METTL3 attenuated bupivacaine-induced neurotoxicity by enhancing m6A-modified BDNF expression

Bupivacaine (BUP), a widely used amide-type local anesthetic, exhibits neurotoxic effects. This study aimed to explore the functions of brain-derived neurotrophic factor (BDNF) and methyltransferase Like 3 (METTL3) in BUP-induced hippocampal neuronal damage. HT22 cells and SH-SY5Y cells were treated with various concentrations of BUP. METTL3 and BDNF were manipulated using either overexpression or knockdown approaches to assess their functional roles. Cell viability, apoptosis, mitochondrial membrane potential and oxidative stress markers (Lactate Dehydrogenase (LDH), Reactive Oxygen Species (ROS), Superoxide Dismutase (SOD), Malondialdehyde (MDA)) were evaluated using Cell Counting Kit-8 (CCK-8), flow cytometry, JC-1 staining and commercial kits. The expression of BDNF, METTL3, Caspase-9, Bax and Bcl-2 was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. The N6-methyladenosine (m6A) modification of BDNF mRNA was assessed using Methylated RNA Immunoprecipitation (Me-RIP) and commercial kits. BUP treatment dose-dependently reduced viability, while increasing oxidative stress and apoptosis in our cellular model. BDNF expression was down-regulated in BUP-induced cells. Additionally, BUP stimulation suppressed both total m6A levels and METTL3 expression in cell models. Overexpression of BDNF ameliorated BUP-induced cell damage. METTL3 stabilized BDNF through m6A modification, and the depletion BDNF reversed the protective effect of overexpressing METTL3 on BUP-induced neurotoxicity. Together, our results indicated that METTL3 attenuated BUP-induced neurotoxicity by enhancing BDNF expression via m6A modification.