Neuroprotection Of Nrf2-ARE Activation Following Spinal Cord Injury In Rats

ChapterⅠIntroductionIn this chapter, the background of this study is introduced, including the reviews of spinal cord injury, structure and activation of Keap1-Nrf2-ARE pathway, neuroprotection of Nrf2, interactions between Nrf2 and NF-κB and pharmacokinetic of sulforaphane. Chapter II Activation of the Nrf2-ARE Pathway is Neuroprotective Following Spinal Cord InjuryThe activation of oxidative damage, neuroinflammation and mitochondrial dysfunction has been implicated in secondary pathomechanisms following spinal cord injury (SCI). These pathophysiological processes lead to cell death and are tightly regulated by nuclear factor E2 related factor 2 antioxidant response element (Nrf2-ARE) signaling. Here, we investigated whether activation of Nrf2-ARE is neuroprotective following SCI. Female Fischer rats were subjected to mild thoracic SCI (T8) using the New York University injury device. As early as 30 min after SCI, levels of Nrf2 transcription factor were increased in both nuclear and cytoplasmic fractions and remained elevated for 3 days. Treatment of injured rats with sulforaphane (SFN), an activator of Nrf2-ARE signaling, significantly increased levels of Nrf2 and glutamate-cysteine ligase (GCL), a rate-limiting enzyme for synthesis of glutathione, and decreased levels of inflammatory cytokines, interleukin-1β(IL-1β) and tumor necrosis factor-a (TNF-α) thus leading to a reduction in contusion volume and improvement in coordination. These results show that activation of the Nrf2-ARE pathway following SCI is neuroprotective and that SFN presents a viable compound for neurotherapeutic intevention in blocking pathomechanisms following SCI. ChapterⅢSulforaphane suppresses ATP-induced nuclear factor-κB activation via Nrf2 in primary rat neuronsPrevious studies have shown treatment with sulforaphane (SFN), an activator of nuclear factor E2-related factor 2 (Nrf2), significantly increased levels of Nrf2 and downstream signalings, and decreased levels of inflammatory cytokines by suppressing nuclear factor-KB (NF-κB) pathway. However, the precise mechanism is not fully documented. The role of this study was to investigate the molecular mechanisms of Nrf2 involved in anti-inflammation by depressing NF-κB in primary rat neurons. Dosage of 1 mM ATP was added for 45 min to induce NF-κB activation. Cells were pretreated without SFN, or with SFN (10μM, 20μM) for 2 hrs. Immunoblotting was performed to detect levels of Nrf2 and downstream signalings, cytokines, and phospho-1κBα. SFN increased levels of Nrf2 and downstream signalings, and attenuated the ATP-induced increase of Caspase-1, IL-1βand TNF-α, in a concentration-dependent manner. Consequently, phosphorylation of IκBαand synthesis of pro-IL-1βwere inhibited, which indicated NF-κB pathway was depressed. This study suggests one possible molecular mechanism of Nrf2 anti-inflammation ability by regulating NF-κB. Chapter IVExpression of Nrf2-ARE in neurons, astrocytes and microglia after injury:an in vitro studyIn Chapter II, it is demonstrated by using laser scanning confocal scanning, that Nrf2 is activated in motor neurons, astrocytes, oligodendrocytes and microglia in the ventral horn of cords 1 day following spinal cord injury (SCI) in rats. However, Nrf2 expression pattern of each cell type after injury remains unknown. In this chapter, a controlled cell injury model was employed to investigate Nrf2 expression in rat neurons, astrocytes and microglia. Immunoblotting was carried out to determine levels of Nrf2 protein and downstream proteins. Nrf2-ARE was activated 15 min after injury in neurons; In astrocytes, trauma decreased Nrf2 levels time-dependently, no significant changes were observed in HO-1 and NQ01; Nrf2 activation is delayed than in neurons following injury. This study described the Nrf2 expression in each cell type after injury, however further studies are needed to accomplish the precise mechanisms of Nrf2 neuroprotection in each cell type and the interactions between cell types after injury.