Study On Protective Effect And Mechanism Of Oviductus Ranae Protein Hydrolysate On Osteoporosis Rats

Objective:To explore the protective effect of ORPH on osteoporosis model rats,establish quantitative proteomics of osteoporosis rats,and conduct bioinformatics analysis to illustrate the mechanism of anti-osteoporosis by ORPH.Methods:The ORPH was prepared by enzymatic hydrolysis,and the related physical and chemical properties of ORPH were identified by UV,IR,protein coloration,SDS-PAGE and amino acid chromatography.The rat model of osteoporosis was replicated by ovarian surgery,BMD was determined by X-ray method,the total RNA was prepared by modified Trizol method,mRNA expression level of bone metabolism related genes was detected by qPCR method,the expression level of bone metabolism related proteins was detected by Western-blot method,Changes in femoral trabecular structure observed by HE staining,the protein expression levels of ALP and BMP2 were evaluated by immunohistochemistry.The optimal concentration and time of ORPH were screened by MTT method,the peptides were hydrolyzed by trypsin using the TMT labeling method,the labeled peptides were fractionated by ultra-high performance liquid chromatography.The peptide precursors and their secondary fragments were detected by LC-MS,protein annotation,domain annotation,KEGG pathway annotation and subcellular localization were performed using bioinformatics methods,and protein function enrichment and cluster analysis were performed.Results:1.The optimal hydrolase prepared by ORPH was determined to be alkaline protease by evaluating the hydrolysis time and the viscosity value of the Oviductus Ranae.The molecular weight distribution of ORPH prepared by buffer extraction or alkaline protease hydrolysis was detected by SDS-PAGE.The molecular weight of ORPH after enzymatic hydrolysis was significantly reduced,which was more easily absorbed by the body.The molecular weight of ORPH obtained after enzymatic hydrolysis is mainly concentrated at 30kD and below,and the protein content is about 60%.The enzymatic method improves the water solubility of the Oviductus Ranae and eliminates the odor of the Oviductus Ranae.2.Conducted the identification of physical and chemical properties of ORPH.The maximum UV absorption wavelength of ORPH is 280-290nm.The IR characteristic absorption functional groups are“-COOH”and“-CONH”ORPH contains 17 kinds of amino acids,the highest content of which is threonine（Thr）,the content of Thr is 4.87%,and the amino acid species have no change before and after hydrolysis.The biuret reaction and the ninhydrin reaction showed positive results in ORPH.3.Osteoblast MC3T3-E1 was injuried by H₂O₂,treatment with ORPH,optimal delivery time（400ug/mL）and concentration（24h）was screened by MTT assay.Observed under the microscope,the cells in the blank control group were spindle-shaped,and the number of cells in the model control group was decreased.Compared with the model control group,the number of cells in the ORPH-administered group increased,and the shrinkage of the cells was improved,the cell body shrinkage phenomenon was significantly improved in the administered group（400ug/mL）.4.The regulation of ORPH on H₂O₂ injured MC3T3-E1 cells related to bone metabolism maker was evaluated.The relative expressions of ALP and BMP2 mRNA in ORPH group were significantly up-regulated（P<0.01）,and the relative expression levels of Osteocalcin and Runx-2 mRNA had an upward trend.The protein expressions of ALP,Runx-2 and Osteocalcin were significantly increased in ORPH group（P<0.01,P<0.05）.BMP2 protein had an upward trend,ORPH could promote osteoblast proliferation and differentiation,have a certain protective effect in the MC3T3-E1 cells injured by H₂O₂.5.Established a rat model of osteoporosis.After 90 days of ORPH treatment,the BMD of the femur and lumbar spine of the rats were significantly reduced compared with that before the model establishment.It was confirmed that the ovarian surgery can successfully replicate the rat model of osteoporosis.At the same time,ORPH can significantly improve the BMD and maximum load of the femur,enhance femoral toughness and reduce fracture risk.However,ORPH had no significant effect on body weight changes in the osteoporotic rats.6.The pathological examination of the relevant organ and femur in rats was carried out.The ovariectomized adult rats were examined for histopathology.Only a few tissues and organs showed mild inflammation,and no significant toxicity changes were observed in the ORPH group.HE staining showed that ORPH can protect the trabecular structure of the femur and promote the orderly and structurally intact trabecular bone,which significantly reduces the risk of fracture,which is consistent with the improvement of BMD results in rats.7.To investigate the regulation of ORPH on bone metabolism maker related to osteoporosis rats.qPCR and Western-blot experiments confirmed that ORPH can up-regulate the relative expression of ALP,Osteocalcin,Runx-2,BMP2 mRNA and protein;while ORPH can inhibit the expression of osteoclast-associated protein TRAP,ORPH has an important regulatory effect in the bone metabolism balance.8.Immunohistochemistry experiments showed that ORPH can significantly promote the secretion and expression of ALP and BMP2 in osteoporosis rats,which affect bone differentiation,growth and participate in bone metabolism.9.A total of 5,654 proteins were identified,of which 4,889 proteins have quantitative information annotations.The variation of the difference multiplier value is more than 1.2times as a significant upward adjustment,less than 1/1.2 as a significant downward adjustment standard,compared with the blank group,there were 195 up-regulated expression proteins and 204 down-regulated expressions compared with the blank group.Compared with the model group,after ORPH intervention,there were 230 up-regulated expression proteins and 299 down-regulated expression proteins.10.Annotate the protein pathway using the KEGG pathway database,annotate the submitted protein using the KEGG online service tool KAAS,and match the annotated protein to the corresponding pathway in the database via the KEGG mapper.The Osteoporosis and Control data were differentially expressed and enriched,and 11 KEGG Pathway were enriched（P<0.05）.The three pathways with the highest degree of enrichment were Ribosome signaling pathway,Focal adhession signaling pathway and ECM-recepertor interaction signaling pathway.The ORPH and Osteoporosis data were analyzed for differentially expressed protein enrichment and enriched into 14 KEGG Pathway.The two pathways with the highest degree of enrichment are the Ribosome signaling pathway and the Complement and coagulation cascades signaling pathway.11.Change the difference value by more than 2 times as a significant upward adjustment,less than 1/2 as a significant downward adjustment standard.A total of 33differential proteins were screened out.Osteoporosis VS Control group,there were 6up-regulated expression proteins and 16 down-regulated expression proteins;ORPH_Drug VS Osteoporosis group,there were 7 up-regulated expressed proteins and 4 down-regulated expressed proteins.Statistics show that differentially expressed proteins caused by castration surgery and differentially expressed proteins caused by ORPH intervention therapy have seven consensus differential proteins,namely Gal,Ighm,Myl3,Tnni1,TnnC1,SerpinA3k,and IgkC.After treatment with ORPH,the expression levels of seven differential proteins such as Gal,Myl3 and SerpinA3k returned to normal levels（P<0.01）.Conclusion:In this study,the rat model of osteoporosis was successfully replicated.ORPH can increase BMD and maximum load,promote osteoblast differentiation,regulate the expression of related bone proteins,and participate in the regulation of bone metabolism.Its possible mechanism of action is to regulate the key proteins of Gal,Myl3and SerpinA3k,et al.activate the corresponding signaling pathways,protect osteoblast differentiation,promote osteoblast growth,promote metabolism and bone regrowth,exert estrogen-like effects,and prevent antioxidant stress damage,maintaining the balance of bone metabolism,thereby exerting an anti-osteoporosis effect.