Study On The Biological Mechanism Of Pilose Antler Extract Regulating Chondrocytes

Objective:The purpose of this study is to explore the biological mechanism of the regulation of pilose antler extract on chondrocytes by means of transcriptome and proteomics.Methods:In this study,the antler in the rapid growth period was used as the research object,and the High-Speed Homogenization mechanism was used to prepare the antler homogenate,and the freeze-dried powder of the antler was obtained by freeze-drying method.The content of protein in pilose antler extract was determined by SDS-PAGE gel electrophoresis.Primary chondrocytes were prepared from costal cartilage of suckling mice born within 72 hours.Chondrocytes were cultured with different concentrations of antler extract(0,0.02,0.04,0.08,0.16,0.32,0.64,1.28 mg/ml).Chondrocytes were counted with CCK-8 and stained with alcian blue 8GX.RNA-seq technology was used to analyze the transcriptome of chondrocytes,total RNA was extracted,the library was constructed,the quality of the library was detected by Agilent 2100 Bioanalyzer,the gene was sequenced by Illumina hiseq 2500 sequencing platform,and the differential expression of genes was analyzed.Serum proteomics was analyzed by iTRAQ technique.Male SD rats were selected as the research object.Drinking water and pilose antler extract were administrated by gavage.iTRAQ was used to label the serum protein of rats.Mascot and proteome discoverer software were used to identify the protein.The results were compared with NCBI and UniProt databases,and the results of differential expression protein were obtained.Functional enrichment and protein interaction were analyzed.Results:1.Pilose antler extract can promote the proliferation of chondrocytes in a concentration dependent manner,and the proliferation rate of chondrocytes reached the peak at the concentration of 0.64mg/ml and 1.28mg/ml.2.There were 639 genes differentially expressed in chondrocytes cultured with antlerextract.The genes which can target regulate the mitotic cycle like Tnfsf11,Bmper,Cebpb,Dbf4,Slc39a14 and Usp3 were up-regulated,and the genes which can promote chondrocyte differentiation like Cilp,Bmp4,Cthrc1,Cilp2,Lect1,Frzb and Tgfb3 were down-regulated.The genes which can improve the anti-inflammatory ability of cells like Ptgs2,Zc3h12 a,Tnip1 and Sbno2 were up-regulated,but the genes which can promote cellular inflammatory response like Trem2,Asic3 and Tsc22d3 were down-regulated,the genes which can improve the antioxidant capacity of chondrocytes like Mafb,Sod3,Hif1 a,Gsr,Nqo1 and Nfkb1 were up-regulated.3.There were 79 differentially expressed proteins identified in the serum proteome of rats by gavage of antler extract.For the 79 proteins,the expression of 47 proteins was significantly up-regulated,and the expression of 32 proteins was significantly down regulated.The main functions of differentially expressed proteins are to regulate immune response,inflammatory response,cytoskeleton dynamics,cell proliferation and differentiation.The up-regulated proteins are concentrated in four domains,while the down-regulated proteins are concentrated in two domains.The up-regulated proteins such as Tpm1,Tpm2,Tpm4,Wdr1,Actn1,DSTN,B2 m,Cxcl1,Gadph,LdhA,Lta4 h and the down-regulated proteins such as A2 m,ApoH,Serpina3 n and Apof form a complex interaction network.Conclusion:1.Pilose antler extract can promote the proliferation of chondrocytes,and promote the synthesis of chondrocytes’ acid polysaccharides.The degree of cell proliferation was positively correlated with the concentration of antler extract.2.Pilose antler extract may regulate chondrocyte proliferation through many ways,such as regulating cell mitotic cycle,inhibiting chondrocyte differentiation,improving cell anti-oxidation ability and anti-inflammatory ability.3.Pilose antler extract can regulate the differential expression of serum proteins,which form a complex interaction network.Pilose antler extract may regulate the metabolism of osteoblasts by regulating immune response,inflammatory response,cytoskeleton dynamics,cell proliferation and differentiation,so as to affect the process of bone metabolism.