Effects Of Dietary Proteins Isolated From Different Meat Products On Colonic Homeostasis Of Mice

Protein has been confirmed to change to varying degrees during the meat processing.These changes not only affect gut digestion and absorption,but also affect other physiological functions.Our previous researches have shown that there were certain differences in in vitro or in vivo digestibility of proteins from different meat products.In addition,gut microbiota and gut-brain axis signal molecules were also influenced.However,little researches and reports have been done on the effects of these changes on gut homeostasis and immune system.especially in immunocompromised organisms.Therefore,this thesis will systematically focus on the influence of continuous intake of meat products'proteins on the normal and adaptive immue-deficient mice colon homeostasis for the first time.We aim to explore the interactions between diet,host,and gut microbiota.And the mechanism of the impact of processed meat proteins on colon health would be revealed,which would provide a new understanding for meat intake,immunity,and intestinal health.At the same time,a certain theoretical basis for correctly guiding normal or immunocompromised people to choose protein sources and cooking methods would be provided.The main research contents and results are as follows:1.Effects of meat product protein diets on physiological or biochemical responses and intestinal homeostasis in mice with different immune status.Rag1^-/-mice and wild-type mice were assigned to one of diets containing 20%casein,cooked pork protein or dry-cured pork protein respectively for three months.Growth indicators(body weight,food intake and fat deposition rate),serum biochemical indicators(LPS,SOD),and histopathological indicaters(mucosal thickness,goblet cell numbers,mRNA expression of Muc2 and inflammatory factor-related genes,and etc.).The results showed that the intake of cooked pork protein and dry-cured pork protein increased the daily dietary intake,weight and fat deposition rate.And the serum LPS increased,while the serum SOD content decreased.In addition,the mRNA expression of inflammation-related genes in the colon tissue increased.However,there were no significant pathological changes judging from the HE staining and AB-PAS staining.What's more,the number of goblet cells and Muc2 mRNA expression had no significant difference among diet groups.In summary,pork product protein diets has more growth-promoting effect than casein diet,but they would induce an increase in low-level inflammation,increased intestinal permeability,and reduced antioxidant capacity in the body.However,until now,no obvious pathological changes can be observed in the colon under the normal dietary intake.2.Effects of meat product protein diets on gut microbiota in mice with different immune statusIn order to investigate the effect of continuous intake of meat product protein diets on gut microbiota in mice with different immune status,the colonic DNA of the mice in each diet group was extracted and 16s rRNA genes in the V4 variable region was amplified by PCR.Then library construction,sequencing,and subsequent bioinformatics analysis were conducted,followed by analysis of gut microbiota composition.The results showed that continuous intake of different meat product protein diets had significant effects on gut microbiota,especially in Rag1^-/-mice.?? or ? diversity:Intake of meat product protein diets(CPP and DPP)can increase the community richness of two genotype mice(P<0.05).And the community diversity of Rag1^-/-mice(P<0.05)was higher than that of wild-type mice(P>0.05).Immune deficiency seemed to magnify the difference in community composition and structure among diet groups.?Community composition:At the phylum level,the relative abundance(RA)of Firmicutes was significantly different in wild type mice(P<0.05),it's significantly higher in WDPP group than in WC and WCPP groups.In Rag1^-/-mice,intake of meat product protein diets reduced the abundance of Actinobacteria.but increased the abundance of Verrucomicrobia.At the genus level,the RA of Clostridium sensu stricto 1 and Blautia decreased after feeding on dietary meat protein(P<0.05).In wild-type mice,meat protein intake increased the RA of Ruminococcaceae UCG-014.In Rag1^-/-mice,meat protein intake also reduced the RA of Rikenellaceae RC9 gut group(P<0.01),Alistipes(P<0.001),and Bifidobacterium(P<0.05)while the RA of Lachnospiraceae NK4A136 group increased.The characteristic bacteria of RCPP group were Ruminococcaceae uncultured and Ruminiclostridium.The characteristic bacteria of RDPP group were Muribacaceae norank,Dubosiella and Akkermansia.At the OTU level,a total of 96 OTUs were significantly different among diet groups in wild-type mice,while there were 110 OTUs in Rag1^-/-mice.Intake of processed protein diets reduced the RA of Clostridium sensu stricto 1(OTU5),Alistipes(OTU7)and Blautia(OTU19,OTU110,OTU 112)related bacteria strains,while the RA of Ruminococcaceae related bacteria strains increased.In the case of acquired immune deficiency,intake of dry-cured pork protein diet also significantly increased the RA of Akkermansia(OTU16).In summary,the continuous intake of processed meat protein diets will reduce the RA of beneficial bacteria in the intestine and increase the RA of Ruminococcaceae-related strains.In addition,gut microbes are more sensitive to diets,especially under immunodeficient conditions.And the abundance of Akkermansia will increase after intake of pork protein diets for 3 months.Therefore,the source of dietary protein and the immune system play a vital role in maintaining the gut microbiota homeostasis.3.Study on the mechanism of meat product protein on the interaction of colon transcriptome and microbiota in mice with different immune statusIn order to further explore how the host responded to intestinal environment changes(dietary digestion products,gut microbiota,and etc.)caused by dietary intake of different proteins under different immune status.Second-generation transcriptome sequencing was performed to analyze the gene expression of the colon tissue.The results showed that the number of differentially expressed genes(DEGs)among the dietary groups in acquired immunodeficiency mice was much higher than that of wild-type mice.GO and KEGG enrichment analysis showed that intake of meat product protein diets affected functions of the transition metal ion homeostasis,signal transduction,and metabolism related biological processes.What's more,pathways about mineral absorption and lipid metabolism are significantly regulated.In addition,continuous intake of meat protein also significantly affected the pathways related to immunity,gaps and adhesion junction in Rag1^-/-mice.Gene function module analysis was then conducted based on functional enrichment analysis and related research reports.It can be seen that most of the DEGs are mainly involved in lipid metabolism,oxidative stress,mucin glycosylation,inflammation,antigen processing and presentation,cells circulation and proliferation,and apoptosis-related functions,and most of them were significantly differentially expressed in the RDPP group of Ragl-/-mice.Key genes such as Homx1,Ppara,and Pparg were higher expressed in the meat product protein groups.Interaction analysis of gut microbiota and transcriptome showed that microbial species in Clostridiales and Bacteroidales played an vital role in regulating genes involved in lipid metabolism,oxidative stress,inflammation,mucin glycosylation,and cell proliferation.And different bacterial strains from the same family may have different functions.In summary,intake of meat product proteins had greater effects on acquired immune deficient mice than that on the normal mice.It induced colonic oxidative stress,low-level inflammation,activated innate immune function,and promoted cell circulation-related and migration-related genes expression in colon.And these changes seemed to be related to a decrease in the RA of Blautia and Alistipes and an increase in the RA of Ruminococcaceae.