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Objectives and significance:Acute lung injury (ALI) is a kind of clinic syndrome with pathological changes in the lung tissue structure caused by various factors such as serious infection, trauma, shock and aspiration, etc. Permeable pulmonary edema is a kind of its common clinic syndrome. The main reason of permeable pulmonary edema can be interpreted that pulmonary microvascular endothelial cells (PMEC) and pulmonary epithelial cells were injured, the liquid balanced of lung was be destructed, the lung water barrier suffers injury, the permeability advances, which caused the decreased water transport function between airspace, interstitial and capillary compartments, and then accumulating water in interstitial and alveolar spaces. 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、5 in 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 Group and model making156 SPF level healthy Wistar rats (weight 150-180g) were randomly divided into seven groups,the control group (ig NS, iv NS)、the acute lung injury group (ig NS, iv LPS 5mg/kg), LPS+Lianggesan three different treatment groups (ig 7.5,15,30g/crude drug/kg, iv LPS 5mg/kg), LPS+DEX treatment group (ig DEX 0.135mg/kg, iv LPS 5mg/kg). Establish rat ALI model by injecting LPS 5mg/kg (control group rats were intravenously injected with NS) into rats vein. Each LPS group was subdivided into five small groups according to the time of endotoxin administrated at 1,2,4,8h and 16h,6 in every group.The course of intragastric administration in all groups lasted 5 days. After the last dose of the drugs, rat ALI model was established by injection of lipopolysaccharide 5mg/kg into tail, control group rats were intravenously injected with NS. All rats were killed corresponding time,the tissue and blood were observed.2 The protective effect of Lianggesan on rat with acute lung injury pulmonary edema induced by endotoxinAccording to the method of establishing the model of ALI, rats in each group were anesthetized by intraperitoneal injection of 10% chloral hydrate after intravenously injected with NS or LPS corresponding time, and then 2ml blood was observed through abdominal aorta in order to determinate the whole blood viscosity in 150s-1,30s-1,5s-1, 1s-1 by blood viscosity tester (R80); Observe and weigh the whole lung, get lung index (LI) by calculate ratio of total lung weight/ body weight; Observe and get the wet weight (W) of part of right lung, then dry it with oven (80℃, 48h) and get the dry weight (D), W/D ratio of right lung was mesaured carefully; Put the left part of right lung into 10% formaldehyde for pulmonary pathology and immunohistochemistry, and save left lung in-80℃refrigerator for Western Blot.3 The effect of Lianggesan on the expression of AQP-1、AQP-5 in the Endotoxin-induced acute lung injury in rat3.1 The changes of AQP-1、AQP-5 expression in the lungs of rats were analyzed by immunohistochemistry (SP)3.2 The changes of AQP-1、AQP-5 expression in the lungs of rats were analyzed by western blot4 Statistics analysisData are analyzed by SPSS13.0 and expressed as means±SE((?)±s). The consequence of Wet-to-dry weight ratio, lung index, immunohistochemical expression and western blot analysis expression of AQP-1、AQP-5 were analyzed by factorial analysis. Whole blood viscosity results were analyzed by repeated measures analysis of variance and then factorial analyzed the two repetition factors which had been fixed. In effect of separate, data were analyzed by One-way ANOVA when meeting the homogeneity of variance, but by Welch when missing homogeneity of variance. In multiple comparison, LSD was used when data in each group met the homogeneity of variance, or Dunnett’s T3 when missed the homogeneity of variance. P value less than 0.05 was considered to be statistically significant.Results:1 The protective effect of Lianggesan on rat with acute lung injury pulmonary edema induced by endotoxin1.1 The change of wet-to-dry weight ratioSignificant difference of W/D ratio existed within each group (F=64.453, P=0.000) and within each time point (F=88.758,P=0.000), and there was interaction between the parameters group and time point. (F=8.286,P=0.000). When compared to the control group, the analysis of the effect of different time-point within each group revealed that the ratio of W/D apparently raised 2 hours after being induced by the LPS and progressively raised later. (P<0.05). Compared to the LPS group, there were significant declines of W/D ratio in the 2h-time-point high dose Lianggesan group and DEX group, in the high dose, middle dose and low dose Lianggesan groups and DEX group at various 4h,8h, and 16hour points (P<0.05).1.2 The change of lung index(LI)Significant difference of lung index existed within each group (F=56.477, P=0.000) and within each time point (F=101.092,P=0.000), and there was interaction between the parameters group and time point (F=5.912,P=0.000). When compared to the control group, the analysis of the effect of different time-point within each group revealed that the LI apparently raised 2 hours after being induced by the LPS and progressively raised later. (P<0.05). Compared to the LPS group, there were significant declines of LI in the 2h-time-point high dose Lianggesan group and DEX group, in the high dose, middle dose and low dose Lianggesan groups and DEX group at various 4h,8h, and 16hour points (P<0.05).1.3 The change of blood viscositySignificant difference existed within groups with various sheer rates (F=3250.550, P=0.000). there were interactions within the parameters of sheer rate, group (F=17.984, P=0.000) and time point(F=54.277, P=0.000). To fix the sheer rate, factorial analyses were performed in different groups and various time points, significant differences existed in different groups(150s-1, F=20.937, P-0.000; 30s-1, F=19.783, P=0.000; 5s-1, F=12.168, P=0.000;1s-1, F=21.364, P=0.000) and in different time points(150s-1, F-84.014, P=0.000; 30s-1, F=77.053, P=0.000; 5s-1, F=65.249, P=0.000; 1s-1, F=70.784, P=0.000), and there was interaction between parameters of group and time point (150s-1, F=7.045, P=0.000; 30s-1, F=6.578, P=0.000; 5s-1, F=5.533, P=0.000; 1s-1, F=6.083, P=0.000). After the damage induced by LPS in rats, the analysis of individual effect in different groups revealed the lh and 2h blood viscosity in the LPS group measured at different sheer rates (150s-1、30s-1、5s-1) was all significantly higher than that of the control group (P <0.05). The 1h blood viscosity measured under the 1s-1 sheer rate are significantly higher than that of the control group(P<0.05). No significant difference of the 4h blood viscosity existed when measured at different sheer rates. In the 8h and 16h point blood viscosity measured under the different sheer rates are significantly lower than that of the control group(P<0.05). Compared to that of the control group, the blood viscosity measured at different sheer rates was significantly higher in the 1h point the high dose, middle dose and low dose Lianggesan groups,2h point high dose and middle dose Lianggesan groups and the 4h point the high dose Lianggesan group. No significant difference of the blood viscosity measured at different sheer rates existed in the 1h,2h,4h point when compared to that of the DEX group. Compared to that of the LPS group, the blood viscosity measured at different sheer rates was significantly higher in the 8h point the high dose, middle dose and low dose Lianggesan groups, the DEX group and the 16h point the high dose, middle dose lianggesan groups, the DEX group, but showed no significant difference to that of the control group.1.4 Situation of histopathology of lungWhole lung observation in LPS group:the lungs in the two sides enlarged with obvious pulmonary edema 2h 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.2 The effect of Lianggesan on the expression of AQP-1、AQP-5 in the Endotoxin-induced acute lung injury in rat2.1 The results of AQP-1 were analyzed by immunohistochemistryOutcomes indicated that the AQP-1 was mainly expressed in the pulmonary vessels and II-type cells of alveolar and secondarily expressed in the bronchial endothelium. In the control group, the AQP-1 was over-expressed with a yellow color. Significant difference of expression of AQP-1 existed within groups (F=15.109,P=0.000), so did that at various time points(F=3.338,P=0.012), there was no interaction between parameter of group and time point (F=0.384,P=0.992). The analysis of the main effect within groups indicated:significant difference of expression of AQP-1 existed between the control group and the LPS group. (P< 0.05). compared to the LPS group, expression of AQP1 was stronger in the 1 high dose, middle dose and low dose Lianggesan groups, and in the DEX group. (P< 0.05).2.2 The results of AQP-5 were analyzed by immunohistochemistryThe outcome showed that AQP-5 was mainly expressed in the membrane of I-type alveolar, and was over-expressed in the control group with a yellow color. Significant difference of expression of AQP-5 existed within groups (F=14.056,P=0.000), so did that at various time points(F=5.831,P=0.000), and there was no interaction between the parameters of group and time point. (F=0.656, P=0.863) The analysis of the main effect within groups indicated:significant difference of expression of AQP-5 existed between the control group and the LPS group. (P<0.05). compared to the LPS group, expression of AQP-5 was stronger in the high dose, middle dose and low dose Lianggesan groups, and in the DEX group. (P<0.05).2.3 The results of AQP-1 were analyzed by western blotSignificant difference of expression of AQP-1 existed within groups (F=22.822, P=0.000), so did that at various time points (F=12.152,P=0.000), and there was no interaction between the parameters of group and time point (F=0.822,P=0.684). The analysis of the main effect within groups indicated:significant difference of expression of AQP-1 existed between the control group(0.65±0.08) and the LPS group. (P<0.05). Compared to the LPS group, expression of AQP1 was stronger in the high dose, middle dose and low dose Lianggesan groups, and in the DEX group. (P<0.05).2.4 The results of AQP-5 were analyzed by western blotSignificant difference of expression of AQP-5 existed within groups(F24.245, P=0.000), so did that at various time points (F=22.809,P=0.000), and there was no interaction between the parameters of group and time point (F=1.299,P=0.684188). The analysis of the main effect within groups indicated:significant difference of expression of AQP-5 existed between the control group and the LPS group. (P< 0.05). compared to the LPS group, expression of AQP-5 was stronger in the high dose, middle dose and low dose Lianggesan groups, and in the dexamethasone group. (P<0.05).Conclusion:1 W/D and LI increased in the acute lung injury induced 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.2 The results of the immuohistochemistry and western blot revealed that expression of AQP-1 and AQP-5 in lung tissue declined in the acute lung injury induced by endotoxin. Lianggesan can improve the fluid transportation and further on alleviate pulmonary edema by up-manipulating the expression of AQP-1 and AQP-5.
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