Protective Effect Of Surface Layer Protein From Lactobacillus On Intestinal Epithelial Barrier

The intestinal epithelial barrier is a physical barrier,whose structural integrity and function are important prerequisites for resisting the invasion of external substances and maintaining intestinal homeostasis.Numerous researches showed that a variety of Lactobacillus could effectively strengthen the function of intestinal epithelial barrier and protect the intestinal epithelial barrier from external substances.As one of the surface substances of Lactobacillus,Surface layer protein(Slp)from some strains was reported to antagonize intestinal cell apoptosis and inflammatory response by competing with pathogen,so as to reduce the damage of intestinal epithelial barrier induced by pathogen.In this paper,we studied whether Slp of various Lactobacillus could protect the intestinal epithelial barrier from TNF-α-induced damage,which further revealing the contribution of Slp to intestinal function,providing a theoretical basis for further development and utilization of Lactobacillus resources,and creating new ideas for intestinal health intervention.Firstly,Slp from four Lactobacillus strains(L.acidophilus NCFM,L.acidophilus ATCC4356,L.crispatus JCM 2009 and L.fermentum CCFM 421)were isolated and purified.After the removal of Slp by Li Cl,the number of four Lactobacillus strains adhered to Caco-2 cells was significantly decreased(P < 0.05),confirming that Slp was involved in the adhesion of Lactobacillus to the intestine,which was also the prerequisite for Lactobacillus to play a probiotic role.Caco-2 cells were used to construct the normal intestinal epithelial barrier model,and the transepithelial resistance(TEER)and the flux of fluorescein isothiocyanate-dextran(FITCdextran)in the paracellular pathway were used as the evaluation indexes.It was found that Slp of the four Lactobacillus strains could significantly inhibit the decrease of TEER and the increase of FITC-dextran flux induced by TNF-α in a concentration-dependent manner(P <0.05),indicating that Slp maintained the tight junction between adjacent intestinal epithelial cells and protected the integrity of intestinal epithelial barrier structure by inhibiting the increase of the permeability of the paracellular pathway.Western blot and immunofluorescence staining showed that Slp from the four Lactobacillus strains could significantly increase the expression levels of tight junction proteins Occludin and ZO-1(P < 0.05),and the abnormal distribution of Occludin and ZO-1 stimulated by TNF-α in the cells was partly restored,confirming that Slp could alleviate the intestinal epithelial barrier damage induced by TNF-α by maintaining the function of tight junction.Using cell viability determination and Hoechst 33342/PI double staining,it was found that Slp could significantly antagonize TNF-α-induced damage on Caco-2 cell viability and apoptosis(P < 0.05),confirming Slp had a protective effect on the viability of intestinal epithelial cells.The secretion of IL-8 in Caco-2 cells was determined by ELISA,and it was found that Slp could significantly inhibit TNF-α-induced IL-8 secretion(P < 0.05),suggesting that Slp could alleviate TNF-α-induced intestinal inflammation.Immunofluorescence staining showed that Slp could inhibit the nuclear translocation of NF-κB p65 subunit induced by TNF-α and keep NF-κB inactivated,indicating that Slp played a protective role in intestinal epithelial barrier by blocking the activation of NF-κB signaling pathway.In conclusion,Slp from four Lactobacillus strains could block the NF-κB signaling pathway and maintain the viability of intestinal epithelial cells,so as to inhibit TNF-α-induced decrease of TEER and the increase of paracellular permeability,regulate the expression of tight junction protein,restore the distribution of tight junction protein,and inhibit intestinal inflammation,which ultimately protect the intestinal epithelial barrier from TNF-α interference.Therefore,this study revealed the positive contribution of Slp to intestinal epithelial barrier,and provided a theoretical basis for the further development and utilization of Lactobacillus and Slp resources in the gastrointestinal tract.