Evaluation Of Antioxidant And Anti-inflammatory Functions Of Rice Active Peptide F2d And Its Molecular Mechanism

The utilization rate of rice residue as a by-product of rice processing has been low,neglecting its high quality protein.And high protein content is necessary for the development of active peptides,therefore,the extraction of antioxidant peptides from rice residue protein is one of the best ways to improve its added value and research significance.Our group has extracted several active peptides from rice pomace protein and measured their in vitro antioxidant activity,and selected a small molecule peptide(F2d)with the highest antioxidant activity for further study.In the present study,we established an oxidative stress model by stimulating human umbilical vein endothelial cells(HUVEC)with H2O2 and high glucose,and used the small molecule active peptide F2d extracted from rice pomace as the experimental material to evaluate the antioxidant activity and anti-inflammatory activity of F2d and explore the molecular mechanism of its action,which provides a theoretical basis for future research on the deep processing of rice by-products and the development and application of small molecule active peptides..Firstly,antioxidant experiments such as free radical scavenging were conducted on rice active peptide F2d,and its antioxidant activity was initially determined.With the increase of F2d content,its ability to eliminate free radicals was improved.Through the detection of intracellular oxidative stress indicators,it was found that the active peptide F2d could significantly reduce the content of intracellular oxygen radical ROS and lipid peroxides MDA,and increase the content of antioxidant metalloenzymes SOD.The ability of F2d to reduce the rate of apoptosis was explored by flow-through apoptosis assay.We used Western blot assay to detect the protein level of key target genes of Nrf2 signaling pathway,and found that F2d could promote the expression of antioxidant core gene Nrf2 as well as downstream genes,leaving Nrf2 in vivo repressor Keap1 at low expression level;further study found that F2d could also promote the phosphorylation degree of Nrf2,making its stability It was further found that F2d could also promote the phosphorylation of Nrf2,increase its stability and content,activate its inherent transcriptional activity,and exert antioxidant effects.And the RT-qPCR results showed that the effect of active peptide F2d on mRNA expression level was consistent with the protein level expression results.To confirm that F2d does exert its antioxidant effect through Nrf2 signaling pathway,the expression of Nrf2 was blocked by ML385,a specific inhibitor of Nrf2,and Nrf2 was confirmed to be blocked by Western blot method at protein level.The antioxidant activity of F2d is through the Nrf2 signaling pathway.To further investigate whether the active peptide F2d has corresponding efficacy on high glucose-induced oxidative stress and inflammation models.We used MTS cytotoxicity assay to determine the modeling concentration of high glucose,and the results showed that the modeling concentration of high glucose was 40 mM;through the detection of intracellular oxidative stress indexes,we found that the active peptide could significantly reduce the content of intracellular ROS and MDA,and upregulate the level of SOD.Flow cytometry detection of apoptosis showed that high glucose could lead to a decrease in cell survival,and the addition of active peptide F2d showed a concentration-dependent increase in the number of viable cells,demonstrating that F2d could significantly improve the apoptotic state.Western blot and RT-qPCR illustrated from protein and mRNA levels,respectively,that with successful modeling of high glucose,active peptide F2d could effectively improve Nrf2 antioxidant response pathway expression,enhanced cellular tolerance and antioxidant capacity,inhibited the expression of inflammatory response factors and attenuated the inflammatory response,while also regulating apoptosis-related factors and reducing apoptosis caused by high glucose.Experimental conclusion:(1)Active peptide F2d can attenuate the oxidative stress produced by H2O2-induced HUVEC cells.(2)Active peptide F2d can attenuate oxidative stress and inflammatory damage produced by high glucose induced HUVEC cells(3)Active peptide F2d can reduce apoptosis caused by oxidative stress and inflammation.(4)Active peptide F2d exerts antioxidant and anti-inflammatory effects that are mediated through the Keap1-Nrf2 signaling pathway and inhibits mitochondrial apoptosis and activation of the downstream Caspase pathway to reduce the expression of inflammatory factors.These findings further contribute to the development of biological functional studies of rice by-products after deep processing and deepen the understanding of the theoretical basis required for the mechanism of action of small molecule active peptides.These studies are necessary to further investigate the molecular mechanisms of antioxidant and anti-inflammation for the production of functional products with rice active peptides.