The Effect And Mechanism Of Lianggesan On Fluid TransPortation In Endotoxin Acute Lung Injury Tissue

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�'�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.