S-allyl Cysteine Activates The Nrf2-dependent Antioxidant Response And Protects Neurons Against Ischemic Injury In Vitro And In Vivo

BackgroundIschemic stroke is a devastating clinical condition for which an effective neuroprotective treatment is currently unavailable, There is increasing evidence showing that oxidative stress plays an important role in cerebral ischemic pathogenesis, reducing oxidative damage is one of the most promising strategies for the treatment of ischemic stroke.The nuclear transcription factor NF-E2-related factor 2 (Nrf2) plays a central role in the regulation of the cellular redox status. As one of the master regulators of endogenous antioxidant defenses, Nrf2 can induce the expression of a variety of cytoprotective and detoxification genes. It has great potential in combating oxidative stress injury. Several of the genes commonly regulated by Nrf2 have been implicated in protection neurons against ischemic damage, such as hemeoxygenase(HO-1), glutathione cysteine ligase regulatory subunit(GCLC) and glutathione cysteine ligase modulatory subunit (GCLM). Therefore, Nrf2 can be a valuable therapeutic target for the treatment of ischemic stroke.Numerous evidence is presented to demonstrate that the Nrf2-ARE pathway plays a critical role in the pathogenesis of ischemic/reperfusion brain injury. Looking for novel and high efficient Nrf2 inducers can provide new and effective candidate drugs for the treatment of cerebral ischemia. Our preliminary data showed that S-allyl cysteine(SAC) isolated from garlic is a robust Nrf2 activator and has potential therapeutic effects against OGD injury through activation of the Nrf2. We used primary neurons to examine the protective potential of SAC in vitro, and test whether it can attenuate OGD injury in cerebral ischemia model of C57BL/6 mice in vivo. The objective of the study was to investigate whether SAC can ameliorate OGD injury in primary cultures and animal models of cerebral ischemia and how Nrf2-ARE pathway is involved. This study will be valuable for future development of new therapeutic strategies for the treatment of cerebral ischemia.ObjectivesThe objective of this study was to investigate whether SAC activate the Nrf2-dependent antioxidant response and protects neurons against ischemic injury in vitro and in vivo and to explore its mechanism of action.Materials and Methods In vitro studies1. Primary neurons were either treated with indicated doses of SAC for 8 h and 24 h or treated with 50 μM of SAC for different time points, western blot was used to detect the expression of Nrf2 protein levels and the downstream (GCLC, GCLM, HO-1) protein levels. EMSA was used to determine the DNA-binding capacity of Nrf2.2. Primary neurons were pretreated with SAC for 2 h at the indicated concentrations and subjected to 60 min OGD followed at 24 h. Then, cell viability was determined by MTT assay. LDH level was examined by the chemical chromogenic assay. Hoechst 33258 and TUNEL staining was used to test cells apoptosis. To detect whether SAC has a protective effect on OGD-induced primary neurons damage, western blot was performed to detect proteins levels involved in apoptosis pathway (p-JNK, p-p38, cleaved-caspase-3).3. To detect whether SAC still has a protective effect after Nrf2 was knocked down, shRNA was used to knock down the Nrf2 expression. SAC (50 μM) was added to the cells, western blot was performed to detect Nrf2 protein levels 8 h later. Downstream (GCLC, GCLM, HO-1) protein levels were tested 24 h later. Primary neurons were pretreated with SAC (50 μM) for 2 h, then were challenged with OGD for 60 min.24 h later, cell viability was determined by MTT assay and LDH assay, while cell apoptosis was observed by Hoechst33258 staining.In vivo studies1. Nrf2+/+ and Nrf2-/- mice were pretreated with 300 mg/kg SAC intraperitoneally 30 min before the onset of MCAO. Immunohistochemical staining was carried out for Nrf2 expression in cerebral ischemic cortex and hippocampus at 24 h after MCAO. Western blot was performed to detect the Nrf2 and downstream (GCLM, HO-1, GCLC) protein expression levels in cerebral ischemic cortex and hippocampus.2. Transient middle cerebral artery occlusion (MCAO) was induced in twelve-week-old male Nrf2-/- and Nrf2+/+ mice. TTC staining was used to calculate the infarct volume and behavioral change was observed by neurological deficit scores, HE staining and Nissl staining were used to show morphological changes of injured neurons in the cerebral cortex and hippocampus.3. Mice were pretreated with 300 mg/kg SAC intraperitoneally 30 min before the onset of MCAO. Whole brain or cortex and hippocampus were taken from the mice which were sacrificed 24 h after reperfusion. TUNEL staining was used to detect the change of the number of apoptotic cells in the cortex and hippocampus. Western blot was performed to detect proteins levels involved in apoptosis pathway (p-JNK, p-p38, cleaved-caspase-3).ResultsThe main conclusions were as follows:1. SAC induced nuclear translocation of Nrf2 and the expression of ARE-regulated genes in primary neurons.2. Compared to vehicle treated control cells, pretreatment of cells with SAC significantly reduced OGD-associated cell death as well as LDH release in a dose-dependent manner. This was further confirmed by Hoechst 33258 staining and TUNEL staining. SAC treatment inhibited the phosphorylation of both JNK and p38 and subsequently inhibited the activation of pro-apoptotic caspase-3 after OGD exposure.3. The Nrf2 shRNA treatment significantly reduced Nrf2 level in nuclear extracts from neuronal cells treated with SAC, and decreased the up-regulation of its downstream genes, HO-1, GCLC and GCLM. The results showed that knockdown of Nrf2 significantly blocked the protective effects of SAC, indicating a contribution of Nrf2 to SAC-dependent protection against oxidative stress in primary neurons.4. SAC was effective at upregulating protein levels of Nrf2 and Nrf2 target genes in Nrf2+/+ mice but not Nrf2-/- mice, as measured by both immunohistochemistry and immunoblot analysis.5. Both the infarct volumes and the neurological deficit scores were significantly less than those in the vehicle-treated mice at 24 h after reperfusion in SAC-treated Nrf2+/+ mice, while no improvement was observed in SAC-treated Nrf2-/- mice. SAC treatment significantly improved neuronal survival and retention of Nissl substance in Nrf2+/+ but not in Nrf2-/- mice.6. The number of TUNEL-positive cells was significantly reduced by SAC treatment in Nrf2+/+ mice, whereas the protective effect of SAC was not obvious in Nrf2-/- mice. SAC pretreatment significantly decreased MCAO-induced activation of JNK and p38 signaling pathways in Nrf2+/+ mice, yet this effect was not observed in Nrf2-/- mice.ConclusionsThe results of our study reveal that SAC activates the Nrf2 antioxidant responses in primary neurons and protects them from ischemic injury in vitro and in vivo.