acute lung injury ( ali ) is a life - threatening hypoxemic respiratory failure showing bilateral infiltrates in the absence of left atrial hypertension , which develops to acute respiratory distress syndrome in the severe stage . causes of ali are divided into direct and indirect injuries , including sepsis , pneumonia , trauma , aspiration , multiple blood transfusions , and drug abuse or overdose [ 1,46 ] . therapeutic methods benefit from the improvement of mechanical ventilation , and positive - pressure ventilation supplemented with oxygen and positive end - expiratory pressure is necessary for almost all ali patients . the efficacy and safety of lung recruitment maneuvers and individualized treatment are still under discussion . acute and widespread inflammation and tissue fibrosis play vital roles in the development of ali . polymorphonuclear neutrophils in the injured lung can not be eliminated by alveolar macrophages in time , thus producing excess inflammatory factors , including interleukin 1 beta ( il-1 ) and tumor necrosis factor alpha ( tnf- ) . other pro - inflammatory factors , such as toll - like receptor 2 ( tlr2 ) , interleukin 6 ( il-6 ) , il-17 , and il-23 , all of which are potential therapeutic targets for treating ali , also accelerate inflammation and fibrosis and thus aggravate ali . in addition , lung fibrosis is promoted in the early phase of ali , manifesting as increased collagen fiber content . thymic stromal lymphopoietin ( tslp ) is an epithelial cell - derived cytokine , mainly expressed in skin , gut , lung , and thymus , that plays pivotal roles in regulating t helper 2-mediated inflammation . tslp and its receptor tslpr mediate pro - inflammatory cytokines and chemokines release , thus contributing to airway inflammation . inhibition of mitogen - activated protein kinases ( mapks ) suppresses the il-1-induced production of tslp in endometrioma stromal cells , indicating the association between tslp and mapk pathways . however , little is known about the regulatory mechanism of tslp in pulmonary fibrosis of ali . in this study we aimed to discover the function of tslp and the potential mechanism in mediating cell fibrosis in ali . human lung fibroblast mrc-5 cells were used and tslp expression was altered to detect fibrosis indicators , including -sma and collagen i. we also analyzed the activation of major mapks to discover the possible mechanism of tslp in cell fibrosis . our study results add to the evidence of a connection between tslp and lung cell fibrosis , and provide a potential target for treating lung fibrosis in ali . human lung fibroblast mrc-5 cells ( atcc , manassas , va ) were cultured in dulbecco s modified eagle s medium supplemented with 10% fetal bovine serum , 100 u / ml penicillin , and 100 g / ml streptomycin ( gibco , carlsbad , ca ) , and maintained in a humidified atmosphere with 5% co2 at 37c . cells were seeded in 6-well plates and divided into 4 groups : control , si - control , tslp , and si - tslp . transfection was performed in serum - free medium using lipofectamine 2000 ( invitrogen , carlsbad , ca ) according to the manufacturer s instructions . cells in the si - control and si - tslp groups were transfected with the negative control for sirna transfection and the tslp - specific sirna designed by ribobio ( guangzhou , china ) . for tslp overexpression in the tslp group , the coding sequence of tslp was cloned into pcdna3.1 vector ( transheepbio , shanghai , china ) and the vector ( 50 m ) was transfected into the cells . after the transfection , cells were recovered in complete medium and incubated at 37c for 48 h before sampling . cells of the 4 groups were collected and lysed in trizol ( invitrogen ) for rna extraction . the dna contamination was removed using dnasei ( invitrogen ) and detected by agarose gel electrophoresis . the quantity and quality of rnas were examined by nanodrop 2000 ( thermo scientific , carlsbad , ca ) , and 1 g rnas of each sample was applied to reverse transcription into complementary dnas ( cdnas ) using the primescript 1 strand cdna synthesis kit ( takara , dalian , china ) . qpcr was conducted on a lightcycler 480 platform with 20-ng cdnas and the specific primers for human tslp ( fw : 5-tat gag tgg gac caa aag tac cg-3 and rv : 5-acg cca caa tcc ttg taa ttg tg-3 ) in each reaction . data were analyzed using 2 method with gapdh ( fw : 5-gaa ggt gaa ggt cgg agt c-3 and rv : 5-gaa gat ggt gat ggg att tg-3 ) as an endogenous reference . protein samples of the cells were extracted by lysing the cells with radio immunoprecipitation assay lysis buffer ( beyotime , shanghai , china ) . the protein samples were quantified using the bca protein assay kit ( beyotime ) and the same amount of samples were loaded for sodium dodecyl sulfate - polyacrylamide gel electrophoresis . protein bands on the gel were transferred to a polyvinylidene fluoride membrane and blocked in 5% skim milk for 2 h at room temperature and then the blot was incubated in the specific primary antibodies for tslp ( ab47943 ) , skeletal muscle actin ( -sma , ab5694 ) , collagen i ( ab6308 ) , mapk7 ( ab40809 ) , extracellular signal - regulated kinase 1 ( erk1 , ab180163 ) , phospho - erk1 ( p - erk1 , ab24157 ) , p38 ( ab7952 ) , p - p38 ( ab178867 ) , c - jun n - terminal kinase 1 ( jnk1 , ab199380 ) , and p - jnk1 ( ab47337 ) , purchased from abcam ( cambridge , uk ) , overnight at 4c . after washing , the blot was incubated in horseradish peroxidase - conjugated secondary antibodies for 1 h at room temperature . positive signals were developed using the ecl plus western blotting substrate ( thermo scientific ) . band densities for each sample were analyzed using imagej 1.49 software ( national institutes of health , bethesda , md ) . all experiments were repeated in triplicate and results are indicated as the mean  standard deviation . p values were calculated by t test in spss ( ibm , new york , usa ) and differences were considered significant at p<0.05 . human lung fibroblast mrc-5 cells ( atcc , manassas , va ) were cultured in dulbecco s modified eagle s medium supplemented with 10% fetal bovine serum , 100 u / ml penicillin , and 100 g / ml streptomycin ( gibco , carlsbad , ca ) , and maintained in a humidified atmosphere with 5% co2 at 37c . cells were seeded in 6-well plates and divided into 4 groups : control , si - control , tslp , and si - tslp . transfection was performed in serum - free medium using lipofectamine 2000 ( invitrogen , carlsbad , ca ) according to the manufacturer s instructions . cells in the si - control and si - tslp groups were transfected with the negative control for sirna transfection and the tslp - specific sirna designed by ribobio ( guangzhou , china ) . for tslp overexpression in the tslp group , the coding sequence of tslp was cloned into pcdna3.1 vector ( transheepbio , shanghai , china ) and the vector ( 50 m ) was transfected into the cells . after the transfection , cells were recovered in complete medium and incubated at 37c for 48 h before sampling . cells of the 4 groups were collected and lysed in trizol ( invitrogen ) for rna extraction . the dna contamination was removed using dnasei ( invitrogen ) and detected by agarose gel electrophoresis . the quantity and quality of rnas were examined by nanodrop 2000 ( thermo scientific , carlsbad , ca ) , and 1 g rnas of each sample was applied to reverse transcription into complementary dnas ( cdnas ) using the primescript 1 strand cdna synthesis kit ( takara , dalian , china ) . qpcr was conducted on a lightcycler 480 platform with 20-ng cdnas and the specific primers for human tslp ( fw : 5-tat gag tgg gac caa aag tac cg-3 and rv : 5-acg cca caa tcc ttg taa ttg tg-3 ) in each reaction . data were analyzed using 2 method with gapdh ( fw : 5-gaa ggt gaa ggt cgg agt c-3 and rv : 5-gaa gat ggt gat ggg att tg-3 ) as an endogenous reference . protein samples of the cells were extracted by lysing the cells with radio immunoprecipitation assay lysis buffer ( beyotime , shanghai , china ) . the protein samples were quantified using the bca protein assay kit ( beyotime ) and the same amount of samples were loaded for sodium dodecyl sulfate - polyacrylamide gel electrophoresis . protein bands on the gel were transferred to a polyvinylidene fluoride membrane and blocked in 5% skim milk for 2 h at room temperature and then the blot was incubated in the specific primary antibodies for tslp ( ab47943 ) , skeletal muscle actin ( -sma , ab5694 ) , collagen i ( ab6308 ) , mapk7 ( ab40809 ) , extracellular signal - regulated kinase 1 ( erk1 , ab180163 ) , phospho - erk1 ( p - erk1 , ab24157 ) , p38 ( ab7952 ) , p - p38 ( ab178867 ) , c - jun n - terminal kinase 1 ( jnk1 , ab199380 ) , and p - jnk1 ( ab47337 ) , purchased from abcam ( cambridge , uk ) , overnight at 4c . after washing , the blot was incubated in horseradish peroxidase - conjugated secondary antibodies for 1 h at room temperature . positive signals were developed using the ecl plus western blotting substrate ( thermo scientific ) . band densities for each sample were analyzed using imagej 1.49 software ( national institutes of health , bethesda , md ) . all experiments were repeated in triplicate and results are indicated as the mean  standard deviation . p values were calculated by t test in spss ( ibm , new york , usa ) and differences were considered significant at p<0.05 . to analyze the function of tslp , we performed overexpression and inhibition of tslp using its specific overexpression vector or sirna , respectively . qpcr and western blot analyses were conducted to verify the successful transfection of the vector and sirna . the tslp mrna level in the si - control group was only slightly changed compared to the control group ( p<0.05 ) , while that of the tslp group was obviously up - regulated and the si - tslp group was significantly down - regulated ( p<0.001 , figure 1a ) . the tslp protein levels showed similar changing patterns , with no obvious change existing between the control group and the si - control group ( p<0.05 ) , but significant up - regulation and down - regulation were found in the tslp group and the si - tslp group ( p<0.001 , figure 1b ) . these results show the effectiveness of tslp overexpression and inhibition , and the cells were thus used for further analysis . elevated fibrosis is usually accompanied by increased -sma and collagen levels ; thus , we tested the protein levels of -sma and collagen i to evaluate the cell fibrosis . their expression levels in the si - control group were nearly unchanged , but were significantly up - regulated when tslp was overexpressed by its overexpression vector ( p<0.001 ) , and inhibited when tslp was suppressed ( p<0.001 , figure 2a , 2b ) . these data suggest that tslp can promote the production of -sma and collagen i , which appear to indicate the accelerated fibrosis in mrc-5 cells . mapk pathways have been reported to respond to the cytokines induced by stimuli and promote the production of collagen , thus contributing to fibrosis . therefore , in this study we analyzed the activation of mapk pathways by detecting the expression of 4 major mapks : mapk7 , erk1 , jnk1 , and p38 . western blot results showed that the expression of mapk7 was significantly promoted by tslp overexpression , and was inhibited in the si - tslp group ( p<0.001 , figure 3a , 3b ) . phosphorylated erk1 , p38 , and jnk1 were all promoted by tslp and inhibited when tslp was inhibited , and their total protein levels were almost unchanged ( figure 3a , 3c3e ) , indicating the activation of erk1 , p38 , and jnk1 by tslp . these results suggest that all the major mapk pathways in mammals are activated by tslp , which might be the functional mechanism of tslp in promoting fibrosis in mrc-5 cells . to analyze the function of tslp , we performed overexpression and inhibition of tslp using its specific overexpression vector or sirna , respectively . qpcr and western blot analyses were conducted to verify the successful transfection of the vector and sirna . the tslp mrna level in the si - control group was only slightly changed compared to the control group ( p<0.05 ) , while that of the tslp group was obviously up - regulated and the si - tslp group was significantly down - regulated ( p<0.001 , figure 1a ) . the tslp protein levels showed similar changing patterns , with no obvious change existing between the control group and the si - control group ( p<0.05 ) , but significant up - regulation and down - regulation were found in the tslp group and the si - tslp group ( p<0.001 , figure 1b ) . these results show the effectiveness of tslp overexpression and inhibition , and the cells were thus used for further analysis . elevated fibrosis is usually accompanied by increased -sma and collagen levels ; thus , we tested the protein levels of -sma and collagen i to evaluate the cell fibrosis . their expression levels in the si - control group were nearly unchanged , but were significantly up - regulated when tslp was overexpressed by its overexpression vector ( p<0.001 ) , and inhibited when tslp was suppressed ( p<0.001 , figure 2a , 2b ) . these data suggest that tslp can promote the production of -sma and collagen i , which appear to indicate the accelerated fibrosis in mrc-5 cells . mapk pathways have been reported to respond to the cytokines induced by stimuli and promote the production of collagen , thus contributing to fibrosis . therefore , in this study we analyzed the activation of mapk pathways by detecting the expression of 4 major mapks : mapk7 , erk1 , jnk1 , and p38 . western blot results showed that the expression of mapk7 was significantly promoted by tslp overexpression , and was inhibited in the si - tslp group ( p<0.001 , figure 3a , 3b ) . phosphorylated erk1 , p38 , and jnk1 were all promoted by tslp and inhibited when tslp was inhibited , and their total protein levels were almost unchanged ( figure 3a , 3c3e ) , indicating the activation of erk1 , p38 , and jnk1 by tslp . these results suggest that all the major mapk pathways in mammals are activated by tslp , which might be the functional mechanism of tslp in promoting fibrosis in mrc-5 cells . the roles and regulation mechanisms of tslp in lung fibrosis in ali have not been previously defined . in this study , we tried to determine them by overexpressing and inhibiting tslp in human lung fibroblast mrc-5 cells and detecting the expression changes of some key factors . -sma is expressed at higher levels in the epithelial cells and periductular collagen fibers of biliary atresia - induced hepatic fibrosis , and its inhibition attenuates mouse lung fibrosis . moreover , a hallmark of fibrosis is the accumulation of fibrillary collagens , especially collagen i. therefore , -sma and collagen i are widely used as indicators of fibrosis , including lung fibrosis [ 1618 ] . in this study , we used these 2 factors to assess the progression of fibrosis in mrc-5 . our findings show that tslp overexpression significantly up - regulated -sma and collagen i levels , both of which were inhibited when tslp was suppressed . based on the indicated roles of -sma and collagen i in fibrosis , these results suggest that the changes in degree of fibrosis are modulated by tslp ; therefore , tslp appears to promote mrc-5 cell fibrosis , suggesting its roles in accelerating lung fibrosis in ali . mapks are serine - threonine kinases that participate in the mediation of various cellular activities . they are mainly divided into 4 subgroups : erk1/2 mapk , jnk mapk , p38 mapk , and erk5 mapk ( mapk7 ) . inhibiting mapks can attenuate lipopolysaccharide - induced ali . with this information , we attempted to analyze the relationship between tslp and mapks in lung fibrosis . results showed that the expression levels of mapk7 and the proportion of phosphorylated erk1 , jnk1 , and p-38 were all up - regulated by tslp overexpression , indicating tslp can activate mapk pathways in mrc-5 cells . in addition , mapks contribute to the progression of fibrosis , as proven in cardiomyopathy   and peritoneal fibrosis . moreover , inhibiting p38 mapk may attenuate the inflammation and fibrosis of lung diseases . therefore , it was reasonable to deduce that the activation of mapk pathways by tslp might be a potential mechanism of tslp - induced mrc-5 cell fibrosis . however , the relationship between tslp and mapk pathways is complicated , and studies have found that activation of mapks can increase tslp expression . the inter - activation between tslp and mapks might help to amplify the signals during ali , but this needs further analysis . tslp inhibition resulted in the down - regulation of -sma and collagen i , as well as the less - activated mapks , suggesting tslp is a potential therapeutic target for treating lung fibrosis in ali . tslp has been shown to contribute to skin fibrosis in atopic dermatitis   and diffuse cutaneous systemic sclerosis . importantly , a previous study proved that inhibition of tslp is beneficial in suppressing lung fibrosis ; nevertheless , it is achieved through regulating the type i receptor of tgf- and the downstream smad family member 2/3 and jnk , rather than the other mapks . it would be of great value if these regulatory pathways were defined in detail and confirmed in further studies . this study indicates that tslp may promote the fibrosis of mrc-5 cells and activate the mapk pathways , which is a possible mechanism of tslp in promoting mrc-5 cell fibrosis . our study will help to define the molecular regulation of fibrosis , but verification in further studies is necessary .
backgroundacute lung injury ( ali ) is a life - threatening hypoxemic respiratory disorder with high incidence and mortality . 
 ali usually manifests as widespread inflammation and lung fibrosis with the accumulation of pro - inflammatory and pro - fibrotic factors and collagen . 
 thymic stromal lymphopoietin ( tslp ) has a significant role in regulation of inflammation but little is known about its roles in lung fibrosis or ali . 
 this study aimed to define the role and possible regulatory mechanism of tslp in lung fibrosis.material/methodswe cultured human lung fibroblast mrc-5 cells and overexpressed or inhibited tslp by the vector or small interfering rna transfection . 
 then , the pro - fibrotic factors skeletal muscle actin alpha ( -sma ) and collagen i , and the 4 mitogen - activated protein kinases ( mapks )  mapk7 , p38 , extracellular signal - regulated kinase 1 ( erk1 ) , and c - jun n - terminal kinase 1 ( jnk1 )  were detected by western blot.resultsresults showed that tslp promoted the production of -sma and collagen i ( p<0.001 ) , suggesting that it can accelerate mrc-5 cell fibrosis . 
 it also activated the expression of mapk7 , p - p38 , p - erk1 , and p - jnk1 , but the total mapk7 , p-38 , erk1 , and jnk1 protein levels were mostly unchanged , indicating the activated mapk pathways that might contribute to the promotion of cell fibrosis.conclusionsthis study shows the pro - fibrotic role of tslp in mrc-5 cells , suggesting tslp is a potential therapeutic target for treating lung fibrosis in ali . 
 it possibly functions via activating mapks . 
 these findings add to our understanding of the mechanism of fibrosis .