The Changes Of Protein Phosphatase 1 And Its DNA Methylation In Hypoxic Preconditioned Mice Brain And Nervous Cell

Objective The aim of this study is to detect the roles of Protein Phosphatase-1? and its DNA methylation in hypoxic neuroprotection using hypoxia preconditioning animal and cell models that treated with 5-aza-2~?deoxycytidine(5-aza-cdR,a hibitor of DNMTs).Methods(1)NG108-15 cell model: NG108-15 cell line was in exponential growth stage and divided into two groups randomly: experimental group(10 ?mol/L 5-aza-cdR)and control group(isopyknic 0.1% BSA).Then they were exposed to hypoxia or hypoxic preconditioning two days later after treatment;First,cells morphologys were observed by inverted microscope;Second,the proliferation of NG108-15 was tested using the RTAC;Third,cell cycles and cell apoptosis were analyzed by flow cytometry analysis;Fourth,Real-time PCR was used to detect the mRNA levels of DNMTs and PP1?;Fifth,PP1? protein level was tested by Western-blot;Last,PP1? transcriptional activity was detected.(2)Mice model: Mice were divided into two groups: experimental group(10 ?mol/L 5 ?L 5-aza-cdR was injected into lateral ventricles)and control group(isopyknic 0.1% BSA).Then they were exposed to hypoxia or hypoxic preconditioning two days later after treatments;First,hippocampus were isolated after mice were hypoxia 0-4 days;Second,mRNA levels of DNMTs and PP1? were tested by Real-time PCR;Last,PP1? protein level was detected by Western-blot.Results Cell model:(1)Cells proliferation was restrained by 5-aza-cdR and hypoxic.(2)5-aza-cdR and hypoxic promoted cell early apoptosis,and have not significant influence on cell later apoptosis.(3)The mRNA level of DNMT1 was up-regulated after hypoxia proconditioning,and the mRNA level of DNMT1 was unchanged in 5-aza-cdR.The mRNA level of DNMT3A was increased after hypoxia,and the mRNA level of DNMT3A was restrained by 5-aza-cdR;the mRNA level of DNMT3B was unchanged by 5-aza-cdR or hypoxia;the mRNA level of PP1? was increased in hypoxia and was decreased by DNA methyltransferases.(4)The protein level of PP1? was down-regulated by DNA methyltransferases.And this change does not be affected by the activity of DNA methyltransferases enzymes.(5)5-aza-cdR down-regulated the transcriptional activity of PP1?.Mice model:(1)The mRNA level of DNMT1 was down-regulated after one day since hypoxia,but those changes do not be affected by the activity of DNA methyltransferases enzymes.It was unchanged in 5-aza-cdR.(2)The mRNA level of DNMT3A was down-regulated after two days hypoxia,but this change doesn't be affected by the activity of DNA methyltransferases enzymes also.It was restrained by 5-aza-cdR.(3)The mRNA level of DNMT3B was down-regulated after one day and hypoxic preconditioning,and this change has something with the activity of DNA methyltransferases enzymes.(4)The mRNA and protein levels of PP1? were down-regulated in hypoxia.And those changes were affected by the activity of DNA methyltransferases enzymes.The mRNA and protein levels of PP1? were increased by 5-aza-cdR,and this up-regulated have relationship with hypoxia time.Conclusions Our results suggested that the reduced PP1? expression is related to learning and memory.It should depend on the change of DNMT3B under the Hypoxic preconditioning condition and have nothing to do with the apoptosis of nerve cells.