Mechanism Of Effect Of Millet Trypsin Inhibitor On Promoting Lactobacillus Reuteri Intestinal Colonization In Alleviating Obesity

Obesity poses a serious threat to human health.Studies have shown that Lactobacillus,as an intestinal probiotic,can effectively alleviate obesity.The colonization of Lactobacillus in the intestine is the basis for its beneficial effects.Due to the impaired intestinal barrier in obese people,the colonization ability of Lactobacillus is inferior.Studies have shown that plant active peptides can be a vital source of anti-obesity functional foods and candidate drugs.Therefore,separating active peptide that promote Lactobacillus colonization on the intestinal is very important for the development of prebiotic products that alleviate obesity.In our previous study,a novel Bowman-Birk type major trypsin inhibitor from foxtail millet bran(named FMB-BBTI)was obtained by in vitro gastrointestinal bionic digestion.We found that FMB-BBTI significantly increased the abundance of Lactobacillus in the intestines of atherosclerotic mice,but the type of strain and the mechanism of how FMB-BBTI to promote strain in intestinal colonization remain unclear.In this study,we isolated the strain by a Lactobacillus specific medium and investigated the promotional effect of FMB-BBTI on intestinal colonization of this strain.Meanwhile,the alleviating effect of FMB-BBTI on obesity was evaluated by high-fat diet(HFD)-induced obesity(DIO)model in mice.In addition,a model of intestinal epithelial inflammation cell induced by lipopolysaccharide(LPS)was used to explore the molecular nutritional mechanism on which FMB-BBTI promotes strain intestinal colonization.The research results were as follows:(1)The strain identification of FMB-BBTI promoting Lactobacillus intestinal colonization.In this study,a novel strain of Lactobacillus reuteri was isolated from the cecal contents of FMB-BBTI treated mice and named L.reuteri SY523.The result of bacterial attachment assay in cell level showed that FMB-BBTI significantly promoted the L.reuteri SY523 adhesion to intestinal epithelial cells in a dose-dependent manner.The results of fluorescence in situ hybridization of colon and qPCR experiments on cecal contents in mice showed that FMB-BBTI could promote L.reuteri SY523 colonization of the intestinal mucosa.The above results suggest that FMB-BBTI has the potential to promote L.reuteri SY523 on intestinal colonization.(2)The mitigatory effect of FMB-BBTI on obesity by promoting L.reuteri SY523 colonization in DIO mice.The findings suggested that the obesity related indexes,such as the body weight,six items of lipid profile,and levels of inflammatory factors in DIO mice,were significantly decreased after co-treatment of FMB-BBTI and L.reuteri SY523.In addition,co-treatment of FMB-BBTI and L.reuteri SY523 effectively alleviated the hepatic steatosis,colonic tissue inflammation,and gut barrier injury in DIO mice.Interestingly,studies of cellular inflammation models found that treatment with FMB-BBTI reduced the levels of inflammatory factors,but without significant differences.These data illustrated that L.reuteri SY523 played a key role in the effect of FMB-BBTI alleviating obesity in DIO mice.(3)Study on the mechanism of FMB-BBTI promoting L.reuteri SY523 colonization.The adhesion factor for L.reuteri SY523 was identified as mucus adhesion promoting protein(MapA)by Biotin pull-down coupled with MOLDI-TOF-TOF mass spectrometry.The specific receptor of L.reuteri SY523 on intestinal epithelial cells is heat shock protein-60(Hsp60).Further findings revealed that FMB-BBTI inhibited the dephosphorylation of Hsp60 by interacting with serine/threonine-protein phosphatase(PPP1CA)inside the cell and induced Hsp60 translocation from the cytoplasm to the cell membrane,which promoted colonization of L.reuteri SY523.In conclusion,in present study,a novel strain of Lactobacillus reuteri was isolated from mice cecal contents,named L.reuteri SY523,and we found that FMB-BBTI could promote L.reuteri SY523 intestinal colonization to alleviate obesity of DIO mice.Meanwhile,it was elucidated that molecular mechanisms of L.reuteri SY523 colonization that FMB-BBTI induced membrane translocation of Hsp60 by interacting with intracellular PPP1 CA.This study provides a theoretical basis for further development of FMB-BBTI into anti-obesity prebiotic product in the future.