acute lung injury ( ali ) is characterized by atelectasis of lung airspaces , reducing the total lung capacity . in ali cases , the lung tissue releases large amounts of inflammatory cytokines , chemokines , and inflammatory media , such as nitric oxide and cyclooxygenase 2 ( cox-2 ) . ali also causes lung cells to secrete more proteases , leading to alveolar cell damage and fluid accumulation in alveoli , causing edema in the lung tissue . alveolar damage results in increased vascular permeability of blood vessels and neutrophil infiltration in the lung tissue , further exacerbating lung inflammation and pulmonary edema and potentially causing respiratory failure and death . ali commonly occurs as an early symptom of sepsis or bacterial infection of the respiratory tract . lipopolysaccharide ( lps ) is a cell wall component of gram - negative bacteria and can induce an innate immunity response , activating immune cells to combat microbial infection . thus , lps is widely used to induce acute lung inflammation in animal models . activated macrophages and prior studies have used lung epithelial cells to evaluate the anti - inflammatory responses of drugs or natural compounds during lps - induced inflammatory response [ 7 , 8 ] . in china and taiwan , the root of sophora flavescens ( leguminosae ) is used to treat fever , jaundice , urinary tract infections , and pyogenic infections . matrine is isolated from s. flavescens and can reportedly attenuate cerebral ischemic injury in mice and induce apoptosis of chronic myeloid leukemia cells , osteosarcoma cells , and cholangiocarcinoma cells [ 9 , 10 ] . zhang et al . previously demonstrated that matrine suppressed proinflammatory cytokine production in lps - stimulated mouse macrophages . previously , our group also found that matrine could improve eosinophil infiltration , airway hyperresponsiveness , and th2-associated cytokine production in asthmatic mice . however , the mechanisms of matrine 's anti - inflammatory activity in lung epithelial cells are largely unclear . in the present study , we investigated whether matrine reduced inflammatory responses and production of intercellular adhesion molecule-1 ( icam-1 ) in lung epithelial cells . we also examined whether matrine protected and prevented acute lung injury in lps - induced mice . matrine ( 99% by hplc ) was purchased from sigma - aldrich ( st . louis , mo , usa ) and was dissolved in normal saline as previously described . the human lung epithelial cell line a549 was purchased from the bioresource collection and research center ( bcrc , taiwan ) . a549 cells were cultured in dmem medium ( invitrogen - gibco , paisley , scotland ) containing 2  mm glutamine , 100  u / ml penicillin and streptomycin , and 10% fetal bovine serum ( biological industries , haemek , israel ) . cells were incubated at 37c in 5% co2 humidified air and were subcultured twice each week . cell viability was assayed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ; sigma ) as previously described . in 96-well culture plates , a549 cells were treated with various matrine concentrations for 24  h. the plates were then washed , mtt solution was added , and the plates were incubated at 37c for 4  h. formazan crystals were dissolved in isopropanol , and cell viability was spectrophotometrically measured at 570  nm with a microplate reader ( multiskan fc ; thermo , waltham , ma , usa ) . female balb / c mice were purchased from the national laboratory animal center in taiwan . the care and housing of the mice were approved by the laboratory animal care committee of chang gung university of science and technology ( iacuc approval number : 2014 - 007 ) . the mice were randomly divided into four groups of 10 mice each : normal control mice ( n group ) , the lps group , 10  mg / kg matrine plus lps ( m10 group ) , and 20  mg / kg matrine plus lps ( m20 group ) . on days 17 of the experiment , mice were intraperitoneally injected with matrine . on day 8 , the mice were anesthetized with isoflurane ( aesica , kent , uk ) and received intratracheal injection of 50  l lps ( 1  g / ml ) or normal saline . four hours after lps administration , the mice were sacrificed and we collected samples of serum , bronchoalveolar lavage fluid ( balf ) , and lung tissue . briefly , the mouse trachea was intubated and the lungs were flushed with 1  ml normal saline . to determine the neutrophil cell count in balf , the cells were stained with liu stain solution ( polysciences , inc . , lung tissues were fixed in 10% formalin , embedded in paraffin , and cut into 6  m sections . serum samples were centrifuged at 6000  rpm for 5  min as previously described , followed by storage at 80c until use . in an in vitro experiment , a549 cells were pretreated with matrine ( 100400  m ) for 1  h in 24-well plates and then they were stimulated with lps ( 1  g / ml ) and cultured for 24  h. we then used specific elisa kits ( r&d systems , minneapolis , mn , usa ) to assay il-8 , il-6 , monocyte chemotactic protein-1 ( mcp-1 ) , ccl5 , and icam-1 production . optical density ( od ) was spectrophotometrically measured at 450  nm using a microplate reader ( multiskan fc , thermo ) . in our in vivo study total rna was extracted from lung tissue using trizol reagent ( life technologies , carlsbad , ca , usa ) . from this rna we then used a real - time pcr system kit ( bio - rad laboratories , hercules , ca , usa ) with a spectrofluorometric thermal cycler ( icycler ; bio - rad ) to detect cdna gene expression . a549 cells were treated with matrine and incubated with lps ( 1  g / ml ) for 24  h. then , neutrophil - like hl-60 cells were treated with calcein am solution ( sigma ) and cocultured with a549 cells for 1  h. we observed the adhesion of hl-60 cells to a549 using fluorescence microscopy ( olympus , tokyo , japan ) . following treatment with matrine for 1  h , a549 cells were stimulated with lps ( 1  g / ml ) for 30  min to detect protein phosphorylation or for 24  h to evaluate total protein expressions . proteins were extracted using protein lysis buffer containing protease and phosphatase inhibitors ( sigma ) . nuclear proteins were extracted using ne - per nuclear and cytoplasmic extraction reagent kits ( pierce , rockford , il , usa ) . equal amounts of protein were separated on 10% sds polyacrylamide gels and then transferred to polyvinylidene fluoride ( pvdf ) membranes ( millipore , billerica , ma , usa ) . the pvdf membranes were incubated overnight at 4c with primary antibodies , including cox-2 , ib- , phosphorylated - ib- , lamin b1 , p65 , phosphorylated - ib- ( santa cruz , ca , usa ) , erk1/2 , p38 , jnk , phosphorylated - erk1/2 , phosphorylated - p38 , phosphorylated - jnk ( millipore ) , icam-1 , and -actin ( sigma ) . after the overnight incubation , the membrane was washed with tbst buffer ( 150  mm nacl ; 10  mm tris , ph 8.0 ; and 0.1% tween 20 ) and incubated with hrp - conjugated secondary antibodies for 1  h. finally , the membranes were washed and processed with luminol / enhancer solution ( millipore ) for detection and quantification of the specific protein using the biospectrum 600 system ( uvp , upland , ca , usa ) . transfected pnfb - luc plasmid evaluated nf-b activity as previously described . briefly , a549 cells were transfected with pnfb - luc plasmid ( stratagene , california , usa ) and cells were pretreated with matrine for 1  h and were stimulated with lps for 4  h. the luciferase activity was assayed using a multi - mode microplate reader ( biotek synergy all data were analyzed using one - way analysis of variance ( anova ) , followed by tukey - kramer method for multiple comparisons . . a p value of < 0.05 was considered to indicate a statistically significant difference . at doses of 400  m , matrine did not affect the viability of a549 cells ( data not shown ) . thus , matrine concentrations of 100400  m were used in the in vitro experiments . first , we investigated how matrine modulated cytokine and chemokine production in lps - stimulated a549 cells ( figure 1 ) . we found that matrine significantly decreased il-6 production in a concentration - dependent manner : il-6 production with lps alone , 179.4  7.9  pg / ml ; with 100  m matrine , 185.4  10.2  pg / ml ( p = 0.28 versus lps alone ) ; 200  m matrine , 150.5  11.2  pg / ml ( p < 0.05 versus lps alone ) ; 400  m matrine , 101.9  11.5  pg / ml ( p < 0.01 versus lps alone ) . matrine also dose - dependently decreased the levels of il-8 , mcp-1 , and ccl5 in lps - stimulated a549 cells . a549 cells were pretreated with matrine , followed by stimulation with lps for 24  h. matrine pretreatment significantly suppressed cox-2 protein expression in a concentration - dependent manner compared with lps - stimulated a549 cells ( figure 2(a ) ) . elisa ( figure 2(b ) ) and western blot analysis ( figure 2(a ) ) also showed that matrine significantly inhibited icam-1 expression : lps alone , 215.9  24.3  pg / ml ; matrine 100  m , 192.9  10.3  pg / ml ( p = 0.12 versus lps alone ) ; matrine 200  m , 163.8  18.4  pg / ml ( p < 0.05 versus lps alone ) ; matrine 400  m , 130.6  17.5  pg / ml ( p < 0.01 versus lps alone ) . correspondingly , we also found that matrine treatment decreased the adherence of hl-60 cells to lps - stimulated a549 cells ( figure 2(c ) ) . upon finding that matrine decreased productions of chemokines , proinflammatory cytokines , and cox-2 in lps - stimulated a549 cells , we further investigated whether matrine attenuated activation of the nf-b and mapk pathways in these cells . in unstimulated cells , ib holds the nf-b complex ( p65 and p50 ) in the cytoplasm , while lps stimulation induces phosphorylation of ib- , increasing the release of p65 into the nucleus . here we found that matrine significantly inhibited ib- phosphorylation and decreased ib- degradation , thus attenuating p65 translocation into the nucleus compared with that observed in lps - stimulated a549 cells ( figures 3(a ) and 3(b ) ) . nf-b activity was evaluated using a promoter luciferase assay , and it was found that matrine could significantly decrease luciferase activity ( figure 3(c ) ) . we also observed that matrine suppressed phosphorylation of erk1/2 , jnk , and p38 compared with lps - stimulated a549 cells ( figure 4 ) . after the sacrifice of mice , lung tissue was fixed with formalin and he stained to analyze the distribution of neutrophils in lung sections . the lps group showed greater neutrophil infiltration compared to the n group ( figure 5 ) . moreover , matrine treatment significantly suppressed neutrophil infiltration in the lung biopsy sections of mice . evaluation of inflammatory cells in balf revealed that matrine suppressed neutrophil infiltration in acute lung injury mice . intraperitoneal injection of matrine in lps - induced acute lung injury mice led to a significant decrease of neutrophils compared with the lps group : lps alone , 4.3  10  6.1  10 neutrophils / ml ; m10 , 2.7  10  7.1  10 neutrophils / ml ( p = 0.08 versus lps alone ) ; m20 , 2.0  10  6.1  10 neutrophils / ml ( p < 0.05 versus lps alone ) ( figure 6(a ) ) . acute lung injury mice treated with matrine exhibited lower il-6 levels : lps alone , 347.8  97.6  pg / ml ; m10 , 199.3  8.3  pg / ml ( p < 0.05 versus lps alone ) ; m20 , 163.9  49.8  pg / ml ( p < 0.01 versus lps alone ) ( figure 6(b ) ) . matrine treatment also reduced tnf- levels in these mice : lps alone , 556.3  111.9  pg / ml ; m10 , 441.0  71.1  pg / ml ( p = 0.11 versus lps alone ) ; m20 , 262.9  69.1  pg / ml ( p < 0.05 versus lps alone ) ( figure 6(c ) ) . using real - time pcr to evaluate gene expression , we found that matrine significantly decreased levels of il-1 , il-6 , tnf- , il-13 , mcp-1 , and ccl5 in lung injury tissue ( figure 7 ) . however , matrine did not suppress the gene expressions of il-4 , ccl11 , or ccl24 . we further found that matrine inhibited the gene expressions of icam-1 , inos , and cox-2 in lung injury tissue . matrine significantly inhibited phosphorylation of p65 compared with that in lps - stimulated lung injury tissue . matrine also suppressed phosphorylation of erk1/2 , jnk , and p38 in the lung injury tissue ( figure 8) . serum analysis revealed significantly reduced il-6 production and tnf- production in the m20 group compared to in the lps group ( figure 9 ) . matrine is a major active component isolated from sophora flavescens ( kushen ) , which reportedly shows many biological and medical properties , including improved inflammatory responses of the central nervous system , antitumor activities , and immunity - regulating effects [ 10 , 19 ] . we previously demonstrated that matrine suppresses eosinophil infiltration in lung tissue and improves airway hyperresponsiveness and suppresses tracheal goblet cell hyperplasia in asthmatic mice . moreover , zhang et al . described lung histopathological changes and inflammatory response at 18  h after lps injection in mice , reporting that matrine decreased myeloperoxidase and malondialdehyde activities and inhibited reactive oxygen species and oxidative stress in balf . in our present study , we investigated whether matrine improved the inflammatory response in acute lung injury mice at 4  h after lps injection into the trachea and also examined inflammatory responses in lps - stimulated human lung epithelial cells . our results showed that matrine significantly suppressed cox-2 expression and productions of il-6 , il-8 , mcp-1 , and ccl5 in lps - stimulated a549 cells . matrine treatment also suppressed icam-1 expressions and , correspondingly , reduced lps - stimulated a549 cell adherence to neutrophils . finally , matrine suppressed components of inflammatory signaling pathways , including the nf-b and mapk pathways . lps is an important component of gram - negative bacteria cell walls . upon infection with gram - negative bacteria , lps release leads to inflammation and fever symptoms , potentially causing severe acute or chronic organ congestion , edema , or even septic shock and death . lps stimulates ib phosphorylation , emancipating nf-b ( containing p50 and p65 ) to translocate into the nucleus . in the nucleus , p65 binds the promoters of inflammatory genes , leading to the expressions of proinflammatory cytokines , chemokines , cox-2 , and icam-1 in lps - stimulated a549 cells [ 21 , 23 ] . our present experiments demonstrated that matrine significantly inhibited the secretion of proinflammatory cytokines and chemokines from lps - induced a549 lung epithelial cells . these results suggest that matrine treatment could improve the inflammatory response in cases of lung bacterial infections . previously , zhang et al . investigated the administration of matrine after 30  min of lps stimulation and reported that matrine reduces lung inflammation and oxidation by inhibiting inflammatory signal pathways . however , their protocol involved lps injected intravenously after 60 hours , and the survival rate was only 20% . mice given 20  mg / kg matrine did not show an increased survival rate ; mice given 100  mg / kg matrine showed a 60% survival rate and decreased lung damage . in contrast , our present experimental model of lung injury involved intratracheal lps injection to induce local inflammatory lung injury , which seems to be more suitable for assessing the inflammatory response . furthermore , our experiment mainly investigated whether matrine could provide protection against lung injury in mice . the mice were treated with intraperitoneal matrine injection for continuous 7 days , followed by intratracheal lps injection on the 8th day , and the mice were sacrificed four hours later . the experimental process did not lead to the death of any mice , and our model is more appropriate for observing the early response of acute lung injury and for evaluating the protective effects of matrine in early lung injury . our results showed that 20  mg / kg matrine could protect and prevent lung injury symptoms in this experimental model . despite the differences between our animal experimental model and the lung injury mouse model used by zhang et al . , both sets of experimental results suggest that matrine can reduce lung injury in mice . in cases of bacterial infection , neutrophils in the lung can ingest invading bacteria and release large amounts of inflammatory mediators to induce an inflammatory response in the lung . our experiments showed that treatment with 20  mg / kg of matrine suppressed neutrophil infiltration in the lung and correspondingly reduced the number of neutrophils in balf . matrine inhibited the productions of il-8 , mcp-1 , and ccl5 in lps - stimulated a549 cells and suppressed mcp-1 and ccl5 gene expressions in mouse lung tissue . inflammatory lung epithelial cells also express icam-1 adhesion molecules to promote neutrophil adsorption [ 18 , 27 ] . overall , our results suggest that matrine may be useful for reducing neutrophil infiltration in the inflammatory response of the lungs . this would be particularly useful , since lung inflammation and lung damage in lps - induced acute lung injury are mainly caused by neutrophil infiltration . on the other hand , matrine did not effectively reduce the gene expressions of ccl11 and ccl24 in the lung and , therefore , matrine did not improve eosinophilic infiltration in this lps - induced lung injury mouse model . matrine also did not effectively suppress the gene expressions of the th2-associated cytokines il-4 and il-13 in lung tissue . in conclusion , our results demonstrate that matrine significantly suppressed the inflammatory response in human lung epithelial cells and blocked icam-1 expression , preventing acute lung injury in lps - induced mice .
matrine is isolated from sophora flavescens and shows anti - inflammatory effects in macrophages . 
 here we evaluated matrine 's suppressive effects on cyclooxygenase 2 ( cox-2 ) and intercellular adhesion molecule-1 ( icam-1 ) expressions in lipopolysaccharide- ( lps- ) stimulated human lung epithelial a549 cells . additionally , balb / c mice were given various matrine doses by intraperitoneal injection , and then lung injury was induced via intratracheal instillation of lps . in lps - stimulated a549 cells , 
 matrine inhibited the productions of interleukin-8 ( il-8 ) , monocyte chemotactic protein-1 , and il-6 and decreased cox-2 expression . 
 matrine treatment also decreased icam-1 protein expression and suppressed the adhesion of neutrophil - like cells to inflammatory a549 cells . in vitro results demonstrated that matrine significantly inhibited mitogen - activated protein kinase phosphorylation and decreased nuclear transcription factor kappa - b subunit p65 protein translocation into the nucleus . in vivo data indicated that matrine significantly inhibited neutrophil infiltration and suppressed productions of tumor necrosis factor- and il-6 in mouse bronchoalveolar lavage fluid and serum . 
 analysis of lung tissue showed that matrine decreased the gene expression of proinflammatory cytokines , chemokines , cox-2 , and icam-1 . 
 our findings suggest that matrine improved lung injury in mice and decreased the inflammatory response in human lung epithelial cells .