Study On The Mechanism Of P25-CDK5-p53 Signaling Pathway In Methanol-induced Apoptosis Of SK-N-SH Cell

Objective To detect the cell vitality,apoptosis,oxidative stress levels and gene and protein expression level of related factors(Calpain2,p35,p25,CDK5,p53)by different concentrations of methanol intervention SK-N-SH cell,and explore the mechanism of p25-CDK5-p53 signaling pathway in methanol-induced apoptosis of SK-N-SH cells,which provides a theoretical basis for the neurotoxicity of methanol.Method Take the different concentrations of methanol(0-2000 mmol/L)intervention SK-N-SH 24 h and 48 h respectively.To detect the cell vitality by CCK-8 and determine methanol concentration which were 0,250,750,and 1250mmol/L(control,low,medium,and high concentration groups).Cell morphology was observed under in microscope;apoptosis was detected by Annexin V-FITC double staining method;ROS level was detected by reactive oxygen species(ROS)detection kit;DNA damage was detected by single cell agarose gel electrophoresis;The gene expression levels of Calpain2,CDK5,p35,p25 and p53 were detected by RT-PCR.The protein expression levels of Calpain2,CDK5,p25,p35,p53 and p53 phosphorylation were detected by Western blot.Results 1.After 24 and 48 hours,the viability decreased from(100.00±0.00)% to(16.03±0.72)% and(9.66±0.37)% respectively.The difference was statistically significant(P < 0.01).Compared with the control group,the cell viability of the methanol group at all concentrations showed statistically significant differences(P<0.05).2.After 24 h,the cells in the control group showed irregular triangular or fusiform growth with longer protrusions,small gaps between cells,tight junctions and they were flat monolayer adherently growing glioma cells.In the low concentration group,individual cells become round.In the medium concentration group,many cells become round and shortened,the shape is somewhat similar to the polygonal shape,and also has a round shape,the gap between the cells increases,and some cells float and lose the anchoring force.In the high concentration group,the cells gap was significantly increased,the cell refractive index became stronger,most of the cells became round,floating in the culture solution and a small number of cells were normally adherent.3.After 24 h,the apoptosis rate in the low concentration group was higher than the control group(P<0.05);the middle and high concentration groups were significantly higher than the control group(P<0.01);the high concentration group was significantly higher than the low and middle concentration groups(P<0.01).4.After 24 h,the ROS level in the middle concentration group was higher than the control group(P<0.05);the high concentration group was higher than the low concentration group(P<0.05);the high concentration was significantly higher than the control group(P<0.01).5.After 24 h,the expression levels of Calpain2,CDK5 and p25 in the high concentration methanol groups were significantly higher than the control groups,low and medium concentration groups(P<0.01).The expression level of p53 gene in the medium and high concentration groups were significantly higher than the control and the low concentration groups(P<0.01).The expression of p35 gene was not statistically significant(P>0.05).6.After 24 h,the protein expression levels of Calpain2,CDK5 and p53-Ser15 in the low concentration groups were higher than the control group(P<0.05),the middle and high concentration groups were significantly higher than the control group(P<0.01).The protein expression of p35 and p25 in the low,medium and high concentration groups were significantly higher than the control group(P<0.01).The protein expression of p53 in middle group was higher than the control group(P<0.05),and the high concentration group was significantly higher than the control group(P<0.01).Conclusion 1.Methanol can inhibit the activity of the SK-N-SH cells.2.Methanol can change the morphology of the SK-N-SH cells.3.Methanol can induce the apoptosis of the SK-N-SH cells.4.Methanol may induce oxidative stress and DNA damage response of the SK-N-SH cells,further activate Calpain2,and increase the protein expression levels of p35,p25 and CDK5,causing accumulation and phosphorylation of p53 protein,and finally inducing SK-N-SH nerve cells undergo apoptosis.