Regulatory Effects And Mechanisms Of Sea Cucumber Peptide On Sex Hormones

Sea cucumbers（Acaudina leucoprocta）are Chinese traditional tonic marine food and economic aquatic products.Sea cucumber peptide（SCP）is obtained from sea cucumber protein by enzymatic hydrolysis.SCP is potential for a dietary supplement,which has various functions such as anti-aging,anti-cancer,anti-fatigue,improving immunity,etc.However,the regulation mechanism of SCP on sex hormones and its improvement effect on diseases caused by sex hormone imbalance is still blank.Therefore,in this study,the normal mice,POF females,and AES mice models were first used to evaluate the effect of SCP on sex hormone regulation.Meanwhile,proteomics and metabolomics transcription were combined to explore the mechanism of SCP in sex hormone regulation,and certain molecular mechanisms were verified in the TM3 Leydig cells.Finally,SCP was purified by different anion and cationic resins.HPLC-MS/MS analysis and molecular docking technology were used to verify and identify the potential active peptide in SCP which could promote testosterone synthesis.This study aimed to provide a theoretical basis for the functional development of SCP and a new perspective for the market-oriented application of SCP.The main results of this study were sorted out as follows:1.SCP is a small molecule peptide,which is rich in glutamic acid,glycine,alanine,proline,and arginine.The molecular weight of 88.32%peptides is less than 1000 Da.SCP was identified using HPLC-MS/MS method,the result indicated that SCP has 227 peptides and was mainly composed of peptides with 5,7,8,9,or 10 amino acid residues.Also,SCP contained a large number of peptides containing Gly-X-Y structure.In vivo,normal female and male mice received different concentrations of SCP（0.2 mg/mL or 0.6 mg/mL）.The results indicated that the body weight,feed intake,and major organ indexes of normal mice were not significantly affected by SCP treatment,as well as the estrous cycle,ovarian and uterine morphology,and ovarian indexes in female mice.The effects of SCP on sex hormones were gender differences,which showed that SCP had no significant effect on serum hormones of female rats,but significantly increased serum testosterone,FSH,and LH of male rats（p<0.05）.In addition,the mating ability of male mice and the expression of Hsd17b2,Hsd17b3,Cyp19a1 and StAR genes in testis were enhanced significantly by SCP intervention（p<0.05）.Meanwhile,the expression of StAR protein was significantly increased（p<0.05）.2.In the experiment of female mice with premature ovarian failure（POF）,SCP intervention significantly improved the estrous cycle disorder and ovarian atrophy caused by POF.Meanwhile,the serum E2,T,and AMH levels were increased（p<0.05）,but FSH level was not affected.Besides,the expressions of StAR and Fshr in the ovary and Mapk1,Esr1 and Gnrh in the hypothalamus were significantly up-regulated by 0.6 mg/mL of SCP（p<0.05）,as well as the contents of GnRH and cAMP in serum（p<0.05）.Compared with the model group,the metabolomics approach demonstrated SCP intervention caused forty-seven different biomarkers,and eight metabolic pathways（including five amino acid synthesis and metabolism pathways,two lipid metabolism pathways,and one aminoacyl t RNA biosynthesis pathway）.3.In male rats with acute exhaustion exercise（AES）,both 0.2 and 0.6 mg/mL SCP intervention significantly（p<0.05）increased penis indexes,prolonged exhaustive swimming time,decreased BUN and LA levels,increased muscle T-AOC and effectively alleviated fatigue.In terms of sexual function,the mating ability of mice,serum T and E2 levels,and the absorption of TCHO and LDL-C in testicular tissue（p<0.05）were significantly increased by SCP administration.In addition,0.6 mg/mL of SCP significantly enhanced the expression of StAR,Hsd17b3 and Cyp17a1 proteins in mouse testicular tissue（p<0.05）,but also significantly up-regulated the expression of StAR,Hsd17b3,Hsd17b2,Ldlr and Cyp19a1 genes（p<0.05）.Moreover,the Western Blot result found that SCP may regulate testosterone synthesis by affecting PKA signaling pathway.Compared with other model mice in this research,the promoting effect of SCP on sex hormone synthesis was more obvious in AES mice.4.Serum metabolomics and testicular tissue transcriptomic studies revealed that AES induced 35 metabolite differential changes（p<0.05）,the differential metabolites were mainly concentrated in GABAergic synapse,proximal tubule bicarbonate reclamation,HIF-1 signaling pathway,ABC transporters,arginine biosynthesis,glucagon signaling pathway,isoflavonoid biosynthesis,glycerophospholipid metabolism,and pyruvate metabolism.It also significantly affected the expression of genes of base excision repair,GABAergic synapse and apoptosis signaling pathway in testicular tissue（p<0.05）.However,SCP intervention（0.6mg/g.bw）significantly reversed metabolite changes induced by AES（p<0.05）to the normal level.Differential expression of lactate,succinate,DAG and T-type calcium channel protein（Cacna1g）induced by SCP intervention（p<0.05）,indicating that the process of SCP intervention in AES mice is closely related to cAMP signaling pathway and calcium signaling pathway,and interacts with GnRH secretion,MAPK,steroid hormone biosynthesis and other signaling pathways,which effectively alleviating fatigue symptoms,regulating sex hormones and restoring normal metabolism in AES mice.5.In the TM3 Leydig cells model,it was found that the intervention of 0.1 and 1 mg/mL SCP significantly increased the T synthesis ability of the cells.In addition,LDL-C and Ca²⁺content were significantly raised by SCP treatment,and the T-type calcium channel protein genes（Cacna1g,Cacna1h,and Cacna1i）,Plce1,Prkca,Prkcb,Prkcg,Prkaca genes,and testosterone synthesis related protease gene StAR,Hsd17b3 and Cyp17a1 were also enhanced（p<0.05）.Consistent with the in vivo results,SCP treatment significantly enhanced the expression of phosphorylated PKA protein（p<0.05）.Moreover,in TM3 Leydig cells with T-type calcium channel inhibition,although the ability of SCP to promote T synthesis was partially inhibited,SCP intervention significantly improved the expression of Cacna1g,Cacna1h,Cacna1i,Plce1,Prkaca and Lhcgr genes.Taken together,SCP regulates sex hormone synthesis by mediating PLC/Ca_v3/PKA signaling but is not solely dependent on the activation of Ca_v3 channels.6.In the TM3 Leydig cells model,Ca²⁺treatment alone effectively up-regulated the expression of Cacna1h,Cacna1i,Plcl1,Prkca,Prkcg,Prkaca,Mapk1,and testosterone synthetase protein genes（p<0.05）.Under the co-treatment of SCP and Ca²⁺,the expressions of T-type calcium channel and PLC/PKA signaling pathway-related genes were more significant（p<0.05）.Moreover,the LDL-C content was also significantly increased by SCP treatment,which effectively enhanced testosterone synthesis.The results showed that Ca²⁺and SCP were directly involved in promoting testosterone synthesis,and SCP could regulate intracellular calcium dynamics by stimulating PLC/Ca_v3/PKA signal and enhancing the activity of the second messenger Ca²⁺in the process of testosterone synthesis.7.SCP was separated and purified into 15 components（F1-F15）using ionic resins（ZGC108DQ gel strong acid positive resin,201×4 strong base negative resin,special D001FD strong acid positive resin,D202FD strong base negative resin and SDN-5 neutral macroporous resin）and elution concentrations（30%,50%and 75%ethanol）.The results showed that the intervention of F8,F9 and F12 effectively enhanced the ability of TM3 Leydig cells to synthesize testosterone（p<0.05）,and significantly increased intracellular Ca²⁺level（p<0.05）.Thirteen common peptides of SCP,Dig-SCP,F8,F9 and F12 were screened out by HPLC/MS/MS identification results.Then,according to the molecular docking results,GPAGPQGPAG（GG-10）,GPTGPAGPR（GR-9）and PGAQGLR（PR-7）were preliminarily identified as potential active peptides in SCP.Based on cell level verification,the results indicated that GR-9 can effectively promote testosterone synthesis,and it may further stimulate the activation of PKA,PKC,PLC and T-type calcium channels after binding to Lhcgr,so as to achieve the regulation of testosterone synthesis.