Retinoic Acid Upregulates TLR4 To Facilitate The Level Of ZO-2 Expression In Intestinal Epithelium Through RAR?

Objective To confirm TLR4 effects on the RA upregulating RAR?to induce the level of ZO-2 expression,which enhances intestinal epithelial barrier function.Methods 1.In vitro,with the RA treatment to the Caco-2 cells,the concentration of zonulin was deteced in the culture medium by ELISA.The subcellular co-localization of RAR?,TLR4 and ZO-2 were observed in the Caco-2 cells treated by RA by laser-scanning confocal microscopy.The levels of RAR? and ZO-2 expressions were tested in the Caco-2 stable cells infected by Lv-siTLR4 with real-time PCR and western blotting.2.In vivo,The vitamin A normal(VAN)and vitamin A deficiency(VAD)mouse models were established in the TLR4 knockout and Wild Type mice,respectively.The levels of serum retinol were detected by high performance liquid chromatography(HPLC)and the transepithelial resistance(TER)of colonic mucosal tissue were tested by Ussing chamber system.The levels of RAR?,TLR4 and ZO-2 expressions in the colonic mucosal epithelium tissue were tested with real-time PCR and western blot.The morphological changes of tight junction in the intestinal epithelium were observed by Transmission Electron Microscope(TEM).Results 1.The concentration of zonulin was significantly decreased in the culture medium of the Caco-2 cells following the RA treatment compared with that of the untreated cells(P=0.043).The expressions of RAR? and TLR4 were co-located in the nucleus and membrane of the Caco-2 cells,and the TLR4 and ZO-2 proteins were co-expressed in the Caco-2 cells membrane.However,little co-expression between the RAR?and ZO-2 proteins was found in the Caco-2 cells with or without RA treatment.Although the level of RAR? protein expression was statistically increased,the level of ZO-2 expression in the Caco-2 cells was significantly decreased by Lv-siTLR4 after RA treatment.2.The levels of serum retinol were significantly decreased in the both Wild Type(WT)and TLR4 knockout(TLR4KO)mice with VAD compared with those of the VAN mice(P<0.0001,P=0.009).The TER of colonic mucosal layer in the WT mice with VAN was statistically higher than that of the VAD mice(P=0.002),while there were no significant differences in the levels of colonic TER of the TLR4 KO mice with VAN or VAD.Moreover,the levels of RAR?,TLR4 and ZO-2 mRNA and protein expressions were significantly decreased in the colon tissues of the WT mice with VAD compared with those of the WT mice with VAN.Although the levels of RAR? mRNA and protein expressions were reduced in the TLR4 KO micewith VAD,there is no statistical difference between the VAN and VAD groups in the levels of ZO-2 expression.Transimission electron microscope displayed that the tight junction of WT mice with VAN was densification and integrity,while loose and imperfect in the WT mice with VAD.However,no obviously changes were found in the tight junction between the TLR4 KO mice with VAN and VAD.Conclusions After RA activates RAR? signaling pathway,siTLR4 downregulates the level of intracellular ZO-2 expression.And the intestinal epithelial barrier function is not affected by the different VA nutritional levels in the TLR4 KO mice.These results suggest that TLR4 plays an important role on the RA inducing RAR? to promote ZO expression level and enhance intestinal epithelial barrier function.