The Role Of TGF-?1 On Intestinal Epithelial Barrier Restoration And Its Signal Pathways In Weaned Piglets

Weaning is the most significant event in the life of pigs as they are abruptly forced to adapt to nutritional,immunological and psychological disruptions.Early weaning has been shown to impair the intestinal epithelial barrier of piglets.It is still unknown whether transforming growth factor beta 1(TGF-?1)plays an important regulatory role in the restoration of intestinal epithelial barrier and its sigal pathways involved in the progress.So this study was aimed to delineate the role of TGF-?1 in intestinal epithelial barrier restoration and its signal pathway in weaned piglets.An in vivo experiment was design to study the developmental changes of TGF-?1 and its receptors,tight junction proteins,smads and MAPKs signaling pathway in weaned pigs.Also,another in vivo was carried out to study the exact role of TGF-?1 and its signal pathways involved in restoration of injured intestinal epithelial barrier by lipopolysaccharide(LPS)in weaned piglets.Basing on the in vivo results,the effects of TGF-?1 on the restoration of damaged intestinal epithelial barrier are determined in vitro cultured IPEC-J2 cells.We further inhibit or knockdown the MAPKs or Smads pathwasys in cultured IPEC-J2 cell to study the effects of TGF-?1 on tight junctions and its receptors,Smads and ERK signaling pathways.Cross-talk between MAPKs and Smad signaling pathways were also investigated.Our study elucidated the mechanism of TGF-?1-mediated Smads and MAPKs signaling pathways on the restoration of intestinal epithelial barrier.It will not only create some theory,but also provide a new target to the nutrition regulation of intestinal barrier function in injured intestine of piglets.1.Developmental changes of TGF-?1,tight junction proteins,smads and MAPKs signaling pathway in intestinal adaption of weaned pigsWeaning stress caused marked changes in intestinal structure and function.TGF-?1 was suspected to play an important regulatory role in the small intestine.In the present study,six litters(Durocx Landrace x Yorkshire,9 to 11 piglets per litter)were selected.At 20 d of age(preweaning stage),1 piglet from each of 6 different litters was killed.At weaning day(21 d of age),3 piglets from each of 6 different litters were allocated to 1 of the 3 experimental groups killed at 3,7,and 14 d postweaning.For each group,6 piglets from 6 different litters were removed from the sow,mixed,and housed in nursery pens.The results showed that a shorter villus and deeper crypt were observed on d 3 and d 7 postweaning and intestinal morphology recovered to preweaning values on d 14 postweaning.Early weaning increased(P<0.05)transepithelial electrical resistance(TER)and decreased(P<0.05)paracellular flux of fluorescein isothiocyanate dextran(FD4)in intestinal mucosa on d 3 and d 7 post-weaning.Compared with the pre-weaning stage,tight junction proteins level of occludin and claudin-1 were reduced(P<0.05)on d 3,7 and 14 post-weaning,and ZO-1 protein was reduced(P<0.05)on d 3 and d 7 post-weaning.An increase(P<0.05)of tumor necrosis factor-a(TNF-a)and interleukin-6(IL-6)mRNA and concentration on d 3 and d 7 postweaning and an increase(P<0.05)of interferon-?(IFN-y)mRNA and concentration on d 3 postweaning were observed compared with preweaning.No significant increase of TGF-?1 and interleukin-10(IL-10)mRNA after weaning was observed.An increase(P<0.05)of TGF-?1 in intestinal mucosa was observed on d3 and d7 and then level down on d14 post-weaning.By using the immunofluorescence,we found that TGF-?1 at the intestinal villus epithelium decreased(P<0.05)significantly 3d after weaning while the staining intensity increased(P<0.05)significantly at the intestinal crypts compared with that in pre-weaning pigs.There was a significant increase(P<0.05)in T?R2 at the intestinal villus epithelium on d3 and decrease(P<0.05)in intestinal crypts on d3 and d7 post-weaning.T?R2 at the intestinal villus epithelium was significantly increased on d3 and d7 post-weaning,however there is no change of T(3R1 at intestinal crypts.Although there was an increase(P<0.05)of T?R2 protein expression in the intestinal mucosa on d3 and d 7,no significant increase of mRNA of T?R1,T?R2,smad2/3,smad4 and smad7 was observed during postweaning(P>0.05).Phosphorylated smad2/smad3 was significantly increased(P<0.05)on day d3 and reach its peak at day7 postweaning(P<0.05),There were no significant changes in smad4 and smad7 protein expression post-weaning(P>0.05).The phosphorylated(activated)ratios of JNK and p38 on d 3 and 7 postweaning and the phosphorylated ratio of ERK1/2 on d 3 postweaning were increased(P<0.05)compared with preweaning.The results indicated that TGF-?1 was associated with the restoration of intestinal morphology and barrier function following weaning stress.The increased intestinal endogenous TGF-?1 can activate the canonical Smads and MAPKs signaling pathways.2.Transforming growth factor-?1 enhances intestinal integrity and influences smads and MAPK signaling pathways in weaned piglets after lipopolysaccharide challengeTGF-?1 has been reported to play an important role in the restoation of epithelial barrier function in the weaning piglets as shown in the previous chapter,however,whether supplyment TGF-?1 in vivo could alleviate the damage of intestine barrier function have not been reported yet.The aim of this study was to investigate the protective effects of TGF-?1 on intestinal epithelial barrier as well as whether TGF-?1 involved in these protection processes in a piglet model after lipopolysaccharide(LPS)challenge.Eighteen weanling pigs were randomly allocated to the following treatment groups:1)non-challenged pigs and control diet;2)LPS challenged pigs and control diet;3)LPS challenged pigs+TGF-?1 diet.After 19-d feeding with control or TGF-?1 diets,pigs were injected with LPS or sterile saline.At 4 h post-injection,pigs were killed to harvest samples and measured.The results showed TGF-?1 improved(P<0.05)intestinal morphology,as indicated by greater villus height and villus height:crypt depth ratio,and intestinal barrier function,which was reflected by increased transepithelial electrical resistance(TER)and decreased mucosal to serosal paracellular flux of dextran(4 kDa),as compared with LPS group.Moreover,TGF-?1 prevented the LPS-induced decrease(P<0.05)in claudin-1,occludin and ZO-1 expressions in the jejunal mucosae.TGF-?1 also attenuated intestinal inflammation,indicated by decreased(P<0.05)mRNA expressions of TNF-a,IL-6,IL-8,IL-1?.Supplementation with TGF-?1 also increased(P<0.05)TGF-?1 protein,phosphorylated-Smad2/3 expression,smad4 and smad7 mRNA expressions and decreased(P<0.05)the ratios of the phosphorylated to total JNK and p38(p-JNK/JNK and p-p38/p38),while it increased(P<0.05)the ratio of ERK(p-ERK/ERK).Collectively,these results suggest that dietary inclusion of TGF-?1 attenuates the LPS-induced intestinal injury by improving mucosa barrier function,alleviating intestinal inflammation and influencing TGF-?1 canonical smads and MAPK signaling pathways.3.Transforming growth factor-?1 protects intestinal integrity and influences Smads and MAPK signal pathways in IPEC-J2 after tumor necrosis factor-a challengeThe aim of this study was to investigate the protective effects of TGF-?1 on intestinal epithelial barrier as well as whether canonical Smads and MAPKs signal pathways involved in these protection processes by a IPEC-J2 model stimulated with TNF-a.IPEC-J2 monolayers were treated without or with TNF-? in the absence or presence of TGF-?1.The results showed that TGF-?1 pretreatment ameliorated TNF-a-induced intestinal epithelial barrier disturbances as indicated by decrease(P<0.05)of transepithelial electrical resistance(TER)and increase(P<0.05)of paracellular permeability measured by FD4 flux.TGF-?1 also drtamatically alleviated TNF-?-induced alteration of tight junction proteins ZO-1 and Occludin(P<0.05)but had no effects on the Claudin-1 expression(P>0.05).Compared with control,TNF-a treatment also lead to the rearrangement of Occludin and ZO-1 proteins(P<0.05)and TGF-?1 pretreatment ameliorated those tight junction proteins disturbance caused by TNF-a when compared with TNF-a group(P<0.05).TNF-? treatment or TGF-?1 alone has on effects on T?R1,T?R2 protein expression and smad2/3 phosphorylation(P>0.05).Moreover,TGF-?1 pretreatment increased(P<0.05)of T?1R2 protein expression in IPEC-J2 monolayers challenged with TNF-a.In addition,a significant increase(P<0.05)of mRNA of smad4 and smad7 was also observed in the TGF-?1 pretreatment after TNF-a challenge compared with the control group.Furthermore,TGF-?1 pretreatment enhanced(P<0.05)p-smad2 and p-smad3 protein activation and the translocation of p-smad2/3.Those results indicated that canonical Smads signaling pathway was activated by TGF-?1 pretreatment.Finally,TGF-?1 pretreatment decreased the ratios of the phosphorylated to total JNK and p38(p-JNK/JNK and p-p38/p38)and increased the ratio of ERK(p-ERK/ERK)(P<0.05).These results indicated that TGF-?1 protects intestinal integrity and influences Smads and MAPK signal pathways in IPEC-J2 after tumor necrosis factor-a challenge.4.The effects of Smads and MAPK signal pathways in mediating the protective effects of TGF-?1 on intestinal epithelial barrier in IPEC-J2 cells challenged with TNF-?The present study was aimed to elucidate the mechanism of TGF-?1 on intestinal epithelial barrier and explore how MAPKs and canonical Smads were involved in the protection process.By using the transwell system model,IPEC-J2 cells were subjected with TNF-? and TGF-?1 stimulation pretreatment with specific MAPK inhibitors(SB203580,SP60025,PD98059)and canonical smads inhibitor(SB431542)to investigate underlying mechanism of TGF-?1 on intestinal epithelial barrier.The results showed that compared with TNF-?+TGF-?1 group,pretreatment with P38 inhibitor(SB203580)or JNK inhibitor(SP60025)did not influence the transepithelial electrical resistance(TER)and the paracellular permeability of FD4 flux(P>0.05),but when pretreatment with MEK/ERK inhibitor(PD98059)or smads inhibitor(SB431542)significantly deteriorated intestinal epithelial barrier indicated by decreased TER and increased FD4 flux(P<0.05)which indicated that the protective effects of TGF-?1 were eliminated by PD98059 and SB431542.Moreover,incubating the cells with P38 inhibitor(SB203580)or JNK inhibitor(SP60025)has no significant effects on tight junction protein Occludin and ZO-1 expression and distribution(P>0.05)but MEK/ERK inhibitor(PD98059)or smads inhibitor(SB431542)decreased the protein expression and distribution induced by TGF-?1(P<0.05).We also evaluated the smads signal pathways and found that MEK/ERK inhibitor(PD98059)or smads inhibitor(SB431542)significantly inhibited(P<0.05)the phosphorylated smad2/smad3 expression and the mRNA expression of smad4 and smad7 compared with TNF-?+TGF-?1 treatment.Also,the smad2/3/4 formation and p-smad2/3 translocation was decreased by PD98059 and SB431542 incubation(P<0.05)but there was no change on T?RI and T?R2 protein expression(P>0.05)after treatment with PD98059.SB431542 significantly decreased(P<0.05)the T?R2 protein expression compared with control group and TNF-?+TGF-?1 group.P38 inhibitor(SB203580)or JNK inhibitor(SP60025)had no influence on the smads signal components expression compared with TNF-?+TGF-?1 treatment.Incubation of SB203580,SP60025 and PD98059 significantly reduced corresponding phosphorylation signal,respectively.However,compared with control and TNF-?+TGF-?1 group,smads inhibitor(SB431542)treatment also suppressed the ratio of ERK(p-ERK/ERK)(P<0.05)induced by TGF-?1 which indicated cross-talk between ERK/MAPKs and Smad signaling pathways exist in mediating the protective effects of TGF-?1.Therefore,these results suggest that inhibiting P3 8 and JNK signal pathways did not influence the TGF-?1 mediated protective effects on intestine barrier function,while inhibiting the MER/ERK could partly abolish the beneficial effects of TGF-?1.Canonical smads pathways played a critical role in protecting the barrier function.Furthermore,Canonical smads pathways cross-talk with MER/ERK pathways was verified in TGF-?1 protecting barrier function in IPEC-J2 cells.5.Smads and ERK/MAPK signal pathways synergistically mediated the protective effects of TGF-?1 on intestinal epithelial barrier in IPEC-J2 cells after TNF-a challengeThe results of experiment 3 and 4 showed that TGF-?1 protected intestinal barrier of IPEC-J2 cells after TNF-a challenged,Smads and MAPK signal pathways plays an important role in mediating the beneficial effects of TGF-?1,however the relationship about the ERK/MAPK and Smads has not been fully elucidated.In the present study,we specifically scilenced the ERKs or Smads genes by Smad2/3 and ERK siRNA of the IPEC-J2 cell with or without TGF-?1 after TNF-? challenged to investigate the underlying mechanism.The results showed that ERK siRNA treatment significantly decreased the intestine barrier function reflected by decreased TER and increased FD4 flux(P<0.05)compared with the TGF-?1 pretreatment group.Smad2 and smad3 siRNA treatment also partly contradict(P<0.05)the beneficial effects of TGF-?1 in restoration on the barrier function.Moreover,compared with the TNF-?+TGF-?1 group,silencing the smad2 and smad3 preventing(P<0.05)the increasing of Occludin and ZO-1 proteins caused by TGF-?1 and silencing ERK mRNA also abolished(P<0.05)the protein expression of Occludin and ZO-1 inducing by TGF-?1.Silencing the smad2,smad3 and ERK had no significant effects on T?RIand T?R2 protein expressions(P>0.05).Compared with TNF-?+TGF-?1,silencing the smad2 and smad3 specifically decreased(P<0.05)the phosphorylated smad2/smad3 expression and translocation in the cells and also inhibit(P<0.05)the formation of the smad2/3/4 complex,while silencing the ERK mRNA expression significantly lead to the decreasing(P<0.05)of phosphorylated smad2/smad3 expression and translocation and the formation of smad2/3/4 was also reduced(P<0.05).Those results indicated that ERK/MAPK cross-talk with canonical smads targeting in smad2/3 phosphorylation in mediating the protective effects of TGF-?1 and they synergistically regulated the barrier function in the IPEC-J2 cells.Smad2/3 was the critical regulation factor in mediating the beneficial effects of TGF-?1 on restoration the compromising epithelium barrier function.