Protective Effect And Mechanism Of Oyster Active Peptides On Reproductive Function Injury In Male Mice

In recent years,the incidence of male infertility is increasing,and the fertility rate in all regions of the world is also decreasing year by year.How to effectively prevent and treat male reproductive dysfunction has become a serious problem that needs to be solved urgently.Oyster meat has high nutritional value,rich in protein,glycogen,amino acid,trace elements and other nutrients.Several studies have focused on the health benefits of oyster meat extract and have demonstrated their antioxidant,immunity-improving,anti-fatigue,anti-skin photoaging,antihypertensive,anti-hyperglycemic and liver-protecting properties.However,there are few reports on the protective effect of oyster meat extract on male reproductive injury,and the relevant mechanism is not clear.Therefore,in this study,oyster meat was used as raw material to prepare oyster enzymatic hydrolysis products,which were separated and purified,and the in vitro anti-reproductive injury activity of the separated components was evaluated synchronously.Meanwhile,the protective effects of the active components on reproductive function injury in mice were evaluated and the mechanism was preliminarily explored.After that,the separated and purified components were identified by mass spectrometry,and their structural characterization and potential activity were analyzed.Finally,peptides with high reliability were artificially synthesized,and the anti-reproductive injury activity of the synthetic peptide was verified by in vitro cell experiments and its possible mechanism of action was further explored.The main research results are as follows:（1）Four UF components(UF1（>10 ku）,UF2（5-10 ku）,UF3（3-5 ku）and UF4（<3 ku）were separated by ultrafiltration.The basic physical and chemical compositions were determined.The protective effect of UF on TM4 cells was evaluated by TP-induced TM4 testicular sertoli cell injury model.The cell survival rate,contents of reduced glutathione（GSH）and propylene glycol（MDA）and intracellular reactive oxygen species（ROS）levels were determined.The results showed that UF was rich in trace metal elements such as Copper（Cu）,zinc（Zn）,manganese（Mn）and selenium（Se）.By ultrafiltration,the hydrolysates of oyster with different molecular weights were effectively separated.The crude protein contents of UF2,UF3 and UF4 were 67.12%,65.06%and 64.25%,and the total amino acids were58.21%,54.11%and 54.75%,respectively,which were much higher than 39.04%and27.47%of UF1.TP-induced TM4 cell model was successfully established by cytotoxicity experiments.The toxicity and oxidative damage of TP-induced TM4 cells were dose-dependent.The survival rate of TM4 cells was only 64.29%when TP-induced TM4 cells were treated at 500 nmol·L^-1for 24 h.UF2,UF3 and UF4could significantly inhibit TP-induced TM4 cytotoxic injury,and the effect was better than UF1.UF4 can effectively resist oxidative damage of TM4 cells caused by TP,improve cell survival rate and intracellular GSH content,reduce intracellular ROS production and MDA content,and its activity is superior to UF2 and UF3.（2）The reproductive injury model of male mice was successfully established by intraperitoneal injection of TP.Sperm parameter analysis,testicular hematoxylin-eosin（HE）staining,testicular histological evaluation,testicular tissue enzyme activity determination,serum reproductive hormone level determination,Td T-mediated d UTP nick-end labeling（TUNEL）staining and Western Blot were used,to evaluate the protective effect of UF4 on TP-induced reproductive injury in male mice and its possible mechanism.The results showed that UF4 significantly improved TP-induced sperm quality deterioration,increased sperm count and motility,and reduced the sperm deformity rate.UF4 alleviated the atrophy and morphological abnormalities of testicular tissue and significantly reduced the vacuolation of testicular tissue.UF4 decreased the content of MDA,increased the activities of superoxide dismutase（SOD）and glutathione peroxidase（GSH-Px）,and alleviated the oxidative stress of testicular tissue in TP-induced mice.In addition,UF4 improved the activities of enzymes（LDH,ALP and ACP）related to energy metabolism in the testis and restored the serum hormone level of mice to normal.UF4 significantly reduced the total number of TP-induced testicular seminiferous cells and inhibited the increase of the number and proportion of apoptotic cells.Furthermore,UF4 promoted the expression of Nrf2 protein,and then increased the expression of antioxidant enzyme regulatory protein（HO-1 and NQO1）in the testis.UF4 inhibited JNK phosphorylation and Bcl2/Bax-mediated apoptosis.（3）UF4 was separated by Sephadex-G25 dextran gel chromatography to get 7components,among which the isolated components F1,F4,F6 and F7 could significantly improve the cell survival rate of TM4 cells after TP injury,significantly reduce the intracellular MDA content and increase the intracellular GSH content.Compared with F1,F4 and F7,F6 showed superior inhibitory activity against oxidative damage of TP on TM4 cells.F6 components were further separated by C18reverse high performance liquid chromatography,and four purified components were collected,among which F6-3 showed the best activity in protecting TM4 from TP-induced cytotoxic damage.F6-3 was identified by UPLC-MS/MS to obtain six peptides SSSDMFR,SDPLYP,YGGA,THPS,VISNVC and YQGMYNW.The six peptides were docked with Keap1 protein.The results showed that the six peptides had a good binding effect with Keap1 target protein with high matching degree,and the binding energy was far less than-5 kcal/mol.All of them could form multiple hydrogen bond interactions with the active pocket of Keap1 protein and form stable complexes.（4）Six identified peptides（P1-P6）were synthesized by solid phase synthesis method.Through cell activity evaluation,it was found that different concentrations of P1-P6 synthetic peptides（0.1-0.4mm）inhibited toxicity and oxidative damage of TP to TM4 cells to varying degrees.P2（SDPLYP）and P4（THPS）significantly increased GSH content in TM4 cells,decreased MDA content in TM4 cells,significantly inhibited TP-induced ROS production and increased apoptosis rate in TM4 cells,activated Nrf2/Keap1 signaling pathway and significantly upregulated Nrf2,Keap1,HO1 and NQO1 protein expressions,and further improve the cellular antioxidant defense ability.Meanwhile,P2（SDPLYP）and P4（THPS）synthetic peptides significantly reduce JNK phosphorylation,reduce the effect of ROS on mitochondrial function,and further promote the expression of anti-apoptotic protein Bcl2 and inhibit the expression of pro-apoptotic protein Bax by affecting the Bax/Bcl2 signaling pathway,and reduce TP-induced apoptosis of TM4 cells.