| Objective:An ovariectomized SD rat model was established to observe the effects of Xuling-jiangu formula on bone density and bone microstructure in the ovariectomized rat model.Proteomics and metabolomics techniques were used to screen the differential expression proteins and differential metabolites of the lumbar spine in the ovarian deactivated rat model after the intervention of Xuling-jiangu formula,and bioinformatics analysis was carried out.From the perspective of metabolism and proteomics,the mechanism of action of Xuling-jiangu formula in the treatment of osteoporosis rats was discussed.Methods:1.The ovariectomized rat were randomly divided into model group and the Xuling-jiangu formula group(hereinafter referred to as the Xuling group),and there were 12 rats in each group.The rats in the sham surgery group was resected the appropriate size of adipose tissue near the ovaries.Gavage administration was started 30 days after modeling,and the dose of administration in the Xuling group was 15.25 g/(kg*d),and each rat in the model group and the sham group was given the same volume of normal saline,and the material was taken after 12 weeks of continuous administration.The efficacy of the rat model of Xuling-jiangu formula in the treatment of osteoporosis was comprehensively evaluated by the change of bone density at the left proximal tibia and the HE staining of the pathological section of the distal left femur.2.Using UHPLC-Q-TOF MS non-targeted metabolomics technology,differential metabolic screening of lumbar tissue metabolites(after multivariate statistical analysis)was performed.Using Metabo Analyst 5.0 analyzed the metabolic pathways involved in the screening of common differential metabolites to find potential target metabolic pathways and detect the metabolic pathways of common differential metabolites.Targeted quantification of non-targeted metabolomic metabolites was achieved by mass spectrometry.3.TMT quantitative proteomics was used to quantify rat lumbar protein,screen out differentially expressed proteins.Using differentially expressed proteins to analyze GO,KEGG pathways,protein interactions,and construct protein interaction networks.4.We performed correlation pathway enrichment analysis of differential metabolites and differential proteins.Western Blotting experiments verified the candidate differentially expressed proteins Aldh3b1,Bid,Tubb,Myl12 a.Results:1.The results of bone density showed that the bone density of rats in the Xuling group was higher than that in the model group(P<0.05),and the difference was statistically significant.After HE staining,it was seen that the trabeculae of the distal femur epiphyseal line were significantly different in each group,and the number of trabeculae in the Xuling group was higher than that in the model group,the distribution was more uniform,and the spacing between the trabeculae was reduced.After Masson staining,the area of mature collagen fibers near the femoral growth plate in the Xuling group was increased compared with that in the model group(P<0.05).This showed that Xuling-jiangu formula can be effective in treating osteoporosis in a rat model.2.A total of 38 common differential metabolites were identified in rat lumbar spine samples,and the Xuling-jiangu formula had a callback effect on 23 of them.Differential metabolites were enriched with 27 related metabolic pathways,of which the three most significant metabolic pathways were Alanine,aspartate and glutamate metabolism,Arginine biosynthesis,and Histidine metabolism all of which were amino acid metabolism.The targeted quantitative verification results showed that some metabolites coincided,that is,the non-targeted metabolomics results were true and reliable.3.A total of 3614 proteins were identified in the lumbar spine samples of rats,and the Xuling-jiangu formula had a callback effect on 32 of the abnormally expressed proteins.GO enrichment analysis showed that the differential proteins of the Xuling group and the model group were mainly involved in mitochondrial regulation and hemoglobin binding,as well as nucleic acid transcription.KEGG enrichment analysis showed that they were mainly involved in important pathways such as Autoimmune thyroid disease,Allograft rejection,Graft-versus-host disease,Type I diabetes mellitus.PPI analysis showed that Myh9,Myl12 a,Unc45a,Ryr1,Myoz1,Birc5 and Bid were located at the nodes of the protein interaction network among the common differential proteins in the Xuling group and the model group.4.In the integrated analysis of the two omics data,differential metabolites and differential proteins were integratedly enriched in 10 metabolic pathways.Four differential proteins such as Aldh3b1,Bid,Tubb,Myl12 a,and three differential such as metabolites histidine,pantothenic acid,arachidonic acid showed a pullback trend.Further Western Blotting experiments confirmed that the expression trend of differential proteins Aldh3b1,Bid,Myl12 a and Tubb was the same as that of proteomics.Conclusion:1.Xuling-jiangu formula could effectively increase the BMD,improve the microstructure of the bone tissue,reduce the loss of bone mass and promote the formation of new bones in ovariectomized rat.2.The Lumbar metabolomics analysis showed that Xuling-jiangu formula recalled 23 differential metabolites and may exert its antiosteoporosis effect by intervening in amino acid metabolism.3.The Lumbar proteomics analysis showed that Xuling-jiangu formula may play a role in bone protection by regulating differential proteins,participating in mitochondrial regulation and hemoglobin binding processes,and regulating immune-related pathways.4.Conjoint analysis of proteomics and metabolomics found that amino acid metabolism was the main pathway for the common enrichment of the two omics,further indicated that the treatment of PMOP with Xuling-jiangu formula was closely related to amino acid metabolism. |
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