Lactobacillus Casei Fermented Blueberry Pomace Regulates The Intestinal Barrier Function Of High Fat Diet Mice And The Underlying Mechanisms

The intestinal barrier dysfunction caused by the continuous intake of high fat diet is the key factor in inducing diseases such as obesity.Functional phytochemical components may effectively alleviate this damage.Blueberry pomace is the by-product during juice processing and is rich in a variety of phytochemical components.Phytochemical components in blueberry pomace will be changed after the lactic acid bacteria fermentation and having the potential in improving the barrier function.But,the undering mechanisms are still unclear.Moreover,systematic researches aimed at improving the barrier function are insufficient,currently.Thus,this thesis aimed to use Lactobacillus casei to ferment blueberry pomace(Fermented blueberry pomace,FBP)as the research object.From the perspective of intestinal mechanical,chemical,microbiological and immune barriers,the influence and possible molecular mechanism of FBP on regulating the barrial functions of both the small and large intestines in high fat diet were further investigated.The main research contents and results were as follows:(1)FBP had the potential in modifying intestinal microbiota.In this part,the activity of FBP during the L.casei fermentation and the influence of FBP on fecal microbiota and short chain fatty acids(SCFA)in in vitro were revealed.The result indicated that the number of viable L.casei,the total polyphenols content(TPC)and the antioxidant capacity of FBP during L.casei(5%,v/v)fermentation(0-6 days)were all stayed at high level;FBP showed effects on inhibiting the proliferation of potential pathogenic bacteria,promoting the abundance of beneficial bacteria and increasing the SCFA concentration,while blueberry pomace fermented by L.casei for 42 h showed the best effect among all the FBP samples with different fermentation time.This chapter revealed that FBP had high antioxidant activity and showed improvement on the intestinal microbiota in the in vitro simulated intestinal system.(2)FBP improved the mechanical and chemical barrier function of the small intestine in high fat diet mice.In this part,the effect of FBP on oxidative stress level and inflammation as well as the regulation of FBP on barrier-associated functional proteins in the small intestine were studied.The result indicated that FBP increased the the activity of antioxidant enzymes while decreased the concentration of serum pro-inflammatory cytokines,and the activity of myeloperoxidase(MPO)in small intestine tissues;FBP also showed an improvement in the morphology and the density of goblet cells of the small intestine in high fat diet mice;FBP significantly promoted the expression of tight junctions,adhesion junctions and mucin(MUC)in the small intestine of high fat diet mice at m RNA and protein levels;further studies found that FBP inhibited the activation process of nuclear factor?B(NF-?B)signaling pathway and the myosin light chain kinase(MLCK)in the small intestine of high fat diet mice.This chapter revealed that FBP improved the mechanical and chemical barrier function in the small intestine of high fat diet mice by inhibiting the signal transduction of the NF-?B-MLCK pathway.(3)FBP improved the mechanical,chemical and microbial barrier function in the large intestine of high fat diet mice.In this part,the expression of functional proteins related to the mechanical and chemical barrier in the large intestine and the influences of FBP on modifying intestinal microbiota,SCFA and SCFA receptors were investigated.It revealed that FBP improved the large intestinal morphology of high fat diet mice;in terms of the mechanical and chemical barriers,FBP improved the expression of functional proteins related to the mechanical and chemical barrier function in the large intestine of high fat diet mice at m RNA level;in terms of the colonic microbial barrier,FBP increased the relative abundance of beneficial microbiota,elevelated the content of SCFA and up-regulatted the genic expression of SCFA receptors in colon of high fat diet mice;FBP effectively inhibited the activation of mitogen-activated protein kinase(MAPK),NF-?B signaling pathway and MLCK.This chapter revealed that FBP promoted the mechanical,chemical and microbial barrier function in the large intestine of high fat diet mice through the MAPK-NF-?B-MLCK signaling pathway.(4)FBP improved the immune barrier function in both small and large intestines of high fat diet mice.In this part,the amount of secreted immunoglobulin A(s Ig A)in the intestine was set as the evaluation target of the intestinal immune barrier function to explore the effect of FBP on immune barrier function in both small and large intestines of high fat diet mice.It revealed that FBP promoted the production of B cells(Ig A~+B)with the ability to secrete immunoglobulin A(Immunoglobulin A,Ig A),the homing process of plasmablasts(Ig A~+plasmablasts)with the ability to secrete Ig A,the generation of Ig A and the migration of s Ig A to promote the secretion of s Ig A in both small and large intestines of high fat diet mice;FBP improved the abundance of microbiota internalizated in Peyer's patches(PPs)in the small intestine as well as the relative abundance of microbiota and the SCFA concentration in cecum content;in addition,the positive correlation between the concentration of SCFA,the abundance of beneficial microbiota and the level of immunoglobulins related that the improvement of intestinal beneficial microbiota was closely associated with the immune barrier function.This chapter revealed that FBP improved the s Ig A production in both the small and large intestines by influencing intestinal microbiota.(5)The polyphenols in FBP(Fermented blueberry pomace phenolic extraction,FBPE)had the potential in regulating barrier function of cells from the small or large intestine.In this part,IPEC-J2 cells from the small intestine and Caco-2 cells from the large intestine were selected and expressions of tight junction proteins in IPEC-J2 cells and Caco-2 cells were calculated to predict the potential function of FBPE in regulating the barrier function of both small and large intestine cells.It revealed that FBPE increase the expression of tight junction proteins in IPEC-J2 cells and Caco-2 cells at m RNA and protein levels;the medium concentration(50-100?g/m L)FBPE showed better effects in regulating the expression of tight junction proteins than that of low concentration(25?g/m L)FBPE and high concentration(200?g/m L)FBPE.This chapter revealed that FBPE had the potential in regulating barrier function of cells from the small or large intestine.In conclusion,this thesis revealed that FBPwas potential in improving the microbiota and SCFA during the simulated intestinal system in in vitro;then,from the perspective of intestinal mechanical,chemical,microbiological and immune barriers,the influence and possible molecular mechanism of FBP on regulating the barrial functions of both the small and large intestines in high fat diet were further investigated;last,it was speculated that FBPE might be the important components for FBP to improve the intestinal barrier function of high fat diet mice,in addition,this speculation was verified by analyzing the expression of tight junction proteins in intestinal cells.