BACKGROUND
diarrheal diseases in animals cause the livestock industry great economic loss and also represent a serious threat to farm animal welfare  <cit> .
one of the main causes of diarrhea is gastrointestinal disorder and intestinal mucosa barrier dysfunction or “leaky gut” is reported to play a vital role in the pathophysiology of a variety of gastrointestinal disorders  <cit> .
the intestinal mucosal barrier is maintained by tight junctions   <cit> , which are multiprotein complexes located around the apical end of the lateral membrane of the epithelial cells and seal the paracellular space between adjacent epithelial cells  <cit> .
tjs function as a selective/semipermeable paracellular barrier, which regulates the transport of ions, water and solutes through the paracellular pathway  <cit> .
the most important and critical components in the structural and functional organization of the tjs are occludin, zonula occluden- <dig>  and claudin- <dig>  <cit> .
the importance of these tj proteins has been demonstrated under many conditions.
for example, increased intestinal permeability, which is thought to increase the load of bacterial and dietary antigens in the lamina propria, has been observed in early weaned pigs  apparently due to aberrant expression of the essential tj proteins  <cit> .
hence, modulation of tj function, particularly through increasing levels of occludin, zo- <dig> and claudin- <dig>  is a target for novel therapeutic or prophylactic treatments against a range of diseases.
probiotics, defined as live microorganisms which, when consumed in adequate amounts as part of food, confer a health benefit on the host  <cit> , are widely used in humans and animals.
since the european union banned the use of antibiotics as growth promoters in  <dig>  the use of probiotic animal feed additives has increased as reported both in vivo  <cit>  and in vitro  <cit> .
ingestion of probiotics has been shown to prevent or treat a variety of gut disorders  <cit> , although the mechanisms through which they function are not completely known.
however, a large amount of evidence suggests that one mechanism may be via protection or augmentation of the intestinal mucosal barrier function  <cit> .
lactobacillus, one of the most commonly used probiotic microorganisms  <cit> , frequently occurs in the intestinal microflora of various vertebrates and exerts lactobacillus-mediated barrier protection via modulation of tjs  <cit> .
previous studies have demonstrated that this genus significantly reduces the incidence and the severity of diarrhea  <cit>  and helps maintain a functional mucosal barrier  <cit> .
to test the hypothesis that natural lactobacillus found in animal intestinal tract may be a potential source of probiotic bacteria for livestock.
in  <dig>  our group isolated thousands of strains of lactobacillus from the gastrointestinal tract git of eleven healthy weaned piglets from different pig farms.
according to criteria including tolerance to heat, low ph, and bile salts, as well as storage stability and antagonism to pathogenic agents, four strains, lactobacillus fermentum i <dig>  l. gasseri s <dig>  l. reuteri i <dig> and l. acidophilus i <dig>  were selected  <cit> .
subsequently, a series of in vivo and in vitro studies demonstrated that l. fermentum i <dig> had the greatest adhesion  <cit> , and showed beneficial effects on growth performance and antioxidative activity, as well as modulation of intestinal microbiota and mucin secretion in piglets  <cit> .
summarily, our work in the past decade indicates that l. fermentum i <dig>  now known as l. reuteri i <dig> according whole-genome sequence of this strain  <cit> , can be used as a potential probiotic.
recently, our group found that oral administration of l. reuteri i <dig> favored intestinal development and maintained the gut epithelial barrier in neonatal piglets  <cit> .
however, the mechanism through which it exerts functions on the gut epithelial barrier is not fully understood.
the aim of the present study was to evaluate l. reuteri i5007-mediated intestinal mucosal barrier augmentation in a newborn piglet model and investigate the mechanism for modulation of tjs in a porcine jejunal epithelial cell line  challenged by lipopolysaccharides .
RESULTS
oral administration of l. reuteri i <dig> enhanced the protein abundance of intestinal epithelial tjs in newborn piglets
twelve male newborn piglets  were orally administrated with  <dig> % sterile peptone solution  or 6 × 109 cfu of l. reuteri i <dig>  daily for 14 days.
the expression of tj protein claudin- <dig>  occludin and zo- <dig> both in the jejunal and ileal epithelium were measured using immunoblotting to investigate the effect of l. reuteri i <dig> on tj function.
compared with control group, no significant difference was observed in the jejunal epithelial claudin- <dig> expression in the l. reuteri i <dig> group, while oral administration of l. reuteri i <dig> significantly increased the protein abundance of occludin and zo- <dig> in jejunal epithelium of piglets .
however, all levels of these tj proteins were distinctly higher in the ileum of piglets administrated with l. reuteri i <dig> than those of control piglets .figure  <dig>
expression of tj proteins in piglets following oral administration with
l. reuteri
i <dig>  four-day-old piglets were orally administrated with 6 × 109 cfu of l. reuteri i <dig> per day for 14 days.
subsequently, proteins were isolated from the intestinal tissues of piglets and then the expression of claudin- <dig>  occludin and zo- <dig> was measured using immunoblotting.
protein levels of occludin and zo- <dig> in jejunal tissue lysates were induced by l. reuteri i <dig>
l. reuteri i <dig> increased the expression of claudin- <dig>  occludin and zo- <dig> in the ileum.
data are represented as mean ± sem, n =  <dig>  *p <  <dig>  compared with control piglets.
l. reuteri i <dig> maintained transepithelial electrical resistance  of ipec-j <dig> cells
ipec-j <dig> cell monolayers cultured for complete confluence were treated with l. reuteri i <dig> cells  and teer values were measured every two hours for 10 h .
when compared with the untreated control, treatment with l. reuteri i <dig> had no effect on teer of ipec-j <dig> cells during 0–2 h, but did lead to significantly higher values of teer during 4–10 h. on the whole, an  <dig> % decline of teer values was caused during 10 h of the measurement, as seen for the control, while co-incubation with l. reuteri i <dig> showed almost no decline  in teer values, indicating that l. reuteri i <dig> had a positive maintaince on teer of ipec-j <dig> cells with time.figure  <dig>
response of transepithelial electrical resistance  to
l. reuteri
i <dig> in ipec-j <dig> cells.
ipec-j <dig> cells were treated with 3 × 107 cfu/ml of l. reuteri i <dig> cells alone for  <dig>   <dig>   <dig>   <dig>   <dig> and 10 h, and teer values were measured, respectively.
treatment with l. reuteri i <dig> induced a nice maintaince on teer of ipec-j <dig> cells with time.
results are represented as mean ± sem.
the experiment was repeated three times with quadruplicate wells in each assay.
*p <  <dig>  vs. control cells.
l. reuteri i <dig> differentially affected lps-induced tnf-α and il- <dig> mrna expression in ipec-j <dig> cells
to further elucidate the effects of l. reuteri i <dig> on modulation of tj function, the mrna expression of inflammatory cytokines including tnf-α and il- <dig> was determined in ipec-j <dig> cells treated with lps for  <dig>   <dig>   <dig>   <dig> or 24 h, following pre-treatment with l. reuteri i <dig> for 2 h. according to a previous study of our group, l. reuteri i <dig> could behave strong adhesion ability to monolayer cells by co-culture with cells for 1 h  <cit> .
resta-lener and barrett  <cit>  showed enteroinvasive escherichia coli evoked fall in teer was prevented if cell monolayers were pretreated with live probiotics, and was also partially attenuated by exposure simultaneously to probiotics and enteroinvasive e. coli, but not if the cells were infected with enteroinvasive e. coli for one hour and then exposed to probiotics.
considering these circumstances, to investigate the protect effects of l. reuteri i <dig> on tj proteins, we appropriately extended the incubation time for pretreatment with l. reuteri i <dig> to 2 h.
figure 3a shows that lps significantly increased tnf-α mrna expression after 4 h, but not at 1 h. no significant differences were observed in the mrna expression of tnf-α at 1 h among different treatments.
however, tnf-α mrna expression in lps-stimulated cells was time-dependently increased compared with the control treatment, while after 12 h, the increase in tnf-α mrna expression reached a plateau.
similarly, the mrna expression of il- <dig>  was increased in ipec-j <dig> cells treated with lps after 8 h and was maintained until 12 h treatment.
collectively, treatment with l. reuteri i <dig> in the absence of lps had no effect on mrna levels of tnf-α and il- <dig> compared with the control group at different time points.
however, in the presence of lps, l. reuteri i <dig> significantly decreased the mrna expression of these two inflammatory cytokines, compared with the lps group after 8 h.figure  <dig>
mrna expression of inflammatory cytokines in lps-activated ipec-j <dig> cells.
ipec-j <dig> cells were pre-treated with either l. reuteri i <dig>  or l. reuteri i <dig> culture supernatant  2 h prior to lps-activation.
quantitative rt-pcr  analysis was performed to determine the mrna expression of inflammatory cytokines after  <dig>   <dig>   <dig>   <dig> or 24 h treatment with lps.
lps affected no change in tnf-α expression at 1 h, but significantly increased tnf-α expression during 4–24 h. however, both l. reuteri i <dig> and its culture supernatant had no effect on tnf-α expression at the different time points in the absence of lps, while they inhibited or prevented lps-induced expression of tnf-α.
there were obvious increases in il- <dig> expression during 8–24 h, but not 1–4 h induced by lps, while l. reuteri i <dig> and its culture supernatant produced no change at any time compared with control cells.
l. reuteri i <dig> and its culture supernatant inhibited or prevented lps-induced il- <dig> expression.
results are represented as mean ± sem, n =  <dig>  *p <  <dig>  vs. control cells, and #p <  <dig>  vs. cells in the lps group.
l. reuteri i <dig> augmented lps-induced tj protein expression in ipec-j <dig> cells
we further investigated the mrna expression of claudin- <dig>  occludin and zo- <dig> in inflamed cultured intestinal cells.
treatment of ipec-j <dig> cells with 1 μg/ml lps had no effect on the mrna expression of claudin- <dig> and zo- <dig> during 1–4 h, but showed a significant decline during 8–24 h .
similarly, compared with the control treatment, lps time-dependently reduced occludin mrna expression.
in detail, no difference at 1 h, but decreases during 4–24 h .
however, there was no significant change in mrna expression of all three tj proteins in the other four groups versus the control group.
pre-treatment with l. reuteri i <dig> significantly mitigated or prevented the reduction of mrna expression of all three tj proteins induced by lps.figure  <dig>
mrna expression of tj proteins in lps-activated ipec-j <dig> cells.
qrt-pcr analysis showed that lps induced a decrease in mrna expression of claudin- <dig> at  <dig>   <dig> and 24 h, while l. reuteri i <dig>  and l. reuteri i <dig> culture supernatant  effectively inhibited lps-induced expression of claudin- <dig>   there was no change in occludin expression at 1 h, but a significant decrease after 4 h treatment with lps.
however, l. reuteri i <dig> and its culture supernatant inhibited the decline.
lps induced a distinct decrease in zo- <dig> expression after 8 h, which was inhibited by l. reuteri i <dig> and its culture supernatant.
results are represented as mean ± sem, n =  <dig>  *p <  <dig>  vs. control cells, and #p <  <dig>  vs. cells in the lps group.
we used immunoblotting to investigate the protein abundance of claudin- <dig>  occludin and zo- <dig>  in agreement with the observation of mrna expression of these tj proteins, there was no significant difference in the levels of these tj proteins in the other four groups  compared with the control group .
however, lps stimulation robustly decreased the levels of claudin- <dig>  occludin and zo- <dig> during 4–24 h , but not at 1 h .
treatment of ipec-j <dig> cells using l. reuteri i <dig> markedly induced higher levels of these proteins than those in cells treated with lps during 4–24 h.figure  <dig>
levels of tj proteins in lps-activated ipec-j <dig> cells at different time points.
proteins were isolated from ipec-j <dig> cells and the expression of tj proteins was assessed using immunoblotting.
there was no significant difference in claudin- <dig>  occludin and zo- <dig> expression among the different treatments at 1 h .
lps produced decreases in all three tj proteins at  <dig> ,  <dig> ,  <dig>  and  <dig>  h. although l. reuteri i <dig>  and its culture supernatant  had no effect on the levels of tj proteins in the absence of lps, they inhibited lps-induced levels of tj proteins.
results are represented as mean ± sem, n =  <dig>  *p <  <dig>  vs. control cells, and #p <  <dig>  vs. cells in the lps group.
l. reuteri i <dig> culture supernatant differentially affected lps-induced inflammatory cytokine and specific tj protein expression in ipec-j <dig> cells
to further elucidate whether the culture supernatant was involved in l. reuteri i5007-mediated improvement in tj function, the culture supernatant of l. reuteri i <dig> was also added to pre-treat ipec-j <dig> cells.
compared with the control group, cells treated with l. reuteri i <dig> culture supernatant in the absence of lps caused no significant change in tnf-α and il- <dig> mrna expression.
however, the culture supernatant of l. reuteri i <dig> did reduce lps-induced inflammatory cytokine expression during 8–24 h .
in general, both mrna expression and protein levels of claudin- <dig>  occludin and zo- <dig> in cells pre-treated with the culture supernatant of l. reuteri i <dig> were higher than those in cells induced by lps after  <dig> or 8 h .
discussion
the defense response of the intestinal epithelium against pathogenic bacteria has been extensively explored, and evidence suggests that interactions between commensal bacteria and the host are involved in this process  <cit> .
thus, much attention has been paid to the effects of probiotics on gut barrier function.
some probiotics might improve or prevent gut barrier dysfunction caused by pathogenic bacteria or other toxic substances  <cit> .
for example, l. plantarum significantly ameliorated phorbol ester-induced zo- <dig> and occludin redistribution and increased permeability in caco- <dig> cells  <cit> .
therefore, although the mechanisms of action of probiotics may vary, the vital mechanism of probiotics is regulating the expression of tj proteins.
the effects of probiotics on gut barrier in animal models are commonly studied in rodents.
for example, l. helveticus and l. rhamnosus prevented chronic stress induced intestinal abnormalities in rats, which received 7 days of these strains in the drinking water prior to either a water avoidance stress or a sham stress for one hour per day for ten consecutive days and remained on these probiotics during the duration of the study  <cit> .
however, reports in pigs, especially in neonatal piglets, are rare.
in the present study, newborn piglets were selected to study the effects of l. reuteri i <dig> on the expression of tj proteins in the intestinal epithelium.
we showed that l. reuteri i <dig> up-regulated the expression of jejunal epithelial occludin and zo- <dig>  and ileal epithelial claudin- <dig>  occludin and zo- <dig> in young piglets, indicating l. reuteri i <dig> effectively improved the intestinal mucosal barrier function of newborn piglets.
this was consistent with an earlier study  <cit>  showing that l. rhamnosus accelerated maturation of the intestinal mucosal barrier in mice via up-regulating the protein level of claudin- <dig>  oral administration of vsl# <dig>  a mixture of streptococcus thermophilus, three strains of bifidobacterium and four strains of lactobacillus, in samp mice, obviously improved ileal epithelial permeability and increased the expression of occludin, but not claudin- <dig> or zo- <dig>  <cit> , which was not entirely consistent with our results.
this might be due to different experimental conditions including strains of probiotic bacteria and animal models.
we next examined the effects of l. reuteri i <dig> on the expression of inflammatory cytokines and specific tj proteins in ipec-j <dig> cells induced by lps.
lps is an integral component of the gram-negative bacteria cell wall, capable of binding to toll-like receptor- <dig> and activating a variety of protein kinase signaling pathways, subsequently generating inflammatory cytokines and other mediators  <cit> .
liu et al.
<cit>  found that treatment of porcine intestinal epithelial cells  and rat intestinal epithelial cells  with lps for 16 h markedly increased the secretion of il- <dig>  while co-culture with l. reuteri robustly inhibited lps-induced overexpression of il- <dig> in ipec-j <dig> cells.
in the present study, lps induced increases in mrna expression of tnf-α and il- <dig> of ipec-j <dig> cells after  <dig> or 8 h, and pre-treatment with l. reuteri i <dig> significantly suppressed lps-induced tnf-α and il- <dig> expression.
in addition, several studies have reported effects of probiotics on tnf-α and il- <dig> expression induced by lps, but the regulatory action differs in different species or even different strains.
for instance, l. reuteri actt  <dig> greatly inhibited the generation of tnf in lps-activated human monocytoid thp <dig> cells, as an immunosuppressive action  <cit> .
in contrast, l. reuteri actt  <dig> stimulated tnf-α production, as an immunostimulatory action  <cit> .
moreover, rachmilewitz et al.
<cit>  observed that co-culture of mice macrophages  with dna of vsl# <dig> bacteria significantly promoted il- <dig> production.
tjs, apical-most component of the junctional complex in epithelial cells, determine the selective permeability along the paracellular pathway.
claudin- <dig>  occludin and zo- <dig> are critical components of the tjs  <cit> .
lps challenge resulted in a distinct decrease in the levels of zo- <dig> and zo- <dig>  and induced the redistribution of zo- <dig> in human corneal epithelial cells  <cit> .
also, deterioration of the tj proteins occludin and zo- <dig> was observed in pulmonary cells of lps-treated mice  <cit> .
in agreement with previous studies, the present study found lps reduced the levels of claudin- <dig>  occludin and zo- <dig> in ipec-j <dig> cells.
our results also showed l. reuteri i <dig> significantly attenuated the lps-induced decline in claudin- <dig>  occludin and zo- <dig> expression.
in fact, one of the effects probiotics exert is to promote the integrity of the intestinal mucosal barrier by affecting the expression and structure of tj proteins.
an earlier study, conducted in dextran sodium sulfate-induced colitis in mice, demonstrated the probiotic mixture vsl# <dig> prevented apoptosis and low expression of tj proteins, and especially increased the zo- <dig> level  <cit> .
khailova et al.
<cit>  studied the effects of bifidobacterium bifidum in a rat model of necrotizing enterocolitis.
the results showed that b. bifidum improved intestinal integrity by normalizing the expression and localization of tj and anherens junction proteins in the ileum compared to rats with neonatal necrotizing enterocolitis.
moreover, lactobacillus and bifidobacterium robustly suppressed e. coli-induced hyperpermeability and decreased zo- <dig> expression in vitro  <cit> .
a previous study showed that lactobacillus and bifidobacterium produced increases in the expression of the tj proteins claudin- <dig> and occludin in normal human epidermal keratinocytes  which had not been treated with any stressor  <cit> .
however, it contrasts with our result that l. reuteri i <dig> had no effect on the expression of specific tj proteins in the absence of lps.
the in-vitro result seemingly showed differences from in-vivo finding that l. reuteri i <dig> augmented tj protein expression in piglets under normal conditions.
it is well-known that git, in which microorganisms, toxins, digestive enzymes and other substances are all likely to cause damage, is the site of the largest and most complex environment in the mammalian host  <cit> .
in this complex environment, piglets orally administrated with l. reuteri i <dig> had higher expression of tj proteins compared with control piglets which may be stimulated by complex factor in the git, indicating that l. reuteri i <dig> exerted protective effects on tjs.
in vitro, l. reuteri i <dig> indeed prevented the lps-induced decline in tj protein expression, which further validated the protective effects of l. reuteri i <dig> on tj protein expression.
although l. reuteri i <dig> behaved no effect in the absence of lps, in-vitro results provide corroborative evidences that l. reuteri i <dig> may play a positive role in protecting tj proteins in dysfunctional or damaged git caused by pathogenic bacteria or other toxic substances  <cit> .
in other words, in-vivo results together with in-vitro results imply that l. reuteri i <dig> performs its protective effects by stabilization of synthesized tj proteins or increasing new synthesis, and studies on a definite mechanism are needed.
teer is a very important parameter of epithelial barrier function, which indicates variation of permeability and integrity of the cell monolayer  <cit> .
in the present study, co-incubation with l. reuteri i <dig> alone showed a nice maintaince on teer of ipec-j <dig> cells, consistent with an earlier study  <cit>  showing that l. rhamnosus gg and b. longum increased teer over control levels in nhek cells.
furthermore, teer in caco- <dig> cells continued to decrease with time when treated with tnf-α, while increased in l. plantarum + tnf-α  <cit> .
we also found the effects of lps and l. reuteri i <dig> on inflammatory cytokines and specific tj protein expression were time dependent.
there was no difference in the expression of tnf-α, il- <dig> and tj proteins among the different groups at 1–4 h. however, tnf-α and il- <dig> mrna expression in lps-mediated cells increased and the levels of tj proteins decreased with time compared with control cells after  <dig> or 8 h. meanwhile, l. reuteri i <dig> prevented the increased expression of tnf-α and il- <dig> and decreased expression of tj proteins induced by lps.
these findings were supported by work conducted by sheth et al.
<cit> , which evaluated the effect of lps in normal rat cholangiocyte monolayers and showed lps produced a time-dependent effect on tj integrity and barrier function.
moreover, in lps-activated caco- <dig> cells model, teer was minimized at 1 h after treatment with lps and was sustained after that, while co-culture of lactobacillus after activation with lps for 3 h boosted teer over time and was maximized at 24 h  <cit> .
in the past, only live microorganisms were thought to exert probiotic effects, which may be why probiotics were defined as ‘live microorganisms which, when consumed in adequate amounts as part of food, confer a health benefit on the host’  <cit> .
as the effective ingredients of probiotics have not been fully revealed, more and more studies are further exploring this issue.
heat-killed preparations of the probiotic l. rhamnosus gg accelerated intestinal barrier maturation and induced claudin- <dig> expression  <cit> .
furthermore, treatment of nhek with lysates of lactobacillus and bifidobacterium for 24 h produced significant increases in teer and levels of tj proteins in nhek, suggesting lysates of microorganisms also can exert probiotic effects  <cit> .
this study also showed peptidoglycan, a major ligand of gram-positive bacteria, induced increased teer in keratinocytes, in agreement with a previous study  <cit> , together raising the interesting possibility that the cell wall components from probiotic lysates may at least partially be responsible for the changes in tj function.
herein, our results showed the culture supernatant of l. reuteri i <dig>  obviously attenuated or prevented high expression of inflammatory cytokines and low levels of tj proteins induced by lps, indicating the culture supernatant of l. reuteri i <dig> played a role in l. reuteri i5007-mediated improvement in tj function.
similarly, secreted bioactive factors from b. infantis significantly increased teer and levels of occludin and zo- <dig> in t <dig> cells  <cit> .
therefore, the culture supernatant of l. reuteri i <dig> had a similar effect of l. reuteri i <dig> on modulation of inflammatory cytokine and tj protein expression, implying that substances secreted by l. reuteri i <dig>  including lactic acid, hydrogen peroxide, bacteriocin and exopolysaccharide, play a role in this effect.
whole genome sequencing result showed that genome of l. reuteri i <dig> encoded two gene clusters for exopolysaccharide biosynthesis  <cit> .
it may be proposed that secreted bioactive factors of l. reuteri i <dig>  especially exopolysaccharide interact with pattern recognition receptors of cells to induce activation of a series of signaling pathways  <cit>  and subsequently regulate barrier function, including tj function.
CONCLUSIONS
taken together, the data presented here suggest that l. reuteri i <dig> could effectively increase the levels of specific tj proteins in the intestinal epithelium, thereby promoting maturation of the intestinal mucosal barrier in formula-fed piglets.
l. reuteri i <dig> maintained teer of ipec-j <dig> cells and mitigated and even prevented lps-induced inflammatory cytokine and tj protein expression, and the effects might be derived from its culture supernatant and were time dependent.
since l. reuteri i <dig> has a positive effect on tj protein expression, it is possible that this stain plays a potential role in treatment or prevention of intestinal epithelial tjs deficiency in mammals or human beings.

 diarrheal diseases in animals cause the livestock industry great economic loss and also represent a serious threat to farm animal welfare. one of the main causes of diarrhea is gastrointestinal disorder and intestinal mucosa barrier dysfunction or “leaky gut” is reported to play a vital role in the pathophysiology of a variety of gastrointestinal disorders. the intestinal mucosal barrier is maintained by tight junctions, which are multiprotein complexes located around the apical end of the lateral membrane of the epithelial cells and seal the paracellular space between adjacent epithelial cells. tjs function as a selective/semipermeable paracellular barrier, which regulates the transport of ions, water and solutes through the paracellular pathway. the most important and critical components in the structural and functional organization of the tjs are occludin, zonula occluden- and claudin-. the importance of these tj proteins has been demonstrated under many conditions. for example, increased intestinal permeability, which is thought to increase the load of bacterial and dietary antigens in the lamina propria, has been observed in early weaned pigs apparently due to aberrant expression of the essential tj proteins. hence, modulation of tj function, particularly through increasing levels of occludin, zo- and claudin- is a target for novel therapeutic or prophylactic treatments against a range of diseases. probiotics, defined as live microorganisms which, when consumed in adequate amounts as part of food, confer a health benefit on the host, are widely used in humans and animals. since the european union banned the use of antibiotics as growth promoters in the use of probiotic animal feed additives has increased as reported both in vivo and in vitro. ingestion of probiotics has been shown to prevent or treat a variety of gut disorders, although the mechanisms through which they function are not completely known. however, a large amount of evidence suggests that one mechanism may be via protection or augmentation of the intestinal mucosal barrier function. lactobacillus, one of the most commonly used probiotic microorganisms, frequently occurs in the intestinal microflora of various vertebrates and exerts lactobacillus-mediated barrier protection via modulation of tjs. previous studies have demonstrated that this genus significantly reduces the incidence and the severity of diarrhea and helps maintain a functional mucosal barrier. to test the hypothesis that natural lactobacillus found in animal intestinal tract may be a potential source of probiotic bacteria for livestock. in our group isolated thousands of strains of lactobacillus from the gastrointestinal tract git of eleven healthy weaned piglets from different pig farms. according to criteria including tolerance to heat, low ph, and bile salts, as well as storage stability and antagonism to pathogenic agents, four strains, lactobacillus fermentum i l. gasseri s l. reuteri i and l. acidophilus i were selected. subsequently, a series of in vivo and in vitro studies demonstrated that l. fermentum i had the greatest adhesion, and showed beneficial effects on growth performance and antioxidative activity, as well as modulation of intestinal microbiota and mucin secretion in piglets. summarily, our work in the past decade indicates that l. fermentum i now known as l. reuteri i according whole-genome sequence of this strain, can be used as a potential probiotic. recently, our group found that oral administration of l. reuteri i favored intestinal development and maintained the gut epithelial barrier in neonatal piglets. however, the mechanism through which it exerts functions on the gut epithelial barrier is not fully understood. the aim of the present study was to evaluate l. reuteri i5007-mediated intestinal mucosal barrier augmentation in a newborn piglet model and investigate the mechanism for modulation of tjs in a porcine jejunal epithelial cell line challenged by lipopolysaccharides.  oral administration of l. reuteri i enhanced the protein abundance of intestinal epithelial tjs in newborn piglets twelve male newborn piglets were orally administrated with % sterile peptone solution or 6 × 109 cfu of l. reuteri i daily for 14 days. the expression of tj protein claudin- occludin and zo- both in the jejunal and ileal epithelium were measured using immunoblotting to investigate the effect of l. reuteri i on tj function. compared with control group, no significant difference was observed in the jejunal epithelial claudin- expression in the l. reuteri i group, while oral administration of l. reuteri i significantly increased the protein abundance of occludin and zo- in jejunal epithelium of piglets. however, all levels of these tj proteins were distinctly higher in the ileum of piglets administrated with l. reuteri i than those of control piglets.figure expression of tj proteins in piglets following oral administration with l. reuteri i four-day-old piglets were orally administrated with 6 × 109 cfu of l. reuteri i per day for 14 days. subsequently, proteins were isolated from the intestinal tissues of piglets and then the expression of claudin- occludin and zo- was measured using immunoblotting. protein levels of occludin and zo- in jejunal tissue lysates were induced by l. reuteri i l. reuteri i increased the expression of claudin- occludin and zo- in the ileum. data are represented as mean ± sem, n =  *p <  compared with control piglets. l. reuteri i maintained transepithelial electrical resistance of ipec-j cells ipec-j cell monolayers cultured for complete confluence were treated with l. reuteri i cells and teer values were measured every two hours for 10 h. when compared with the untreated control, treatment with l. reuteri i had no effect on teer of ipec-j cells during 0–2 h, but did lead to significantly higher values of teer during 4–10 h. on the whole, an % decline of teer values was caused during 10 h of the measurement, as seen for the control, while co-incubation with l. reuteri i showed almost no decline in teer values, indicating that l. reuteri i had a positive maintaince on teer of ipec-j cells with time.figure response of transepithelial electrical resistance to l. reuteri i in ipec-j cells. ipec-j cells were treated with 3 × 107 cfu/ml of l. reuteri i cells alone for and 10 h, and teer values were measured, respectively. treatment with l. reuteri i induced a nice maintaince on teer of ipec-j cells with time. results are represented as mean ± sem. the experiment was repeated three times with quadruplicate wells in each assay. *p <  vs. control cells. l. reuteri i differentially affected lps-induced tnf-α and il- mrna expression in ipec-j cells to further elucidate the effects of l. reuteri i on modulation of tj function, the mrna expression of inflammatory cytokines including tnf-α and il- was determined in ipec-j cells treated with lps for or 24 h, following pre-treatment with l. reuteri i for 2 h. according to a previous study of our group, l. reuteri i could behave strong adhesion ability to monolayer cells by co-culture with cells for 1 h. resta-lener and barrett showed enteroinvasive escherichia coli evoked fall in teer was prevented if cell monolayers were pretreated with live probiotics, and was also partially attenuated by exposure simultaneously to probiotics and enteroinvasive e. coli, but not if the cells were infected with enteroinvasive e. coli for one hour and then exposed to probiotics. considering these circumstances, to investigate the protect effects of l. reuteri i on tj proteins, we appropriately extended the incubation time for pretreatment with l. reuteri i to 2 h. figure 3a shows that lps significantly increased tnf-α mrna expression after 4 h, but not at 1 h. no significant differences were observed in the mrna expression of tnf-α at 1 h among different treatments. however, tnf-α mrna expression in lps-stimulated cells was time-dependently increased compared with the control treatment, while after 12 h, the increase in tnf-α mrna expression reached a plateau. similarly, the mrna expression of il- was increased in ipec-j cells treated with lps after 8 h and was maintained until 12 h treatment. collectively, treatment with l. reuteri i in the absence of lps had no effect on mrna levels of tnf-α and il- compared with the control group at different time points. however, in the presence of lps, l. reuteri i significantly decreased the mrna expression of these two inflammatory cytokines, compared with the lps group after 8 h.figure mrna expression of inflammatory cytokines in lps-activated ipec-j cells. ipec-j cells were pre-treated with either l. reuteri i or l. reuteri i culture supernatant 2 h prior to lps-activation. quantitative rt-pcr analysis was performed to determine the mrna expression of inflammatory cytokines after or 24 h treatment with lps. lps affected no change in tnf-α expression at 1 h, but significantly increased tnf-α expression during 4–24 h. however, both l. reuteri i and its culture supernatant had no effect on tnf-α expression at the different time points in the absence of lps, while they inhibited or prevented lps-induced expression of tnf-α. there were obvious increases in il- expression during 8–24 h, but not 1–4 h induced by lps, while l. reuteri i and its culture supernatant produced no change at any time compared with control cells. l. reuteri i and its culture supernatant inhibited or prevented lps-induced il- expression. results are represented as mean ± sem, n =  *p <  vs. control cells, and #p <  vs. cells in the lps group. l. reuteri i augmented lps-induced tj protein expression in ipec-j cells we further investigated the mrna expression of claudin- occludin and zo- in inflamed cultured intestinal cells. treatment of ipec-j cells with 1 μg/ml lps had no effect on the mrna expression of claudin- and zo- during 1–4 h, but showed a significant decline during 8–24 h. similarly, compared with the control treatment, lps time-dependently reduced occludin mrna expression. in detail, no difference at 1 h, but decreases during 4–24 h. however, there was no significant change in mrna expression of all three tj proteins in the other four groups versus the control group. pre-treatment with l. reuteri i significantly mitigated or prevented the reduction of mrna expression of all three tj proteins induced by lps.figure mrna expression of tj proteins in lps-activated ipec-j cells. qrt-pcr analysis showed that lps induced a decrease in mrna expression of claudin- at and 24 h, while l. reuteri i and l. reuteri i culture supernatant effectively inhibited lps-induced expression of claudin- there was no change in occludin expression at 1 h, but a significant decrease after 4 h treatment with lps. however, l. reuteri i and its culture supernatant inhibited the decline. lps induced a distinct decrease in zo- expression after 8 h, which was inhibited by l. reuteri i and its culture supernatant. results are represented as mean ± sem, n =  *p <  vs. control cells, and #p <  vs. cells in the lps group. we used immunoblotting to investigate the protein abundance of claudin- occludin and zo- in agreement with the observation of mrna expression of these tj proteins, there was no significant difference in the levels of these tj proteins in the other four groups compared with the control group. however, lps stimulation robustly decreased the levels of claudin- occludin and zo- during 4–24 h, but not at 1 h. treatment of ipec-j cells using l. reuteri i markedly induced higher levels of these proteins than those in cells treated with lps during 4–24 h.figure levels of tj proteins in lps-activated ipec-j cells at different time points. proteins were isolated from ipec-j cells and the expression of tj proteins was assessed using immunoblotting. there was no significant difference in claudin- occludin and zo- expression among the different treatments at 1 h. lps produced decreases in all three tj proteins at,, and h. although l. reuteri i and its culture supernatant had no effect on the levels of tj proteins in the absence of lps, they inhibited lps-induced levels of tj proteins. results are represented as mean ± sem, n =  *p <  vs. control cells, and #p <  vs. cells in the lps group. l. reuteri i culture supernatant differentially affected lps-induced inflammatory cytokine and specific tj protein expression in ipec-j cells to further elucidate whether the culture supernatant was involved in l. reuteri i5007-mediated improvement in tj function, the culture supernatant of l. reuteri i was also added to pre-treat ipec-j cells. compared with the control group, cells treated with l. reuteri i culture supernatant in the absence of lps caused no significant change in tnf-α and il- mrna expression. however, the culture supernatant of l. reuteri i did reduce lps-induced inflammatory cytokine expression during 8–24 h. in general, both mrna expression and protein levels of claudin- occludin and zo- in cells pre-treated with the culture supernatant of l. reuteri i were higher than those in cells induced by lps after or 8 h. discussion the defense response of the intestinal epithelium against pathogenic bacteria has been extensively explored, and evidence suggests that interactions between commensal bacteria and the host are involved in this process. thus, much attention has been paid to the effects of probiotics on gut barrier function. some probiotics might improve or prevent gut barrier dysfunction caused by pathogenic bacteria or other toxic substances. for example, l. plantarum significantly ameliorated phorbol ester-induced zo- and occludin redistribution and increased permeability in caco- cells. therefore, although the mechanisms of action of probiotics may vary, the vital mechanism of probiotics is regulating the expression of tj proteins. the effects of probiotics on gut barrier in animal models are commonly studied in rodents. for example, l. helveticus and l. rhamnosus prevented chronic stress induced intestinal abnormalities in rats, which received 7 days of these strains in the drinking water prior to either a water avoidance stress or a sham stress for one hour per day for ten consecutive days and remained on these probiotics during the duration of the study. however, reports in pigs, especially in neonatal piglets, are rare. in the present study, newborn piglets were selected to study the effects of l. reuteri i on the expression of tj proteins in the intestinal epithelium. we showed that l. reuteri i up-regulated the expression of jejunal epithelial occludin and zo- and ileal epithelial claudin- occludin and zo- in young piglets, indicating l. reuteri i effectively improved the intestinal mucosal barrier function of newborn piglets. this was consistent with an earlier study showing that l. rhamnosus accelerated maturation of the intestinal mucosal barrier in mice via up-regulating the protein level of claudin- oral administration of vsl# a mixture of streptococcus thermophilus, three strains of bifidobacterium and four strains of lactobacillus, in samp mice, obviously improved ileal epithelial permeability and increased the expression of occludin, but not claudin- or zo-, which was not entirely consistent with our results. this might be due to different experimental conditions including strains of probiotic bacteria and animal models. we next examined the effects of l. reuteri i on the expression of inflammatory cytokines and specific tj proteins in ipec-j cells induced by lps. lps is an integral component of the gram-negative bacteria cell wall, capable of binding to toll-like receptor- and activating a variety of protein kinase signaling pathways, subsequently generating inflammatory cytokines and other mediators. liu et al. found that treatment of porcine intestinal epithelial cells and rat intestinal epithelial cells with lps for 16 h markedly increased the secretion of il- while co-culture with l. reuteri robustly inhibited lps-induced overexpression of il- in ipec-j cells. in the present study, lps induced increases in mrna expression of tnf-α and il- of ipec-j cells after or 8 h, and pre-treatment with l. reuteri i significantly suppressed lps-induced tnf-α and il- expression. in addition, several studies have reported effects of probiotics on tnf-α and il- expression induced by lps, but the regulatory action differs in different species or even different strains. for instance, l. reuteri actt greatly inhibited the generation of tnf in lps-activated human monocytoid thp cells, as an immunosuppressive action. in contrast, l. reuteri actt stimulated tnf-α production, as an immunostimulatory action. moreover, rachmilewitz et al. observed that co-culture of mice macrophages with dna of vsl# bacteria significantly promoted il- production. tjs, apical-most component of the junctional complex in epithelial cells, determine the selective permeability along the paracellular pathway. claudin- occludin and zo- are critical components of the tjs. lps challenge resulted in a distinct decrease in the levels of zo- and zo- and induced the redistribution of zo- in human corneal epithelial cells. also, deterioration of the tj proteins occludin and zo- was observed in pulmonary cells of lps-treated mice. in agreement with previous studies, the present study found lps reduced the levels of claudin- occludin and zo- in ipec-j cells. our results also showed l. reuteri i significantly attenuated the lps-induced decline in claudin- occludin and zo- expression. in fact, one of the effects probiotics exert is to promote the integrity of the intestinal mucosal barrier by affecting the expression and structure of tj proteins. an earlier study, conducted in dextran sodium sulfate-induced colitis in mice, demonstrated the probiotic mixture vsl# prevented apoptosis and low expression of tj proteins, and especially increased the zo- level. khailova et al. studied the effects of bifidobacterium bifidum in a rat model of necrotizing enterocolitis. the results showed that b. bifidum improved intestinal integrity by normalizing the expression and localization of tj and anherens junction proteins in the ileum compared to rats with neonatal necrotizing enterocolitis. moreover, lactobacillus and bifidobacterium robustly suppressed e. coli-induced hyperpermeability and decreased zo- expression in vitro. a previous study showed that lactobacillus and bifidobacterium produced increases in the expression of the tj proteins claudin- and occludin in normal human epidermal keratinocytes which had not been treated with any stressor. however, it contrasts with our result that l. reuteri i had no effect on the expression of specific tj proteins in the absence of lps. the in-vitro result seemingly showed differences from in-vivo finding that l. reuteri i augmented tj protein expression in piglets under normal conditions. it is well-known that git, in which microorganisms, toxins, digestive enzymes and other substances are all likely to cause damage, is the site of the largest and most complex environment in the mammalian host. in this complex environment, piglets orally administrated with l. reuteri i had higher expression of tj proteins compared with control piglets which may be stimulated by complex factor in the git, indicating that l. reuteri i exerted protective effects on tjs. in vitro, l. reuteri i indeed prevented the lps-induced decline in tj protein expression, which further validated the protective effects of l. reuteri i on tj protein expression. although l. reuteri i behaved no effect in the absence of lps, in-vitro results provide corroborative evidences that l. reuteri i may play a positive role in protecting tj proteins in dysfunctional or damaged git caused by pathogenic bacteria or other toxic substances. in other words, in-vivo results together with in-vitro results imply that l. reuteri i performs its protective effects by stabilization of synthesized tj proteins or increasing new synthesis, and studies on a definite mechanism are needed. teer is a very important parameter of epithelial barrier function, which indicates variation of permeability and integrity of the cell monolayer. in the present study, co-incubation with l. reuteri i alone showed a nice maintaince on teer of ipec-j cells, consistent with an earlier study showing that l. rhamnosus gg and b. longum increased teer over control levels in nhek cells. furthermore, teer in caco- cells continued to decrease with time when treated with tnf-α, while increased in l. plantarum + tnf-α. we also found the effects of lps and l. reuteri i on inflammatory cytokines and specific tj protein expression were time dependent. there was no difference in the expression of tnf-α, il- and tj proteins among the different groups at 1–4 h. however, tnf-α and il- mrna expression in lps-mediated cells increased and the levels of tj proteins decreased with time compared with control cells after or 8 h. meanwhile, l. reuteri i prevented the increased expression of tnf-α and il- and decreased expression of tj proteins induced by lps. these findings were supported by work conducted by sheth et al., which evaluated the effect of lps in normal rat cholangiocyte monolayers and showed lps produced a time-dependent effect on tj integrity and barrier function. moreover, in lps-activated caco- cells model, teer was minimized at 1 h after treatment with lps and was sustained after that, while co-culture of lactobacillus after activation with lps for 3 h boosted teer over time and was maximized at 24 h. in the past, only live microorganisms were thought to exert probiotic effects, which may be why probiotics were defined as ‘live microorganisms which, when consumed in adequate amounts as part of food, confer a health benefit on the host’. as the effective ingredients of probiotics have not been fully revealed, more and more studies are further exploring this issue. heat-killed preparations of the probiotic l. rhamnosus gg accelerated intestinal barrier maturation and induced claudin- expression. furthermore, treatment of nhek with lysates of lactobacillus and bifidobacterium for 24 h produced significant increases in teer and levels of tj proteins in nhek, suggesting lysates of microorganisms also can exert probiotic effects. this study also showed peptidoglycan, a major ligand of gram-positive bacteria, induced increased teer in keratinocytes, in agreement with a previous study, together raising the interesting possibility that the cell wall components from probiotic lysates may at least partially be responsible for the changes in tj function. herein, our results showed the culture supernatant of l. reuteri i obviously attenuated or prevented high expression of inflammatory cytokines and low levels of tj proteins induced by lps, indicating the culture supernatant of l. reuteri i played a role in l. reuteri i5007-mediated improvement in tj function. similarly, secreted bioactive factors from b. infantis significantly increased teer and levels of occludin and zo- in t cells. therefore, the culture supernatant of l. reuteri i had a similar effect of l. reuteri i on modulation of inflammatory cytokine and tj protein expression, implying that substances secreted by l. reuteri i including lactic acid, hydrogen peroxide, bacteriocin and exopolysaccharide, play a role in this effect. whole genome sequencing result showed that genome of l. reuteri i encoded two gene clusters for exopolysaccharide biosynthesis. it may be proposed that secreted bioactive factors of l. reuteri i especially exopolysaccharide interact with pattern recognition receptors of cells to induce activation of a series of signaling pathways and subsequently regulate barrier function, including tj function.  taken together, the data presented here suggest that l. reuteri i could effectively increase the levels of specific tj proteins in the intestinal epithelium, thereby promoting maturation of the intestinal mucosal barrier in formula-fed piglets. l. reuteri i maintained teer of ipec-j cells and mitigated and even prevented lps-induced inflammatory cytokine and tj protein expression, and the effects might be derived from its culture supernatant and were time dependent. since l. reuteri i has a positive effect on tj protein expression, it is possible that this stain plays a potential role in treatment or prevention of intestinal epithelial tjs deficiency in mammals or human beings. 
tight junctions maintain the intestinal mucosal barrier, dysfunction of which plays a vital role in the pathophysiology of a variety of gastrointestinal disorders. in addition, ipec-j cells were stimulated with 1 μg/ml lipopolysaccharide for or 24 h, following pre-treatment with l. reuteri i or its culture supernatant for 2 h. the results showed that lps time-dependently induced the expression of tnf-α and il- and decreased tj proteins, which was reversed by pre-treatment of l. reuteri i or its culture supernatant. l. reuteri i had beneficial effects on the expression of tj proteins in newborn piglets and the in-vitro results showed this strain had a positive effect on teer of cells and inhibited the reduction of tj proteins expression induced by lps. in vitro, treatment with l. reuteri i alone maintained the transepithelial electrical resistance of ipec-j cells with time. previously, we have shown that l. reuteri i maintained the gut epithelial barrier in newborn piglets. we found that l. reuteri i significantly increased the protein abundance of intestinal epithelial claudin- occludin and zonula occluden- in newborn piglets. lactobacillusnewborn pigletstight junctionipec-j2lipopolysaccharidesissue-copyright-statement© the author 2015 these findings indicated l. reuteri i may have potential roles in protection tj proteins in tj-deficient conditions.
 tight junctions maintain the intestinal mucosal barrier, dysfunction of which plays a vital role in the pathophysiology of a variety of gastrointestinal disorders. previously, we have shown that l. reuteri i maintained the gut epithelial barrier in newborn piglets. here we aimed to decipher the influence of l. reuteri i on tight junction protein expression both in vivo and in vitro.  we found that l. reuteri i significantly increased the protein abundance of intestinal epithelial claudin- occludin and zonula occluden- in newborn piglets. in vitro, treatment with l. reuteri i alone maintained the transepithelial electrical resistance of ipec-j cells with time. in addition, ipec-j cells were stimulated with 1 μg/ml lipopolysaccharide for or 24 h, following pre-treatment with l. reuteri i or its culture supernatant for 2 h. the results showed that lps time-dependently induced the expression of tnf-α and il- and decreased tj proteins, which was reversed by pre-treatment of l. reuteri i or its culture supernatant.  l. reuteri i had beneficial effects on the expression of tj proteins in newborn piglets and the in-vitro results showed this strain had a positive effect on teer of cells and inhibited the reduction of tj proteins expression induced by lps. these findings indicated l. reuteri i may have potential roles in protection tj proteins in tj-deficient conditions. keywords lactobacillusnewborn pigletstight junctionipec-j2lipopolysaccharidesissue-copyright-statement© the author 2015 