Study On Lactobacillus Reuteri Accelerating Lgr5~+ ISCs Regeneration To Protect The Integrity Of Intestinal Epithelial Barrier

Pig intestine is the largest digestive and absorbing organ in pigs.In recent years,intestinal inflammation caused by various reasons has caused a large number of pig farm to have lower survival rate and lower yield of livestock-related products,bringing serious economics to the breeding industry.Health and safety issues are also receiving more and more attention from all walks of life.The pathogenesis of animal enteritis is still unclear,but it has been reported that the disorder of intestinal mucosal immune system and dysbacteriosis play an important role in the pathogenesis of animal enteritis.Little is known about the regulatory effect of microbiota on proliferation and regeneration of pig ISCs.In this study,we used a mouse model to explore the mechanism of lactohacillus repairing pathogen-induced damage,and laid the foundation for exploring the mechanism of lactobacillus to improve intestinal inflammation in piglets.Here,we found that both C.rodentium and Salmonella can induce intestinal inflammation and crypt hyperplasia in mice,and L.reuteri and L.acidophilus protected the integrity of intestinal mucosal barrier through moderate modulation intestinal epithelial proliferation to repair epithelial damage and reduce pro-inflammatory cytokines secretion in intestine in colon.We also found that L.reuteri stimulated proliferation of intestinal epithelial through increased expression of R-spondins and thus activation of Wnt/β-catenin pathway.The proliferation-stimulating effect of Lactobacillus on repair is further enhanced under TNF-α damage to intestinal mucosa,as well as maintaining the number of Lgr5+cells.Moreover,We also found that L.reuteri can regulate intestinal proliferation and immune function in piglets to maintain intestinal mucosal barrier.This study demonstrated that Lactobacillus is effective in maintaining intestinal epithelial regeneration and homeostasis as well as in repairing intestinal recovery during pathological injury,which is a promising alternative therapeutic method for intestinal inflammation.1 Lactobacillus regulates the proliferation of intestinal stem cell to maintain intestinal barrierIn order to explore the protective effect of Lactobacillus on intestinal inflammation,we selected two strains of Lactobacillus,L.reuteri D8 and L.acidophilus4356,two classical mouse intestinal pathogens,Citrobacter and Salmonella typhimurium SL1344.First,we found that the antagonism of L.reuteri D8 was better for Citrobacter,and the antagonistic effect of L.acidophilus 4356 on SL1344 was more obvious by Bacteriostatic test in vitro.Therefore,we used L.reuteri D8 to investigate the repair of colitis caused by Citrobacter in the in vivo,and the condition of L.acidophilus 4356 on the repair of colitis caused by SL1344.To confirm the protective effect of L.reuteri D8 on ameliorating intestinal inflammation in vivo,C57BL/6 mice were orally administered with L.reuteri D8.Firstly,we found that colonization of L.reuteri was detected with immunofluorescence microscopy and reached its peak at 16h through CFU counting length shorten by C.rodentium infection The colon and ileum of C.rodentium-infected mice became air-blowing and transparent.However,L.reuteri D8 greatly decelerated the pathological damage,as well as maintained normal crypt depth to avoid crypt hyperplasia both in colonand ileum.The pathological changes in intestinal morphology caused by C.rodentium infection were consistent with the increased concentration of LPS in serum and secretion of proinflammatory cytokines-TNF-α and IL-1β in intestine,while L.reuteri protected the function of intestinal mucosa by reducing LPS and proinflammatory cytokines levels.C.rodentium increased mRNA expressions of Lgr5 and PCNA,which is consistent with the increased crypt depth and crypt hyperplasia.However,L.reuteri reduced the mRNA expression level of Lgr5 and PCNA to avoid over-activation.At the same time,we explored the protective effect of Lactobacillus on alleviating changes in the intestinal barrier caused by Salmonella infection.We found that S.Typhimurium colonized the colon,damaged colonic mucosa.However,L.acidophilus ATCC4356 alleviated the colitis caused by Salmonella infection.Moreover,S.Typhimurium infection caused colonic crypt hyperplasia with increased PCNA+cells,while L.acidophilus administration resolved these pathological changes.2 L.reuteri D8 increased the proliferation of intestinal organoids by activating Wnt/β-catenin pathway under physiological conditionsTo assess the stimulatory effect of L.reuteri D8 on intestinal epithelia,we successfully isolated crypts from small intestine of mice and cultured them in Matrigel as indicated in the schematic diagram.The organoids began to bud on the third day,at which point they were passaged and cultured for 1 d and then treated with D8(106 CFU)for 48 h.The morphology and surface areas of intestinal organoids were increased significantly under L.reuteri D8 treatment.However,L.reuteri stimulated intestinal epithelial proliferation with increased mRNA expressions of c-Myc,cyclin and Ki67 significantly,which was also verified with enhanced proliferating cells stained with 5-ethynyl-2’-deoxyuridine(EdU)in crypt significantly.The results demonstrated that L.reuteri D8 did activate Wnt/β-catenin pathway by increasing mRNA expression levels of Wnt3 and Lrp5 significantly,which was also further verified with increased β-catenin and active β-catenin expression.As expected,L.reuteri D8 induced clear nuclear accumulation of active β-catenin.After activation of Wnt/β-catenin pathway by D8,the mRNA expression levels of ISCs marker genes,such as active ISCs markers(Lgr5,Olfm4,Ascl2)and quiescent ISCs markers(Bmil,Msi1)were also increased in organoids co-cultured with L.reuteri D8.Moreover,compared to control group,increased Lgr5 protein level and Lgr5+immunofluorescence cells were detected in organoids treated with L.reuteri.We also further found that without R-Spondins in the cultural medium,the activation of Wnt/β-catenin pathway was inhibited.Interestingly,L.reuteri D8 could still increase the mRNA expressions of Wnt3,Lgr5 and c-Myc,even in the lack of r-Spondins.We also found that L.reuteri D8 could increase the mRNA expressions of R-spondin-1,R-spondin-2 and R-spondin-3.This phenomenon was also verified with increased EdU+cells in organoids.Moreover,the stimulatory effect of live L.reuteri on proliferation was more obvious than heat killed Lactobacillus.3 L.reuteri D8 ameliorate the intestinal inflammation induced by TNF-αSince the model of the pig organoid has not yet matured,we established a co-culture model of L.reuteri D8 and mouse organoids.In addition to assess stimulatory effect of L.reuteri D8 in physiological conditions,we also detected whether L.reuteri D8 possesses an intestinal epithelial repair function in pathological states,such as intestinal inflammation caused by TNF-α in intestinal organoids.Obvious damage to intestinal organoids was observed after TNF-α treatment,and more than 80%organoids were disrupted within 36 h.However,L.reuteri D8 significantly reduced percentage of disrupted organoids and TNF-αexpression,which was consistent with the reduced apoptotic cells.L.reuteri also saved the loss of EdU+cells caused by TNF-α treatment to maintain the proliferative ability to repair the damaged epithelial.Furthermore,L.reuteri reversed reducing expression of c-Myc,cyclin and Ki67 after TNF-α treatment.Compared to L.reuteri D8,the ability of L.salivarius C5 to accelerate intestinal epithelial proliferation,as measured by the mRNA expression of c-Myc,Lgr5 and EdU staining,was weaker than that of L.reuteri in the pathological state.These results indicated that the protective effect of Lactobacillus on intestinal mucosa is species-specific.Compared to control group,L.reuteri D8 up-regulated Wnt3 and Lrp5 expressions significantly,which was consistent with the enhanced protein expression of β-catenin.Interestingly,TNF-α inhibited mRNA expression of the active stem cell marker Lgr5,Olfm4,Ascl2,and increased expression of quiescent stem cell marker Msi1.Moreover,L.reuteri significantly improved Lgr5,Olfm4,Ascl2 and Bmi1 expressions.The maintenance of ISCs was also further verified by the increased protein expression of Lgr5 and Lgr5+cells in crypt.These results demonstrated that L.reuteri maintained activation of Wnt/β-catenin pathway inhibited by TNF-α and thus stimulated the proliferation of intestinal epithelia.TNF-α significantly decreased the number of Paneth cells in crypt and lysozyme expression compared to those in the control group,which indicates the dysfunction of Paneth cells.However,L.reuteri D8 restored the number of Paneth cell and lysozyme expression after organoids damage by TNF-α.4 L.reuteri D8 regulates intestinal proliferation and immune function in piglets to enhance intestinal mucosal barrierThree-day-old piglets were randomly divided into two groups.The control group was continuously administered with sterile PBS for 5 days,2 mL each time.L.reuteri D8 was continuously administered 10^9 CFU dissolved into2 mL sterile PBS for 5 days.The piglets were weighed daily and the results showed that there was a significant difference between the L.reuteri D8 treatment group and the control group on the second and third days.HE staining showed that L.reuteri D8 treatment can increase the height of intestinal villi and the depth of crypt in the jejunum of piglets,and the statistical results are consistent with the results of HE pictures.The results of PCNA immunohistochemistry showed that L.reuteri D8 treatment significantly increased the number of PCNA-positive cells in each crypt of piglets compared to control group.At the same time,the proliferative gene c-Myc was up-regulated after treatment with L.reuteri(P=0.056),and L.reuteri D8 also significantly up-regulated the expression of Wnt-β-catenin signaling pathway target gene Tcf4.This result suggests that L.reuteri D8 may promote the proliferation of intestinal epithelium in piglets by activating the Wnt-β-catenin signaling pathway.We tested Muc-2 and Lyz-1 in the jejunum of piglets and found that L.reuteri D8 increased the gene expression levels of Muc2 and Lyz-1 in piglets.Morever,HE statistics show that L.reuteri D8 treatment can significantly increase the area of ileal Peyer’s patch in piglets.RT-PCR show that L.reuteri D8 can increase the cytokine IFN-y and IL-4 mRNA expression.The above results indicated that L.reuteri D8 promoted intestinal epithelial proliferation,activated secretion of antimicrobial peptides and regulated mucosal immune function by activating Wnt-β-catenin signaling pathway,and ultimately maintained intestinal mucosal barrier function.