Investigation On The Mechanism Of Anti-Tumor And Intestinal Barrier Function Regulation By Exopolysaccharides From Lactobacillus Plantarum NCU116

Lactic acid bacteria(LAB)is an order of bacteria that can ferment glucose to produce large amounts of lactic acid.It is widely distributed in the whole world,and is also a common flora in the human intestinal tract,which is closely related to human health.Numerous studies have shown that Lactobacillus and its secondary metabolite,exopolysaccharide(EPS),play an important role in anti-cancer,regulating immunity and optimizing intestinal microecological environment.However,little research has been done on the specific molecular mechanisms and detailed signaling pathways of anti-tumor and intestinal epithelial barrier function regulation.Therefore,we took exopolysaccharides(EPS116)from Lactobacillus plantarum NCU116 discovered in our laboratory as materials to explore its anti-tumor and intestinal barrier regulation functions and underlying molecular mechanisms.It provided a theoretical basis for the development of a new type of health food.The main research contents and results were summarized as follows:1.The exopolysaccharide of Lactobacillus plantarum NCU116(EPS116)was isolated,and its composition and properties were studied.EPS116 was separated by solvent fractional precipitation.The ultraviolet spectrum analysis showed that EPS116 was a proteoglycan composed of polysaccharides and proteins.Ion chromatography analysis revealed that the polysaccharides were mainly composed of five monosaccharides,including mannose,glucose,glucuronic acid,glucosamine and galactosamine.2.Different types of cancer cell lines were used to evaluate the anti-cancer ability of EPS116 and investigate its potential molecular mechanisms.Experiments of cell proliferation and apoptosis showed that EPS116 inhibited the proliferation of cancer cells in a cell type dependent manner,and significantly repressed the survival of CT26 cells by inducing apoptosis.RT-qPCR and Western blot results showed that EPS116 increased the expression of death receptor Fas and its ligand Fasl,transcription factor c-Jun,and induce c-Jun phosphorylation in CT26 cells.Caspase enzyme activity test and Western blot results showed that EPS116 induced the proteolysis and activation of Caspase-3 or Caspase-8.In addition,EPS116 also up-regulated the expression of TLR2(Toll like receptor 2)in CT26 cells.The results of RNAi showed that the defect of TLR2 gene resulted in a significant decrease in the sensitivity of CT26 cells to EPS116 and the expression of apoptotic genes Fas,Fasl and c-Jun,suggesting that the antitumor activity of EPS116 might depend on TLR2.TLR2 forms heterodimers with TLR1 or TLR6,facilitating interaction with different microbial components.Therefore,EPS116 induced apoptosis may depend on the functional interaction between TLR2 and TLR1 or TLR6.Knockdown of TLR1 or TLR6 genes showed that TLR2 synergy with TLR1 promoted EPS116 to induce apoptosis of CT26 cells.In conclusion,the inhibitory effect of EPS116 on the proliferation of CT26 cells might be mediated by TLR2 and activated c-Jun-dependent Fas/FasL-mediated apoptotic signaling pathway.3.The role of EPS116 in regulating colonic mucosal barrier and its underlying mechanism were studied.The results of animal experiments and HE staining showed that EPS116 alleviated colon inflammation induced by DSS in vivo(including weight loss,fecal blood,diarrhea,shortening of colon and the score of colon tissue section).FITC-dextran permeability test showed that EPS116 repaired DSS induced intestinal barrier dysfunction in mice.RT-qPCR and Western blot results showed that EPS116 promoted the expression of tight junction proteins Claudin-1,Occludin and ZO-1 in colon tissue of mice,and decreased the expression of leakage proteins Claudin-2.The levels of TNF-alpha,IFN-gamma and IL-6 in serum of mice were significantly increased by DSS treatment,while administration of EPS116 decreased the level of them.In addition,EPS116 could decrease the permeability of Caco-2 monolayer cells and up-regulate the expression of tight junction proteins Occludin and ZO-1 in vitro.In addition,the chromatin immunoprecipitation data showed that EPS116 promoted the binding of STAT3 to the promoters of tight junction protein genes Occludin and ZO-1.Furthermore,RNAi assay showed that EPS116 significantly decreased the expression of Occludin and ZO-1 in STAT3-deficient Caco-2 cells,and increased the permeability of monolayer cells,suggesting that the regulation of EPS116 on epithelial barrier function should depend on STAT3.Therefore,our study reveals a new mechanism by which EPS116 inhibits enteritis by regulating intestinal barrier function.In conclusion,this study confirmed the anti-tumor activity and the regulation of intestinal mucosal barrier of exopolysaccharide from Lactobacillus plantarum NCU116(EPS116).The anti-tumor activity of EPS116 is mediated by TLR2 and depended on the c-Jun and Fas/FasL signaling pathways.Its intestinal mucosal barrier regulation ability depended on the transcription factor STAT3,which promoted the expression of tight junction proteins.