Fig. 5: Upregulating Ndufa1 rescues Hcy-induced mitochondrial dysfunction.

A–E The levels or activities of complex I, ATP, ROS, MDA, and SOD in the hippocampus of rats were measured using the corresponding kits. F The procedure of plasmid (Vector-pcDNA3.1 or Ndufa1-pcDNA3.1) transfection was carried out according to the instructions of the Lipo2000 agent. G, H The protein levels of Ndufa1 were detected and quantified by western blotting (n = 3 for each group). I The related kits tested relatively complex I activity after overexpressing Ndufa1 or Vector in N2a cells. J Respiratory oxygen consumption rate (OCR, pmol/min/500 cells) determined by Seahorse real-time cell metabolic analysis in N2a cells treated with/without Hcy and with/without OE-Ndufa1 plasmid. The cells were stimulated with 1.5 μM oligomycin (Oligo), 2 μM FCCP, and 0.5 μM antimycin A (Ant A) every 20 min during the test. K Measurement of basal respiration, ATP-linked respiration, non-mitochondrial respiration, proton leakage, maximum respiration, and spare respiration was analyzed by the software Wave Pro (Agilent, USA). L, M Representative flow cytometric analysis of mean content of ROS targeted by FITC-A from N2a cells. The data were analyzed by FlowJo (v7.5, USA) (n = 5 for each group). N, O MitoSOX was used to detect mitochondrial superoxide in N2a cells. Q, R MMP in N2a cells was measured by JC-1 dye. Red fluorescence (Aggregate) indicated higher MMP and green fluorescence (Monomer) indicated lower MMP (n = 5 for each group). P, S The related kits tested relative ATP and MDA levels after overexpressing Ndufa1 or Vector in N2a cells. Data were presented as mean ± SEM. One-way ANOVA followed by Bonferroni’s post hoc was used to analyze the data (*P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001, ns no significance; n = 6 for each group).