| In this study,the structure of feruloyl oligosaccharides from fermented wheat bran(FOs-FWB)was studied by ultraviolet spectrum,infrared spectrum,high-performance gel permeation chromatography and Gel permeation chromatography.Further,in vivo and in vitro methods were used to explore the antioxidant activity of FOs-FWB and to analyze its mechanism from Nrf2/Keap1 signaling pathway.The main research contents and results are as follows:Experiment 1: structural characterization and in vitro antioxidant activity of FOs-FWBIn this experiment,the structure of FOs-FWB purified by Amberlite XAD-2macroporous adsorbent resin and Sephadex LH-20 dextran gel was characterized by using ultraviolet spectrum,infrared spectrum,high-performance gel permeation chromatography and Gel permeation chromatography.The results showed that the weight average molecular weight(MW)of FOs-FWB was11.81 k Da,which was composed of mannose,ribose,rhamnose,glucuronic acid,galacturonic acid,glucose,galactose,xylose,arabinose and fucose,and the molar ratio was1.69:0.38:1.02:0.53:0.05:29.79:3.19:33.42:29.81:0.12.Experiment 2: Evaluation of antioxidant activity of FOs-FWB based on IPEC-J2 cells modelIn this experiment,the in vitro antioxidant activity of FOs-FWB was evaluated using IPEC-J2 cell model.The safe dose of FOs-FWB was determined by the effect of different concentrations(25,50,100,200,400 and 800 μg/m L)of FOs-FWB on the proliferation rate of IPEC-J2 cells,and the intracellular antioxidant-related indexes and gene expression of IPEC-J2 cells were detected.On this basis,LPS(10 μg/m L for 24 h)was used to establish the IPEC-J2 cell oxidative damage model was established using LPS(10 μg/m L for 24 h),and the effects of FOs-FWB on the proliferation,antioxidant-related indexes and chemical gene expression of IPEC-J2 cells under oxidative stress conditions were determined to explore the protective mechanism of FOs-FWB on IPEC-J2 cells along the Nrf2/Keap1 pathway.The results showed:(1)when FOs-FWB was applied for 24 h,the cell proliferation rates of 50,100,200 and 400 μg/m L FOs-FWB groups increased to 107.76 %,109.68 %,116.69 % and 105.94 %,respectively,while FOs-FWB significantly reduced intracellular ROS production and MDA levels(P<0.05),and increased intracellular SOD,CAT,GSH-Px activity and GSH content in IPEC-J2 cells(P<0.05),and upregulated the m RNA expression of GCLC,GCLM,NQO1,HO-1,and Nrf2(P<0.05).(2)The cell proliferation rate decreased to 75.63%(P<0.05)after 10 μg/m L LPS attack for 24 h,while the intracellular ROS production and the levels of MDA,PC,and 8-OHd G were significantly elevated(P<0.05),so an oxidative stress model of IPEC-J2 cells was established using LPS(10 μg/m L,24 h).(3)The proliferation rate of cells pre-protected by FOs-FWB was significantly restored to 103.35 %(P<0.05),and significantly inhibited the production of ROS and MDA in the oxidative stress model of IPEC-J2 cells induced by LPS(P<0.05),increased the levels of CAT,SOD,GSH-Px activity and GSH(P<0.05),upregulated the intracellular GCLC,GCLM,NQO1,HO-1,and Nrf2 m RNA expression(P<0.05).It indicates that the addition of FOs-FWB can reduce the LPS-induced ROS and MDA levels in IPEC-J2 cells by activating the Nrf2/Keap1 signaling pathway and attenuate the LPS-induced decrease in antioxidant enzyme activity,thus alleviating the oxidative damage in cells.Experiment 3: Evaluation of antioxidant activity of FOs-FWB based on zebrafish embryo modelIn this experiment,zebrafish embryo model was used to evaluate the in vivo antioxidant activity of FOs-FWB.The safe dose of FOs-FWB was determined by the effect of adding different concentrations(50,100,200,400 and 800 μg/m L)of FOs-FWB on the survival rate of zebrafish embryos;after incubation with 50,100,200 and 400 μg/m L FOs-FWB for 1 h,the antioxidant-related indexes of zebrafish embryos were measured;on this basis,an oxidative damage model was established using AAPH(15 mmol/m L for 24 h)to determine the effect of FOs-FWB on zebrafish embryos under oxidative stress conditions.The effects of FOs-FWB on the antioxidant-related indexes of zebrafish embryos under oxidative stress conditions were determined.The results showed that(1)The safe doses of FOs-FWB in zebrafish embryos ranged from 50 to 400 μg/m L;the simultaneous addition of 50 to 400 μg/m L FOs-FWB significantly reduced ROS production,cell death and lipid peroxidation levels in zebrafish embryos(P<0.05);and increased the activity of antioxidant enzymes SOD and GSH-Px(P<0.05).(2)Cell death,ROS production,and lipid peroxidation levels in zebrafish embryos induced by AAPH significantly increased to 121.88 %,128.26 %,and 131.75 %,respectively,compared with the control group(P<0.05),and the addition of 50～400 μg/m L FOs-FWB inhibited cell death and lipid peroxidation levels in zebrafish induced by AAPH(P<0.05).levels caused by AAPH(P<0.05),while 100～400 μg/m L FOs-FWB significantly inhibited ROS production in zebrafish embryos(P<0.05);meanwhile,50～400 μg/m L FOs-FWB significantly increased CAT,SOD and GSH-Px activities in zebrafish embryos compared with the AAPH group.It indicates that FOs-FWB can effectively inhibit cell death,ROS production and lipid peroxidation levels in zebrafish embryos caused by AAPH by increasing CAT,SOD and GSH-Px activities,thus alleviating oxidative damage in zebrafish embryos.In summary,this study provides a preliminary characterization of the structure of FOs-FWB and further explores the antioxidant effect and mechanism of FOs-FWB using a combination of in vivo and in vitro methods.That is,FOs-FWB is mainly composed of glucose(29.79 %),xylose(33.42 %)and arabinose(29.81 %),and can promote the expression and secretion of downstream detoxification/antioxidant enzymes by activating the Nrf2/Keap1 signaling pathway,thereby improving the antioxidant capacity of the body. |
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