Study On The Role And Mechanism Of TDCS-Regulated NSD3 In Ischemic Brain Injury

Objective: To investigate the effect of transcranial Direct Current Stimulation(tDCS)on NSD3 and pineal melatonin synthesis in vivo model of ischemic brain injury.Combined with in vitro and in vivo models of ischemic brain injury,the effects of endogenous melatonin synthesis promoted by tDCS on MT1/NSD3/NRF2 signaling pathway were studied to explore the mechanism of neuroprotective effect of tDCS,providing a new potential target for the treatment of ischemic brain injury.Methods: Oxygen glucose deprivation(OGD)model and middle cerebral artery occlusion(MCAO)model were used as in vivo and in vitro models of ischemic brain injury.(1)To determine the changes and effects of histone methyltransferase NSD3 after ischemic brain injury.Western Blot(WB)was used to detect the protein expression level of NSD3 at various timepoints after OGD injury in SH-SY5 Y cells and 6 h postMCAO in the penumbra of mouse.The fluorescence intensity of NSD3 in the penumbra was detected by immunofluorescence staining.Primary neurons insulted by OGD were treated with BI-9321,an NSD3 inhibitor,and the effect on cell survival was detected by CCK-8.(2)To determine whether tDCS plays a neuroprotective role by NSD3.The effect of BI-9321,an inhibitor of NSD3,on infarction volume which was detected by triphenyl tetrazole chloride(TTC)staining in mice 24 h post-MCAO.(3)To determine whether tDCS plays a neuroprotective role by regulating melatonin synthesis and NSD3 related pathways.The levels of NSD3 in the penumbra and H3K36me2 in the nucleus were detected by WB.Quantitative PCR was used to detect the effect of tDCS on the transcription level of AANAT gene,which encodes the rate-limiting enzyme of pineal melatonin synthesis.(4)To determine whether regulation of melatonin receptor(MT1)affects NSD3/NRF2 signaling pathway and plays a neuroprotective role.The neurons insulted by OGD were treated with melatonin,and the fluorescence intensity of NSD3 was detected by immunofluorescence staining and the protein expression level of NSD3 was detected by WB.After injecting MT1 receptor agonist and NSD3 inhibitor into the tail vein of MCAO insulted mice,the expression level of NRF2 was detected by WB and the infarct ion volume was detected by TTC staining.NSD3 inhibitor and NRF2 inhibitor were injected into the tail vein of MCAO insulted mice after injection of MT1 receptor agonist.Modified Neurological Severity Scores(m NSS)were used to evaluate the motor function of mice.Results:(1)Compared with the Non-OGD group,the expression level of NSD3 began to decrease at 6 h after OGD injury in SH-SY5 Y cells.Compared with the sham group,the expression level of NSD3 protein in penumbra was significantly decreased 6 h after ischemic brain injury,and the fluorescence intensity of NSD3 in neurons in penumbra was significantly decreased 6 h after ischemic brain injury.After treatment with BI-9321,the survival rate of primary neurons decreased significantly.(2)tDCS intervention decreased the infarction volume of mice with ischemic brain injury,whereas the injection of BI-9321 increased the infarction volume of mice,suggesting that the neuroprotective effect of tDCS may be mediated by NSD3-related pathways.(3)Following ischemic brain injury,tDCS upregulated the expression level of NSD3 protein in penumbra and the level of H3K36me2 in the nucleus,and significantly increased the transcription level of pineal AANAT gene.(4)Melatonin treatment upregulated the expression level of NSD3 after OGD injury in primary neurons;Compared with MCAO+Vehicle group,MT1 receptor agonist 2-Iodomelatonin promoted NRF2 expression in penumbra,reduced infarction volume(31.09±5.25% vs.21.39±4.90%,P<0.05))and improved the motor function of mice 14 days post-MCAO.At the same time of 2-Iodomelatonin intervention NSD3 injection or NRF2 inhibitors after each timepoint in mice the m NSS was no significant difference compared with MCAO group.Conclusions:(1)The expression of NSD3 decreased at 6 h after ischemic brain injury,and the survival rate of primary neurons was decreased by BI-9321 treatment.(2)tDCS decreased infarction volume post-MCAO,which may be achieved by regulating melatonin synthesis and up-regulating NSD3.(3)Melatonin,through its receptor MT1,plays a neuroprotective role by upregulating the expression of NSD3 and NRF2,reducing infarction volume and improving the motor function of mice post-MCAO.