traumatic injury is the leading cause of morbidity and mortality , which affects many parts of the body , including the brain , extremities , and internal organs . the incidence of life - threatening complications , such as systemic inflammatory response syndrome ( sirs ) and acute lung injury ( ali ) , in severely injured trauma patients remains between 30% and 50% [ 1 , 2 ] . sirs induced by severe trauma or other injuries is a clinical syndrome initiated by dysregulation of inflammation , which could lead to various tissue injuries culminating in multiple organ dysfunction / failure syndrome ( mods / mof ) . bone marrow - derived mesenchymal stem cells ( bmscs ) , also referred to as marrow stromal cells , are a multipotential lineage characterized by the capacity for extracorporeal expansion and the ability to differentiate into bone , cartilage , and adipose tissues . numerous studies indicated that bmscs can be an effective modality for cell - based immunomodulatory therapies in various diseases , such as sepsis , endotoxemia , diabetes , and lung injury [ 39 ] , especially for reducing of inflammation [ 4 , 5 , 10 ] . several clinical trials assessing the efficacy of bmscs in immune - mediated diseases are currently underway . quick and efficient response to microbial infections is driven by recognition of molecules broadly shared by a variety of pathogens , which are distinguishable from host molecules named pathogen associated molecular patterns ( pamps ) . pattern - recognition receptors ( prrs ) that contain membrane - bound prrs , including toll - like receptors ( tlrs ) and other cytoplasmic proteins , recognize the pamps . upon binding with the pamps , tlr2 and tlr4 then activate nf-b and subsequently promote cytokine synthesis , which include il-1 , il-6 , and tnf- [ 11 , 12 ] . the network of tlrs and prrs mediates the response of bmscs to inflammatory stimuli , such as lps [ 13 , 14 ] . however , how tlrs modulate the bmsc activity is not clearly elucidated . since tlr2 and tlr4 response to acute otitis through activation of nf-b , we hypothesized that bmscs ameliorate the inflammation damage of alveolar macrophages and huvecs through the inhibition of tlr2 and tlr4 mediated nf-b pathways . louis , mo ) supplemented with 10% fetal bovine serum ( fbs ) ( gibco , carlsbad , ca , usa ) and 1% penicillin / streptomycin ( solarbio , beijing , china ) at 37c in a humidified atmosphere of 5% co2 . ( suzhou , china ) were cultured in 10% fbs - dmem - lg ( gibco , carlsbad , ca , usa ) . the surface markers for bmscs differentiation of adipocytic , osteogenic , and chondrogenic lineages were characterized as previously reported . monolayer permeability was quantitated by spectrophotometric measurement of the flux of evans blue - albumin across huvecs as described previously . huvecs ( 1  10 ) were seeded in 24-well transwell inserts ( 5  m pore size , corning incorporated , ny , usa ) and cultured with 10% fbs dmem overnight and then changed with serum - free medium for another 24 hours . the various numbers of bmscs were seeded at the bottom of the same 24-well plate with serum - free media . lps was added to the transwell inserts at a final concentration of 100  ng / ml for different time points ( 1 , 3 , 6 , 12 , and 24  h ) . after that , the bmscs were digested by trypsin - edta solution to terminate the effects of bmscs and 100  l hank 's solution containing evans blue- ( eb- ) conjugated bovine serum albumin ( final concentration 0.67  mg / ml ) was added to the transwell inserts , while 600  l 4% bsa was added to the lower plate chamber . after incubation at 37c for 1 hour , 100  l bsa solution from the lower chamber was harvested and measured for the absorbance at 620  nm . the huvecs permeability capacity was calculated as follows : evans blue dye - labeled albumin ( eb - albumin ) leak  rate = od620leaked  eb - albumin / od620total  eb - albumin  100% . alveolar macrophages or huvecs ( 1  10/well ) were seeded in a 6-well plate , after 24-hour incubation , bmscs ( 2  10/well ) were seeded in the 6-well transwell inserts ( 5  m pore , corning incorporated , ny , usa ) at the indicated time points ( 1 , 3 , 6 , 12 , and 24  h ) . lps ( 100  ng / ml ) was then added to the lower chamber for 1  h. alveolar macrophages or huvecs lysate was collected for rna and protein analyses , and the cell - free supernatants were collected for elisa detection . / huvecs were lysed in the ripa buffer ( thermo scientific , rockford , usa ) supplemented with 1% pmsf and 1% protein phosphatase inhibitor mixture ( p1260 , applygen , beijing , china ) . the concentration of the proteins was measured with the bicinchoninic acid kit ( thermo scientific , rockford , usa ) . rabbit anti - tlr2 , tlr4 , and phosphorylated p65 unit of nf-b antibodies were obtained from cell signaling technology . horseradish peroxidase- ( hrp- ) conjugated goat anti - rabbit antibodies were purchased from bio - rad . ecl - plus chemiluminescent reagent ( thermo scientific , rockford , usa ) was used to visualize the specific proteins . relative concentration of proteins was quantitated using the image j software ( national institutes of health , bethesda , usa ) . total rna was isolated from cells using the trizol rna isolation reagents ( life technologies ) . the first - strand cdnas were converted using the superscript iii reverse transcriptase ( invitrogen , carlsbad , ca ) according to the manufacturer 's protocols . real - time rt - pcr analysis was performed on abi 7500 sequence detection system using the ultrasybr mixture ( cw bio co. ltd , beijing , china ) . the pcr primer sequences are gapdh forward 5-tggagtctactggcgtctt-3 , reverse 5-tgtcatatttctcgtg gttca-3 ; tlr4 forward 5-ctgcatagaggtagttcct-3 , reverse 5-tccagcc actgaagttctga-3 ; tlr2 forward 5-ggagactctggaagc atg-3 , reverse 5-gcatcct gaagcctgtg-3. the supernatants collected from coculture system were centrifuged for 10 minutes at 1000  g ( 4c ) to remove cell debris . the levels of tnf- and il10 were measured using commercially available enzyme - linked immunosorbent assay ( elisa ) kits ( bd biosciences , san diego , ca , usa ) according to the instructions provided by the manufacturer . spss 17.0 software was used to perform the statistical analysis and values are presented as the means  standard deviations ( sd ) ; all experiments were repeated at least three times . the one - way anova was used to determine the significance of differences between various groups . to assess how bmscs affected huvecs permeability induced by lps , huvecs with or without coculture with bmscs were stimulated by lps for the indicated times . without coculture with bmscs , the eb - albumin leaking rate ranged from 5.35% ( at 1  h ) to 7.72% ( at 24  h ) in the lps group ( figure 1 ) . compared with the lps group , the eb - albumin leaking rates in the bmsc coculture group were reduced in a bmsc cell number- and time - dependent manner . the eb - albumin leaking rate in the 1  10/ml bmscs group was decreased 3 hours after coculture . the effects were less significant in the group with 1  10/ml bmscs and more significant in the group with 1  10/ml bmscs ( figure 1 ) . to determine whether lps stimulated expression of tlr2 , tlr4 , and p65 in macrophages and huvecs was affected by bmscs , alveolar macrophages and huvecs cells were cocultured with bmscs for the indicated time followed by lps treatment . western blot ( figure 2 ) and real - time rt - pcr analyses ( figure 3 ) showed that lps administration increased expression of tlr2 , tlr4 , and p65 within 1 hour after the treatment . however , the activity of lps was attenuated by coculture with bmscs in both alveolar macrophages and huvecs ( figures 2 and 3 ) . to determine whether bmscs regulated the release of tnf- and il10 from alveolar macrophages and huvecs in response to lps stimulation , the conditioned medium was collected from the coculture system and the concentrations of il10 and tnf- it was clear that concentration of tnf- in the medium was increased after lps stimulation within 1 hour and the effects lasted for 24 hours . in contrast , il10 was reduced by lps stimulation , while coculture with bmscs increased medium il10 production . the results indicate that bmscs suppress lps - induced inflammation by decreasing tnf- and increasing il10 releases ( figure 4 ) . bmscs treatments have been shown to reduce inflammatory response in human and animal models in response to injury . the results also showed that bmscs restricted the inflammatory responses of huvecs and alveolar macrophage stimulated by lps and therefore protected huvecs from damage induced by the endotoxin . the data indicate that attenuating lps - induced tnf- pathway activation in huvecs and macrophage by bmscs contributes to the protection of huvecs from endotoxin - induced damage by bmscs . our data demonstrated that lps increased the permeability of huvecs monolayers in a time - dependent manner . although the extent of the activity was lower than that after coculture for a longer time , the protection effects were detectable within one hour . since bmscs and huvecs were cultured separating by the transwell membrane , the effects of bmscs must be mediated by soluble factors released from bmscs . however , we did not rule out the possibility that bmscs sequestrated lps and therefore protected the endothelial cells . although lps still induced huvecs damage in the coculture , the eb - albumin leaks were reduced compared with the group without coculture with bmscs . we observed that the protective effect of bmscs on huvec was cell number - dependent . it appeared that the bmscs released sufficient protective molecules to protect endothelial cells within one hour . therefore , immediately administration of bmscs to sirs patients shall have beneficial effects . furthermore , the results were in line with previous reports that lps leads to persistent damage of huvecs [ 18 , 19 ] . however , it was different from the report that the damage of huvec by lps is not time - dependent . we cultured the cells for 24 hours to reach the confluence , followed by serum starvation for 24 hours and lps treatment . this method is different from the published one where the cells were cultured for 4 days prior to the experiments . it is well documented that cells use tlr2 and tlr4 to recognize pamps expressed by bacteria . activation of tlr2 and tlr4 leads to activation of mapk and nf-b pathways and production of inflammatory cytokines , including il-1 , il-6 , and tnf- [ 2022 ] . in this study , we showed that lps promoted expression of tlr2 and tlr4 in alveolar macrophages and huvecs . however , how expression of tlr2 expression was increased by lps is unknown and deserves future investigations . tnf- and furthermore , the lps effects were diminished when the huvecs and macrophages were cultured with bmscs . tnf- is a potent inflammatory factor and il10 is a key anti - inflammatory cytokine . downregulation of tnf- and upregulation of il10 in huvecs and macrophage indicate that the inflammatory reactions were suppressed . the results suggest that bmscs have the ability to alleviate pathogen - induced inflammation damage in huvecs and macrophages . in conclusion , the results showed that bmscs attenuated the activity of lps on the inflammatory reaction of huvecs and alveolar macrophages through inhibiting upregulation of tlr2 , tlr4 , and p65 expression , as well as downregulating tnf- release and upregulating il10 release .
background . 
 systemic inflammatory response syndrome ( sirs ) accompanied by trauma can lead to multiple organ dysfunction syndrome ( mods ) and even death . 
 early inhibition of the inflammation is necessary for damage control . 
 bone marrow mesenchymal stem cells ( bmscs ) , as a novel therapy modality , have been shown to reduce inflammatory responses in human and animal models . 
 methods . in this study , we used western blot , quantitative pcr , and enzyme - linked immunosorbent assay ( elisa ) to assess the activity of bmscs to suppress the inflammation induced by lipopolysaccharide ( lps ) in human umbilical cord endothelial cells ( huvecs ) and alveolar macrophages . results . 
 our results demonstrated that lps caused an inflammatory response in alveolar macrophages and huvecs , increased permeability of huvec , upregulated expression of toll - like receptor ( tlr ) 2 , tlr4 , phosphorylated p65 , downregulated release of il10 , and promoted release of tnf- in both cells . 
 coculture with bmscs attenuated all of these activities induced by lps in the two tested cell types . conclusions . together 
 , our results demonstrate that bmscs dosage dependently attenuates the inflammation damage of alveolar macrophages and huvecs induced by lps .