carrageenan , a high molecular weight sulfated polysaccharide derived from a red seaweed [ 13 ] , is used as a food additive to improve the formation of food products [ 4 , 5 ] . despite its wide application , earlier work demonstrated that intake of carrageenan may cause anabasis or cancer of the gut in animal models [ 1 , 611 ] . moreover , carrageenan has been used to study mediators of inflammation [ 6 , 9 , 12 ] . on the other hand , reports from many agencies emphasize that food grade carrageenan is safe to use as a carrageenan has been demonstrated to trigger innate immune responses through the interaction between an -d - gal-(13)-d - gal epitope on carrageenan and the tlr4 [ 1820 ] . the role of tlr4 in the innate immune response to carrageenan has also been demonstrated using a tlr4/ mouse . furthermore , carrageenan has been indicated to activate nf-b and ap-1 pathways through tlr4 in macrophages , leading to the upregulation of tumor necrosis factor alpha ( tnf- ) secretion . however , in these studies , the degree of inflammation caused by carrageenan is low . for example , 10  g / ml -carrageenan only increases the il-8 secretion level in ncm460 human colonic epithelial cells 4-fold , with even lower responses for - and -carrageenan . by comparison , 1  ng / ml lipopolysaccharide ( lps ) can induce > 10-fold increase in il-8 in the same cell line and can trigger a hundredfold upregulation for the tnf- expression in macrophages [ 23 , 24 ] . on the basis of these observations , investigators have pondered whether the inflammatory effect of carrageen is plenty to trigger severe inflammatory reactions in vivo . in our previous studies , we found that epithelial monolayers composed of differentiated caco-2 cells cocultured with thp-1 cells were easily damaged by carrageenan . because the intestinal tract is in constant contact with microorganism , including pathogens , the intestinal tract may undergo low levels of chronic inflammation , especially if pathogens accumulate . in this situation , the inflammation provoked by the pathogens might be enhanced by carrageenan . this situation may partially explain why carrageenan has been found to induce colonic inflammation in some studies , but not in others . therefore , we considered the possibility that carrageenan may act as a conditional inflammatory agent . according to our hypothesis , when the gut is in an inflammatory state , carrageenan will collaboratively aggravate the colitis . lps is an important constituent of the external wall of gram - negative bacteria , and lots of the inflammatory signals were triggered through lps component of gram - negative bacteria . therefore , in this study , we used lps as an inflammatory agent to study the synergistic effects of carrageenan on the lps - induced cellular inflammatory response . furthermore , we developed a citrobacter freundii ( dbs100)-induced nih mouse inflammation model to verify whether -carrageenan could aggravate bacterial - induced colitis in the mice intestinal tract . our findings provide an evidence to support the conditional impact of carrageenan in vitro and in vivo . the human colonic carcinoma cell line ht-29 was purchased from the china center for type culture collection and was incubated with eagle 's medium containing 10% fetal bovine serum at 37c in an atmosphere with 5% co2 . cells were cultured to 6080% confluence and then treated with -carrageenan or lps at the different dose and for the indicated time . the average molecular weight of 1  10 of -carrageenan was used in this experiment . the -carrageenan was dissolved in ultrapure water at different concentrations ( 1 , 10 , 20 , 40 , and 60  g / ml ) prior to experimentation . ht-29 cells were treated with or without indicated dose of lps ( 0.01 , 0.1 , and 1  g / ml ) for 0.5  h , 1  h , 2  h , 4  h , 6  h , 12  h , and 24  h. the secretion of il-8 was detected according to the elisa kit ( r&d systems , minneapolis , mn ) . ht-29 cells were incubated with or without indicated dose of -carrageenan for 24  h or were pretreated with the different dose of -carrageenan for 1  h and then add lps ( 1  g / ml ) for 24  h. the secretion of il-8 was detected according to the elisa kit ( r&d systems , minneapolis , mn ) . ht-29 cells were incubated with the different dose of -carrageenan for 24  h or were pretreated with indicated dose of -carrageenan for 1  h and then lps ( 1  g / ml ) is added for incubating 24  h. the cells were collected and mixed on ice with 1  g pe - tlr4 antibody of against human . these cells were washed twice in pbs after 25  min and then examined by beckman gallios flow cytometer ( beckman coulter , inc . , ht-29 cells were incubated with the different dose of -carrageenan for 24  h and treated with lps or -carrageenan for 1  h and then ht-29 cells were harvested with trypsin - edta . these cells were mixed with 1  g / ml fitc - lps for 25  min and analyzed on a flow cytometer . for the in vitro analyses , we incubated ht-29 cells in the different dose of -carrageenan for 24  h or pretreated with indicated dose of -carrageenan for 1  h and then added them to lps ( 1  g / ml ) for 24  h. then we harvested cells and isolated total rna with rnaiso plus reagent ( takara , dalian , china ) . for the in vivo analyses , we extracted total rna from gut mucosa using rnaiso plus reagent . after rna extraction , 0.4  g of total rna was reverse - transcribed into cdna in a 10  l reverse transcription reaction , and 2  l of the synthesized cdna was used as template for pcr amplification . the primers used for the amplification of md-2 , cd14 , and the reference gene , human-actin ( for in vitro experiments ) or tlr4 , nf-b , and the reference gene , mouse-actin ( for in vivo experiments ) are listed in table 1 . amplification reactions ( 20  l ) included sample cdna , primers and sybr premic ex taq ii mix ( takara , dalian , china ) . rt - qpcr was conducted on the mastercycler ep realplex real - time pcr system ( eppendorf , hamburg , germany ) using sybr - green i in accordance with the manufacturer 's instructions . the reaction mixtures were incubated at 95c for 300  sec and then at 95c for 10  sec , 60c for 1  min , and 72c for 15  sec for 40 cycles . the relative gene expression was determined by calculating the threshold cycle value , which was compared to the value for-actin using the relative quantification method . ht-29 cells were stimulated with the different concentrations of -carrageenan for 24  h or were pretreated with the indicated concentrations of -carrageenan for 1  h and then added to lps ( 1  g / ml ) for 24  h. nuclear extracts were prepared using the ne - per nuclear and cytoplasmic extraction kit ( pierce , rockford , il , usa ) according to manufacturer 's protocol . proteins ( 30  g ) from nuclear extracts and whole cell lysates were separated by 10% sodium dodecylsulfate - polyacrylamide gel electrophoresis and were measured with antibodies of against tlr4 , bcl10 , nf-b ( cst , danvers , ma , usa ) , or phospho - ib ( abcam , cambridge , uk ) , evaluated using the relevant hrp - linked mouse anti - human igg ( abcam , cambridge , uk ) . the immunoreactive proteins were visualized by enhanced chemiluminescence ( ecl ) kit ( santa cruz biotechnology , santa cruz , ca , usa ) . the results were quantified using nih image program by measuring the band intensity and compared to corresponding band intensity of -actin . to measure the impact of -carrageenan on nf-b activation , we transiently cotransfected with firefly luciferase reporter plasmid p - nf-b - luc and renilla luciferase plasmid p - rl into ht-29 cells and used x - treme gene hp dna transfection reagent for transfection . after transfection 1012  h , cells were treated with -carrageenan for 24  h or treated with -carrageenan for 1  h and then stimulated with lps ( 1  g / ml ) for a further 24  h. the luciferase activities measurement of cell was used by a previously described method . male and female nih ( s ) mice ( 3-week - old , weighing 1520  g ) were housed in pathogen - free conditions . all the mice were placed in clear plastic cages with free access to food and water , and each experimental group consisted of five males and five females . all experimental procedures were performed in accordance with the guide for the care and use of laboratory animals of the national institute of health , and the ethical committee of animal use and protection has approved animal protocols . these indicated concentration ( 1.7  mg / kg , low ; 8.3  mg / kg , med ; or 41.7  mg / kg , hig ) of -carrageenan were selected based on preliminary experiments in a related study [ 12 , 27 ] . to measure the aggressive impact of -carrageenan on c. freundii dbs100-induced inflammation , -carrageenan was orally administered for 1 week prior to c. freundii dbs100 treatment . the experimental model of inflammation was established using a previously described method with minor modifications . the bacterial strain used in this study ( dbs100 , citrobacter freundii biotype 4280 ) was purchased from the american type culture collection ( atcc ) . bacteria were grown at 37c in lysogeny broth ( lb ) ( american bioanalytical ) or on macconkey lactose agar plates . water in the mouse cages was supplemented with streptomycin ( 5  mg / ml ) for 48  h prior to bacterial inoculation . infection was performed by oral gavage with 10  cfu / mouse of c. freundii dbs100 grown overnight in lb broth ( 100  g / ml ) . these symptoms of diarrhea , weight loss , and death were monitored after 10 days following the dbs100 administration . to determine the ratio of regulatory t cells ( tregs , cd4+cd25+cd127dim ) and cytokine levels , blood and serum were collected from the mice through exsanguination by cardiac puncture . the severity of intestinal histopathology was graded and the entire colon was used to evaluate the severity of colonic tissues and expressions of nf-b , tlr4 at the mrna level , whereas the paraformaldehyde - fixed ( merck , darmstadt , germany ) or glutaraldehyde - fixed portion was used for further analyses , including histological examination , immunohistochemical analysis , and scanning electron microscopy ( sem ) . 80 mice were divided into eight groups , and each group have ten mice , including the blank group ( sterile lb broth ) : the dbs100 group ; the -carrageenan - groups ( low ; med ; hig ) ; and the -carrageenan + dbs100 groups ( low + dbs100 , med + dbs100 , and hig + dbs100 ) . the colonic tissues were dissected , fixed with 4% paraformaldehyde , and then were paraffined and cut into 4  m thick ; we used hematoxylin and eosin ( h&e ) to stain them . the colitis severity was quantified using the histological activity index ( hai ) [ 29 , 30 ] , as previously described   and as detailed in table 3 . to identify morphology changes on the epithelial cell surface , intestinal segments were processed as previously described . 100  l of peripheral blood was added to efluor450 , pe , apc , and efluor710 labeled anti - mouse cd3 , cd4 , cd25 , and cd127 , respectively ( all from ebioscience , california , usa ) . then 1  ml of erythrocyte lysis solution was added for incubating 20  min . these cells were then centrifuged at 200  g for 5  min and added to 500  l pbs . the levels of proinflammatory cytokines , including interleukin 6 ( il-6 ) , tnf- , th1 ( il-2 , interferon ifn- ) , th2 ( il-4 , il-10 ) , and th17 ( il-17 ) cytokines , were measured using the cytokine kit ( bd biosciences , nj , usa ) . these serum samples and standards were measured by flow cytometer . for immunocytochemistry , colonic tissues were steeped with 5% paraformaldehyde and then were paraffined and cut into 4  m thick . then anti - tlr4 antibody and anti - nf-b antibody ( 1  :  200 , abcam , cambridge , uk ) were used in this experiment . nonimmunized mouse igg at equal concentrations was used as a negative control for each primary antibody . rabbit anti - rabbit igg and goat anti - mouse igg ( 1  :  200 , zsgb - bio , peking , china ) were used as the secondary antibodies . images were analyzed using image pro plus 6.0 software ( media cybernetics uk , marlow , uk ) . the immunological histological chemistry ( ihc ) indexes for tlr4 and nf-b were determined as average integral optical densities according to the following equation : aiod = ( positive area / total area )  iod . spss software , version 16.0 ( spss inc . , chicago , il , usa ) , was used for statistical analyses . to determine the effect of lps on time - course and dose - response , ht-29 cells were treated with or without different concentrations of lps ( 0.01 , 0.1 , and 1  g / ml ) for 0.5  h , 1  h , 2  h , 4  h , 6  h , 12  h , and 24  h. as shown in figure 1(a ) , in 0.01 and 0.1  g / ml of lps , the secretion of il-8 experienced almost no change compared with the untreated sample ( p > 0.05 ) . however , ht-29 cells , treated with 1  g / ml lps for 0.524  h , had approximately 7.5%50.6% increase in the levels of il-8 secretion as compared to untreated cells . the greatest marked effect was observed with 1  g / ml lps ( 1.5-fold higher expression than untreated cells ; p < 0.01 ) at 24  h. our data showed that ht-29 cells treated with 0.01 and 0.1  g / ml lps for 212  h were not effective . therefore , in the present studies , at firstly we add different dose -carrageenan to ht-29 cells for 1  h and then add lps for incubating 24  h. to study whether-carrageenan may have differential effects depending on the inflammatory state of the cell , we treated ht-29 cells with-carrageenan in the presence and absence of lps . as shown in figure 1(b ) , in the absence of lps , il-8 production was not significantly different among the untreated sample and the samples treated with different doses of-carrageenan ( p > 0.05 ) . however , samples treated with 1  g / ml lps had approximately 1.58-fold increase in the levels of il-8 expression as compared to untreated cells ( p < 0.01 ) . moreover , pretreatment with increasing concentrations of -carrageenan further increased the il-8 levels induced by lps . the greatest synergistic effect was observed with 1  g / ml lps + 40  g / ml -carrageenan ( 1.45-fold higher expression than with lps alone ; p < 0.01 ) . these results suggest that -carrageenan does not stimulate il-8 on its own but enhances lps - stimulated il-8 expression in ht-29 cells . to explore the mechanism by which -carrageenan increases lps - dependent activation of il-8 , we assessed its effects on the expression and activity of tlr4 , which is known to function as an inflammatory signaling receptor for lps and other bacterial components . treatment with increasing dose of -carrageenan did not significantly promote the protein expression of tlr4 as assessed by western blotting ( figure 2(a ) ) . furthermore , pretreatment with 40 or 60  g / ml -carrageenan promoted additional enhancement of lps - induced tlr4 expression ( p < 0.01 ) ( figure 2(b ) ) . in agreement with the western blot results , no significant changes in tlr4 expression could be detected by flow cytometry after treatment with -carrageenan alone , but tlr4 levels were increased by 54% after exposure to 1  g / ml lps , with an additional increase observed for samples treated with -carrageenan + lps ( 38  8.18% additional increase for 40  g / ml + lps ; 35  2.88% additional increase for 60  g / ml + lps ; p < 0.05 ) ( figures 2(c ) and 2(d ) ) . while lps triggers innate immune signaling by reciprocating with tlr4 , lbp , md-2 , and cd14 are known to be required for the interaction between lps and tlr4 [ 3335 ] . therefore , we performed rt - qpcr to determine whether -carrageenan may also modulate the expression of md-2 and cd14 . our results showed that treatment with -carrageenan alone can not significantly alter the expression of either gene . however , lps alone significantly increased the expression of both genes ( md-2 : ~8-fold increase ; cd14 : ~2-fold increase ) . furthermore , pretreatment with -carrageenan before lps promoted additional enhancement of the expression of both factors ( 2.3-fold increase for both genes , compared with lps alone ) ( figure 3(a ) ) . these results suggest that -carrageenan synergizes with lps to increase the expression of both md-2 and cd14 . because il-8 is known to be activated by lps signaling through tlr4 , we considered the possibility that the increased levels of tlr4 , md-2 , and cd14 could provide an explain for the effects of -carrageenan on il-8 secretion . to measure whether the enhanced increase of tlr4 , md2 , and cd14 through -carrageenan increases the binding of lps to the cell surface , after 24  h treatment with -carrageenan , we applied fitc - lps to ht-29 cells . -carrageenan treatment for 24  h can not upregulate fitc - lps combined with the ht-29 cells ( figure 3(b ) ) . as a potential explanation for these findings , we tested whether -carrageenan might also interplay with tlr4 , therefore competitively inhibiting the connection with fitc - lps . to determine this hypothesis , we use lps or -carrageenan - treated cells for 1  h and then add fitc - lps to the cells . our results revealed that adding either 1  g / ml lps or -carrageenan downregulated the bonding of fitc labeled lps ( figure 3(c ) ) , which indicates that -carrageenan binds to the same target as lps . the decrease occurred in a concentration - dependent manner , which further verifies our results . therefore , these experiments rule out the possibility that the upregulation of tlr4 or its signaling partners can explain the enhancement of il-8 levels by -carrageenan in lps - treated ht-29 cells . because -carrageenan failed to promote the binding of lps to tlr4 , we considered other possible explanations for its ability to cooperate with lps to increase the il-8 secretion . to determine whether -carrageenan could enhance bcl10-mediated lps - induced activation in ht-29 cells , we performed western blotting assays . as shown in figure 4(a ) , -carrageenan alone mediated a slight nonstatistical increase in the expression of bcl10 , the phosphorylation of ib , and the nuclear expression of nf-b in ht-29 cells at high doses ( p > 0.05 ) . additionally , 1  g / ml lps increased bcl10 expression by 48.89% and ib phosphorylation by 59.10% ( p furthermore , -carrageenan pretreatment markedly enhanced the lps - dependent increase in bcl10 expression and ib phosphorylation in a dose - dependent manner ( p < 0.01 , figure 4(b ) ) . the expression of nf-b was also enhanced significantly at lower -carrageenan concentrations ( 4060  g / ml ; p < 0.01 ) . to confirm the induction of nf-b transcriptional activity by -carrageenan and lps , we performed luciferase assays using a nf-b reporter plasmid . consistent with our other findings , -carrageenan treatment induced a low - level increase in the transcription activity of nf-b , but the increase was not statistically significant . however , treatment with lps significantly increased the transcriptional activity of nf-b ( p < 0.01 ) , and pretreatment with -carrageenan potentiated this lps - stimulated transcriptional activity of nf-b in ht-29 cells . these findings are coincident with previously published results suggesting that -carrageenan potentiated lps - stimulated activation of nf-b mediated by bcl10 . lps is an important constituent of the external wall of gram - negative bacteria , and lots of the inflammatory signals were triggered through lps component of gram - negative bacteria . based on our in vitro findings , we postulated that the inflammatory response induced in vivo by a gram - negative bacterium might also be enhanced by -carrageenan . to explore this possibility , we established a c. freundii dbs100-induced inflammation model in mice . the effect of -carrageenan pretreatment on c. freundii dbs100-induced gut damage was investigated by measuring survival rate and weight loss . as shown in figure 5(a ) , any comparisons between the groups had marked differences , other than -carrageenan alone - treated mice ( low , med , and high ) on days 1 to 10 ( p < 0.01 ) . however , the body weights of the c. freundii dbs100-treated mice were markedly decreased at day 1 , with a more significant difference at day 10 ( p < 0.01 ) ; in addition , the -carrageenan + dbs100-treated mice had significantly greater decreases in body weight than the dbs100 mice . the hig + dbs100 group showed the most dramatic result , with the body weight decreasing by 5.1%   1.01 ( for dbs100 mice ) and by 15.7%   1.43 ( for hig + dbs100 mice ) at day 10 ( p < 0.01 ) . at the end of the experiment ( day 10 ) , no mice in the blank or the -carrageenan groups died ; however , one mouse in the c. freundii dbs100-treated group died ( 1/10 ) at day 7 ( figure 5(b ) ) . additionally , treatment with -carrageenan prior to bacterial infection obviously increased the number of dead mice , even starting at day 1 ; there were 1 , 2 , and 3 dead mice in the low + dbs100 , med + dbs100 , and hig + dbs100 groups . by day 10 , the number of dead mice in the hig + dbs group reached 5 ( i.e. , 50% mortality ) . thus , these findings demonstrate the exacerbatory effect of -carrageenan towards bacterial - induced inflammatory gut disease in the mouse . to further explore the in vivo effects of -carrageenan in exacerbating c. freundii dbs100-induced gut inflammation , we examined the intestinal mucosal gut morphology of the eight groups of mice . the symptoms of inflammation were obvious after exposure to c. freundii dbs100 for 10 days ( figure 6 , see ( b ) versus ( a ) ) . the pathologic changes included a slight increase in the rigidity of the colon and signs of colonic thickening , which are consistent with previously reported symptoms for the entire colon after exposure to c. freundii dbs100 . furthermore , -carrageenan treatment alone had no detectable impact on the colons of the low , med , and hig groups of animals , which exhibited apparently normal colonic mucosa ( figures 6(c ) , 6(e ) , and 6(g ) ) . however , pretreatment of high - dose -carrageenan in the hig + dbs100 group led to more severe inflammation ( figure 6(h ) ) . this group of mice showed invasive hyperemia , bleeding ulceration , and focal mucosal necrosis . the macroscopic inflammation scores for colon damage supported our observations ( figure 6(i ) ) . therefore , our results verify that -carrageenan potentiates c. freundii dbs100-stimulated colitis . to further examine the effects of -carrageenan on c. freundii dbs100-induced gut inflammation at the animal level , we performed histological analysis of gut tissue sections ( figure 7 ) . the blank group indicated regular architecture , with abundant of goblet cells and an unbroken epithelial layer and crypts ( a ) . however , compared with the blank group , colonic sections from dbs100-infected mice indicated crypt elongation , goblet cell loss , and mild inflammation ( ( b ) versus ( a ) ) . similar to the blank group , the colonic tissues of the -carrageenan - treated groups also showed no obvious inflammatory presentations ( ( c ) , ( e ) , and ( g ) ) . however , -carrageenan aggravated the microscopic colon damage induced by dbs100 for the hig + dbs100 group ( h ) . this group possessed symptoms of architectural crypt distortion , epithelial damage , mucosal inflammation with inflammatory cell infiltration in the lamina propria , reduced numbers of goblet cells , and submucosa edema . these results were supported by the hai scores , which were 2.08-fold higher for the hig + dbs100 group than for the dbs100 group ( p < 0.01 ; figure 7(i ) ) . to verify these findings , we performed scanning electron microscopy ( sem ) ( figure 8) . the blank group exhibited a normal appearance , with intact microvilli and normal epithelial cell surface and crypt architecture ( a ) . however , the colons from the dbs100 group had injury to the mucosa skin and effacement of the microvilli ( b ) . -carrageenan treatment can not impact the morphology in the absence of c. freundii dbs100 ( ( c ) , ( e ) , and ( g ) ) ; however , for mice infected with bacteria , -carrageenan exacerbated the morphological variation of the colon in a concentration - dependent manner ( ( d ) , ( f ) , and ( h ) ) . there was more pronounced injury and diffuse ulcers on the mucosa surface in the hig + dbs100 group ( h ) . collectively , these results verify at both the macroscopic and the microscopic levels that -carrageenan aggravates c. freundii dbs100-induced gut inflammation . tregs are subpopulation of t cells that reduce inflammatory processes by maintaining tolerance to self - antigens . to examine the impact of -carrageenan on the treg subpopulation in mice treated with or without c. freundii , we performed flow cytometric analysis ( figure 9 ) . our results show that the ratio of tregs in systemic circulation was markedly reduced in the dbs100 group compared to the blank group ( 7.21%   0.54 versus 8.77%   0.38 ; p furthermore , -carrageenan alone did not affect the treg ratio , but pretreatment with -carrageenan promoted further decrease in dbs100-infected mice . the decrease was the greatest in the peripheral blood from the hig + dbs100 , which showed a 24.17% reduction relative to the dbs100 group . these findings demonstrate that -carrageenan inhibits the activity of regulatory t cells , which may further aggravate the imbalance of the immune response . we assessed the levels of the proinflammatory ( tnf , il-6 ) , th1 ( ifn- and il-2 ) , th2 ( il-4 , il-10 ) , and th17 ( il-17a ) cytokines in serum samples of mice ( table 4 ) . the levels of tnf , il-6 , ifn- , il-2 , and il-17a were elevated in the dbs100 group compared with blank group ( p < 0.05 or p < 0.01 ) ; in contrast , the levels of il-10 , which mediates anti - inflammatory t cell responses , were significantly decreased ( p < 0.05 ) , while il-4 levels were below the detection limit . some slight increases in the cytokine levels were observed in the serum of -carrageenan alone groups , but these increases were not significant ( p > 0.05 ) . however , the -carrageenan + dbs100 groups tended to have higher levels of cytokines than the dbs100 group . this was especially noted for the hig + dbs100 group compared to the c. freundii dbs100 group ( p < 0.05 ) . on the other hand , the administration of high - dose -carrageenan led to markedly collaborative downregulation for the il-10 levels ( 4.61  0.671  pg / ml versus 8.95  1.01  pg / ml for the dbs100 group ; p < 0.05 ) . to determine whether in the inflammatory mouse model , like in ht-29 cells , -carrageenan enhances inflammation - dependent secretion of tlr4 and nf-b , we examined the effects of -carrageenan on tlr4 and nf-b secretion in the gut mucosa . the secretion of tlr4 and nf-b in the colonic epithelium was induced by c. freundii dbs100 ( p < 0.01 ) . induction was not observed for -carrageenan alone , but -carrageenan caused further upregulation in mice treated with c. freundii dbs100 in a concentration - dependent manner ( figure 10(a ) , table 5 ) . the most significant upregulation was observed in the hig + dbs100 group , which showed a 1.29-fold upregulation in tlr4 levels and a 1.32-fold upregulation in nf-b levels compared with the dbs100 group ( p < 0.01 ) . we also examined the expression of tlr4 and nf-b at the mrna level by quantitative rt - pcr . as shown in figure 10(b ) , a similar pattern of regulation was observed for these mrnas . these results verify that -carrageenan enhances the c. freundii dbs100-dependent induction of tlr4 and nf-b in the intestinal mucosa of infected mice . an important role of the intestinal tract is to absorb nutrients while resisting entry of invasive pathogens that are involved in the inflammatory response . when the intestinal tract is infected by pathogens , microbe - associated molecular patterns , such as lps , upregulate the level of proinflammatory cytokines in the colon through classical inflammatory signaling pathways [ 3739 ] . considering the controversy surrounding the safety of carrageenan and its inflammatory properties in animal models , the present study focused on the potential ability of -carrageenan to enhance lps - mediated inflammatory pathway in human colonic cells . to achieve this purpose , we used a c. freundii dbs100-induced intestinal inflammation model to determine the impact of -carrageenan on aggravate inflammatory of the intestinal tract . a previous study demonstrated the impact of carrageenan on the production of il-8 in the human colonic epithelial cell line ncm460 . treatment with 1  g / ml - , - , or -carrageenan was shown to result in 2.03-fold , 1.59-fold , or 1.44-fold increase in il-8 secretion by ncm460 cells . in the present study , we observed only about 1.12-fold increase in il-8 secretion in ht-29 cells after treatment with 60  g / ml -carrageenan alone . this difference between the two studies may relate to differences in the degree of differentiation of the colon epithelial cells that were used . some reports have shown that carrageenan could induce cancer and ulcer of gut [ 40 , 41 ] . although ncm460 comes from the normal human intestinal epithelial cells , it was transformed minimally to be an immortalized cell stain , and it can express tlr4 by itself ; whereas ht-29 is moderately differentiated colon epithelial cell , the expression of tlr4 is very low in ht-29 cell . in our research , tlr4 is one of the targets of carrageenan and lps . the low secretion of tlr4 in ht-29 cells will be a better cell model to research the synergy impact of carrageenan on lps - stimulated expression and activity of tlr4 . ht-29 cells also express lots of proinflammatory cytokines including interferon- , tnf- , and il-1 , which will induce the release of other inflammatory mediators , such as il-8 , which are increased in ibd [ 45 , 46 ] . in this research , it is obvious that the amount of il-8 induced with -carrageenan alone is not enough to activate an inflammatory response . however , a common finding of our study is that -carrageenan was found to synergize with lps to trigger an enhanced inflammatory response , though the extent of the synergy appears to be high for ht-29 cells . pretreatment with carrageenan also has been demonstrated to enhance lps - induced tnf- production in whole blood from mice . thus , -carrageenan may either increase the signaling within the lps - induced inflammatory signaling pathway or provide a parallel pathway with a similar end result of enhancing inflammatory response . when lps interacts with tlr4 , a cascade is activated that can lead to systemic inflammatory response [ 20 , 48 , 49 ] . we demonstrated that -carrageenan alone can mediate a slight increase in the secretion of tlr4 in ht-29 cells and that lps can mediate a significant increase but that the levels were the greatest after treatment with both lps and -carrageenan . furthermore , the levels of the auxiliary proteins md-2 and cd14 , which are needed for tlr4 signaling , were synergistically increased . thus , our data seems to be consistent with a model in which -carrageenan enhances lps - induced secretion of il-8 by enhancing tlr4 signaling . however , -carrageenan pretreatment can not promote the cell surface fitc - lps combined with tlr4 and in fact functioned as a competitive inhibitor of lps , which is consistent with the demonstration that -carrageenan can bind tlr4   and suggests that the synergy may occur at other targets in the signaling pathway . our data indicated that treatment of ht-29 with lps significantly increases bcl10 , phospho - ib , and nf-b expression , which is consistent with previous studies . bcl10 is known to be a potential mediator of lps - induced activation of nf-b . additionally , -carrageenan mediates prolonged activation of both bcl10 and nf-b in ht-29 and ncm460 cells [ 48 , 50 ] . however , we found that high doses of -carrageenan alone could only mediate a slight , nonstatistical upregulation of the expression of bcl10 , phosphorylated ib , and nf-b . interestingly , when -carrageenan was applied before lps treatment , the expression of bcl10 , phosphorylation of ib , nuclear level of p65 subunit , and transcription activity of nf-b were significantly increased . these results clearly demonstrated that -carrageenan enhances lps - induced secretion of il-8 not through enhancing tlr4 signaling , because -carrageenan can not promote the cell surface fitc - lps combined with tlr4 and suggests that -carrageenan can competitively inhibit lps combining with tlr4 receptor . however , -carrageenan enhances lps - induced il-8 secretion by stimulating the bcl10-nf-b pathway in ht-29 cells . given that lps is derived from gram - negative bacteria , we also asked whether -carrageenan could promote the inflammatory response induced by a gram - negative pathogen in vivo . to answer this question , c. freundii dbs100 was used to establish an intestinal inflammation model , because its virulence factors are similar to those of enteropathogenic escherichia coli of human [ 28 , 51 ] . furthermore , c. freundii dbs100 induces inflammatory processes that are similar to those of inflammatory bowel disease in humans . our data show no weight loss , no mortality , and similar histological appearance as in untreated mice for the -carrageenan alone treatment groups , which indicates that -carrageenan alone does not cause inflammation in vivo . this conclusion is consistent with studies demonstrating that carrageenan does not produce intestinal ulcerations at doses of up to 5% . carrageenan has also been demonstrated not to be carcinogenic in long - term bioassays in rodents . our results showed that administration of c. freundii dbs100 resulted in a minimal weight loss and histological damage , with 10% mortality , which is close to the values from the literature . however , -carrageenan pretreatment before c. freundii dbs100 infection resulted in significantly weight loss , with serious colon damage and 2050% mortality , depending on the -carrageenan dose . morphological and histological data were consistent with the possibility that intragastric administration of -carrageenan can aggravate the c. freundii dbs100-induced inflammatory death of mice . previous studies have addressed the ability of carrageenan to increase the proinflammatory cytokines tnf , il-8 , and il-6 [ 19 , 20 , 53 ] . in our study , il-6 and tnf were not activated by -carrageenan alone but were significantly increased by -carrageenan in conjunction with c. freundii . we also examined the upregulation of cytokines produced by th1 cells , including ifn- and il-2 , and observed an analagous pattern of regulation . several reports have demonstrated that il-17a is a major driver of intestinal inflammation   through its ability to activate t cell production . consistently , -carrageenan provided a similar synergistic increase in the level of il-17a in c. freundii dbs100-treated mice . thus , our findings support the ability of -carrageenan to conditionally exacerbate the inflammatory response . tregs are important for maintaining homeostasis in the intestine and controlling inflammation by inhibiting the activation and proliferation of cd4 and cd8 cells . in this study , we found that -carrageenan did not significantly affect treg levels on its own but significantly reduced the ratio of tregs in a dose - dependent manner in mice exposed to c. freundii dbs100 , which is consistent with our other findings and suggests a mechanism of humoral immune tolerance activated by -carrageenan under inflammatory environmental conditions . finally , we examined the c. freundii dbs100 inflammatory model to determine whether the in vivo exacerbatory effects of -carrageenan are mediated through the bcl10-nf-b signaling pathway . the different doses of -carrageenan pretreated mice indicated a marked upregulation in the dbs100-stimulated secretion of nf-b , indicating that nf-b might constitute a key component in the -carrageenan intestinal inflammatory exacerbation . tlr4 was also increased ; however , whether increased tlr4 plays a role in the inflammation requires further investigation . in conclusion , we demonstrated enhanced effects of -carrageenan on the inflammatory reaction in vitro and in vivo . -carrageenan increased the expression of tlr4 receptor but competitively bound tlr4 , blocking lps binding , which rules out the enhanced tlr4 levels as an explanation for the synergistic effects of -carrageenan . however , our results also showed that -carrageenan can participate in the bcl10-nf-b - mediated pathway to enhance lps stimulated secretion of il-8 in ht-29 cells . the c. freundii dbs100-induced intestinal inflammation model further verified that -carrageenan could aggravate the inflammatory reaction of the colon to pathogen exposure -carrageenan modulated cytokine production , downregulated the proportion of tregs , and upregulated nf-b , all of which are likely to contribute to the exacerbatory effects of -carrageenan . collectively , our results suggest that -carrageenan serves as a potential inflammatory agent that magnifies existing intestinal inflammation .
background . 
 the dietary usage of carrageenan as common food additive has increased observably over the last 50 years . 
 but there is substantial controversy about its safety 
 . methods . 
 we investigated whether the -carrageenan could enhance lipopolysaccharide - induced il-8 expression by studying its actions on the tlr4-nf-b pathway . 
 the aggravating effect of -carrageenan on citrobacter freundii dbs100-induced intestinal inflammation was also investigated in a mouse model . 
 results . 
 our data show that -carrageenan pretreatment promoted lps - induced il-8 expression in ht-29 cells . although cd14 , md-2 , and tlr4 were upregulated , the binding of lps was not enhanced . 
 however , the pathway of bcl10-nf-b was triggered . 
 interestingly , -carrageenan competitively blocked the binding of fitc - lps . 
 furthermore , pretreatment with -carrageenan for one week previous to gavage with c. freundii dbs100 markedly aggravated weight loss , mortality , and colonic damage . 
 the secretion of cytokines was unbalanced and the ratio of tregs was decreased significantly . in addition , 
 -carrageenan , together with c. freundii dbs100 , enhanced the transcription and secretion of tlr4 and nf-b . conclusions . 
 -carrageenan can synergistically activate lps - induced inflammatory through the bcl10-nf-b pathway , as indicated by its aggravation of c. freundii dbs100-induced colitis in mice . 
 general significance . 
 our results suggest that -carrageenan serves as a potential inflammatory agent that magnifies existing intestinal inflammation .