Study On The Underlying Mechanism Of Anti-osteoporosis Of Yak(Bos Grunniens) Bone Collagen Peptides

Yak(Bos grunniens)is a precious stock resource in China and yak bones are rich in collagen(about 21.5%).The supplementation of collagen peptides can increase the regularity and firmness of the bone structural collagen fiber grid.In-depth research on the deep processing technology of yak bones and making full use of the rich collagen in yak bone to develop functional foods are of great significance to improve the added value of yak,promote the social & economic development of pastoral areas and increase the income of herdsmen in Tibetan areas and stabilize the frontiers.In this study,yak bone was used as the research object,and high-purity yak bone collagen was prepared by acid-soluble enzyme extraction technique,and its structure was also identified.Based on the targeted enzymatic hydrolysis technique,the effects of six common commercial proteases including trypsin,alcalase,flavourzyme,protamex,papain and neutrase on the proteolytic activity of yak bone collagen were studied,and the degree of hydrolysis and osteoblast proliferation-promoting activity are the indicator of the most suitable enzyme.Response surface methodology(RSM)was used to optimize the optimal process parameters for the preparation of osteogenic activity peptides from enzymatic hydrolysis of yak bone collagen by neutrase.Sequential purification of yak bone collagen hydrolysates was achieved by ultrafiltration,size exclusion chromatography and semi-preparative reverse phase high-performance liquid chromatography.The digestion,absorption and transport mechanism of yak bone collagen peptides were examined by simulating intestinal digestion and Caco-2 cell monolayer membrane transport model.Based on Real-time qPCR,Western blot and Molecular docking technology,the present study explored the potential mechanism of yak bone collagen peptides on promoting osteoblast proliferation.By using bone turnover biochemical markers,bone biomechanical indexes and bone morphological mechanical as indicators,we also examined the anti-osteoporosis activity effect of yak bone collagen peptides.The non-targeted metabolomics technology was used to systematically explored the mechanism of anti-osteoporosis activity of yak bone collagen peptides.The main conclusions are as follows:(1)By comparing and analyzing the different osteoblast proliferation rate of yak bone collagen hydrolysates form the six kinds of proteases,neutrase was selected as the most suitable enzyme for enzymatic hydrolysis yak bone collagen to prepare osteoblast proliferation active peptides.The combination of optimum enzymatic hydrolysis process conditions was: enzymatic hydrolysate temperature 54 °C,enzymatic hydrolysis time 3.6 h,enzyme bottom ratio(E/S)5637 U/g,initial pH 6.12.(2)The osteoblast proliferation-promoting peptides were isolated and purified by ultrafiltration,size exclusion chromatography and reversed-phase high performance liquid chromatography.Among them,the component UF-IV(Mw < 3 kDa)collected after ultrafiltration showed higher osteoblast proliferation activity.After separation by SuperdexTM peptide 10/300 GL column(size exclusion chromatography),component SP-III showed higher osteoblast proliferation activity,and the osteoblast proliferation rate was up to 160.6% at 0.5 mg/mL.After purified by RP-HPLC,component SP-III was further separation and analyzed by Innoval C18 and component F8 showed strong osteoblast proliferation activity(190.6%).The amino acid sequence was determined by reverse liquid chromatography tandem mass spectrometry combined with Mascot 2.4 search engine,and 35 peptides were identified.Based on the relative abundance,molecular weight,comparison standard score and other indicators,9 peptides with higher potential for osteoblast proliferation were screened and the bioactivity in vitro was verified by chemical synthesis.The results showed that GPAGPPGPIGNV(GP-12)peptide showed a higher osteoblast proliferation activity(142.7%)compared with other eight peptides segment.(3)Simulated gastrointestinal digestive test results show that GP-12 peptide has certain resistance to gastric-intestinal digestion,although some GP-12 peptides are hydrolyzed into GPAGPPGPIGN(GP-11)and GPAGPPGPI(GP-9)by intestinal endopeptidase.After digestion,however,they still have osteoblast proliferation-promoting activity.The results of the Caco-2 cell monolayer membrane assay showed that the GP-12 peptide was further hydrolyzed by the epithelial membrane peptidase to GPPGPIGNV(GV-9)and AGPPGPIGNV(AV-11)peptides.Some GP-12 peptides were still intact and can transport through the Caco-2 cell monolayer membrane,but the absorption rate is very low,and its absorption mechanism is absorbed by the cell bypass pathway.(4)The osteoblast proliferation effect of GP-12 peptide may be related to the activation of cross-talk between Wnt/?-catenin signaling pathway and epidermal growth factor receptor(EGFR)signaling pathway.In vitro molecular docking results showed that the amino acid residue binding sites of GP-12 peptide and epidermal growth factor receptor EGFR were Asn12,Lys13,Leu14,Thr15,Gln16,Leu17,Gly18,Tyr45,Leu325 and Phe357,and the binding force was mainly Hydrogen bonding and van der Waals forces.The potential mechanism of action of GP-12 peptide may be that after activation of epidermal growth factor receptor EGFR,the activated EGFR can further phosphorylate GSK-3?,which can prevent ?-catenin degradation,thereby activating Wnt/?-catenin signaling pathway to induce osteoblasts proliferation process.(5)The yak bone collagen peptide(<3kDa)has good osteoblast proliferation activity and exhibits a good dose-effect relationship.The proportion of polypeptides with molecular weight below 1 kDa in the extracted yak bone collagen peptide(<3 kDa)was as high as 96.8%.The osteoblast proliferation activity of yak bone collagen peptide(<3 kDa)may be attributed to the interaction of GPSGPAGKDGRIGQPG(GP-16),GDRGETGPAGPAGPIGPV(GD-18)or a polypeptide containing a similar amino acid sequence with EGFR.(6)Yak bone collagen peptide(<3kDa)can significantly improve the expression of bone formation-related biochemical markers(osteocalcin,serum bone-specific alkaline phosphatase)and reduce the expression of bone resorption-related biochemical markers(serum tartaric-resistant acid phosphatase,serum C-terminal telopeptide cross-linking of type I collagen,urinary deoxypyridinoline).Compared with the model group,the bone elastic load and fracture load of the yak bone collagen peptide(<3kDa)and 17?-estradiol treatment groups showed a significant increase(P>0.05).In addition,yak bone collagen peptide(<3kDa)and 17?-estradiol significantly increased rat trabecular bone mineral density(Tb.BMD),bone volume fraction(bone volume/total volume,BV/TV),trabecular thickness(Tb.Th),number of trabecular bone(Tb.N)and reduction of trabecular bone spacing(Tb.Sp)(P<0.05).In summary,yak bone collagen peptide(<3kDa)has a better effect on improving osteoporosis in rats and exhibits a dose-effect relationship.(7)Eleven potential biomarkers including 1-stearyl-2-hydroxy-sn-glycero-3-phosphocholine,1-palmitoyl-2-hydroxy-sn-glycerol-3-phosphoethanolamine,taurine,arachidonic acid,(4Z,7Z,10 Z,13Z,16 Z,19Z)-4,7,10,13,16,19-docosahexaenoic acid,sphingosine-1-phosphate,taurodeoxycholic acid,taurocholate,taurochenodeoxycholate,L-citrulline and serotonin were screened out.The pathogenesis of osteoporosis in ovariectomized rats is closely related to the body's phospholipid metabolism,amino acid metabolism and fatty acid metabolism disorder.Yak bone collagen peptides protect against ovariectomized-induced osteoporosis in rats through intervening various metabolic pathways such as Membrane transport(ABC transports),Digestion system(protein digestion and absorption,Mineral absorption),Translation(Aminoacyl-tRNA biosynthesis),Amino acid metabolism(Arginine and proline metabolism,Valine,leucine and isoleucine degradation),Lipid metabolism(Primary bile acid biosynthesis,Taurine and hypotaurine metabolism),and cellular immunity,nervous system,carbon metabolism and endocrine system.Multiple signaling pathways,multiple links,multiple targets,and multi-dimensional interventions were occured in the prevention,improvement,and treatment of osteoporosis in rats at the same time.