Hyperactivation of microglia in response to pathogens or injuries is crucial in inflammation of the brain. This process is implicated in the release of proinflammatory mediators and neuronal damage. Nuclear factor erythroid 2- related factor (Nrf2) has emerged as a key factor in regulating inflammation in the central nervous system (CNS) in response to microglia activation by regulating genes transcription involving Heme-oxygenase-1 (HO-1) and NAD(P)H-Quinone-oxidoreductase-1 (NQO-1) thereby producing cytoprotective proteins. This potential crosstalk between neuroinflammation and Nrf2 activation has resulted in the identification of small molecules which can activate Nrf2 to prevent or reduce excessive release of pro-inflammatory mediators. The focus of this research was to investigate the potential inhibitory properties of two Nrf2 activators, oltipraz and cheirolin on the release of pro- inflammatory mediators in lipopolysaccharide (LPS)-stimulated BV- 2 microglia. Oltipraz (5, 10, and 20 µM) or cheirolin (1.65, 2.5, 5 µM) were used to pre- treat LPS- stimulated BV-2 microglia for 24 hours. Cultured supernatants were used to quantify the levels of pro-inflammatory cytokines using ELISA, while the Griess assay was used to detect nitrite production. PGE2 levels in supernatants were determined using enzyme immunoassay (EIA). Lysates obtained from cells were also assayed for levels of inducible nitric oxide synthase (iNOS), phospho-p65 proteins, and cyclooxygenase (COX-2) using mouse ELISA. Results showed that oltipraz and cheirolin inhibited the released of pro-inflammatory mediators in LPS-activated BV-2 microglia. Oltipraz 10 µM produced a significantly (p<0.0001) reduction in interleukin (IL)-6 and tumour necrosis factor- alpha (TNF-α) production while also markedly (p<0.0001) reducing the release of nitrite. Analyses of culture supernatants showed that oltipraz markedly (p<0.0001) reduced prostaglandin E2 (PGE2) at all concentrations investigated. Also, oltipraz (10 µM) showed significant (p<0.0001) reduction of iNOS and COX-2 protein. Cheirolin 2.5 µM significantly (p<0.0001) decreased IL-6 and TNF-α production while also significantly (p<0.0001) reduced the nitrite released. Furthermore, cheirolin markedly reduced (p<0.0001) PGE2 levels and significantly (p<0.0001) inhibited iNOS protein expression as concentration increased following LPS activation of BV- 2 microglia. Investigation of COX-2 protein revealed that cheirolin (1.65 µM) did not produce significant suppression when compared with LPS-induced enhanced COX-2 expression. However, on increasing the concentration to (2.5, and 5 µM), there was a significant (p<0.05) suppression of COX-2 protein. These effects of oltipraz and cheirolin were shown to be mediated through significant (p<0.0001) inhibition of nuclear factor kappa B (NF- κB) phospho-p65 signalling. The outcome of this study suggests that Nrf2 activators, oltipraz and cheirolin inhibit neuroinflammation in LPS- activated BV-2 microglia.
| Date of Award | 15 Feb 2024 |
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| Original language | English |
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| Supervisor | Olumayokun Olajide (Main Supervisor) |
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