Study On The Separation Of Velvet Antler Protein And Its Mechanism Of Inhibiting The Activity Of PC-3M Cells

As a traditional precious animal medicine,velvet antler is warm in nature,sweet and salty,and it returns to the kidney and liver meridian.It has traditional functions such as strengthening the kidney yang and replenishing the essence.The protein components in deer antler are important components for its pharmacological activity.Modern pharmacological studies have found that velvet antler protein(VAP)has a certain effect on the treatment of prostate cancer,but the active components of the protein have not been clear,and the mechanism of action has not been fully elucidated.Therefore,this study aimed to explore the active protein components of velvet antler protein that inhibit prostate cancer cells PC-3M and their molecular mechanisms.The velvet antler protein(VAP)was separated by dextran gel filtration chromatography and the velvet component proteins VAP4 and VAP5 were used as the research objects.The velvet antler protein components were analyzed by liquid chromatography tandem mass spectrometry combined with proteomics and biological Informatics methods screen out the material basis and molecular mechanism that inhibit the growth of cancer cells.It was detected and identified by mass spectrometry that there were 14 credible proteins in VAP4.The molecular weight of the protein was mainly distributed between 35.9-66.1k Da,and the isoelectric point was between 5.33-9.82.There were 13 credible proteins in VAP5.The molecular weight is mainly distributed between 8.66-71.4k Da,and the isoelectric point is between 4.74-9.6.Through GO and KEGG analysis,the sequence alignment,three-dimensional structure establishment and phylogenetic tree analysis of the common proteins of interest in VAP4 and VAP5 were carried out.The results showed that VAP4 and VAP5 may inhibit the growth of PC-3M cells by regulating the PI3K/AKT signaling pathway.To study the inhibitory effect of deer antler component protein on PC-3M cell growth and its molecular mechanism.The effects of VAP,VAP4 and VAP5 on the survival rate of PC-3M cells were determined by MTT method and RTCA real-time cell analysis technology.Plate cloning experiments were carried out to study the effects of VAP,VAP4 and VAP5 on the proliferation and cloning ability of PC-3M cells.Transwell assay to determine the changes in the migration ability of PC-3M cells,Western Blot method was used to determine the protein expression levels of PI3K/AKT signaling pathway in PC-3M cells p-PI3K/PI3 K,p-AKT/AKT,VEGF,MMP-2,MMP-9.The results showed that administration of VAP,VAP4 and VAP5 significantly inhibited the cell survival rate of PC-3M cells(P<0.01);The plate cloning experiment showed that it significantly reduced the proliferation ability of PC-3M cells,that is,the colony forming ability;In the Transwell experiment,the migration ability of PC-3M cells,that is,the number of cell migration per well,was significantly inhibited;Finally,Western Blot results showed that the protein expression levels of p-PI3 K,p-AKT,VEGF,MMP-2 and MMP-9 were significantly reduced(P<0.01),thereby inhibiting the growth of PC-3M cells.To study the antler component proteins inducing PC-3M cell apoptosis and autophagy and their molecular mechanisms.Detect the apoptosis rate and nuclear morphology of PC-3M cells by flow cytometry and Hoechst 33258 staining to determine the level of cell apoptosis;Acridine orange staining was used to judge the effect on autophagy;Western Blot method was used to determine p-m TOR/m TOR,Beclin-1,LC3-I,LC3-II,Bax,p53,Bcl in each group of PC-3M cells-2,Caspase 3 and Cleaved-Caspase 3 protein expression levels.The results showed that the administration of VAP,VAP4 and VAP5 significantly increased the degree of nuclei fragmentation in Hoechst 33258 staining;The results of flow cytometry showed that administration of VAP,VAP4 and VAP5 significantly increased the apoptosis rate of PC-3M cells(P<0.01);Acridine orange staining showed that administration of VAP,VAP4 and VAP5 significantly improved the autophagy ability of PC-3M cells(P<0.01);Finally,Western Blot showed that the protein expression levels of p-m TOR and Bcl-2 were significantly reduced(P<0.01),and the protein expression levels of Beclin-1,Bax,p53,Cleaved-Caspase 3/Caspase3 were significantly increased(P<0.01).These results indicate that velvet antler protein promotes autophagy and apoptosis of PC-3M cells.Combined with the results of proteomics and pharmacological tests,it can be seen that the velvet proteins VAP,VAP4 and VAP5 can effectively inhibit the PI3K/AKT signaling pathway to inhibit the growth of PC-3M cells,and promote their apoptosis and autophagy,thereby inhibiting the occurrence and development of prostate cancer,provide theoretical basis for antler clinical treatment of cancer.