Preparation And Hepatoprotective Properties In Vitro Of Bioactive Peptide From Three-Spot Seahorse

Three-spot seahorse?Hippocampus trimaculatus Leach,1814?,which belongs to the Syngnathidae family,is a small and rare marine bony fish that combines medicinal,edible and ornamental properties,and is widely distributed in the eastern Indian Ocean and the northwestern Pacific Ocean.Three-spot seahorse is rich in biologically active ingredients such as proteins and phospholipids that are beneficial to human health.Therefore,it possesses health-enhancing functions such as enhancing sexual function,nourishing kidney,antioxidant,and antithrombotic.At present,various types of liver diseases such as alcoholic liver disease are still a worldwide health problem.Animal and in vitro hepatocyte experiments have indicated the hepatoprotective effects of bioactive peptides?e.g.,bradykinin,marine collagen peptides,chicken peptides,and corn peptides?.In view of this,three-spot seahorse?10-13 cm?was used as the raw material,and a bioactive peptide was isolated from it by alkaline protease digestion,dialysis,and continuous chromatography.The liver-protecting properties of three-spot seahorse bioactive peptide in vitro were studied by human hepatic L-O2 cell ethanol injury model.The main conclusions are as follows:1.Isolation,purification,and characterization of bioactive peptide from three-spot seahorseAn alkaline protease digestion,dialysis,Sephadex G-25 chromatography,and RP-HPLC were used to obtain a bioactive from three-spot seahorse.Mass spectrometer identified the primary structure of three-spot seahorse bioactive peptide as Pro-Ala-Gly-Pro-Arg-Gly-Pro-Ala?PAGPRGPA,721.39 Da?.Its ACE inhibitory activity was measured in vitro with IC₅₀ value of 7.90?M.Circular dichroism analysis confirmed that the bioactive peptide was composed of 6.0%?-helix,31.5%?-sheet?26.6%anti-parallel,4.9%parallel?,31.4%?-turn,and 35.6%random coils.2.Hepatoprotective properties of three-spot seahorse bioactive peptide in vitro?1?In DPPH and ABTS assays,the IC₅₀ values of bioactive peptide were 367.63?g/m L and 467.38?g/m L,respectively.Compared to Trolox,the equivalent antioxidant capacity was 16 mg Trolox/g dw and 14 mg Trolox/g dw,respectively.?2?A model of alcoholic hepatocyte injury in vitro was established with human hepatic L-O2 cells.It was found that the survival rate of cells was significantly reduced after incubation with 200 m M ethanol for 24 h,and different concentrations of three-spot seahorse bioactive peptide had no toxic effect on cells.300?g/m L three-spot seahorse bioactive peptide and 50?M Trolox pre-cultured cells for 6 h can significantly restore the reduced cell viability caused by ethanol,and Trolox was better than bioactive peptide.Ethanol can induce hepatocellular toxicity,which was mainly manifested by significantly increased levels of ALT,AST,and LDH.Three-spot seahorse bioactive peptide inhibited the increase of these indicators and showed a concentration-dependent manner?P<0.05?.The effect of 50?M Trolox was more significant.SOD,GSH,MDA,and NO were used to detect the level of oxidative stress induced by ethanol.The results showed that different concentrations of three-spot seahorse bioactive peptide could significantly inhibit the oxidative stress induced by ethanol,and showed a concentration-dependent manner?P<0.05?.The effect of Trolox was more obvious.When ethanol was used,the levels of TNF-?and IL-6 in cells were significantly increased,while three-spot seahorse bioactive peptide and Trolox could significantly inhibit the release of inflammatory factors.A similar concentration-dependent manner was obtained by flow cytometry for apoptosis,ROS,and intracellular Ca²⁺level.These results showed that three-spot seahorse bioactive peptide could remove intracellular ROS,restore SOD and GSH levels,reduce oxidative stress damage to cells,and thereby protect cells from oxidative ethanol damage.?3?Western blot results demonstrated that three-spot seahorse bioactive peptide?300?g/m L?mainly activated the p38 MAPK signaling pathway and cooperated with PKC to activate the Keap1/Nrf2 signaling pathway to regulate the expression of the antioxidant enzyme HO-1,thereby reducing ethanol-induced oxidative damage to cells.?4?Based on the existing results,we used non-targeted metabolomics to study the possible metabolic pathways and biomarkers of three-spot seahorse bioactive peptide?300?g/m L?and ethanol?200 m M?from a molecular perspective.The results revealed that three-spot seahorse bioactive peptide?300?g/m L?could significantly promote the production of alanine and reduce the creatine content in cells.After incubation with ethanol?200 m M?,the contents of arginine,proline,and choline in the cells were significantly increased,and the contents of branched chain amino acids?leucine,isoleucine,and valine?,pantothenic acid,methionine,and creatine were significantly reduced.When the three-spot seahorse bioactive peptide and ethanol worked together,the change trend was similar.These amino acids may be biomarkers or therapeutic targets for ethanol-induced liver cell damage in vitro.