema2h after the LPS-induced damage on gross inspection. With the extension of time, the extent of pulmonary edema aggravated and red spotted hemorrhagic focus appeared extensively on the surface. Alveolar septum was seen to thicken, and pulmonary intersititum and alveolar was seen to swollen with large amounts of inflammatory cells immersed, and some extent of atelectasis and structural damage of alveolar septum was observed under light microscopic inspection. The reduction of infiltration of inflammatory cells in various extent could be observed in different Lianggesan grouPs and the DEX grouP. The structure of the alveolar is intact, distinct and exudation-free in the control group.The LPS can lead to the higher pathology scores in rats. Compared with the control group, the LPS group showed significantly lower higher and was gradually on the rise in the4h,8h,16h (P<0.05); The Pathology scores was lower in the high dose and low dose Lianggesan grouPs, and in the DEX group.(P<0.01, P<0.01, P<0.01).11. The effect of Lianggesan on the expression of α-rENaC、β-rENaC、γ-rENaC, Na+-K+-ATase和AQP-3in the LPS-induced acute lung injury in rats.11.1The results of α、β、γ-rENaC were analyzed by immunohistochemistryThe α、β、γ-rENaC are protein membrane proteins, they distributes in the cell membrpane and cytoplasm. In the control group, the α、β、γ-rENaC was over exPressed with a yellow color. Significant difference of exPression of α、β、γ-rENaC existed within groups(F=110.376,P<0.01;F=132.29,P<0.01;F=826.509,P<0.05). The analysis of the main effect within grouPs indicated:significant difference of expression of α、β、γ-rENaC existed between the control group and the LPS group.(P <0.05). compared to the LPS group, expression of α、β、γ-rENaC was stronger in the high doseand low dose Lianggesan groups, and in the DEX group.(P<0.05、P<0.01, P<0.05).11.2The results of a-Na+-K+-ATPase were analyzed by immunohistochemistryThe a-Na+-K+-ATPase distributes in the cell membr ane and cytoplasm. In the control group. Significant difference of expression of a-Na+-K+-ATPase existed withi-n groups (F=568.495,P<0.05). The analysis of the main effect within groups indicated:significant difference of expression of α、β、γ-rENaC existed between the control group and the LPS group.(P<0.05). comPared to the LPS group, expression of α、β、γ-rENaC was stronger in the high doseand low dose Lianggesan groups, and in the DEX group.(P<0.05、P<0.01、P<0.05).11.3The results of α、β、γ-rENaC were analyzed by westeblottingSignificant difference of expression of α、β、γ-rENaC existed within groups (F=493.841, P<0.01; F=370.7,42, P<0.01; F=6.526, P<0.01). The analysis of the main effect within grouPs indicated:significant difference of expression of α、β、 γ-rENaC existed between the control group and the LPS group (P<0.01,P<0.01). compared to the LPS group, expression of α、β、γ-rENaC was stronger in the high doseand low dose Lianggesan groups, and in the DEX group.(P<0.01、P<0.05、 P<0.05).11.4The results of AQP-3were analyzed by westeblottingThe LPS can lead to rise AQP-3in rats. Significant difference of expression of AQP-3existed within groups(F=86.189,P<0.01).Compared with the control group, the LPS group showed significantly lower in the8h (P<0.01); Compared with ALI group, The expression of AQP-3was significantly higher in the high dose and low dose Lianggesan groups, and in the DEX group.(P<0.01, P<0.01, P<0.01).11.5The results of α-Na+-K+-ATPase were analyzed by westeblottingIn the control group. Significant difference of expression of α-Na+-K+-ATPase existed within groups (F=73.925, P<0.01). The analysis of the main effect within groups indicated:significant difference of expression of α-Na+-K+-ATPase existed between the control group and the LPS group.(P<0.01、P<0.05). compared to the LPS group, expression of α-Na+-K+-ATPase was stronger in the high doseand low dose Lianggesan groups, and in the DEX group.(P<0.05, P<0.01, P<0.05).12. The results of α,β,γ-rENaC and Na+-K+-AtPase were analyzed by qRT-PCR12.1The results ofα,β,γ-rENaC were analyzed by qRT-PCRThe result of qRT-PCR had shown that gene expression of α、β,γ-rENaC mRNA in Lung of AL1male rats grouP was significantly lower than control group (F=430.776,P<0.01; F=569.578, P<0.01; F=111.832, P<0.01). A,β,γ-rENaC mRNA expression of Lianggesan groups had significantly higher than ALI group (P<0.05, P<0.01, P<0.05). The more dose of Lianggesan, the more exPression of α、β、γ-rENaC mRNA.12.2The results of a-Na+-K+-ATPase were analyzed by qRT-PCRThe result of qRT-PCR had shown that gene expression of a-Na+-K+-ATPase mRNA in Lung of ALI male rats grouP was significantly lower than control grouP (P <0.01). Significant difference of expression of a-Na+-K+-ATPase existed within grouPs (F=651.907,P<0.01).a-Na+-K+-ATPase mRNA expression of Lianggesan and DEX groups had significantly higher than ALI group (P<0.01, P<0.05). The more dose of Lianggesan, the more expression of a-Na+-K+-ATPase mRNA.Conclusion:1. The Study of Lianggesan’s Influence to male rats with ALI and the protective effect on Lung. The respiratory frequency, PaO2,W/D, LPI and EVLW increased in the acute lung injury,but the respiratory cycle and PaO2asignificant reduct in the respiratory cycle and PaO2induced by the endotoxin, which can be inhibited by Lianggesan, demonstrating the protective function of it to the acute lung injury and acute pulmonary edema induced by endotoxin.Pathology scores 2. The results of the immuohistochemistry,western blot and qRT-PCR revealed that expression of α、β、γ-rENaC,α-Na+-K+-ATPase and AQP-3in lung tissue declined in the acute lung injury induced by endotoxin. Lianggesan can imProve the fluid transportation and further on alleviate pulmonary edema by upmaniPulating the expression of α、β、γ-rENaC,α-Na+-K+-ATPase and AQP-3.

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