Neuroprotective mechanisms of DNA methyltransferase in a mouse hippocampal neuronal cell line after hypoxic preconditioning

Hypoxic preconditioning has been shown to improve hypoxic tolerance in mice,accompanied by the downregulation of DNA methyltransferases(DNMTs)in the brain.However,the roles played by DNMTs in the multiple neuroprotective mechanisms associated with hypoxic preconditioning remain poorly understood.This study aimed to establish an in vitro model of hypoxic preconditioning,using a cultured mouse hippocampal neuronal cell line(HT22 cells),to examine the effects of DNMTs on the endogenous neuroprotective mechanisms that occur during hypoxic preconditioning.HT22 cells were divided into a control group,which received no exposure to hypoxia,a hypoxia group,which was exposed to hypoxia once,and a hypoxic preconditioning group,which was exposed to four cycles of hypoxia.To test the ability of hypoxic preadaptation to induce hypoxic tolerance,cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium assay.Cell viability improved in the hypoxic preconditioning group compared with that in the hypoxia group.The effects of hypoxic preconditioning on the cell cycle and apoptosis in HT22 cells were examined by western blot assay and flow cytometry.Compared with the hypoxia group,the expression levels of caspase-3 and spectrin,which are markers of early apoptosis and S-phase arrest,respectively,noticeably reduced in the hypoxic preconditioning group.Finally,enzyme-linked immunosorbent assay,real-time polymerase chain reaction,and western blot assay were used to investigate the changes in DNMT expression and activity during hypoxic preconditioning.The results showed that compared with the control group,hypoxic preconditioning downregulated the expression levels of DNMT3A and DNMT3B mRNA and protein in HT22 cells and decreased the activities of total DNMTs and DNMT3B.In conclusion,hypoxic preconditioning may exert anti-hypoxic neuroprotective effects,maintaining HT22 cell viability and inhibiting cell apoptosis.These neuroprotective mechanisms may be associated with the inhibition of DNMT3A and DNMT3B.

Potassium bisperoxo(1,10-phenanthroline) oxovanadate suppresses proliferation of hippocampal neuronal cell lines by increasing DNA methyltransferases

Bisperoxo(1,10-phenanthroline) oxovanadate(BpV) can reportedly block the cell cycle. The present study examined whether BpV alters gene expression by affecting DNA methyltransferases(DNMTs), which would impact the cell cycle. Immortalized mouse hippocampal neuronal precursor cells(HT_(22)) were treated with 0.3 or 3 μM BpV. Proliferation, morphology, and viability of HT_(22) cells were detected with an IncuCyte real-time video imaging system or inverted microscope and 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium, respectively. mRNA and protein expression of DNMTs and p21 in HT_(22) cells was detected by real-time polymerase chain reaction and immunoblotting, respectively. In addition, DNMT activity was measured with an enzyme-linked immunosorbent assay. Effects of BpV on the cell cycle were analyzed using flow cytometry. Results demonstrated that treatment with 0.3 μM BpV did not affect cell proliferation, morphology, or viability; however, treatment with 3 μM BpV decreased cell viability, increased expression of both DNMT3B mRNA and protein, and inhibited the proliferation of HT_(22) cells; and 3 μM BpV also blocked the cell cycle and increased expression of the regulatory factor p21 by increasing DNMT expression in mouse hippocampal neurons.