Effects Of GLP-2 On The Small Intestinal Tight Junction Barrier In Stressed Weaned Piglets And The Mechanism Studies

After piglets weaned,various stress(including weaning itself)may cause enteritis,resulting in intestinal dysfunction and further blockage of growth.The main reason for that may be the increased intestinal epithelial permeability which facilitates invasion of protein antigens and bacteria/endotoxin from gut into submucosa,triggering an immune response.Tight junction(TJ),which serves as the basis of intestinal structural barrier,its expression and protein ultrastructural distribution variation may be the root cause for the change of intestinal permeability.The contraction of peri-junctional actinomyosin filaments caused by myosin light-chain(MLC)phosphorylation,as well as decreased TJs protein expression,may lead to the opening of the TJ structure and enhance the permeability of the intestinal barrier.Glucagon-like peptide 2(GLP-2),whose endogenous level decreased abruptly after weaning,is an intestinal peptide hormone with specific intestinotropic effects.However,it has not been clear that whether GLP-2 regulates the intestinal barrier permeability in weaned piglets and its molecular mechanism.Therefore,in this study,injected exogenous GLP-2 in vivo to investigate its effects on growth performance in weaned piglets and further investigation of intestinal barrier function state was carried out.The effect of GLP-2 on the expression and distribution of TJ was investigated through in vivo and in vitro experiments.In vitro,the possible signaling pathways from extracellular,intracellular,signaling nuclear translocation,to protein phosphorylation,which were possibly involved in intestinal TJ stabilization,were evaluated to reveal the molecular mechanism of GLP-2 regulation in the intestinal epithelial barrier in weaned piglets systematically.1 The effect of GLP-2 on intestinal development and growth performance in weaned pigletsForty healthy DLY piglets weaned at the age of 28 d with an similar BW were assigned to four treatments:(i)non-challenged control;(ii)LPS-challenged control;(iii)LPS + low GLP-2;and(iv)LPS+ high GLP-2.Piglets were s.c.injected with PBS supplemented with h[Gly2]GLP-21-34 at doses of 0,0,2 and 10 nmol/kg BW per day for seven consecutive days.Piglets were challenged with i.p.administration of Escherichia coli lipopolysaccharide(LPS)at a dose of 100 μg/kg on d 14 to induce intestinal damage.Results are as follows:High GLP-2 treatment significantly increased the duodenal,jejunal and ileal weight,as well as the gross weight of the small intestine(SI),and the SI weight index(P<0.05).GLP-2 also significantly increased the villus height and the villus height/crypt depth ratio(VCR)of the duodenum,jejunum,and ileum(P<0.05).Histological examination revealed that in GLP-2-treated protected the villus against LPS-induced damage,and maintained their integrity.GLP-2 significantly increased the activity of alkaline phosphatase(AKP),y-glutamyltranspeptidase(γ-GT),and pancreatic lipase in the duodenum and jejunum(P<0.05).GLP-2 treatment also significantly increased the average daily gain(ADG)and G:F of piglets at 0 to 7,7 to 14,as well as 0 tol4 d(P<0.05),resulting in a significant increase of final BW in high GLP-2 pigs(P<0.05).The results suggested that exogenous GLP-2 improved the growth of weaned piglets and protected them against LPS-induced intestinal damage by promoting the development of small intestine,relieving the stress-induced damage on intestinal villi and enhancing the activity of intestinal digestive enzymes.2 The effect of GLP-2 on intestinal mucosal barrier injury in lipopolysaccharide-challenged weaned piglets On the basis of experiments 1,this study further evaluated the protective effect of GLP-2 on LPS-induced intestinal barrier injury after weaning and its possible mechanism.The results as follows:High dose of GLP-2 pretreatment restoring the expression(P<0.05)and ultrastructural distribution of tight junction proteins zona occluden-1(ZO-1)and occludin,significantly decreased the LPS concentration,D(-)-lactate and diamine oxidase(DAO)activity in plasma(P<0.05),and DAO activity in duodenum and jejunum(P<0.05),and reduced the LPS-induced serum level(P<0.05)and mRNA expression of pro-inflammatory cytokines including IL-1β,IL-6,IL-8 and TNF-a in small intestines(P<0.05).GLP-2 dose-depedently prevented the LPS-induced increase in the gene and protein expression of myosin light-chain kinase(MLCK)and the increase in phosphorylated MLCThr18/Ser19(pMLCThr18/Ser19)levels in the duodenum,jejunum,and ileum.These results indicated that GLP-2 treatment alleviated intestinal barrier injury and inflammation in LPS-challenged weaned piglets by regulation of the expression and distribution of tight junction proteins.3 The effect of GLP-2 action to regulate the LPS-challenged IPEC-J2 paracellular permeability and the mechanism study3.1 The building of research model(1)The identification of GLP-2R expression in IPEC-J2The first trial was conducted to evaluate whether IPEC-J2 cell line could be used for the mechanism research of GLP-2 effect through the GLP-2R expression identification.Real-time PCR,western blot,and immunofluorescence techniques were used to detect the expression and subcellular location of GLP-2R in the IPEC-J2 cell line.The results showed that GLP-2R was expressed in IPEC-J2 cell,and located in the cell membrane and cytoplasm.(2)LPS-induced paracellular permeability in IPEC-J2The trial was devided into 5 treatment groups to determine the effects of LPS exposure(0,]0,50,100,150 μg/mL of medium for 24 h)on IPEC-J2 paracellular permeability in vitro.The results indicated that with increasing levels of LPS exposure,the transepithelial electrical resistance(TER)gradually decreased,and the penneability to HRP 24 h after LPS exposure gradually increased consistently.When LPS level increased up to 100 μg/mL,TER and permeability to HRP were significantly higher(P<0.05),cell viability significantly decreased(P<0.05).Microscope observasion showed that the cell morphologic characteristics had been changed more obviously,dead cells increased,but the integrity of cell monolayer was sustained.However,when LPS increased up to 150 μg/mL,much more dead cells increased to destroy the cell monolayer architecture,and the morphocytology was completely impaired with the cell border line fractured.The results suggested that LPS enhanced the IPEC-J2 paracellular permeability.The stress dose was established to be 100 μg/mL LPS.(3)Treatment with GLP-2 reduced LPS-induced cell monolayer permeability in IPEC-J2This trial was devided into six treatments,to investigate the effect of GLP-2 at different doses on the paracellular permeability.The IPEC-J2 cells were pre-incubated with 1×10-10～1×10-7mol/L GLP-2 for 1 h and then exposed to 100 μg/mL LPS for 24 h.The results showed that treatment with 1 × 10-8 and 10-7 mol/L GLP-2 significantly inhibited the LPS-induced TER reduction and the increase of permeability to HRP(P<0.05),maintained the morphocytology,and decreased the number of dead cells.Compared with LPS group,the cell viability was gradually increased with the increase of GLP-2 level.When GLP-2 increased up to 1×10-9 mol/L,the difference compared with the control group was not significant(P<0.05).The results suggested that GLP-2 treatment attenuated the LPS-enhanced intestinal paracellular permeability,the suitable dose of GLP-2 was 1 × 10-8 mol/L.3.2 The effect of GLP-2 on TJ protein expression and structural rearrangement in LPS-challenged IPEC-J2 and the mediated effect of MLCK/pMLC signal pathwayTest(1)was devided into three treatments:control,LPS,and GLP-2+LPS,to investigate whether GLP-2 modulates the cell monolayer permeability after LPS challenge by affecting the expression and ultrastructural distribution of tight junction proteins.Test(2):IPEC-J2 cell monolayer was pretreated with MLCK inhibitor(ML-9)and GLP-2 for 1 h,and then exposed to LPS for 24 h.The activation of MLCK/pMLC signaling after LPS treatment for 45 min,and the expression and distribution of TJ proteins at 24 h after LPS exposure was detected to investigate whether MLCK/pMLC signaling was modulated by GLP-2 in the initial period of stress,leading to the TJ ultrastructure redistribution.The test was devided into five treatments:control,LPS,GLP-2+LPS,ML-9+LPS,ML-9+GLP-2+LPS.The results indicated that 1 ×10-8 mol/L GLP-2 significantly inhibited the LPS-induced decrease of ZO-1 and occludin protein expression(P<0.05),the increase of MLCK gene and protein expression and the phosphorylation level of pMLCThr18/Ser19(P<0.05).ML-9 treatment had no effect on LPS-downregulated ZO-1 and occludin protein expression.ML-9+GLP-2 significantly stimulated the TJ proteins expression(P<0.05).GLP-2,ML-9,and ML-9+GLP-2 treatments significantly inhibited the LPS-induced hyperphosphorylation of pMLCThr18/Ser19(P<0.05)and cellular monolayer permeability to HRP(P<0.05);however,the permeability to HRP of GLP-2,ML-9+GLP-2 treatment groups were significantly lower than ML-9 treatment.Immunofluorescence staining showed that LPS diminished the"honeycomb-like" structure of ZO-1 and occludin,increased the granular form of occludin staining in cytoplasm;GLP-2 attenuated the dyslocation of ZO-1 and occludin induced by LPS,by maintaining the intact "honeycomb-like" structure and decreasing the cytoplasmic occludin staining.Although the"honeycomb-like" structure on local cell membrane was restored in ML-9 treatment group,the staining line at the cell border was fractured and incomplete.ML-9+GLP-2 treatment restored the intercellular location and distribution of ZO-1 and occludin similar to the control.The results demonstrate that:1)the mechanism of GLP-2 amelioration of intestianl epithelial permeability is closely related with the stability of TJ architecture.GLP-2 modulation of TJ involves:①stimulation of TJ proteins expression;②preventing the TJ proteins redistribution by inhibition of MLC hyperphosphorylation at the initial stage of LPS stress.2)MLC phosphorylation mediated by MLCK doesn’t contribute to TJ protein expression.3.3 The signial pathways mediates GLP-2 regulation of TJsThe possible signaling pathways,which mediated the GLP-2 action of TJ expression and ultrasturcture distribution,and the relationships between each others were investigated by the following 4 experiments.(1)Role of NF-κB signaling pathway in GLP-2 modulation of MLC hyperphosphorylationThe experiment was carried out to investigate whether NF-κB signaling acted as upstream to mediate the GLP-2 modulation of MLCK/pMLC pathway.IPEC-J2 cell monolayer was treated with NF-κB inhibitor(PDTC)and GLP-2 for 1 h,and then exposed to LPS for 24 h.The test was devided into five treatments:control,LPS,GLP-2+LPS,PDTC+LPS,and PDTC+GLP-2+LPS.The results showed that LPS caused pIKKαThr23 and pIκBSer32/36 rapid phosphorylated,significantly decreased the cytoplasmic IκB level,activated the p65NF-KB nuclear translocation,and increased the MLCK transcription level and protein expression.GLP-2,PDTC,PDTC-HGLP-2 treatment inhibited the NF-κB activation and p65NF-KB nuclear translocation,dowm-regulated the MLCK gene and protein expression,and abolished pMLCThr18/Ser19 hyperphosphorylation.The results indicates that LPS-activated NF-κB signaling pathway is as the upstream of MLCK/pMLC to be inhibited by GLP-2.(2)Role of ROCK/MLCP signaling pathway in GLP-2 modulation of MLC phosphorylationThe experiment was to investigate whether GLP-2 modulated MLC phosphorylation level by regulating ROCK/MLCP-dependent MLC dephosphorylation,besides inhibitation of MLCK-dependent MLC phosphorylation.IPEC-J2 cell monolayer was treated with MLCP inhibitor(Calyculin A),ROCK inhibitor(Y27632,MLCP agonist)and GLP-2 for 1 h,and then exposed to LPS for 24 h.The test was devided into five treatments:control,LPS,GLP-2+LPS,Calyculin A+GLP-2+LPS,and Y27632+GLP-2+LPS.The results showed that LPS inreased the phosphorylation level of MLCP regulatory subunit pMYPT1Thr696,significantly reduced the MLCP activity(P<0.05).GLP-2 or Y27632+GLP-2 inhibited the LPS-induced pMYPT1Thr696 phosphorylation,significantly increased the MLCP activity(P<0.05),and blocking the subsequent pMLCThr18/Ser19 hyperphosphorylation.However,Calyculin A+GLP-2 group had higher pMYPT1Thr696 phosphorylation level,and significantly lower MLCP activity than GLP-2-treatment group.Calyculin A partially abolished the down-regulation effect of GLP-2 on pMLCThr18/Ser19 phosphorylation.The results indicates that GLP-2 can inhibit LPS-induced MLC hyperphosphorylation via inhibition of pMYPT1Thr696 phosphorylation and subsequent maintainance of MLCP activity.(3)The effect of MEK/ERK1/2 pathway on GLP-2 modulation of TJThe experiment was carried out to investigate whether MEK/ERK1/2 signaling mediated the GLP-2 action of TJ by using MEK specific inhibitor U0126.The test was devided into five treatments:control,LPS.GLP-2+LPS,Y0126+LPS,and U0126+GLP-2+LPS.Results indicated that LPS increased the phosphorylation level of ERK1/2Thr202/Tyr204 and MYPT1Thr696,down-regulated the protein expression of HSP70,ZO-1,and occludin,and MLCP activity as well.U0126,GLP-2,or U0126+GLP-2 treatment inhibited the ERK1/2Thr22/Tyr204 phosphorylation.However,compared to LPS treatment,U0126 treatment had no effect on HSP70,ZO-1,and occludin protein expression,MYPT1hr696 phosphorylation level,and MLCP activity(P>0.05);GLP-2 or U026+GLP-2 treatments up-regulated the HSP70,ZO-1,and occludin protein expression,inhibited the increase in MYPT1hr696 phosphorylation,and significantly increased the MLCP activity(P<0.05).U0126,GLP-2 or U0126+GLP-2 treatments inhibited the activation of NF-κB signaling,the up-regulation of the MLCK protein expression and the pMLCThr18/Sser19 phosphorylation.The results demonstrate that:under LPS stress,1)GLP-2 promotes the TJ proteins ZO-1 and occludin expression independently by ERK1/2 pathway;2)GLP-2 inhibits the activation of NF-κB/MLCK signaling by repressing the ERK1/2 pathway;3)GLP-2 is independent of ERK1/2 pathway to modulate the ROCK/MLCP signaling;4)GLP-2 stimulates the HSP70 protein expression independently by ERK1/2 signaling.(4)The effect of G-protein coupled receptor mediated cAMP/PKA signaling on GLP-2 modulation of TJThe experiment was carried out to investigate whether GLP-2R-coupled G protein mediated MEK/ERK1/2 signaling mediated cAMP/PKA signaling on GLP-2 modulation of TJ by using PKA agonist(Forskolin)and inhibitor(H89).The test was devided into seven treatments:control,LPS,GLP-2+LPS,Forskolin+LPS,Forskolin+GLP-2+LPS,H89+LPS,and H89+GLP-2+LPS.Results indicated that LPS had no significant effect on intracellular cAMP production and PKA activity(P>0.05);GLP-2,Forskolin or Forskolin+GLP-2 treatment significantly elevated intracellular cAMP production(P<0.05)and PKA activity(P<0.05),and inhibited the increase in monolayer permeability to HRP(P<0.05).GLP-2,Forskolin or Forskolin+GLP-2 treatment alleviated the decrease in TJ proteins ZO-1 and occludin protein expression,promoted the HSP70 protein expression,down-regulated the key kinases phosphorylation of ERK1/2/IKK/NF-κB/MLCK and ROCK/MLCP signaling pathway,maintained the MLCP activity and blocked the pMLCThr18/Ser,9 hyperphosphorylation.H89 or H89+GLP-2 treatment had no significant effect on the epithelial permeability to HRP,H89 abolished the GLP-2 action of relative kinases.The results indicate that GLP-2 can activate G-protein coupled receptor mediated cAMP/PKA signaling,promote HSP70 and TJ protein expression,inhibite ERK1/2/IKK/NF-κB/MLCK signaling pathway to allevate the MLC phosphorylation process induced by LPS,and block the ROCK mediated MYPThr696 phosphorylation to modulate the pMLC dephosphorylation.Summarily,exogenous injection of 10 nmol·kg-1·d-1 GLP-2 can improve the growth performance in weaned piglets by improving the intestinal development and the enzymatic activity for digestion and absorption,and allevating the intestinal barrier injury and the occurrence of intestinal inflammation.The protective effect of GLP-2 on intestinal barrier function are related to the improvement of the expression and distribution of TJ proteins ZO-1 and occludin;As to the modulation of epithelial paracellular permeability,on the one hand,GLP-2 repressed the MLC hyperphosphorylation at the early stage of LPS stress,and prevented the consequent contraction of peripheral actin ring leading to the intercellular TJs redistribution.On the other hand,GLP-2 stimulated the TJ protein expression to stablize the TJs distribution at the cell-cell contacts.GLP-2 modulated the MLC phosphorylation level by inhibiting ERK1/2/IKK/NF-κB mediated MLCK gene transcription and protein expression to allevate the MLC phosphorylation process and preventing ROCK mediated MYPThr696 phosphorylation to enhance to dephosphorylation of MLC.The action of GLP-2 on the upper signaling pathways and TJ protein expression is dependent on the G-protein coupled receptor mediated cAMP/PKA signaling.