Protective Mechanism Of Milk Phospholipids On Intestinal Epithelium In Mice Based On Intestinal Organoids

The structure and function of intestinal epithelium is determined by intestinal stem cells at the base of the crypts.As a unique bioactive substance in breast milk,milk phospholipids plays a key role in maintaining intestinal epithelial homeostasis.However,due to the lack of suitable intestinal stem cell research techniques in vitro,previous studies on the intestinal biological effects of milk phospholipids have not penetrated into the level of intestinal stem cells.In recent years,with the advent of three-dimensional(3D)intestinal organoid technology,it has become possible to maintain the self-renewal and differentiation of intestinal stem cells in vitro for a long time.Based on the in vitro culture of mouse intestinal organoids and combined with the traditional mouse(disease)animal model,the protective mechanism of milk phospholipids on intestinal epithelium in normal mice and mice with different pathological conditions(enteritis and Salmonella infection)was systematically studied in this study,which provided further theoretical foundation for the functional application of milk phospholipids.The main research contents and results are as follows:1.Mouse intestinal organoids cultured in vitro.Successful in vitro 3D culture of mouse enteroids(duodenum,jejunum and ileum)and colonoids was achieved by screening appropriate EDTA concentration for effective crypt isolation,appropriate concentration of R-spondin1 in culture medium and efficient passage method,and technical operation procedures for mouse intestinal organoids culture were established.The average primary formation rate of primary enteroids and colonoids were about 70%and 55%,respectively,for secondary formation rate about 90%and 70%,respectively.The average expanding fold of individual enteroid and colonoid was about 10 and 12,respectively,and the average budding number of individual enteroid was about 7-9.The proteins of cell type marker genes(Chg A,DCLK1,Muc2,Lyz and Villin)of intestinal epithelium were identified using immunofluorescence staining,which confirmed that the obtained intestinal organoids had all cell types of intestinal epithelium.Meanwhile,utilizing the 3D enteroids,the 2D monolayer culture of intestinal epithelium with milk phospholipids absorption and mucus secretion function of proliferative and diverse cell types was established with optimization.2.Effects of milk phospholipids on intestinal epithelial homeostasis in normal mice.Healthy mice were supplemented with 25 mg/kg milk phospholipids daily for 24 consecutive days,no significant effects on the structure of small intestine and colon,intestinal epithelial proliferation,and intestinal epithelial cell lineage differentiation were observed(p>0.05).In colonoids,milk phospholipids also had no direct significant effects on the proliferation and differentiation of colonic stem cells(p>0.05).3.Protective effect and mechanism of milk phospholipids on colitis-induced colonic epithelial injury in mice.Milk phospholipids supplementation(25 mg/kg/day for 24consecutive days)significantly attenuated DSS-induced acute colitis in mice,manifested as:the average weight loss decreased from 17.5%to 7.2%(p<0.05),the average disease activity index decreased from 2.89 to 1.5(p<0.05),the average score of colonic epithelial injury decreased from 10.3 to 6.5(p<0.05),the activity of myeloperoxidase(MPO)and the expression level of inflammatory factors such as tumor necrosis factor-?(TNF-?)were all significantly decreased(p<0.05).Milk phospholipids significantly reduced goblet cell depletion induced by acute colitis through significantly reducing the overexpression of Dll4,the ligand of Notch receptor,in intestinal submucosal myofibroblasts(from 4.8-fold to2.3-fold,p<0.05),and thus restored the level of overactivated Notch signaling pathway in the colonic epithelium(p<0.05).The number of goblet cells in a single crypt increased from an average of 10.2 to 19.3,the average diameter of individual goblet cell increased from 10.4to 14.5?m.Meanwhile,Muc2 secretion level of colonic epithelium was significantly elevated by nearly 11 times(p<0.05),the gene expression level of Reg3?,Reg3?and Ang4antimicrobial peptide was significantly elevated by 3 to 8 times(p<0.05),which enhanced the non-specific immunity of intestinal mucosa and played a protective role against intestinal inflammatory colonic epithelial injury.Utilizing colonoid model and myofibroblasts and colonoids co-culture system,it was found that milk phospholipids had no significant effect on the proliferation of colonic stem cells(p<0.05),but significantly promoted the goblet cell differentiation of colonic stem cells(p<0.05):the average number of goblet cells in a single colonoid increased from 6.8 to 17.6.The mediation of myofibroblasts,as a factor of colonic stem cell niche,was indispensable for the regulation of colonic stem cell differentiation by milk phospholipids,which indicated an indirect regulation mode.4.Effect and mechanism of milk phospholipids on the repair of enteritis-induced ileal epithelial injury in mice.Supplementation of milk phospholipids(25 mg/kg/day)during the repair period of enteritis-induced ileal epithelial injury promoted the repair process of ileal epithelium,which was manifested as:the recovery score of ileal epithelial structure injury increased from 1.2 to 2.8(p<0.05),significant recovery of tight junction protein ZO-1and E-Cadherin expression(p<0.05),significantly increased the proliferation rate of intestinal epithelial cells during the repair of ileal epithelial injury by about 1.8-fold(p<0.05),and migration speed about 1.5-fold(p<0.05),but there was no significant effect on apoptosis(p>0.05).In the ileal organoids injured by TNF-?,milk phospholipids could directly act on ileal stem cells.By up regulating Wnt/?-catenin signaling pathway,the number of+4 reserve ileal stem cells(Bmi1~+)was up-regulated by about 3.2 times(p<0.05),and then the reduced number of Lgr5~+crypt-base columnar cells was increased by 2.8 times(p<0.05).At the same time,milk phospholipids could balance the imbalance of differentiation of ileal stem cells and thus promote the repair and homeostasis of intestinal epithelium in enteritis.5.Effect and mechanism of milk phospholipids on intestinal mucosal microbial barrier in Salmonella infected mice.The colonization resistance of Salmonella enterica serovar typhimurium mediated by intestinal microflora was significantly increased by supplementation of 0.05%milk phospholipids in mice.The results showed that the average weight loss of mice decreased from 25.7%to 12%(p<0.05),the contents loss of colon and cecum decreased from 56.3%to 26.6%(p<0.05),the histological damage of colonic epithelium significantly ameliorated(p<0.05),the activity of MPO and the expression level of inflammatory factors such as TNF-?decreased significantly(p<0.05),the amount of Salmonella excretion(p<0.05)and tissue colonization(p<0.05)were significantly reduced by about 2 and 1 orders of magnitude,respectively.However,excessive supplementation of milk phospholipids(0.25%)exacerbated Salmonella infection in mice.The intestinal bacterial communities were analyzed using high-throughput sequencing of 16S r RNA gene,and the results showed that:In Salmonella infected mice,the moderate-dose milk phospholipids supplementation only significantly affected the composition of intestinal flora(?-diversity,p<0.05),but had no significant effect on the?-diversity(Chao1 index and Simpson index)of intestinal flora(p<0.05).LEf Se analysis and random forest supervised learning analysis showed that Porphyromonadaceae,Alloprevotella and Bacteroides in Bacteroides groups,which had the highest predictive power,could be predictive features of microbiotas under moderate-dose milk phospholipids supplementation.The level of propionate in colon of mice was significantly increased by 3 times(p<0.05).Spearman's correlation analysis showed that Bacteroides spp.and propionate were significantly negatively correlated with the colitis(p<0.05).Therefore,the significant increase in the relative abundance of Bacteroides spp.and propionate may be one of the main reasons for the alleviation of Salmonella infection in mice with moderate-dose milk phospholipids supplementation.It was further confirmed that milk phospholipids had no direct and significant effect on Salmonella colonoids colonization of(p>0.05).Both Bacillus fragilis and propionate significantly inhibited Salmonella colonoids colonization(p<0.05).Propionate also significantly promoted the differentiation of colonic stem cells to goblet cells in Salmonella-infected colonoids(p<0.05):the average number of goblet cells in a single colonoid increased from 7.6 to 19.3.These results suggested that the intestinal Bacteroides spp.and propionate regulated by moderate-dose milk phospholipids supplementation could directly reduce the colonic epithelial colonization of Salmonella,which may be one of the possible mechanisms that the appropriate amount of milk phospholipids supplementation could significantly alleviated Salmonella infection in mice.In conclusion,the biological effects of milk phospholipids on intestinal epithelium strongly depend on the state of the body,and have different effects on intestinal epithelium and intestinal stem cells in normal mice and mice under different pathological conditions,and the mechanism of stem cell influence on intestinal epithelium of different intestinal segments is also different.The experimental results obtained from the intestinal organoids constructed in this study in vitro are highly consistent with those of donor animals in vivo,and this two models can complement and deepen each other,which may provides a new research platform for the food nutrition biology research.