Methionine Restriction Affects Expression And Function Of Caco-2 Intestinal Epithelial Tight Junction Barrier

Inflammatory bowel disease(IBD),encompassing Crohn's disease(CD),ulcerative colitis(UC)and unclassified IBD(IBDU),is characterized by chronic relapsing intestinal inflammation.Although the etiology of IBD remains largely unknown,it involves a complex interaction between the genetic,environmental or microbial factors and the immune responses.Over the past decades,there has been an increasing recognition of an association between the pathogenesis of IBD and the intestinal barrier function.Barrier dysfunction can predispose and contribute to the occurrence and progression of IBD,and increase the risk of IBD patients relapse.Dietary methionine restriction(MR)has been found to affect the epithelial barrier function.Specifically,MR has been found to modify the protein composition of tight junctional complexes surrounding individual epithelial cells in a manner that renders the complexes less leaky.This has been observed in both a renal epithelial cell culture model and in gastrointestinal tissue.In both cases,MR increased the transepithelial electrical resistance across the epithelium,while decreasing passive leak of small nonelectrolytes.In our previous study in vivo,we found that low methionine diet leads to improve epithelial barrier function and decrease the extent and severity of intestinal inflammation.This improvement may be related to MR diet-induced alteration of gastrointestinal TJ claudin composition.However,the mechanisms of the improvement and protection on the damaged barrier function are completely unknown.In this study,we took a further effort to investigate the protective actions of MR on barrier function and the underlying mechanisms in Caco-2 monolayers challenged with TNF-a.Objective:Based on our previous study in vivo,the objective of this study is to make further investigation of the protective actions of MR on barrier function and the underlying mechanisms in Caco-2 monolayers challenged with TNF-?.Methods:Caco-2 monolayers were cultured with completed culture medium and medium with reduction of methionine by 90%.Then Caco-2 monolayers were treated with or without TNF-a for 48h.The cells were grouped as follows:AA group,MR group,AA+TNF-a group,MR+TNF-a group.Both transepithelial electrical resistance(TEER)and paracellular permeability were measured to evaluate barrier function.Ultrastructural features of Caco-2 monolayers were observed by a transmission electron microscope(TEM).The expression and distribution of tight junction proteins Claudin-1,Occludin and ZO-1 were respectively analyzed by real-time Q-PCR,immunoblot or immunofluorescence.The expressions of phosphorylated myosin light chain(pMLC),MLC kinase(MLCK),phosphorylated MYPT1(p-MYPT1)and Rho kinase were determined by immunoblot to respectively analyze the effect of MLCK-P-MLC and Rho/ROCK signaling pathway.Results:(1)Cell morphology,TEER,LY permeability and cell polarity results showed that the model of intestinal epithelial barrier was successfully established with Caco-2 cells in vitro.(2)The results of barrier function study showed that TNF-a decreased the TEER(P<0.01)and increased the LY permeability(P<0.01),compared with that of AA group;MR had a significant increase on the TEER(P<0.01)and cell polarity(P<0.05),compared with that of AA group;MR significantly dampened the TEER drop and lowered the increase of LY permeability elicited by TNF-?(P<0.05).(3)The results of TEM indicated that the tight junctions and microvilli in AA group and MR group had intact structures;TNF-a treatment resulted in disruption of normal TJ morphology and partly shedding of the microvilli;MR+ TNF-? group had a lessened extent of the disruption of the TJ and microvilli,compared with that of AA+TNF-a group.(4)The results of qPCR and Western Blot revealed that the mRNA and protein expression levels of TJ proteins including Claudin-1,Occludin and ZO-1 were not significantly altered by the treatment of Caco-2 monolayers cultured with completed medium or low methionine medium with or without TNF-?.(5)In the study of the localization of TJ proteins,we found that,in control Caco-2 monolayers,the TJ proteins Claudin-1,Occludin and ZO-1 were respectively localized to the intercellular tight junctions,along the edge of the cells.These regular distributions were not obviously changed in Caco-2 monolayers cultured with low methionine medium.Treatment of Caco-2 monolayers with TNF-a for 48 hours induced pronounced reorganization of TJ proteins Claudin-1,Occludin and ZO-1 such that the distribution profiles became irregular and discontinuous.Low methionine treatment ameliorated TNF-a-induced altered localization of TJs.(6)After treatment of Caco-2 monolayers with TNF-a for 48 h,there was a significant increase in both the expression of MLCK and phosphorylation level of MLC compared with AA group(P<0.05);MR significantly inhibited the increase of both the expression MLCK and phosphorylation level of MLC compared with AA+ TNF-a group.(7)After treatment of Caco-2 monolayers with TNF-a for 48 h,there was a significant increase in the expression of p-MYPT1,ROCK1 and ROCK2 compared with AA group(P<0.05);MR had no effect on the expression of p-MYPT1,ROCK1 and ROCK2 compared with AA+ TNF-? group(P>0.05).Conlusions:(1)The joint evaluation of cell morphology,TEER,LY permeability and cell polarity is an effective way to establish the repeatability and reliability of the model of intestinal epithelial barrier with Caco-2 cells in vitro.(2)TNF-a increases Caco-2 intestinal epithelial permeability and alters localization of TJ proteins.(3)MR improves the intestinal barrier function and has a protective effect on TNF-a induced barrier dysfunction.(4)TNF-a activates both the MLCK-P-MLC and Rho/ROCK signaling pathway.MR significantly attenuates TNF-a-induced activation of MLCK-P-MLC signaling pathway.