Analysis And Identification Of Bioactive Compounds From Four Portions Of Velvet Antlers In Sika Deer(Cervus Nippon)based On Proteomics And Metabolomics

Velvet antlers are a bone tissue model that can fully regenerate in annual cycles.Different portions from the tip to the base correspond to diverse bone development stages.The tip of antler growth center contains reserve mesenchyme which differentiates into four distinctive layers of precartilage,transitional zone,cartilage,and then bone through endochondral ossification.In addition,velvet antlers are the most important derived-animal medicine and are used to treat many diseases.It is fundamental to understand the differences of portions in antler before studying efficacy.Fresh standard three-branched velvet antlers were collected and each divided into four portions(wax-like,blood-color,honeycomb-like and bone)based on sensory index from Chinese agricultural industry standard(NY/T1162-2006)and digitized division method established by our velvet antlers engineering research center in Jilin province.It is known that big differences between antler portions not only reflect in the price,but treatment objects as well as efficacy determined by the variety and content of bioactive compounds.The methods of label-free proteomics and untargeted metabolomics were conducted to identify the proteins and metabolites and the acquired data were analyzed using bioinformatics tools.The main contents and results are as follows:1.The total protein of each portion sample was extracted and digested enzymatically into peptides.After identification and quantification of the peptide mixture,the resultant data was analyzed by using PCA model and other bioinformatic tools.The results showed that there were 2039,1790,1454 and 1358 proteins identified in the wax-like,blood-color,honeycomb-like and bone portions,respectively.PCA model could unambiguously distinguish these four portions.Highly expressed proteins were those include extracellular matrix proteins,skeleton proteins and blood proteins,as well as regulatory factors involved in antler growth and development such as POSTN,Matn4,ACPM and ACCD.The differences between honeycomb-like and bone portions were small and they should be reclassified into one portion,whereas that between wax-like portion and the other three portions were relatively bigger.A total of 659 proteins were found to be differentially expressed in these four portions,of these proteins 190 were fitted to 9 clusters based on the similar expression pattern.2.The total of metabolites of each portion sample was extracted and separated.After identification and quantification of the metabolites,212 metabolites were qualified in ESI positive mode and 148 in ESI negative mode.PCA and OPLS-DA analyses were performed to detect differences in metabolites expressed in comparisons.All metabolites were analyzed using the network pharmacology analysis platform.The PCA results showed the distinct difference between the wax-like portion and the other three portions.OPLS-DA model effectively differentiated two neighboring portions and the differences were mainly contributed by lipids,amino acids,nucleosides and saccharides.There were 56 difference in metabolites between the wax-like portion and the other three portions.Moreover,betaine was found to be decreased from the wax-like portion to the bone portion.Nervonic acid was highly expressed in the bloodcolor portion.Velvet antlers have the potential to treat many diseases,particularly neurodegenerative diseases.This is the first proteomic and metabolomic study to investigate the commercially defined four portions of velvet antlers.It is reasonable to evaluate the quality of antler portion based on the price.Taken together,the results of this study laid the foundation for discovering the molecular mechanisms of rapid antler growth and ossification,and it was of great significance for further study of bioactive compounds and the pharmacological effects for velvet antlers.