The Role Of RNA Demethylase FTO Regulates The Effect Of EAAT3 On Manganese-induced Excitotoxicity Of SH-SY5Y Cells

Objective:Manganese(Mn)is one of the essential trace elements of the human body,but prolonged exposure to manganese will cause manganese to accumulate in the body and cause manganese poisoning.In the early stage of manganese poisoning,the symptoms mainly include slight paroxysmal dizziness and pain on the head,soreness in the limbs,inconvenience,degeneration of movement,etc.,and the emotional performance is unstable and irritable.Excessive manganese accumulation in the brain can increase glutamate(Glu)between neurons and induce excitotoxicity.In recent years,as a research hotspot,N6-methyladenosine(m~6A)methylation modification plays an important biological function in the nervous system,and fat mass and obesity-associated protein(FTO)is important The mRNA demethylase of serotonin plays a very important biological function in the nervous system.Nuclear factor kappa B(Nuclear factor kappa beta,NF-κB),as a nuclear transcription family,maintains its normal transcription function and plays an irreplaceable role in limiting the expression of harmful genes in the body.This study aims to study the role of FTO in manganese-induced excitotoxicity of SH-SY5Y cells,provide new theories and new strategies for the prevention and treatment of manganese poisoning,and lay a laboratory basis for exploring the mechanism of neurodegenerative diseases induced by environmental factors.Methods:SH-SY5Y cells are used for the experiment.MnCl2 exposed for 24h to establish a cell infection model.The FTO inhibitor MA2 was used to establish a FTO inhibitory cell model.Lentiviral transfection was used to construct FTO overexpression cell model.After treatment with manganese and intervention agent,immunofluorescence observation of NF-κB into the nucleus,observation of the cell structure and morphology of each group under a microscope;detection of LDH release of each group;extraction of cell protein to detect NF-κB,FTO and EAAT3 Protein expression.Cell mRNA was extracted to detect the mRNA expression of NF-κB,FTO and EAAT3.Me RIP-PCR detects the expression of EAAT3 m~6A mRNA;extracts the cell culture fluid and cell suspension of each group to detect the Glu content.Results:After the manganese staining treatment,it was found under the microscope that the efficiency of NF-κB into the nucleus increased with the increase of the manganese staining dose,the expression of P-NF-κB protein increased,the expression of NF-κB protein decreased,and the expression of P-NF-κB/NF-κB ratio decreased;LDH release increased;extracellular Glu content increased;Western blotting and q RT-PCR results showed that manganese can inhibit the expression of FTO and EAAT3,FTO inhibitor MA2 constructed FTO inhibitory cell model and lentivirus construction The FTO overexpression cell model further verified the important role of FTO in regulating EAAT3 in manganese-induced excitotoxicity of SH-SY5Y cells.Conclusion:1.Mn can activate NF-κB in SH-SY5Y cells to down-regulate the expression of FTO.2.Mn can up-regulate the expression of EAAT3 m~6A mRNA in SH-SY5Y cells through FTO.3.Mn can down-regulate EAAT3 protein and mRNA expression.