Protective Effects Of Lianggesan On LPS-induced Acute Lung Injury In Rats And Studies On Regulation Inflammatory Mechanism

Objective Acute lung injury(ALI) and acute respiratory distress syndrome(ARDS) are common disease with high mortality and greatly threaten patients' health.The earlier symptoms of ARDS reflect,the permeability between pulmonary capillary endothelial cell and alveolar epithelium barrier is increased,and both lung alveolar and interstitium are filled with massive edema fluid,which is rich in protein and a variety of inflammatory cells.At present,as the effective and safe means of prevention and treatment are not available,ALI becomes one of the difficulties in Critical Care Medicine.There are advantages of the integrated traditional Chinese and western medicine to prevent and treat ALI.According to some scholars,Chinese medicine theory and method have shown a potential prospect in treating SIRS,ALI and ARDS.With the development of traditional Chinese medicine theory of seasonal febeile disease pathogenesis and modern pathophysiology,people become aware of the progression of ALI and its prevention and treatment,for example,it provides new strategy and methods to treat and prevent ALI.Clinical investigations have suggested that lung was the most sensitive target organ.ALI and ARDS appear early with the highest incidence.Except for factors directly damage to lung,the reasons for pulmonary dysfunction include a variety of cells in lung tissue,all the blood of systemic circulation,capillary network,a number of endothelial structures and various harmful factors and mediators in blood.Similar to the pathogenesy of ALI caused by various reasons,It is a inflammatory reponse based on acute injury of alveolar-capillary membrane.The basis of ALI is SIRS,and ALI also promotes SIRS.The main cause of SIRS and ALI is the accumulation and activation of inflammatory cells,the excessive synthesis and release of inflammatory mediators,immune dysfunction,and out-of-control inflammation.In essence,inflammation is the defensive reaction,which could damage its own cells when out-of-control,whether inflammation keep relative balance depends on response between inflammation and anti-inflammatory.ALI as the consequences of excessive inflammatory response,the key to reduce mortality and prevent the occurrence of ALI was regulation inflammatory response.So far,many medicines are in clinical trials,such as glucocorticoids,cyclooxygenase inhibitors,thromboxane synthetase inhibitors,phosphodiesterase inhibitor,anti-endotoxin and cytokines,such as monoclonal antibodies or antagonists et al.However,these drugs has not had positive effects in clinical trials of large sample,so it still lacks a specific and effective treatment.The pathogenesis of ALI was not yet clear,but the recognition that macrophages were activated to raise many nuclear leukocyte(PMN) caused to the pulmonary inflammatory response plays a dominant role.SIRS and compensatory anti-inflammatory response syndrome(CARS) induced LPS after GNB infection, excessive SIRS is the development of multiple organ dysfunction syndrome(MODS) which can induce ALI.When acute lung injury happened,intrapulmonary almost all types of cells are involved in the inflammatory response and the release of many inflammatory mediators,however,pulmonary vascular endothelial cells and lung epithelial cells is the effector cell as well as target cell which is a pathogenic factor. There are two tyes of cytokines:proinflammatory cytokines include:TNF-α,IL-1, IL-6,PAF,and PLA2,etc.and anti-inflammatory cytokines:IL-4,IL-10,IL-13, IL-1ra and so on,whose balance can maintain the stability of environment.But unbalance in two types of cytokines would lead to further amplification of inflammation cascade and eventually caused tissue or organ lesion. It have many targets,and the traditional Chinese medicine has unique advantages on lung injury induced by LPS.In Warm Disease theory,toxin is an important factor and pathological core in determination of the occurrence, development and turnover of disease.It either direct intrusion from outside,or influence by the "six exopathogens".The main characteristics of the syndrome caused by "toxin" are as follows:acute pathogenic,heavy pathogenetic condition,faster transmission of a disease and so on.Chinese medicine deintoxication is the most frequent treatment for Warm Disease especially syndrome pyretic toxicity.It has been enriched and developed at the long-term medical practice,which results in methods for a heat-clearing and detoxifying,cooling blood detoxification,cooling yingfen detoxification,Tongfu detoxification,increasing gas detoxification,etc,and the scope of its application have been expanded.We believe that detoxification methods used early could alleviate the condition in ALI including many pyretic toxicity syndromes,the so-called "detoxification appropriate early." Research of Chinese medicine detoxification role and mechanism mainly embodied in the original disease-resistant microorganisms,the anti-toxin,the immune regulatory,the relieve fever,the anti-inflammatory effects,improving the microcirculation,and anti-free radical damage and so on.Lianggesan(contained Song "He Ji Ju Fang") is the common famous complex prescription of warm disease,with the effect of kiyokami,purgation and detoxication and elimination of toxicant.In previous studies,we studied the mechanism of detoxification from the signal transduction of effector cells in the production of cytokines regulation,in which we found Lianggesan can inhibit the enhancement of expression of endotoxin-induced murine macrophage CD14, p38MAPK,nuclear transcription factor—κB(NF-κB),while inhibition of the expression of inflammatory cytokines TNF-αand IL-6 in mice,reducing the liver, lung tissue damage.Based on the above understanding,we propose:guided by the theory of epidemic febrile disease,the use of traditional Chinese medicine detoxification method may be an effective method to intervent ALI pathological process.Lianggesan maintains the dynamic equilibrium of systemic inflammatory and anti-inflammatory response through many effective components,multi-signal transduction pathway and inflammatory response regulation by a number of effect target,while this comprehensive therapeutic effects are beneficial to treat ALI.Methods:1.group and model makingWistar rats were randomly divided into six groups,the control group,the acute lung injury group(Group LPS),LPS+Lianggesan three different treatment groups(group7.5,15,30g/crude drug/kg),LPS+DEX treatment group(group 0.135mg/kg).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.All rats were killed corresponding time,the tissue and blood were observed.3.Detection index3.1 Arterial blood PaO₂3.2 Wet-to-dry weight ratio and lung index determination3.3 Lung permeability index determination3.4 Situation of histopathology of lung3.5 Content of TNF-α,IL-1β,IL-10 determination3.6 Th1/Th2 cytokine determination4.Statistics analysisData are expressed as means±SE((?)±s).The consequence of Arterial blood PaO₂,Wet-to-dry weight ratio,lung index,Lung permeability index and Content of cytokine was analyzed by One-way ANOVA and multiple comparison,and P value less than 0.05 was considered to be statistically significant. Results1.The change of arterial blood PaO₂(mmHg)Concentration of oxygen inhalation is 21%in all rats.Comparison by PaO₂ instead of oxygenation index.In LPS group the levels of PaO₂ at 4,8,16h was significantly decreased in comparison with the control group(P＜0.01).and PaO₂ was increased in DEX and three doses of Lianggesan groups compared with the LPS group(P＜0.05)2.The change of wet-to-dry weight ratioIn LPS group the ratio of W/D at 4,8,16h was significantly increased in comparison with the control group(P＜0.01).and W/D was decreased in DEX and three doses of Lianggesan groups compared with the LPS group(P＜0.05,P＜0.01)3.The change of lung index.In LPS group the levels of lung index at 4,8,16h was significantly increased in comparison with the control group(P＜0.01).and LI was decreased in DEX and three doses of Lianggesan groups compared with the LPS group(P＜0.05,P＜0.01)4.The change of lung permeability indexIn LPS group the levels of lung permeability index at 4,8,16h was significantly increased in comparison with the control group(P＜0.01).and LPI was decreased in DEX and high dose and mid-dose Lianggesan groups compared with the LPS group (P＜0.05).5.Situation of histopathology of lungLight microscopic observation:In blank control group,pulmonary alveoli presented continuous structure and intact wall,without obvious exudation.After 4h whole lung observation in LPS groups:lung volume increased,punctate hemonhage on the surface.The manifestation of pulmonary edema and bleeding point on the surface increasing at 8,16h.interalveolar septum thickening,hemorrhage,edema and infiltration of lots of inflammatory cells were observed.The lesion degree and effusion were weaker in DEX and three doses of Lianggesan groups than LPS group.6.Content of TNF-α,IL-1β,IL-10 determinationIn LPS group the levels of TNF-αbegan to increase at 1h,which reached the peaks at 2 h,then declined significantly;IL-1βexpression tended to elevate at 2 h, peaked at 8 h,then declined dramatically;IL-10 expression significantly enhanced at 2 h.But the level of TNF-α,IL-1βwere significantly decreased and level of IL-10 was increased in DEX and three doses of Lianggesan groups compared with the LPS group(P＜0.05,P＜0.01).7.Th1/Th2 cytokine determinationDuring or after 4h observation in LPS groups,we determined Th1 in peripheral blood and in BALF was significantly decreased and Th2 was significantly increased in comparison with the control group(P＜0.05,P＜0.01),indicating that LPS makes CD4+T cells drift from Th1 to Th2,and the ratio of Th1/Th2 cytokine is close to a normal level in DEX and high dose and mid-dose Lianggesan groups(P＜0.05,P＜0.01).Conclusion1.Lianggesan could significantly decreased wet-to-dry weight ratio,lung permeability and increased PaO₂ induced by LPS.Results from light microscopy showed that it could reduce inflammatory cell accumulation,alveoli of lung deteleotasis and consolidation of lung.2.Lianggesan could significantly decreased the expression of mediators of inflammation TNF-α,IL-1βand increased the expression of IL-10 induced by LPS. 3.Lianggesan could significantly enhanced the content of Th1 and decreased the expression of Th2 induced by LPS,and the ratio of Th1/Th2 cytokine is close to a normal level.The results suggest that Lianggesan plays a protective role in the endotoxin-induced ALI.The mechanisms of actions may be attributed to its anti-inflammatory activity that inhibits the release of pro-inflammatory cytokines, rectification imbalance between inflammatory and anti-inflammatory.