The Relationship Between Apoptotic Effect Of Alcohol On The PCl2Cells And Sphingomyelin Cycle

Objective In this study, we establish the alcohol-induced apoptosis in PC12cellsmodel,observation of alcohol on PC12cells sphingomyelin cycle activity and its mRNAexpression.To analyze the role of the loop in neuronal apoptosis, and lay the foundation forfurther study of neuronal apoptosis mechanism.Methods Measured by MTT assay of alcohol on the inhibition of the proliferation ofPC12cels; Nuclei morphological changes observed with PI/Hoechst33342staining; DNAagarose gel electrophoresis to detect the degree of DNA fragmentation; By RT-PCR detectionof alcohol on PC12cells SMS1are to SMS2and N-SMase mRNA expression; TLC methodfor determination of the activity of the SMS; Fluorescence spectrophotometric method ofN-SMase activity;To adopt Rhl23/Hoechst33342detection of mitochondrial membranepotential; Western-blotting detection of the wine exquisite PC12cells during apoptosis,apoptotic key proteins caspase-3’s and caspase-9’s change.Results MTT assay results showed that100mmoL/L, and200mmoL/L,300mmoL/L,and400mmoL/L and800mmoL/L, the concentration of alcohol treatment in PC12cells (toserum-free medium24h), the cell survival rates weresimply go to85.66%of the serum,74.28%,62.47%,54.14%and50.35%, a concentration dependent manner, compared with thecontrol group, significant differences (P <0.05). Nuclei morphological changes observed withthe PI/Hoechst33342staining, typical apoptotic phenomenon, such as karyotype shrinkage,chromatin condensation, and so on. DNA-ladder experiment visible100-400mmoL/Lconcentration of the alcohol treatment group there are different degrees of DNAfragmentation with DNA typical of apoptotic cells ladder. RT-PCR to detect the influence ofalcohol on PC12cells SMS and N-SMase mRNA expression. The results show that theinfluence of alcohol of different concentrations of0.5h, SMS1are to the amount of expressionin PC12cells did not change significantly, when the reaction time for more than1h, SMS1areto expression levels were significantly increased; of SMS2the mRNA expression leveldecreased expression at8h. The influence of alcohol of different concentrations of N-SMaseactivity in PC12cells within one hour, no significant change in duration of action to achieve2h N-SMase activity was significantly increased. Different concentrations of alcohol treatment in PC12cells2h, total SMS activity increased in the cells. Different concentrationsof ethanol treatment in PC12cells2h, N-SMase activity was increased. Rhl23/Hoechst33342detection of mitochondriaL membrane potentiaL, and high content results show that alcoholcan make PC12cells mitochondrial membrane potential decline and was measured andtime-dependent. Western-blotting detection of the wine exquisite PC12cells during apoptosis,apoptosis key protein Caspase-3and Caspase-9activation fragment enhanced expression.ConcLusionsAlcohol can induce apoptosis in PC12cells, apoptosis increased with the ethanolconcentration increased, dose-dependent manner.Alcohol-induced PC12cell apoptosis and the sphingomyelin cycle. The influence ofalcohol SMS1and N-SMase mRNA expression in PC12cells increased the activity enhancedSMS2mRNA expression in decline..Alcohol induced apoptosis in PC12cells related to the mitochondrial pathway.