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
|
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。
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 rats
3. The Study of Lianggesan's Influence to male rats with ALI and the protective Effect on Lung
3.1The observation Common signs of in the rat
3.2The 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 index
3.6The change of extravascular lung water
3.7The Lung tissue histoPathological examination and pathological evaluation
4. The study of Lianggesan on the protective Effect in the LPS-induced acute lung injury in rat
4.1The 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+-ATase
expression in the lungs of rats were analyzed by western blot
4.3The 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 rat
We 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 frequency
The 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 cycle
The 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 PaO2
The 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 PaCO2
The 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 LPI
The 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 EVLW
The 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 scores
Whole 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 immunohistochemistry
The 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 immunohistochemistry
The 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 westeblotting
Significant 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 westeblotting
The 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 westeblotting
In 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-PCR
12.1The results ofα,β,γ-rENaC were analyzed by qRT-PCR
The result of qRT-PCR had shown that gene expression of α、β,γ-rENaC m
RNA 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-PCR
The 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.
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。
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 rats
3. The Study of Lianggesan's Influence to male rats with ALI and the protective Effect on Lung
3.1The observation Common signs of in the rat
3.2The 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 index
3.6The change of extravascular lung water
3.7The Lung tissue histoPathological examination and pathological evaluation
4. The study of Lianggesan on the protective Effect in the LPS-induced acute lung injury in rat
4.1The 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+-ATase
expression in the lungs of rats were analyzed by western blot
4.3The 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 rat
We 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 frequency
The 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 cycle
The 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 PaO2
The 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 PaCO2
The 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 LPI
The 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 EVLW
The 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 scores
Whole 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 immunohistochemistry
The 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 immunohistochemistry
The 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 westeblotting
Significant 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 westeblotting
The 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 westeblotting
In 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-PCR
12.1The results ofα,β,γ-rENaC were analyzed by qRT-PCR
The result of qRT-PCR had shown that gene expression of α、β,γ-rENaC m
RNA 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-PCR
The 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.
引文
[1]Lei H, Minghao W, Xiaonan Y, etal. Acute lung injury in patients with severe acute pancreastitis[J]. Turk J Gastroenterol.2013,24(5):502-507.
[2]Teke HU, Behret O, Teke D. etal. Transfusion related acute lung injury[J]. Hematol lood Transfus.2013,30(1):56-58.
[3]陈作宾,白蛋白对创伤性休克大鼠肺脏保护机制研究[J].浙江大学学报,2009,12(11)：87-89.
[4]中华医学会重症医学分会,急性肺损伤/急性呼吸窘迫综合征诊断和治疗指南(2006)[J].中华急诊医学杂志,2007,16(4)：343-349.
[5]耿耘,魏星.急性呼吸窘迫综合征的中医发病机理探讨[J].江西中医药,2002,33(5)：11-12.
[6]陈军,内毒素性急性肺损伤的研究进展[J].国外医药抗生素分册,2005,26(3)：131-147.
[7]Rubenfeld G D,Herridge M S. Epidemiology and outcomes of acute lung injury [J]. Chest,2007,131(2):554-562.
[8]张爱琴,马胜林,王跃珍,等.鱼腥草注射液防治86例放射性肺损伤的临床观察[J].中华放射医学与防护杂志,2006,26(4)：411-412.
[9]刘福成,崔志永,薛芳,等.大承气汤治疗严重创伤呼吸窘迫综合征的实验与临床研究[J].中国中西医结合杂志,1992,12(9)：541-542.
[10]陈军,内毒素性急性肺损伤的研究进展[J].国外医药抗生素分册,2005,26(3)：131-147
[11]张宏伟,急性肺损伤与细胞因子平衡的研究进展[J].Medical Reca Pitulate, 2006,12(2):1411-1414.
[12]Rubenfeld G D,Herridge M S. Epidemiology and outcomes of acute lung injury[J]. Chest,2007,131(2):554-562.
[13]Valentine SL, Sapru A, Higgerson R A. Fluid balance in critically ill children with acute lung injury [J]. Crit Care Med.,2012,40(10):2883-2889.
[14]Hong CM1, Xu DZ, Lu Q,etal. Systemic inflammatory response does not correlate with acute lung injury associated with mechanical ventilation strategies in normal lungs[J]. Anesth Analg,2012,115(1):118-121.
[15]Ma T,Fukuda N, Song Y, etal. Lung fluid transport in aquaporin-5 knockout mice [J]. Clin Invest,2000,105 (1):93-100.
[16]Reutershan J etal. Sequential recruitmentofneutrophils into lung and ronchoalveolar lavagefluid in LPS-induced acute lung injury [J]. Am J Physiol Lung Cell Mol Physio,2005,11(5):807-815.
[17]Fudala R,Krupa A, Stankowska D, etal. Antiinterleukin-8 autoantibody: interleukin-8 immune complexes in acute lung injury/acute respiratory distress syndrome[J].Clin Sci(Lond),2008,114:403-412.
[18]谢俊然,等.地塞米松的抗氧化作用及其对兔内毒素性肺损伤的保护效果[J].徐州医学院学报,2001,21(3)：181-183.
[19]李波,等.水通道蛋白1、3、4、5在内毒素性急性肺损伤小鼠肺组织中的表达[J].第二军医大学学报,2008,29(2)：131-135.
[20]张维,等.肺泡上皮细胞与急性肺损伤水肿液清除的研究进展[J].中国呼吸与危重监护杂志.2008；7(1)：74-76.
[21]Qiu HB, Sun HM, Yang Y, etal. Effect of beta-adrenergic agonist on alveolar fluid clearance in acute lung injury:an experiment with rats [J]. Zhonghua Yi Xue Za Zhi,2008,86(3):187-191.
[22]Migneault F, Boncoeur E, Morneau F, etal. Cycloheximide and lipopoly saccha ride downregulate aENaC mRNA via different mechanisms in alveolar epithelial cells[J].Physiol Lung Cell Mol Physiol.2013,305(10):747-755.
[23]李王平,张芳,李萌等.肺泡上皮细胞钠钾ATP酶与海水淹溺型肺水肿[J].现代生物医学进展,2008,8(1)：158-160.
[24]Sathya M, Kokilavani R, Teepa K S, etal,Biopotency of Acalypha indica Linn on Membrane Bound ATPases and Marker Enzymes urolithic Rats. Anc Sci Life[J].2011,31(1):3-9.
[25]陈正堂.急性呼吸窘迫综合征发病机制及诊治进展[J].中华急诊医学杂,2003,12(1)：65-66.
[26]钱小顺,牛庆磊,杨洁,等.老年和青年大鼠肺泡巨噬细胞凋亡的初步研究[J].中华医学杂志,2005,85(13)：253-256.
[27]李和泉,张合平,李进,等.急性呼吸窘迫综合征的发病机制[J].小儿急救医学.2000,7(3)：113-115.
[28]王士雯,钱小顺.老年人多器官功能衰竭肺启动的研究进展[J].中华老年医学杂志,2005,24(4)：313-316.
[29]扬红,斯琴,孙仁宇等.肺血管内皮细胞在大鼠急性肺损伤发生中的作用[J].中国病理生理杂志,2000,16：831-834.
[30]徐启勇.急性呼吸窘迫综合征(ARDS)[J].实用乡村医生杂,1999,6(5)：5-7.
[31]裘庆元.珍本医书集成[M].北京：中国中医药出版社,1999：103-105.
[32]孔立,卢笑晖,江涛.全身炎症反应综合征的根本病机是气机逆乱[J].中国中西医结合急救杂志,2005,12(2)：68-70.
[33]孙秋生,李华,张梦君等,谈肺通调水道与水液停聚[J].河南中医,1995,12(2)：73-76.
[34]唐·王冰.补注黄帝内经素问[M].北京：人民卫生出版社,1956：71.
[35]明·马莳.黄帝内经素问注证发微[M].清·嘉庆十年右歙鲍氏慎余堂刻本.124.
[36]张余森,彭代国,白朝勇,等.“肺与大肠相表里”动物模型的制做及其机理初探[J].中国兽医医药杂志,1987,12(2)：1-4.
[37]Whitehead T, Slutsky A,The pulmonary physician in critical care:Ventilator Indused lung injury[J].Thorxa,2002,57(7):635-642.
[38]Donorfio D,Chimuello D,etal.Pulmonary and exrtpaulmony acute respiratory distress syndlrome arc different [J],Eur Respir,2003,22(42):48-56.
[39]Fan YM, Huang XL, Dong ZF,etal. Hydrogen sulfide reduces lipopolysa ccharideinduced acute lung injury and inhibits expression of phosphorylated p38 MA-PK in rats [J], Sheng Li Xue Bao,2012,64(6):666-672.
[40]Ma T, Liu Z.Functions of aquaporin 1 and a-epithelial Na+channel in rat acute lung injury induced by acute ischemic kidney injury[J]. Int Urol Nephrol. 2013,45(4):1187-1196.
[41]Cho JS, Kim YD, Shin N, Lee CH, etal. Effect of transpleural perfusion with oxygenated perfluorocarbon in a rat model of acute lung injury [J]. Exp Lung Res.2013,39(1):32-38.
[42]Koksoy FN, Yankol Y, Oran ES, etal. Acute lung injury in patients with severe acute pancreatitis[J]. Turk J Gastroenterol.2013,24(5):495-501.
[43]Shi Q Q, Wang H, Fang H, etal. Dose-response and mechanism of protective functions of selective alpha-2 agonist dexmedetomidine on acute lung injury in rats[J].Saudi Med.2012,33(4):375-381.
[44]Yang JJ, Ji MH. Re:Erbuyun K, et al. Effects of levosimendan and dobutamine on experimental acute lung injury in rats [J]. Acta Histochem 2009,11(1):404-414.
[45]吴英林,赵晓琴.急性肺损伤的药物治疗进展[J].蛇志,2007,19(2)：136-141.
[46]Hagio T, Nakao S, Matsnoka H, etal.Inhibition of neutrophilekestase activity atennates complemnt-mediated lung injury in the hamaster[J].Eur J Pharmac-ol,2001,42(6):1371-1382.
[47]Hoshi K,Kurosawa S,Kato M,etal.Sivel estat,a neutrophil elastase inhibitor, red-ucees mortality rate of critically ill patients[J].Tohoku J Exp Med,2005,207 (2):143-1481.
[48]Tamakuma S,Ogawam,Aikawa N,etal.Relationship between neutrophilelastase and acute lung injury in humans[J].Pulm Pharmacol Ther,2004,17(5):271-2791.
[49]Latere P F,Wittebolex, Dhainaut J F.Anticoagulant therapy in acute lung injury [J]Crit Care M.2003,4(4):315-3211.
[50]洪辉华,蔡宛如.急性肺损伤发病机制及中医药治疗的实验研究进展[J].浙江中医药大学学报,2007,131(1)：133-135.
[51]谢玉宝,李梅.急性肺损伤的中医药治疗进展[J].实用临床医药杂志,2008,12(2)：113-115.
[52]太平惠民和剂局方[M],人民出版社(第3版)：1078-1085.
[53]陈晓丽,李丹,张晓,等.凉膈散在临床的应用探讨[J].中华实用中西医杂志,2005,18(5)：517-519.
[54]余林中,林慧,胡孔友等.凉膈散对家兔内毒素温病模型体温、血浆TNF-α及脑脊液PGE2、cAMP含量的影响[J].中国中医药科技,1996,3(4)：26-27.
[55]江爱达,余林中.凉膈散对LPS诱导的巨噬细胞丝裂素活化蛋白激酶p38活性的影响[J].中国中医基础医学杂志,2005,11(7)：511-513,518.
[56]林慧,余林中等.凉膈散含药血清对内毒素刺激RAW264.7细胞CD14mRNA表达的影响[J].中药药理与临床,2005,21(3)：1-3.
[57]江爱达,余林中,胡孔友等.凉膈散药物血清对脂多糖诱导体外培养巨噬细胞核转录因子-κB的影响[J].第一军医大学学报,2005,25(6)：619-622.
[58]余林中.凉膈散对内毒素血症小鼠肝脏库普弗细胞CD14和清道夫受体表达 的影响[J].中国中药杂志,2006,31(3)：220-223.
[59]余林中.凉膈散对家兔内毒素温病模型的解毒作用研究[J].中药药理与临床,1996,12(5)：4-6.
[60]余林中.凉膈散对小鼠内毒素血瘀模型微循环的影响[J].中药药理与临床,1996,12(2)：1-3.
[61]余林中,林慧,江爱达,等.凉膈散对家兔内毒素温病模型的化瘀作用研究[J].中药药理与临床,1998,14(1)：7-9.
[62]林慧,余林中.凉膈散对内毒素肺损伤小鼠肺组织核因子-κB活性的影响[J].四川中医,2004,22(7)：16-18.
[63]胡孔友,余林中,刘建新,等.凉膈散对内毒素致大鼠急性肺损伤的保护作用[J].中药药理与临床,2008,24(5)：3-5.
[64]胡孔友,刘建新,余林中,等.凉膈散对内毒素大鼠急性肺损伤炎症调控的影响[J].中华中医药杂志,2009,24(11)：1433-1436.
[65]胡孔友,刘建新,余林中,等.凉膈散对内毒素致大鼠急性肺损伤Th1/Th2漂移的调节作用[J].中医杂志,2009,12(1)：34-37.
[66]侯建林,王波,李生,等.凉膈散加味治疗大叶肺炎[J].实用中医内科杂志,1995,9(4)：20-21.
[67]张洁,胡锐,郭琪,等.凉膈散加味治疗小儿病毒性脑炎32例[J].辽宁中医杂志,1993,20(11)：29.
[68]刘璇,魏娜.凉膈散加减治疗小儿癫痫植物神经性发作[J].江西中医药,2007,38(7)：55-57.
[69]李少松,王博,李晟,等.中药治疗急性脑出血42例[J].中国中医急症,200312(4)：362-364.
[70]李素云,王立芹,郑稼琳,等.自由基与衰老的研究进展[J].中国老年学杂志,2007,27(20)：2046-2048
[1]Pelosi P, Donofio D,Chimuello D,etal.Pulmonary and extrapulmonary acute respiratory distress syndlrome are different[J],Eur Respir,2003,22(42):48-56.
[2]Michael AM, Guy AZ, Charles E, etal. Future research directions in acute lung Injury'.summary of a National Heart,Lung, and Blood Instiutte Working Group[j].Am J Respir Crti Care Med,2003,167(7):1027-1035.
[3]刘建新,胡孔友,余林中,等.凉膈散对内毒素致大鼠急性肺损伤信号转导与转录激活子3表达的影响[J].中药药理与临床,2009,25(6)：1-3.
[4]余林中,刘建新,胡孔友,等.凉膈散对内毒素致急性肺损伤大鼠信号转导与转录激活子1表达的影响[J].南方医科大学学报,2010,30(1)：43-46.
[5]Sartori C, Matthay MA. Alveolar epithelial fluid transport in acute lung injury: new insights[J]. Eur Respir J,2002,20(5):1299-1313.
[6]汪东颖,杨爱东,郭永洁,等.急性肺损伤的中医证治探讨及思考[J].辽宁中医杂志,2008,35(7)：1010-1012.
[7]王祥瑞,杭燕南.急性肺损伤-基础与临床[M].中国协和医科大学出版社,2005.
[8]李新甫,汪建新.急性肺损伤动物模型研究进展[J].国外医学呼吸系统分册,2005,25(7)：506-511.
[1]Valentine SL, Sapru A, Higgerson R A. Fluid balance in critically ill children with acute lung injury [J]. Crit Care Med.,2012,40(10):2883-2889.
[2]李珊,张华,崔晓建,等.中华医学会重症医学分会,急性肺损伤／急性呼吸窘迫综合征诊断和治疗指南(2006)[J].中华急诊医学杂志,2007,16(4)：343-349.
[3]耿耘,魏星.急性呼吸窘迫综合征的中医发病机理探讨[J].江西中医药,2002,33(5)：11-12.
[4]陆晶,张林丽,余书勤,等.氧化苦参碱对兔油酸型急性肺损伤的保护作用研究[J].中国新药杂志,2007,16(14)：1090-1095.
[5]李春盛,桂培春,李萌,等.大黄对内毒素诱导急性肺损伤大鼠的保护作用[J].基础医学与临床,2001,21(1)：65-68.
[6]丁成志,聂贞蕴,钱克俭,等.不同肺复张模式对急性呼吸窘迫综合征患者呼吸功能及血管外肺水指数的影响[J].实用临床医学,2012,13(1)：137-138.
[7]耿耘,魏星.急性呼吸窘迫综合征的中医发病机理探讨[J].江西中医药,2002,33(5)：11-12.
[8]Teke HU, Behret O, Teke D. etal. Transfusion-related acute lung injury[J]. Hematol lood Transfus.2013,30(1):56-58.
[9]Lei H, Minghao W, Xiaonan Y, etal. Acute lung injury in patients with severe acute pancreastitis[J]. Turk J Gastroenterol.2013,24(5):502-507.
[10]陈作宾.白蛋白对创伤性休克大鼠肺脏保护机制研究[J].浙江大学学报,2009,12(11)：87-89.
[11]中华医学会重症医学分会,急性肺损伤／急性呼吸窘迫综合征诊断和治疗指南[J].中华急诊医学杂志,2007,16(4)：343-349.
[12]太平惠民和剂局方,人民出版社(第3版)：1078-1085.
[13]陈晓丽,李繁盛,张栋,等.凉膈散在临床的应用探讨[J].中华实用中西医杂志,2005,18(5)：517-519.
[14]余林中,林慧,胡孔友等.凉膈散对家兔内毒素温病模型体温、血浆TNF-α及 脑脊液PGE2、cAMP含量的影响[J].中国中医药科技,1996,3(4)：26-27.
[15]江爱达,余林中.凉膈散对LPS诱导的巨噬细胞丝裂素活化蛋白激酶p38活性的影响[J].中国中医基础医学杂志,2005,11(7)：511-513,518.
[16]林慧,余林中.凉膈散含药血清对内毒素刺激RAW264.7细胞CD14mRNA表达的影响[J].中药药理与临床,2005,21(3)：1-3.
[17]江爱达,余林中,胡孔友等.凉膈散药物血清对脂多糖诱导体外培养巨噬细胞核转录因子-κB的影响[J].第一军医大学学报,2005,25(6)：619-622.
[18]余林中,林慧.凉膈散对内毒素血症小鼠肝脏库普弗细胞CD14和清道夫受体表达的影响[J].中国中药杂志,2006,31(3)：220-223.
[19]余林中.凉膈散对家兔内毒素温病模型的解毒作用研究[J].中药药理与临床,1996,12(5)：4-6.
[20]余林中.凉膈散对小鼠内毒素血瘀模型微循环的影响[J].中药药理与临床,1996,12(2)：1-3.
[21]余林中,林慧,江爱达,等.凉膈散对家兔内毒素温病模型的化瘀作用研究[J].中药药理与临床,1998,14(1)：7-9.
[22]林慧,余林中.凉膈散对内毒素肺损伤小鼠肺组织核因子-κB活性的影响[J].四川中医,2004,22(7)：16-18.
[23]胡孔友,余林中,刘建新,等.凉膈散对内毒素致大鼠急性肺损伤的保护作用[J].中药药理与临床,2008,24(5)：3-5.
[24]胡孔友,刘建新,余林中,等.凉膈散对内毒素大鼠急性肺损伤炎症调控的影响[J].中华中医药杂志,2009,24(11)：1433-1436.
[25]胡孔友,刘建新,余林中,等.凉膈散对内毒素致大鼠急性肺损伤Th1/Th2漂移的调节作用[J].中医杂志,2009,12(1)：34-37.
[26]Weifeng Song,Sadis Matalon,etal,Modulation of alveolar fluid clearance by reactive oxygen-nitrogen intermediates[J]. Am J Physiol Lung Cell Mol Physiol. 2007,293(12):855-858.
[27]Gavin d perkins, Fang Gao and David R Thickett, etal. In Vivo and In vitro effects of Salbutamol upon alveolar epithelial repair in acute lung injury [J]. Published online.2007,Thorax,doi:10.1136/thx.2007.080382.
[28]Coates G,Brodovich H,Jefferies AL,etal. Gray GW. Effects of exercise on lung lymph flow in sheep and goats during normoxia and hypoxia[J]. Clin Invest.1984;74:133-141.
[1]张卫琴.免疫组化技术在病理诊断中的应用[J].安徽医药,2012,16(11)：1700-1703.
[2]Hardiman KM, Matalon S. Modification of sodium transport and alveolar fluid clearance by hypoxia:mechanisms and physiological implications[J]. Am J Respair Cell Mol Biol,2001,25(5):538-541.
[3]Matalon S, Lazrak A, Lucky J,etal. Lung clearance:20 years of progress invited review:Biophysical properties of sodium channels in lung alveolar epithelial cells[J]. J Appl Physiol,2002,93:1852-1859.
[4]Amin MS, Reza E, Shahat E,etal.Enhanced expression of epithelial sodium channels in the renal medulla of Dahl S rats[J]. Can J Physiol Pharmacol. 2011,89(3):159-68.
[5]Modelska K, Matthay MA, Pittet JE,etal.The effect of endogenous angiotensin Ⅱ on alveolarfluid clearance in rats with acute lung injury[J],Can Respir,2012, 19(5):311-318.
[6]Mairbaurl H, Mayer K, Kim KJ,etal.Hypoxia decreases active Na+transport across primary rat alveolar epithelial cell monolayers[J]. Am J Physiol Lung Cell Mol Physiol,2002,282(4):L659-665.
[7]Todd C, Carpenter S, Schomber G,et al. Hypoxia reversibly in-hibits epithelial sodium transport but does not inhibit lung ENaC or Na+-K+-ATPase expression[J]. Am J Physiol Lung Cell MolPhysiol,2003,284:L77-L83.
[8]李王平,张芳,李萌,等.肺泡上皮细胞钠钾ATP酶与海水淹溺型肺水肿[J].现代生物医学进展,2008,8(1)：158-160.
[9]Sathya M, Kokilavani R, Teepa K S, etal,Biopotency of Acalypha indica Linn on Membrane Bound ATPases and Marker Enzymes urolithic Rats[J]. Anc Sci Life.2011,31(1):3-9.
[1]张维,等.肺泡上皮细胞与急性肺损伤水肿液清除的研究进展.中国呼吸与危重监护杂志,2008；7(1)：74-76.
[2]Qiu HB, Sun HM, Yang Y, etal Effect of beta-adrenergic agonist on alveolar fluid clearance in acute lung injury:an experiment with rats [J]. Zhonghua Yi Xue Za Zhi,2008,86(3):187-191.
[3]Knowels M R, Boucher RC. Mucus clearance as a primary innatedefense mechanism for mammalian airways[J]. J Clin Invest,2002,109(5):571-577.
[4]Hardiman K M, Matalon S. Modification of sodium transport and alveolar fluid clearance by hypoxia:mechanisms and physiological implications [J]. Am J Respair Cell Mol Biol,2001,25(5):538-541.
[5]Kellenberger S, Gautschi I, Schild L. Mutations in the outer pore disrupt amiloride block by increas-ing its dissociation rate[J]. Mol Pharmacol,2003, 64(4):848-856.
[6]Nyder PM. Na+ -K+-ATPase:cell surface insertion and retrival in Na+homeostasis and hypertension[J]. Endocr Rev,2002,23(2):258-275.
[7]DOnorfio D,Chimuello D,et al.Pulmonary and exrtpaulmony acute respiratory distress syndl-rome arc different [J],Eur Respir,2003,22(42):48-56.
[8]Fan YM, Huang XL, Dong ZF,et al. Hydrogen sulfide reduces lipopolysaccharide-induced acute lung injury and inhibits expression of phosphorylated p38 MAPK in rats [J], Sheng Li Xue Bao,2012,64(6):666-672.
[9]Fan YM, Huang XL, Dong ZF,et al. Hydrogen sulfide reduces lipopolysa ccharid -einduced acute lung injury and inhibits expression of phosphorylated p38 MA-PK in rats [J], Sheng Li Xue Bao,2012,64(6):666-672.
[10]郭亮,李楠.水通道蛋白与急性肺损伤[J].华南国防医学杂志,2009,23(1)：81-84.
[11]许海波.水通道蛋白-3的研究进展[J].四川解剖学杂志,2006,14(2)：28-31.
[12]李波,陈东,王桂芳,等.水通道蛋白1、3、4、5在内毒素性急性肺损伤小鼠肺组织中的表达[J].第二军医大学学报,2008,29(2)：131-135.
[13]Sathya M, Kokilavani R, Teepa K S, etal,Biopotency of Acalypha indica Linn on Membrane Bound ATPases and Marker Enzymes urolithic Rats[J]. Anc Sci Life.2011,31(1):3-9.
[1]Kahn HJ, Yang LY, Blondal J, etal. RT-PCR amplification of CK19mRNA in the blood of breast cancer patients:correlation with established prognostic parameters [J]. Reast Cancer ResTreat,2000,60(2):143-151.
[2]Klein D. Fredriksson R1, Schioth HB.Quantification using real-time PCR technology applications and limitations[J].Trends Mol Med,2002,8(6):257-260
[3]冯志新,姜平.猪细胞因子SYBR Green实时PCR检测方法的建立[J].现代生物医学进展,2007,7(1)：108-121.
[4]陈琳,姜泊,张亚历,等.运用qRT-PCR和westernblot检测elaudin-1在人类大肠癌细胞株的表达[J].广西医科大学学报,2Q07,24(2)：205-207.
[5]习梅,王立荣,王战勇,等.用SYBR Green I实时荧光PCR结合融解曲线分析 法诊断a-地中海贫血[J].中华医学遗传学杂志,2007,24(2)：192-196.
[6]陈英剑,胡成进,赵苗青.SYBR-GreenI实时荧光定量PCR技术平台的建立[J].实用医药杂志,2004,21(11)：997-1000.
[7]Przybycien-Szymanska MM, Rao YS, Prins SA, etal, Parental binge alcohol abuse alters fl generation hypothalamic gene expression in the absence of direct fetal alcohol exposure[J]. PLoS One.2014,9(2):1154-1164.
[8]Alsio J, Rask-Andersen M1, Chavan RA, etal, Exposure to a high-fat high-sugar diet causes strong up-regulation of proopiomelanocortin and differentially affects dopamine D1 and D2 receptor gene expression in the brainstem of rats[J]. Neurosci Lett.2014,13(1):125-131.
[9]Pires-Oliveira M1, Maragno AL, Parreiras-e-Silva LT, etal, Testosterone represses ubiquitin ligases atrogin-1 and Murf-1 expression in an androgen-sensitive rat skeletal muscle in vivo.[J]. Appl Physiol. 2010,180(2):266-273.
[10]Liu FF1, Ma ZY, Li DL, etal, Differential expression of TRPC channels in the left ventricle of spontaneously hypertensive rats [J]. Mol Biol Rep..2010, 37(6):2645-2651.
[2]Teke HU, Behret O, Teke D. etal. Transfusion related acute lung injury[J]. Hematol lood Transfus.2013,30(1):56-58.
[3]陈作宾,白蛋白对创伤性休克大鼠肺脏保护机制研究[J].浙江大学学报,2009,12(11)：87-89.
[4]中华医学会重症医学分会,急性肺损伤/急性呼吸窘迫综合征诊断和治疗指南(2006)[J].中华急诊医学杂志,2007,16(4)：343-349.
[5]耿耘,魏星.急性呼吸窘迫综合征的中医发病机理探讨[J].江西中医药,2002,33(5)：11-12.
[6]陈军,内毒素性急性肺损伤的研究进展[J].国外医药抗生素分册,2005,26(3)：131-147.
[7]Rubenfeld G D,Herridge M S. Epidemiology and outcomes of acute lung injury [J]. Chest,2007,131(2):554-562.
[8]张爱琴,马胜林,王跃珍,等.鱼腥草注射液防治86例放射性肺损伤的临床观察[J].中华放射医学与防护杂志,2006,26(4)：411-412.
[9]刘福成,崔志永,薛芳,等.大承气汤治疗严重创伤呼吸窘迫综合征的实验与临床研究[J].中国中西医结合杂志,1992,12(9)：541-542.
[10]陈军,内毒素性急性肺损伤的研究进展[J].国外医药抗生素分册,2005,26(3)：131-147
[11]张宏伟,急性肺损伤与细胞因子平衡的研究进展[J].Medical Reca Pitulate, 2006,12(2):1411-1414.
[12]Rubenfeld G D,Herridge M S. Epidemiology and outcomes of acute lung injury[J]. Chest,2007,131(2):554-562.
[13]Valentine SL, Sapru A, Higgerson R A. Fluid balance in critically ill children with acute lung injury [J]. Crit Care Med.,2012,40(10):2883-2889.
[14]Hong CM1, Xu DZ, Lu Q,etal. Systemic inflammatory response does not correlate with acute lung injury associated with mechanical ventilation strategies in normal lungs[J]. Anesth Analg,2012,115(1):118-121.
[15]Ma T,Fukuda N, Song Y, etal. Lung fluid transport in aquaporin-5 knockout mice [J]. Clin Invest,2000,105 (1):93-100.
[16]Reutershan J etal. Sequential recruitmentofneutrophils into lung and ronchoalveolar lavagefluid in LPS-induced acute lung injury [J]. Am J Physiol Lung Cell Mol Physio,2005,11(5):807-815.
[17]Fudala R,Krupa A, Stankowska D, etal. Antiinterleukin-8 autoantibody: interleukin-8 immune complexes in acute lung injury/acute respiratory distress syndrome[J].Clin Sci(Lond),2008,114:403-412.
[18]谢俊然,等.地塞米松的抗氧化作用及其对兔内毒素性肺损伤的保护效果[J].徐州医学院学报,2001,21(3)：181-183.
[19]李波,等.水通道蛋白1、3、4、5在内毒素性急性肺损伤小鼠肺组织中的表达[J].第二军医大学学报,2008,29(2)：131-135.
[20]张维,等.肺泡上皮细胞与急性肺损伤水肿液清除的研究进展[J].中国呼吸与危重监护杂志.2008；7(1)：74-76.
[21]Qiu HB, Sun HM, Yang Y, etal. Effect of beta-adrenergic agonist on alveolar fluid clearance in acute lung injury:an experiment with rats [J]. Zhonghua Yi Xue Za Zhi,2008,86(3):187-191.
[22]Migneault F, Boncoeur E, Morneau F, etal. Cycloheximide and lipopoly saccha ride downregulate aENaC mRNA via different mechanisms in alveolar epithelial cells[J].Physiol Lung Cell Mol Physiol.2013,305(10):747-755.
[23]李王平,张芳,李萌等.肺泡上皮细胞钠钾ATP酶与海水淹溺型肺水肿[J].现代生物医学进展,2008,8(1)：158-160.
[24]Sathya M, Kokilavani R, Teepa K S, etal,Biopotency of Acalypha indica Linn on Membrane Bound ATPases and Marker Enzymes urolithic Rats. Anc Sci Life[J].2011,31(1):3-9.
[25]陈正堂.急性呼吸窘迫综合征发病机制及诊治进展[J].中华急诊医学杂,2003,12(1)：65-66.
[26]钱小顺,牛庆磊,杨洁,等.老年和青年大鼠肺泡巨噬细胞凋亡的初步研究[J].中华医学杂志,2005,85(13)：253-256.
[27]李和泉,张合平,李进,等.急性呼吸窘迫综合征的发病机制[J].小儿急救医学.2000,7(3)：113-115.
[28]王士雯,钱小顺.老年人多器官功能衰竭肺启动的研究进展[J].中华老年医学杂志,2005,24(4)：313-316.
[29]扬红,斯琴,孙仁宇等.肺血管内皮细胞在大鼠急性肺损伤发生中的作用[J].中国病理生理杂志,2000,16：831-834.
[30]徐启勇.急性呼吸窘迫综合征(ARDS)[J].实用乡村医生杂,1999,6(5)：5-7.
[31]裘庆元.珍本医书集成[M].北京：中国中医药出版社,1999：103-105.
[32]孔立,卢笑晖,江涛.全身炎症反应综合征的根本病机是气机逆乱[J].中国中西医结合急救杂志,2005,12(2)：68-70.
[33]孙秋生,李华,张梦君等,谈肺通调水道与水液停聚[J].河南中医,1995,12(2)：73-76.
[34]唐·王冰.补注黄帝内经素问[M].北京：人民卫生出版社,1956：71.
[35]明·马莳.黄帝内经素问注证发微[M].清·嘉庆十年右歙鲍氏慎余堂刻本.124.
[36]张余森,彭代国,白朝勇,等.“肺与大肠相表里”动物模型的制做及其机理初探[J].中国兽医医药杂志,1987,12(2)：1-4.
[37]Whitehead T, Slutsky A,The pulmonary physician in critical care:Ventilator Indused lung injury[J].Thorxa,2002,57(7):635-642.
[38]Donorfio D,Chimuello D,etal.Pulmonary and exrtpaulmony acute respiratory distress syndlrome arc different [J],Eur Respir,2003,22(42):48-56.
[39]Fan YM, Huang XL, Dong ZF,etal. Hydrogen sulfide reduces lipopolysa ccharideinduced acute lung injury and inhibits expression of phosphorylated p38 MA-PK in rats [J], Sheng Li Xue Bao,2012,64(6):666-672.
[40]Ma T, Liu Z.Functions of aquaporin 1 and a-epithelial Na+channel in rat acute lung injury induced by acute ischemic kidney injury[J]. Int Urol Nephrol. 2013,45(4):1187-1196.
[41]Cho JS, Kim YD, Shin N, Lee CH, etal. Effect of transpleural perfusion with oxygenated perfluorocarbon in a rat model of acute lung injury [J]. Exp Lung Res.2013,39(1):32-38.
[42]Koksoy FN, Yankol Y, Oran ES, etal. Acute lung injury in patients with severe acute pancreatitis[J]. Turk J Gastroenterol.2013,24(5):495-501.
[43]Shi Q Q, Wang H, Fang H, etal. Dose-response and mechanism of protective functions of selective alpha-2 agonist dexmedetomidine on acute lung injury in rats[J].Saudi Med.2012,33(4):375-381.
[44]Yang JJ, Ji MH. Re:Erbuyun K, et al. Effects of levosimendan and dobutamine on experimental acute lung injury in rats [J]. Acta Histochem 2009,11(1):404-414.
[45]吴英林,赵晓琴.急性肺损伤的药物治疗进展[J].蛇志,2007,19(2)：136-141.
[46]Hagio T, Nakao S, Matsnoka H, etal.Inhibition of neutrophilekestase activity atennates complemnt-mediated lung injury in the hamaster[J].Eur J Pharmac-ol,2001,42(6):1371-1382.
[47]Hoshi K,Kurosawa S,Kato M,etal.Sivel estat,a neutrophil elastase inhibitor, red-ucees mortality rate of critically ill patients[J].Tohoku J Exp Med,2005,207 (2):143-1481.
[48]Tamakuma S,Ogawam,Aikawa N,etal.Relationship between neutrophilelastase and acute lung injury in humans[J].Pulm Pharmacol Ther,2004,17(5):271-2791.
[49]Latere P F,Wittebolex, Dhainaut J F.Anticoagulant therapy in acute lung injury [J]Crit Care M.2003,4(4):315-3211.
[50]洪辉华,蔡宛如.急性肺损伤发病机制及中医药治疗的实验研究进展[J].浙江中医药大学学报,2007,131(1)：133-135.
[51]谢玉宝,李梅.急性肺损伤的中医药治疗进展[J].实用临床医药杂志,2008,12(2)：113-115.
[52]太平惠民和剂局方[M],人民出版社(第3版)：1078-1085.
[53]陈晓丽,李丹,张晓,等.凉膈散在临床的应用探讨[J].中华实用中西医杂志,2005,18(5)：517-519.
[54]余林中,林慧,胡孔友等.凉膈散对家兔内毒素温病模型体温、血浆TNF-α及脑脊液PGE2、cAMP含量的影响[J].中国中医药科技,1996,3(4)：26-27.
[55]江爱达,余林中.凉膈散对LPS诱导的巨噬细胞丝裂素活化蛋白激酶p38活性的影响[J].中国中医基础医学杂志,2005,11(7)：511-513,518.
[56]林慧,余林中等.凉膈散含药血清对内毒素刺激RAW264.7细胞CD14mRNA表达的影响[J].中药药理与临床,2005,21(3)：1-3.
[57]江爱达,余林中,胡孔友等.凉膈散药物血清对脂多糖诱导体外培养巨噬细胞核转录因子-κB的影响[J].第一军医大学学报,2005,25(6)：619-622.
[58]余林中.凉膈散对内毒素血症小鼠肝脏库普弗细胞CD14和清道夫受体表达 的影响[J].中国中药杂志,2006,31(3)：220-223.
[59]余林中.凉膈散对家兔内毒素温病模型的解毒作用研究[J].中药药理与临床,1996,12(5)：4-6.
[60]余林中.凉膈散对小鼠内毒素血瘀模型微循环的影响[J].中药药理与临床,1996,12(2)：1-3.
[61]余林中,林慧,江爱达,等.凉膈散对家兔内毒素温病模型的化瘀作用研究[J].中药药理与临床,1998,14(1)：7-9.
[62]林慧,余林中.凉膈散对内毒素肺损伤小鼠肺组织核因子-κB活性的影响[J].四川中医,2004,22(7)：16-18.
[63]胡孔友,余林中,刘建新,等.凉膈散对内毒素致大鼠急性肺损伤的保护作用[J].中药药理与临床,2008,24(5)：3-5.
[64]胡孔友,刘建新,余林中,等.凉膈散对内毒素大鼠急性肺损伤炎症调控的影响[J].中华中医药杂志,2009,24(11)：1433-1436.
[65]胡孔友,刘建新,余林中,等.凉膈散对内毒素致大鼠急性肺损伤Th1/Th2漂移的调节作用[J].中医杂志,2009,12(1)：34-37.
[66]侯建林,王波,李生,等.凉膈散加味治疗大叶肺炎[J].实用中医内科杂志,1995,9(4)：20-21.
[67]张洁,胡锐,郭琪,等.凉膈散加味治疗小儿病毒性脑炎32例[J].辽宁中医杂志,1993,20(11)：29.
[68]刘璇,魏娜.凉膈散加减治疗小儿癫痫植物神经性发作[J].江西中医药,2007,38(7)：55-57.
[69]李少松,王博,李晟,等.中药治疗急性脑出血42例[J].中国中医急症,200312(4)：362-364.
[70]李素云,王立芹,郑稼琳,等.自由基与衰老的研究进展[J].中国老年学杂志,2007,27(20)：2046-2048
[1]Pelosi P, Donofio D,Chimuello D,etal.Pulmonary and extrapulmonary acute respiratory distress syndlrome are different[J],Eur Respir,2003,22(42):48-56.
[2]Michael AM, Guy AZ, Charles E, etal. Future research directions in acute lung Injury'.summary of a National Heart,Lung, and Blood Instiutte Working Group[j].Am J Respir Crti Care Med,2003,167(7):1027-1035.
[3]刘建新,胡孔友,余林中,等.凉膈散对内毒素致大鼠急性肺损伤信号转导与转录激活子3表达的影响[J].中药药理与临床,2009,25(6)：1-3.
[4]余林中,刘建新,胡孔友,等.凉膈散对内毒素致急性肺损伤大鼠信号转导与转录激活子1表达的影响[J].南方医科大学学报,2010,30(1)：43-46.
[5]Sartori C, Matthay MA. Alveolar epithelial fluid transport in acute lung injury: new insights[J]. Eur Respir J,2002,20(5):1299-1313.
[6]汪东颖,杨爱东,郭永洁,等.急性肺损伤的中医证治探讨及思考[J].辽宁中医杂志,2008,35(7)：1010-1012.
[7]王祥瑞,杭燕南.急性肺损伤-基础与临床[M].中国协和医科大学出版社,2005.
[8]李新甫,汪建新.急性肺损伤动物模型研究进展[J].国外医学呼吸系统分册,2005,25(7)：506-511.
[1]Valentine SL, Sapru A, Higgerson R A. Fluid balance in critically ill children with acute lung injury [J]. Crit Care Med.,2012,40(10):2883-2889.
[2]李珊,张华,崔晓建,等.中华医学会重症医学分会,急性肺损伤／急性呼吸窘迫综合征诊断和治疗指南(2006)[J].中华急诊医学杂志,2007,16(4)：343-349.
[3]耿耘,魏星.急性呼吸窘迫综合征的中医发病机理探讨[J].江西中医药,2002,33(5)：11-12.
[4]陆晶,张林丽,余书勤,等.氧化苦参碱对兔油酸型急性肺损伤的保护作用研究[J].中国新药杂志,2007,16(14)：1090-1095.
[5]李春盛,桂培春,李萌,等.大黄对内毒素诱导急性肺损伤大鼠的保护作用[J].基础医学与临床,2001,21(1)：65-68.
[6]丁成志,聂贞蕴,钱克俭,等.不同肺复张模式对急性呼吸窘迫综合征患者呼吸功能及血管外肺水指数的影响[J].实用临床医学,2012,13(1)：137-138.
[7]耿耘,魏星.急性呼吸窘迫综合征的中医发病机理探讨[J].江西中医药,2002,33(5)：11-12.
[8]Teke HU, Behret O, Teke D. etal. Transfusion-related acute lung injury[J]. Hematol lood Transfus.2013,30(1):56-58.
[9]Lei H, Minghao W, Xiaonan Y, etal. Acute lung injury in patients with severe acute pancreastitis[J]. Turk J Gastroenterol.2013,24(5):502-507.
[10]陈作宾.白蛋白对创伤性休克大鼠肺脏保护机制研究[J].浙江大学学报,2009,12(11)：87-89.
[11]中华医学会重症医学分会,急性肺损伤／急性呼吸窘迫综合征诊断和治疗指南[J].中华急诊医学杂志,2007,16(4)：343-349.
[12]太平惠民和剂局方,人民出版社(第3版)：1078-1085.
[13]陈晓丽,李繁盛,张栋,等.凉膈散在临床的应用探讨[J].中华实用中西医杂志,2005,18(5)：517-519.
[14]余林中,林慧,胡孔友等.凉膈散对家兔内毒素温病模型体温、血浆TNF-α及 脑脊液PGE2、cAMP含量的影响[J].中国中医药科技,1996,3(4)：26-27.
[15]江爱达,余林中.凉膈散对LPS诱导的巨噬细胞丝裂素活化蛋白激酶p38活性的影响[J].中国中医基础医学杂志,2005,11(7)：511-513,518.
[16]林慧,余林中.凉膈散含药血清对内毒素刺激RAW264.7细胞CD14mRNA表达的影响[J].中药药理与临床,2005,21(3)：1-3.
[17]江爱达,余林中,胡孔友等.凉膈散药物血清对脂多糖诱导体外培养巨噬细胞核转录因子-κB的影响[J].第一军医大学学报,2005,25(6)：619-622.
[18]余林中,林慧.凉膈散对内毒素血症小鼠肝脏库普弗细胞CD14和清道夫受体表达的影响[J].中国中药杂志,2006,31(3)：220-223.
[19]余林中.凉膈散对家兔内毒素温病模型的解毒作用研究[J].中药药理与临床,1996,12(5)：4-6.
[20]余林中.凉膈散对小鼠内毒素血瘀模型微循环的影响[J].中药药理与临床,1996,12(2)：1-3.
[21]余林中,林慧,江爱达,等.凉膈散对家兔内毒素温病模型的化瘀作用研究[J].中药药理与临床,1998,14(1)：7-9.
[22]林慧,余林中.凉膈散对内毒素肺损伤小鼠肺组织核因子-κB活性的影响[J].四川中医,2004,22(7)：16-18.
[23]胡孔友,余林中,刘建新,等.凉膈散对内毒素致大鼠急性肺损伤的保护作用[J].中药药理与临床,2008,24(5)：3-5.
[24]胡孔友,刘建新,余林中,等.凉膈散对内毒素大鼠急性肺损伤炎症调控的影响[J].中华中医药杂志,2009,24(11)：1433-1436.
[25]胡孔友,刘建新,余林中,等.凉膈散对内毒素致大鼠急性肺损伤Th1/Th2漂移的调节作用[J].中医杂志,2009,12(1)：34-37.
[26]Weifeng Song,Sadis Matalon,etal,Modulation of alveolar fluid clearance by reactive oxygen-nitrogen intermediates[J]. Am J Physiol Lung Cell Mol Physiol. 2007,293(12):855-858.
[27]Gavin d perkins, Fang Gao and David R Thickett, etal. In Vivo and In vitro effects of Salbutamol upon alveolar epithelial repair in acute lung injury [J]. Published online.2007,Thorax,doi:10.1136/thx.2007.080382.
[28]Coates G,Brodovich H,Jefferies AL,etal. Gray GW. Effects of exercise on lung lymph flow in sheep and goats during normoxia and hypoxia[J]. Clin Invest.1984;74:133-141.
[1]张卫琴.免疫组化技术在病理诊断中的应用[J].安徽医药,2012,16(11)：1700-1703.
[2]Hardiman KM, Matalon S. Modification of sodium transport and alveolar fluid clearance by hypoxia:mechanisms and physiological implications[J]. Am J Respair Cell Mol Biol,2001,25(5):538-541.
[3]Matalon S, Lazrak A, Lucky J,etal. Lung clearance:20 years of progress invited review:Biophysical properties of sodium channels in lung alveolar epithelial cells[J]. J Appl Physiol,2002,93:1852-1859.
[4]Amin MS, Reza E, Shahat E,etal.Enhanced expression of epithelial sodium channels in the renal medulla of Dahl S rats[J]. Can J Physiol Pharmacol. 2011,89(3):159-68.
[5]Modelska K, Matthay MA, Pittet JE,etal.The effect of endogenous angiotensin Ⅱ on alveolarfluid clearance in rats with acute lung injury[J],Can Respir,2012, 19(5):311-318.
[6]Mairbaurl H, Mayer K, Kim KJ,etal.Hypoxia decreases active Na+transport across primary rat alveolar epithelial cell monolayers[J]. Am J Physiol Lung Cell Mol Physiol,2002,282(4):L659-665.
[7]Todd C, Carpenter S, Schomber G,et al. Hypoxia reversibly in-hibits epithelial sodium transport but does not inhibit lung ENaC or Na+-K+-ATPase expression[J]. Am J Physiol Lung Cell MolPhysiol,2003,284:L77-L83.
[8]李王平,张芳,李萌,等.肺泡上皮细胞钠钾ATP酶与海水淹溺型肺水肿[J].现代生物医学进展,2008,8(1)：158-160.
[9]Sathya M, Kokilavani R, Teepa K S, etal,Biopotency of Acalypha indica Linn on Membrane Bound ATPases and Marker Enzymes urolithic Rats[J]. Anc Sci Life.2011,31(1):3-9.
[1]张维,等.肺泡上皮细胞与急性肺损伤水肿液清除的研究进展.中国呼吸与危重监护杂志,2008；7(1)：74-76.
[2]Qiu HB, Sun HM, Yang Y, etal Effect of beta-adrenergic agonist on alveolar fluid clearance in acute lung injury:an experiment with rats [J]. Zhonghua Yi Xue Za Zhi,2008,86(3):187-191.
[3]Knowels M R, Boucher RC. Mucus clearance as a primary innatedefense mechanism for mammalian airways[J]. J Clin Invest,2002,109(5):571-577.
[4]Hardiman K M, Matalon S. Modification of sodium transport and alveolar fluid clearance by hypoxia:mechanisms and physiological implications [J]. Am J Respair Cell Mol Biol,2001,25(5):538-541.
[5]Kellenberger S, Gautschi I, Schild L. Mutations in the outer pore disrupt amiloride block by increas-ing its dissociation rate[J]. Mol Pharmacol,2003, 64(4):848-856.
[6]Nyder PM. Na+ -K+-ATPase:cell surface insertion and retrival in Na+homeostasis and hypertension[J]. Endocr Rev,2002,23(2):258-275.
[7]DOnorfio D,Chimuello D,et al.Pulmonary and exrtpaulmony acute respiratory distress syndl-rome arc different [J],Eur Respir,2003,22(42):48-56.
[8]Fan YM, Huang XL, Dong ZF,et al. Hydrogen sulfide reduces lipopolysaccharide-induced acute lung injury and inhibits expression of phosphorylated p38 MAPK in rats [J], Sheng Li Xue Bao,2012,64(6):666-672.
[9]Fan YM, Huang XL, Dong ZF,et al. Hydrogen sulfide reduces lipopolysa ccharid -einduced acute lung injury and inhibits expression of phosphorylated p38 MA-PK in rats [J], Sheng Li Xue Bao,2012,64(6):666-672.
[10]郭亮,李楠.水通道蛋白与急性肺损伤[J].华南国防医学杂志,2009,23(1)：81-84.
[11]许海波.水通道蛋白-3的研究进展[J].四川解剖学杂志,2006,14(2)：28-31.
[12]李波,陈东,王桂芳,等.水通道蛋白1、3、4、5在内毒素性急性肺损伤小鼠肺组织中的表达[J].第二军医大学学报,2008,29(2)：131-135.
[13]Sathya M, Kokilavani R, Teepa K S, etal,Biopotency of Acalypha indica Linn on Membrane Bound ATPases and Marker Enzymes urolithic Rats[J]. Anc Sci Life.2011,31(1):3-9.
[1]Kahn HJ, Yang LY, Blondal J, etal. RT-PCR amplification of CK19mRNA in the blood of breast cancer patients:correlation with established prognostic parameters [J]. Reast Cancer ResTreat,2000,60(2):143-151.
[2]Klein D. Fredriksson R1, Schioth HB.Quantification using real-time PCR technology applications and limitations[J].Trends Mol Med,2002,8(6):257-260
[3]冯志新,姜平.猪细胞因子SYBR Green实时PCR检测方法的建立[J].现代生物医学进展,2007,7(1)：108-121.
[4]陈琳,姜泊,张亚历,等.运用qRT-PCR和westernblot检测elaudin-1在人类大肠癌细胞株的表达[J].广西医科大学学报,2Q07,24(2)：205-207.
[5]习梅,王立荣,王战勇,等.用SYBR Green I实时荧光PCR结合融解曲线分析 法诊断a-地中海贫血[J].中华医学遗传学杂志,2007,24(2)：192-196.
[6]陈英剑,胡成进,赵苗青.SYBR-GreenI实时荧光定量PCR技术平台的建立[J].实用医药杂志,2004,21(11)：997-1000.
[7]Przybycien-Szymanska MM, Rao YS, Prins SA, etal, Parental binge alcohol abuse alters fl generation hypothalamic gene expression in the absence of direct fetal alcohol exposure[J]. PLoS One.2014,9(2):1154-1164.
[8]Alsio J, Rask-Andersen M1, Chavan RA, etal, Exposure to a high-fat high-sugar diet causes strong up-regulation of proopiomelanocortin and differentially affects dopamine D1 and D2 receptor gene expression in the brainstem of rats[J]. Neurosci Lett.2014,13(1):125-131.
[9]Pires-Oliveira M1, Maragno AL, Parreiras-e-Silva LT, etal, Testosterone represses ubiquitin ligases atrogin-1 and Murf-1 expression in an androgen-sensitive rat skeletal muscle in vivo.[J]. Appl Physiol. 2010,180(2):266-273.
[10]Liu FF1, Ma ZY, Li DL, etal, Differential expression of TRPC channels in the left ventricle of spontaneously hypertensive rats [J]. Mol Biol Rep..2010, 37(6):2645-2651.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |