| As an important line of defense to maintain the homeostasis of the intestinal environment,the normal function of the intestinal mucosal barrier is a prerequisite for ensuring the health of the body.The proliferation and differentiation of small intestinal stem cells(ISC)is the cellular basis for the normal function of the intestinal mucosal barrier and plays an important role in maintaining intestinal environmental homeostasis.Autophagy,as a widespread pattern of programmed cell death during small intestinal stem cell renewal,is closely related to the function of the intestinal mucosal barrier.In this study,the effects of nine branched-chain amino acid(BCAA)dipeptide on the proliferation and differentiation of small ISC were analyzed.The intestinal epithelial barrier protection function of the target BCAA dipeptide,leucine dipeptide(Leu-Leu,LL),was studied by using the porcine small intestinal organoid epithelial injury model.Furthermore,the possible mechanism of LL protecting intestinal mucosal barrier function from the perspective of promoting proliferation and differentiation of small ISC and regulating autophagy function was further explored by using the rat model of intestinal barrier injury.The results of the analysis of the biological activity of BCAA dipeptide showed that LL at the study concentration could not only promote the proliferation of IPEC-J2,but also had a lower LDH-releasing ability than other test dipeptides,and low toxicity to the test cells.The results of the effect on the proliferation and differentiation of small ISC showed that 2 mM LL,LI(Leu-Ile),LV(Leu-Vla),II(Ile-Ile),VL(Val-Leu)had no adverse effects on the morphology and proliferation of porcine small intestinal organoids.Among them,LL can promote the mRNA expression level of small ISC,secretory cells and absorptive cell signature genes.LL may have the potential to improve intestinal mucosal barrier function by regulating small ISC proliferation and differentiation.The effect of LL on the epithelial barrier function of small intestinal organoids in small intestines was studied by TNF-α-induced porcine organoid epithelial injury model as an in vitro model.The results of TNF-α concentration screening showed that 100 ng/m L TNF-αsignificantly inhibited the proliferation of porcine small intestine organoids,increased the expression of IL-8 and IL-6 mRNA,and significantly increased the expression level of zo-2 mRNA in tight junction(TJ)protein(p < 0.05)and the expression of claudin-2 mRNA significantly decreased(p < 0.05).The results of LL pretreatment on the epithelial barrier of small intestinal organoids showed that the transepithelial electrical resistance(TEER)(p < 0.05)of 2D cultures of porcine small intestinal organoids was significantly reduced in the TNF-αtreatment group compared with the normal group,while the TEER of LL pretreatment group was significantly increased(p < 0.05),and the epithelial structure of porcine small intestine organoids was more compact and complete.LL pretreatment also restored the mRNA expression of zo-2 and claudin-2 to the normal normal group.The results of the analysis of autophagy in porcine small intestinal organoid 2D cultures showed that TNF-α reduced the occurrence of autophagy in porcine small intestinal organoid 2D cultures.LL pretreatment increased the expression level of the autophagy-related gene LC3B mRNA and improved the number of autophagosomes.Autophagy activators significantly increased the expression levels of ERK and LC3B mRNA(p < 0.05);LL pretreatment also significantly improved the decrease in ERK mRNA expression levels caused by TNF-α(p < 0.05),which had the same effect trend as autophagy activators;LL may improve TNF-α-induced reduction of autophagy levels in small intestines through ERK signaling pathway.Autophagy activators also significantly upregulated the expression levels of Chg A mRNA,a signature gene of secretory cells,while there was no significant change in the expression levels of Lgr5,a signature gene of stem cells,and the expression of Villin-1,a signature gene of absorptive cells.The above results showed that LL regulated the autophagy function of secretory cells through the ERK signaling pathway and improved the epithelial barrier function.The results of the study on the mechanism of LL improving intestinal mucosal barrier damage in rats with dextran sodium sulfate(DSS)-induced enteritis as an animal model showed that gavage of 500 mg/(kg bw)of LL could improve the growth rate and disease activity index of rats caused by DSS,and reduce serum diamine oxidase levels.Further analysis found that LL improved DSS-induced structural damage to the colonic mucosa and jejunal villi in rats,increased the number of autophagosomes,and restored the expression level of TJ protein zo-2.The analysis of the correlation between jejunal histiocyte lineage and autophagy in rats with enteritis showed that gavage 500 mg/(kg bw)LL significantly improved the DSS-induced increase in the expression level of mRNA of stem cell and absorptive cell signature gene(p <0.05),while the expression level of secretory cell signature gene mRNA did not change significantly.Gastric gavage 500 mg/(kg bw)LL also enhanced the migration and differentiation ability of stem cells and the co-expression of secretory cell signature genes and autophagy genes,and promoted the differentiation of stem cells into secretory cells and autophagy to participate in the regulation of the intestinal mucosal barrier.The results of analysis of the expression levels of key proteins of the relevant signaling pathways showed that gavage 500 mg/(kg bw)LL improved the increase of the protein expression of p-ERK,autophagy gene LC3Ⅱ / LC3Ⅰ,myosin p-ezrin expression and the decrease of PKCα protein expression caused by DSS.The above results showed that LL promoted the transformation of stem cells into secretory cells and changed the autophagy ability of secretory cells through the ERK/PKCα signaling pathway to achieve the repair effect of the intestinal mucosal barrier.In summary,the results of in vitro experiments using small intestinal organoids as models and in vivo studies using rats as animal models were revealed that LL ameliorated the proliferation and differentiation process of small intestinal stem cells caused by intestinal mucosal barrier damage,and affected the expression level of intestinal inflammatory factors and tight junction proteins by promoting the differentiation of small intestinal stem cells into secretory cells and regulating the autophagy process through ERK/PKCα signaling pathway,which alleviated the damage of TNF-α or DSS to the intestinal mucosal barrier so that it achieves a protective effect on the intestinal mucosal barrier. |
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