Effects Of Oxidized Rice Bran Protein On Gut Microbiota And Inflammation And Underlying Mechanism

China is extremely rich in rice bran resources whose annual output reaches more than 14 million tons with rice processing.In the defatted rice bran after oil extraction,the protein content of rice bran can reach 15%-20%.As a natural protein resource with low allergenicity and high nutrition,rice bran protein has not been effectively used.The main reason is that there are extremely active lipolytic enzymes in rice bran,which can quickly catalyze the lipid hydrolysis of rice bran to form free fatty acids from the beginning of rice milling.These free fatty acids are extremely unstable and are easily oxidized under the action of endogenous lipoxygenase to generate lipid free radicals and active lipid oxidation products,which in turn induce the oxidation of rice bran protein,and finally lead to the changes of structure and function properties of rice bran protein,as well as the digestive properties.However,the effect of oxidized rice bran protein on the body's organs like intestines is still unclear.In this paper,the effects of oxidized rice bran protein on intestinal redox state,barrier function,inflammation,gut microbiota and their metabolites in mice were studied by administration with different degrees of oxidative rice bran for 12 weeks,and seek for its underlying inflammatory mechanism.According to the rice bran stored for 0,3,and 10 days,the rice bran protein with low,medium and high degree of oxidation was prepared,and the mice were divided into 9 groups as experimental group according to the three different doses of low,medium and high doses and different oxidation degrees of oxidative rice bran protein.Mice given the same amount of 0.9%saline intragastrically served as the control group.By calculating the weekly weight gain and feed intake of mice to study the effect of oxidized rice bran protein on the basic growth performance of mice.Indicators like ROS,MDA,carbonyl,AOPP,GSH,GSSG,SOD,T-AOC in ileum and colon tissues were tested to study the effect of oxidized rice bran protein on the intestinal oxidative damage and redox state of mice.The results of the study found that the average feed intake and final body weight of the mice fed with oxidized rice bran protein were significantly lower than those of the control group.With the increase of the time of administration,the content of ROS,MDA,AOPPs and carbonyl in the ileum and colon of mice with high oxidation degree of oxidized rice bran protein increased,GSH/GSSG ratio,GSH and SOD content decreased.The results showed that oxidized rice bran protein can reduce the weight of mice,and also cause the accumulation of oxidation products in the intestines of mice,causing oxidative damage,resulting in an imbalance in the redox state,and the high degree of oxidation of rice bran protein can significantly affect the intestinal redox of mice.Mice ileum and colon slices were observed to study the effect of oxidized rice bran protein on the histological morphology of ileum and colon of mice.Pro-inflammatory factors IL-1? IL-6,TNF-? and anti-inflammatory factor IL-10 were detected to study the effect of oxidized rice bran protein on inflammatory factors in mic serum,ileum and colon.the relative expression level of mRNA of colonic tight junction proteins occludin,claudin and ZO-1 were detected by RT-PCR,to study the effect of rice bran protein with different oxidation degrees on the intestinal barrier function of mice.The results of the study found that after twelve weeks of administration oxidized rice bran protein,oxidized rice bran protein with middle and high degree oxidation destroyed the integrity of mice ileum and colon tissues.With the increase of dose and oxidation degree of oxidized rice bran protein,the levels of IL-6 and TNF-? in mice ileum,and colon rised.The inflammatory response of mice with high oxidation degree of oxidized rice bran protein was more obvious.Oxidized rice bran protein increased the relative expression level of occludin,claudin and ZO-1 in colon of mice.But as the degree of oxidation increased,its expression level decreased.The results show that oxidized rice bran protein would damage the structure of intestinal tissues.Rice bran protein with high degree oxidation would increase the inflammatory factors in the intestine and cause inflammation.In addition,rice bran protein might have protecting effect on the intestinal barrier,but with degree of oxidation increases,the protection function would decrease.The composition of gut microbiota was measured by high-throughput sequencing to study the effect of oxidized rice bran protein on gut microbiota.To explore the possible signaling pathways and potential mechanisms of oxidized rice bran protein leading to intestinal inflammation,the levels of LPS and LBP in the colon content and the expression of related genes of CD14,TLR-4,MyD88 and NF-?B were studied.To study the effect of oxidized rice bran protein on intestinal microbial metabolite by using GC-MS to quantitatively detect short-chain fatty acids in the intestines.This study found that oxidized rice bran protein changed the diversity of gut microbiota in mice colon.Rice bran protein with medium and high oxidation degree increased the relative abundance of Bacteroides,Akkermansia,Lactobacillus,Roseburia and other bacteria,while high oxidation degree rice bran protein reduced the abundance of these bacteria and increased the abundance of gram negative and potentially pathogenic bacteria.After administrated with high oxidized rice bran protein,LBP levels increased,and the expression levels of TLR4 and NF-?B increased.The results showed that high oxidation degree rice bran protein changed the structure of gut microbiota,increased the abundance of Gram-negative and potentially pathogenic phenotypes,which poses a potential threat to intestinal health.In addition,the high oxidation degree rice bran protein may increase the binding of LPS produced by microorganisms to LBP through the TLR4-MyD88-NF-?B signaling pathway,which activates NF-?B and ultimately leads to inflammation.The content of formate,acetate,propionate and butyrate in the colon content did not change significantly with the intake of oxidized rice bran protein,indicating that the influence of oxidized rice bran protein on the intestinal environment is not reflectesd by changing the content of SCFA.