Research On Protection And Mechanism Of Bovine Lactoferricin On Intestinal Mucosal Barrier

The intestinal tract is the main place where the body is exposed to foreign microorganisms,toxins and other antigenic substances;Under normal conditions,external toxic and harmful substances blocked by intestinal mucosal barrier cannot enter the intestinal cavity into the body and affect intestinal health;however,once the intestinal mucosal barrier is damaged that will lead to intestinal function dysfunction,different degrees of threat to the body health.Bovine Lactoferricin(LfcinB)is a 25-amino acid residues obtained by enzymatic hydrolysis of bovine lactoferrin under the action of pepsin.Many experiments have demonstrated that LfcinB has significant antibacterial,antiviral,inhibiting tumor cell growth,immune regulation and other physiological functions.However,the role of LfcinB in studies on whether it can improve intestinal health by improving intestinal barrier function have not been reported in detail.Therefore,based on this background,The animal model(5%DSS)and organoid model was used to investigate the protective function and related mechanism of LfcinB on intestinal mucosal barrier in vivo and in vitro.The main research contents and results are as follows:(1)Study on the protective effect of LfcinB on intestinal mucosal barrier.The effects of LfcinB on the mechanical,immunological,chemical and microbial barriers of intestinal mucosa were studied using animal models.The experimental group consisted of control group C,DSS group(5% DSS),LfcinB group(5 mg/kg LfcinB),and LBD group(DSS+LfcinB).The results showed that LfcinB significantly reduced the disease activity index(DAI)and diamine oxidase(DAO)content(p<0.05),and reduced the intestinal barrier mechanical barrier damage in rats with enteritis.Intestinal tissue section results showed that LfcinB could improve the destruction of intestinal tight junction,loss of microvilli,extensive ulcer,edema,intestinal wall recess injury,inflammatory cell infiltration and other inflammatory phenomena,and protect the integrity of intestinal tissue morphology in rats with enteritis.Moreover,LfcinB alleviated the decrease of mucus secretion and inflammatory infiltration in rats with enteritis.The sequence analysis of colon microbial 16 S rRNA gene revealed that LfcinB improved the microbial species richness and diversity of enteritis rats significantly(p<0.05),making the intestinal flora community structure more similar to that of normal rats.And increase the abundance of the beneficial bacteria of short-chain fatty acids(SCFA)such as Rumenococcus,by 27%.The above results indicate that LfcinB can alleviate intestinal mucosal barrier damage and maintain intestinal health by improving intestinal tissue morphology,mucus secretion,macrophage infiltration,and regulating microflora distribution.(2)Explore the possible mechanism of LfcinB protection of intestinal mucosal barrier.analysis of expression levels of tight junction protein,goblet cell secreted mucin(Muc2)and intestinal trefoil factor(TFF3)in rat colon tissue.LfcinB Significantly relieved abnormal expression of ZO-1,Occludin,Muc2,TFF3 caused by intestinal inflammation,which increased the mRNA expression of Muc2 and TFF3 by 5.6-fold and 4.6-fold,respectively.And reduce the content of IgA and myeloperoxidase(MPO)in colon tissue of colitis rats(p<0.05).Through the regulation of NF-?B/NLRP3 signaling pathway,the expression of pro-inflammatory factor IL-6,IL-1? and TNF-? mRNA in colon tissue of rats with enteritis was reduced by 1.9-fold,1.6-fold,1.3-fold(p<0.05),thereby inhibiting the occurrence and development of inflammation and realizing the protection of intestinal mucosal barrier function.(3)Preliminary study on the damage and repair effect on intestinal cells through in vitro simulated digestion of LfcinB and its digestion products(short peptides).The results of in vitro simulated gastric digestion revealed that LfcinB mainly produced MT10(met-lys-lys-leu-glyala-pro-ser-ile-thr)short peptides after gastric digestion.The effects of MT10 on the proliferation and differentiation of inflammatory injury induced by TNF-? were further studied using an intestinal organoid model.The experimental set up control group C,TNF-?(200 ng/mL),MT10 Group(1 ?g/mL),MT10 + TNF-? group.The analysis of the proliferation and differentiation of organoid cells indicated that MT10 can prevent the abnormal proliferation of organoid and improve the imbalance of organoid differentiation(p<0.01).Analysis of the expression and distribution of tight junction protein ZO-1 indicated that MT10 can attenuate the destruction of the structure of tight junction proteins by TNF-?.The above results revealed that the main product short peptide MT10 produced by LfcinB after gastric digestion can prevent TNF-? damage to organoid cells and maintain stable growth of organoid cells.In conclusion,LfcinB can significantly alleviate the damage of DSS on colon tissue morphology,intestinal permeability,mucus secretion,inflammatory infiltration state,intestinal flora distribution in rats with enteritis,and its mechanism may be through improve the expression of tight junction proteins and mucins to improve the physical and chemical barriers of the intestinal mucosa,And regulate the expression and secretion of inflammatory factors through NF-?B/NLRP3 signaling pathway to inhibit intestinal inflammation,alleviate intestinal inflammatory infiltration,and increased the abundance of beneficial bacteria to protect the intestinal mucosal barrier.The results of in vitro simulated digestion and organoid model study revealed that the protective effect of LfcinB on intestinal mucosal barrier may be closely related to the improvement of the abnormal proliferation and differentiation of organoid and the improvement of tight junction protein expression and distribution by gastric digestive product MT10.