Maintenance Of Intestinal Mucosal Barrier By Lactobacillus Acidophilus Via Modulating Wnt/?-catenin Signaling Pathway

The intestinal mucosal barrier consists of mucosal epithelial cells,normal intestinal microbiota and other links,which can not only ensure the efficient absorption of nutrients,but also keep off the external environment by constantly updating the shedding epithelium.The intestinal mucosa relies on the continuous proliferation and differentiation of the stem cells at the intestinal crypts to replace the intestinal epithelium that damages the shedding to maintain intestinal homeostasis.However,the intestinal epithelium is constantly stimulated by microorganisms,breaking the intestinal homeostasis and inducing intestinal inflammation.Salmonella is one of the most common pathogenic bacteria in the breeding industry.It causes intestinal inflammation with severe diarrhea and brings huge economic losses to the breeding industry.With the deepening of research and application of probiotics,more and more researchers have confirmed that Lactobacillus can effectively resist the invasion and infection of pathogenic bacteria.However,due to the complex in vivo environment and the lack of appropriate research models,the mechanism by which Lactobacillus resists the maintenance of intestinal epithelial barrier integrity by pathogenic bacteria has not yet been clarified,and the relationship between regulation of Lactobacillus and stem cells has not been reported domestically.In order to clarify this mechanism,this experiment explored the mechanism of Lactobacillus through the Wnt/?-catenin pathway to regulate intestinal epithelial proliferation to repair the mucosal barrier of Salmonella injury through the intestinal organoids model and animal experiments.This study was first grouped into a blank control group,Lactobacillus acidophilus ATCC4356 group and Lactobacillus protection group.Then the enteritis model was successfully constructed using Salmonella SL1344.The content of experimental research mainly includes the following three parts:1 Lactobacillus regulates proliferation of intestinal cells through Wnt/?-catenin pathway and alleviates intestinal mucosal damage in Salmonella-infected mice.Lactobacillus plays a more and more important role in the intestinal immunity and antagonism of pathogenic bacteria.In order to explore the role of Lactobacillus in repairing intestinal mucosal damage caused by pathogenic bacteria,four-week-old C57BL/6 mice were selected as the study object.Lactobacillus ATCC4356(l×108CFU)was administration continuously for one month and Salmonella SL1344 was infected(1×107CFU).Salmonella induced severe death of mice,severe intestinal tissue(HE)injury,intestinal villi hemorrhage,atrophy,and Intestinal hyperplasia PCNA+(TA area:transit amplify cell).Immunohistochemistry(IF)detected the number of intestinal secretory cells(Pan-based cells UEA-1+,goblet cells Lysozyme+,and tuft cells(DCLK1+)and found that Salmonella infection caused mice intestinal epithelial secretory cells expansion.All the indicators of mice per-treatment with Lactobacillus pretreated mice:survival rates,pathological morphology of the intestine,pathological hyperplasia of the crypts,expansion of secretory cells were all significantly improved.The power of proliferation and differentiation of the intestinal epithelium was derived from crypt stem cells,so the mRNA levels of Lgr5+intestinal stem cell were detected by qPCR.The results showed that compared with the Lactobacillus pretreatment group,the number of stem cells after Salmonella infection was drastically reduced.The Axin2 mRNA expression were abnormally highly expressed using qPCR,which are the downstream transcription factors of wnt/?-catenin signaling pathway.In order to further explore the protective effect of Lactobacillus against Salmonella infected mice,it was found that Lactobacillus could reduce the intestinal inflammatory factors(IL-1? and TNF-?)by ELISA and intestinal bacteria load.Lactobacillus prevents Salmonella invasion and slows down the inflammation due to invasion.TLRs are pattern recognition receptors that first identify pathogen-associated molecular motifs.(PAMPs),which modulate the inflammatory response of the intestine,so they serve as an important part of the body's defense mechanism.Therefore,in order to verify the role of TLR in the regulation of Lactobacillus protection,the changes of TLR expression were detected by fluorescence PCR of in the intestine,and found that Lactobacillus inhibited LTR2 and TLR4 and slows down Salmonella-induced inflammation,slows pathological hyperplasia of crypts,and eventually reverses the fate of stem cells(Lgr5)exhausted.The above results demonstrate that Lactobacillus improved the damaged intestinal tract.The microenvironment regulates the number of intestinal stem cells in mice and the proliferation rate of the TA area,maintains the rhythm of intestinal renewal,and guarantees normal regulatory mechanisms of the cells themselves.2 Establishment of a co-model of Lactobacillus-intestinal organoids-Salmonella in vitro.The intestinal organoids are a three-dimensional cell culture system that can be stably cultured in vitro to form a crypt-villus structure.The system is highly similar to the intestinal tissue structure of the body.Intestinal organoids have intestinal stem cells,which can replicate and differentiated cell types:absorptive cells and secretory cells,and facilitate spatial expansion and long-term culture with the support of Matrigel.In this study,2 mM EDTA were used to isolate the intestinal crypts of mice,responsible for the continuous regeneration of intestinal mucosa in the habitat of intestinal stem cells.In the primary culture process,we cultured the acquired cells in a support medium(Matrigel)so that it can maintain three-dimensional growth.For the long-term culture and stable passage of the gut organoids,500 ng/ml R-spondin,100 ng/ml Noggin,and 50 ng/mL EGF were added to the medium to provide signals and stimulation which suproted microenvironment of the stem cells i.This experiment used a mature intestinal organoids containing villous structures to explore the mechanism of Lactobacillus infection against pathogenic bacteria.Firstly,the intestinal organoids were isolated and cultured,and then a co-culture system of Lactobacillus acidophilus-intestinal organoids-Salmonella was established:Lactobacillus ATCC 4356(1×108 CFU)was added to the supernatant for 12 h.Subsequently,the crypt cells were dissipated,and Salmonella SL1344(1×107CFU)was added and infected for 1h to fully invade the free surface.After 1h,the cells were mixed with Matrigel and plated in 24 wells for 24 h.The result found that salmonella infection decreased buding of intestinal argonids and induce inflamation cytokines prodution.Conversally,Lactobacillus treatment can promote buding and inhibited inflamantion.This is the first time to establish a model of Lactobacillus-intestinal organoids-Salmonella in vitro.3 Lactobacillus modulates proliferation of intestinal cells via wnt/p-catenin and slows down the damage of intestinal organoids.In order to further verify the results in vivo,this experiment used the model of intestinal organoids to mimic the complex environment in vivo.Salmonella was used to infect the intestinal organoids of mice for 1 h.The infected cells were then resuspended with Matrigel for 24 h.Observation under light microscope at different time points:the germination rate and formation rate of organoids were greatly reduced caused by Salmonella infection,and the proliferation of intestinal organoids(EdU)was induced.In order to deal with the invasion of pathogenic bacteria,intestinal organoidss differentiated a certain number of goblet cells and Paneth cells.These secretory cells can produce antibacterial peptides and other substances and produce high concentrations of inflammatory factors.However,changes in the composition and differentiation of cells affect the intestinal homeostasis,and the expression level of Lgr5,the intestinal stem cell marker,is significantly reduced,and the Axin2 and Wnt3 mRNA expression are abnormally high,causing irreversible damage to the intestine.However,after pretreatment with Lactobacillus,the germination rate and the formation rate of the intestinal organs of Salmonella-infected mice were both increased,and the level of inflammatory factors(TNF-?)caused by pathogenic bacteria was alleviated.Lactobacillus can also cause certain levels of secretory cells to protect against pathogens,but at the same time it can regulate the differentiation level of cells,stabilize the normal proliferation level of cells and ensure the normal regulation mechanism of cells themselves.In this study,the expression of TLR2,TLR4 and downstream MyD88 were detected by real-time quantitative PCR.The results showed that the expression of TLR4,TLR2 and MyD88 in Salmonella infection group was significantly higher than that in healthy control group.Compared with the infected group,the pretreatment of Lactobacillus group showed a significant decrease in the expression levels of TLR4 and TLR2 mRNA.This result is consistent with the in vivo results,and together clarified that the mechanism of probiotics maintaining epithelial homeostasis may be related to the expression of TLR.By preventing the use of TLR by Salmonella,it can slow the excessive proliferation of intestinal crypts,deplete the stem cells,and finally stabilize intestinal stability.