The Effect And Mechanism Of Oxymatrine On Pneumonia Induced By Pseudomonas Aeruginosa Lipopolysaccharide

Objective:Pneumonia is an acute pulmonary inflammation caused by various factors,including pathogenic microorganism,physical and chemical factors,immune injury,allergy and drugs,and the bacterial pneumonia is the most common one.Pseudomonas aeruginosa(PA),as one Gram-negative pathogens,is widely recognized as the main nosocomial pathogens for patients with pneumonia in intensive care unit.As an opportunistic pathogen,PA can lead to lung inflammation in delay and the damage of lung structure by stimulating respiratory epithelial cells,which can cause respiratory persistence pathological damage,thereby resulting in serious respiratory tract symptom and pulmonary dysfunction in PA infectious patients.The morbidity of PA pneumonia trends to raise,while excessive or poor treatments for PA pneumonia exist in clinical practice,and the main reasons are:(1)PA may colonize in damp environment,such as perineum,axillary and ears,and PA can directly enter into the lower respiratory tract through mechanical ventilation or nasogastric tube;(2)PA has natural resistance to multiple antibiotics mediated by chromosome,and transfer drug resistance between or within species by the intracytoplasmic resistance plasmid,which may induce multi-drug resistance due to easy variation during antibiotic therapy.Thus,it is essential to invietigate the mechanism of PA induced lung injury.Lipopolysaccharide(LPS)from PA can activate a variety of immune cells such as natural killer cell,macrophage and lymphocyte,which can stimulate the secretion of various cytokines from host cells,including colony stimulating factor,interferon,tumor necrosis factor-?(TNF-?)and interleukin,thereby regulating the immune function of the body.Currently,acute lung injury animal models induced by LPS have been confirmed and widely applied into mechanism research of acute lung injury.However,effective drug treatments are still lacking for PA-LPS induced lung injury.Oxymatrine(OMT)is one alkaloid extractd from the root of traditional Chinese medicine sophora,which has a variety of important biological functions,and can effectively attenuate fibrosis and inflammation as well as inhibit the proliferation of cancer cells.Recently,many studies have focused on the curative effect of OMT on pulmonary disease.Previous studies have revealed that OMT exerts an anti-asthmatic effect in allergic asthma mice model,protects against from paraquat induced lung injury.However,the effect and mechanism of OMT on PA-LPS induced lung injury are still unclear.A study reveales that the transport and clearance of alveolar fluid can regulated by aquaporins(AQPs),Na~+-K~+-ATPase and epithelial Na channel(ENaC).ENaC in alveolar epithelial cells is essential for the effective treatment of pulmonary edema.Previous study find that PA and LPS from PA can induce decreased expression and activity of ENaC in alveolar epithelial cells.Meanwhile,the expression of ENaC is significantly reduced in LPS-induced acute lung injury model.Thus,we speculated that PA-LPS may influence the balance of sodium flow and inflammation by regulating ENaC.Previous studies reveal that OMT exerts an inhibitory effect on the activation of mitogen-activated protein kinase(MAPK)pathway in myocardial tissue and microglial cell,while the inhibitor of MAPK can block the down-regulation of ENaC caused by LPS.MAPK is a class of serine/threonine protein kinase,and play a crucial role in regulating various signaling pathways related to cell proliferation and inflammation.Therefore,we speculated that OMT might regulate the activity of ENaC via MAPK pathway,and then influenced PA-LPS induced lung injury.To verify the above hypothesis,we established injured alveolar epithelial cells model induced by PA-LPS and PA-LPS induced lung injury mice model,meanwhile these model were pre-treated with OMT.Under different treatments,the degree of lung edema,the lung tissue morphology,the contents of inflammatory cells in bronchoalveolar lavage fluid and the expressions of inflammatory factors were detected.In addition,pneumonia mice were interfered with the inhibitor of ENaC,and then we detected the expressions of ENaC and MAPK related proteins,which aimed to explore the mechanism of OMT in PA-LPS induced lung injury.Methods:The establishment of pneumonia mice model was induced by PA-LPS,meanwhile,pneumonia mice received the administration of OMT with various doses(12.5,25,50 mg/kg).After treatment with LPS for 6 h,the degree of lung edema was detected in 3 randomly-selected mice in each group by lung wet/dry weight method.Meanwhile,the lung tissues were collected in each group,then lung histological morphologic changes and inflammatory injury were observed using HE staining;the contents of inflammatory cells in bronchoalveolar lavage fluid were measured by Wright-Giemsa staining;the expressions of TNF-?,C-reactive protein(CRP),interleukin1?(IL1?),and IL6 were detected in lung tissue using ELISA.Furthermore,primary alveolar epithelial cells were separated from SD rats,and identified by cell immunofluorescence.Then,primary rat alveolar epithelial cells were treated with LPS and/or OMT with various doses(250,500,1000?g/ml).Cell proliferation was measured after treatment for 6 h,12 h,and 24 h using MTT assay.After treatment for 24 h,the cells in each group were collected,then the mRNA levels of ENaC related genes were detected using real-time PCR;the protein levels of ENaC and MAPK related proteins were detected using western blotting.Statistical analyses were performed by the SPSS 17.0software.Data in various goups were expressed as mean±SD.The differences between groups were evaluated by a one-way analysis of variance(ANOVA)followed by pair-wise comparison with the Student-Newman-Keuls test.P<0.05 was considered statistically significant.Results:1.Pneumonia mice models induced by PA-LPS were successfully established.Compared with normal mice,the ratios of lung wet/dry weight were elevated,inflammatory cell infiltration was increased,the contents of inflammatory cells in bronchoalveolar lavage fluid as well as the expressions of TNF-?,CRP,IL1?,and IL6were increased in PA-LPS induced pneumonia mice.However,the pre-treatment of OMT showed decreased ratios of lung wet/dry weight,weakened inflammatory cell infiltration,the reduced contents of inflammatory cells in bronchoalveolar lavage fluid as well as the expressions of TNF-?,CRP,IL1?,and IL6.2.Primary type II alveolar epithelial cells were successfully separated from SD rats.Compared with the control group,LPS treatment inhibited the cell proliferation.The results in vivo and in vitro revealed that compared with the control group,LPS treatment significantly inhibited the mRNA and protein levels of?ENaC,?ENaC,?ENaC,AQP-1and AQP-5 while OMT could increase the levels of these proteins.Conversely,Benzamil,the inhibitor of ENaC,could inhibited the role of OMT.3.The results in vivo and in vitro revealed that compared with the control group,LPS treatment significantly promoted the protein levels of p-ERK,p-JNK and p-p38 MAPK,while OMT treatment could inhibit the l protein levels of p-ERK,p-JNK and p-p38MAPK.Conclusion:1.PA-LPS induced pulmonary edema and neutrephil infiltration,as well as increased the contents of inflammatory and the expression of TNF-?,CRP,IL1?,and IL6in bronchoalveolar lavage fluid in mice.2.OMT inhibit pneumonia induced by PA-LPS in mice in concentration-dependent manners.3.OMT can promote the up-regulation of ENaC and AQP,thereby alleviating alveolar epithelial cell injury and pneumonia induced by PA-LPS in mice.4.Benzamil,the inhibitor of ENaC,can neutralize the anti-pneumonia effect of OMT,indicating that the anti-pneumonia effect of OMT is depend on ENaC.