Upregulation of antioxidant and autophagy pathways via NRF2 activation protects spinal cord neurons from ozone damage

Ozone therapy can relieve multiple types of pain but exhibits potential neurotoxicity, the mechanism of which is unclear. The present study aimed to identify the role of nuclear factor (erythroid-derived-2)-related 2 (NRF2) in preventing spinal cord injury caused by ozone overdose. Primary neuronal cells were extracted from newborn Wistar rats and authenticated by immunofluorescence using anti-microtubule-associated protein 2 as a cell type-specific marker. Cell viability assay with different ozone concentrations (0, 10, 20, 30 and 40 µg/ml) was used to determine the concentration that caused primary neuron injury; 30 min of 40 µg/ml ozone therapy notably decreased cell viability to 71%. In order to test the effects of ozone, the cells were divided into five treatment groups [0-, 30- and 40 µg/ml ozone, tert-butylhydroquinone (tBHQ) + 40 µg/ml ozone (T40) and tBHQ (T0)]. Cells in the T40 and T0 groups received 40 µmol/l tBHQ on the fifth day of SCN cultivation. Reverse transcription-quantitative PCR and western blotting showed that protein expression levels of heme oxygenase-1 (HO-1) and mRNA expression levels of HO-1 and NRF2 were decreased. NRF2, ubiquitin-binding protein p62 and microtubule-associated proteins 1A/1B light chain 3B expression levels were decreased following treatment with 40 µg/ml ozone. Immunofluorescence showed that NRF2 nuclear expression levels also decreased following 40 µg/ml ozone treatment. However, cells in the T40 group did not display decreased NRF2 nuclear expression levels. Normal/Apoptotic/Necrotic Cell Detection kit revealed that necrosis rate increased following treatment with 40 µg/ml ozone; however, the T40 group did not exhibit this increased necrosis. At 40 µg/ml, ozone increased spinal cord neuron (SCN) death in vitro. Moreover, treatment with 40 µg/ml ozone damaged SCNs. The p62/NRF2/antioxidant response element pathway prevented such injury. tBHQ activated this pathway, upregulated autophagy and increased local nuclear NRF2 concentration, thus enhancing the antioxidant system to protect SCNs from injury caused by high concentrations of ozone.