Effect Of Diallyl Disulfide On Proliferation And Apoptosis Of MDCC-MSB-1 Cells

Diallyl Disulfide(DADS)is the main active ingredient in garlic,which induced cell cycle arrest and apoptosis as the main regulatory mechanisms to inhibit various cancer cells proliferation.Marek's disease(MD)is a highly contagious lymphoproliferative neoplastic disease induced by Marek's disease virus(MDV),causing huge economic losses in poultry farming.At the same time,with vaccine failure and increased viral virulence,active ingredients in natural plant extracts have become a hot spot anti-tumor research.As the chicken Marek's disease lymphoproliferative tumor cell line,MDCC-MSB-1 cells were commonly used to study the mechanism of anti-tumor action model.In this study,the cell cycle arrest and apoptosis of MDCC-MSB-1 cells were induced by diallyl disulfide,and the main signaling factors in the mitochondrial apoptotic pathway in apoptosis were further explored.It provides a theoretical basis for the development of new drugs which could reduce the economic losses caused by Marek's disease.In this experiment,CCK-8 method was used to confirm the effect of diallyl disulfide on the viability of MDCC-MSB-1 cells,and the morphology of apoptosis was observed by fluorescence staining with Acridine Orange(AO);Rates of cell cycle arrest and apoptosis detected by flow cytometry;When verifying the mitochondrial apoptotic pathway,we detected changes in mitochondrial membrane potential by JC-1 staining and tested Bax protein expression by western blot,then used activity assay kit for measuring caspase-9 and caspase-3 protein activity,quantitative analysis for the relative expression levels of caspase-9 and caspase-3 mRNA by Q-PCR.Compared with the blank group,50?100 and 150 ?mol/L diallyl disulfide concentrations treated MDCC-MSB-1 cells for 24 hours could dose dependent reduce the viability of MDCC-MSB-1 cells,at the same time,fluorescence staining showed that the cell cytoplasm changed from homogenized to fragmented as the drug concentration increased,at 150 ?mol/L,the degree of edge aggregation was clearly visible.Diallyl disulfide induced an increase in the number of MDCC-MSB-1 cells arrested in the G2/M phase and Bax protein expression,reducing mitochondrial membrane potential,and activated enhancement of caspase-9?caspase-3,causing caspase-9?caspase-3 mRNA transcription upregulated.Diallyl disulfide induced MDCC-MSB-1 cell arrest in G2/M phase,and mediated mitochondrial membrane potential decreased by by Bax protein overexpression,leading to increase mitochondrial membrane permeability,then released pro-apoptotic factors to activate its downstream protein caspase-9.Finally,internal apoptotic hub caspase-3 activated and mitochondrial apoptosis pathway completed.Overexpression of caspase-9 and caspase-3 mRNA indicates that diallyl disulfide could regulate the apoptotic protein of MDCC-MSB-1 cells at the transcriptional level.