Study On The MAO-A Inhibitory Activity Of Hairtail Peptides And Their Antidepressant Mechanism

Depression is a chronic central nervous system disease,which is the biggest cause of non-fatal health loss.However,depression is a heterogeneous disorder with a highly variable course,an inconsistent response to treatment and currently available agents have low efficiency and obvious side effects.These deficiencies highlight a significant unmet need for the new rapid-acting and safe antidepressants to treat depression.In this study,hairtail（Trichiurus japonicas）was used as raw material,after simulated gastrointestinal digestion in vitro,monoamine oxidase A（MAO-A）inhibitory peptides were identified directly from ultrafiltration components,and then in silico technique was used to assist screening.Moreover,the interaction mechanism between hairtail peptides and MAO-A were explored.Furthermore,model of DEX induced-stress in SH-SY5Y cells were established to evaluate the mechanism of peptide promoting neural plasticity and antidepressant,so as to provide scientific basis for the development of hairtail functional food ingredients with MAO-A inhibitory activity and antidepressant effect.Hairtail protein hydrolysates（PDH and GIH） were prepared by pepsin digestion and simulated gastrointestinal digestion（pepsin-pancreatin）in vitro,and their MAO-A inhibitory activity,molecular weight distribution,protein recovery rate,degree of hydrolysis and total amino acid content were analyzed.Results showed that the MAO-A inhibitory activities of PDH and GIH were significantly higher than that of hairtail crude protein,and the protein recovery rate of PDH and GIH were more than 60%.With the progress of intestinal digestion,the degree of hydrolysis,the proportion of components<4k Da and the content of aliphatic amino acids of GIH increased gradually,but the inhibition rate of MAO-A decreased by 8.5%,which suggested that the inhibitory activity of MAO-A may be negatively correlated with the content of aliphatic amino acids.Ultrafiltration purification of PDH and GIH showed that components of PDH-III and GIH-III had the highest MAO-A inhibitory activity with IC₅₀ values of 0.61 and 2.54 mg/m L,respectively.Effects of PDH-III and GIH-III on the activity of MAO-A in SH-SY5Y cells were analyzed,both of them could significantly reduce the activity of MAO-A in SH-SY5Y cells.Finally,33 and 31 peptide sequences were identified from PDH-III and GIH-III by LC-MS/MS,respectively.HPEPDOCK was used to screen the peptide sequences with high scores for synthesis,and Pearson’s correlation analysis showed that there was a significant correlation between docking scores and MAO-A inhibitory activity（r=0.6709,p<0.05）.Four peptide sequences（VFEVFW,LPNSLYQQ,LPNSLYQK and FADAME）with high MAO-A inhibitory activity were selected to further study their interaction mechanism with MAO-A.Autodock Vina showed that the peptides VFEVFW and LPNSLYQQ could enter the active sites of MAO-A through hydrogen bonding and hydrophobic interaction,and interact with the active sites Val93,Val210 and Leu97of MAO-A.Circular dichroism indicated that the ratio ofα-helix in MAO-A decreased by about one time and the ratio of antiparallelβ-fold increased by three times after the addition of VFEVFW（2 m M）.Compared with the other three peptides,the ratio ofα-helix,β-fold andβ-turn of MAO-A changed more significantly after incubation with VFEVFW.Besides,enzymatic kinetics exhibited that VFEVFW and FADAME were mixed inhibitors,LPNSLYQQ and LPNSLYQK were competitive and noncompetitive inhibitors,respectively.Effects of different hairtail synthetic peptides（VFEVFW,LPNSLYQQ,LPNSLYQK and FADAME）and positive control drugs on the proliferation of SH-SY5Y cells and the activity of MAO-A in SH-SY5Y cells were determined.The four peptides could significantly reduce the activity of MAO-A located on the mitochondrial membrane of SH-SY5Y cells.In order to further explore the neuroprotective effect and mechanism of hairtail peptides on SH-SY5Y cells,the stress/injury model of SH-SY5Y cells was established by using 400μM DEX with the viability of SH-SY5Y cells was 52.6±1.1%.When peptides incubated with DEX,results of MTT and Hoechst 33342 showed that peptides could significantly improve the viability of SH-SY5Y cells by 20-30%and alleviate the cell damage induced by DEX.Besides,JC-1fluorescence staining exhibited that peptides could significantly reduce the influence of DEX on the ratio of red/green fluorescence intensity and restore the mitochondrial membrane potential of SH-SY5Y cells induced by DEX.q RT-PCR results showed that DEX could induce SH-SY5Y cell stress by activating MAO-A signaling pathway and inhibiting the expression of BDNF m RNA,and low concentration（0.1 mm）of VFEVFW could effectively alleviate the effect of DEX on the expression of BDNF m RNA in SH-SY5Y cells.In addition,peptides VFEVFW（0.1 mm）and LPNSLYQQ could up-regulate the expression levels of BDNF/CREB/Bcl-2 m RNA in SH-SY5Y cells（compared with the model group）,indicating that VFEVFW and LPNSLYQQ have potential antidepressant effects.These results indicated that hairtail peptides have good MAO-A inhibitory activity,neuroprotective effect and antidepressant effect.Further research and development should be carried out so that hairtail peptides can be used as functional food ingredients or drugs to improve neural plasticity or produce antidepressant effect.