Silent information regulator 1 (SIRT1) exerts neuroprotection in many neurodegenerative diseases. However, it is not clear if SIRT1 has protective effects after intracerebral hemorrhage (ICH)-induced brain injury in rats. Thus, our goal was to examine the influence of SIRT1 on ICH injuries and any underlying mechanisms of this influence. Brain injury was induced by autologous arterial blood (60 μL) injection into rat brains, and data show that activation of SIRT1 with SRT1720 (5 mg/kg) restored nuclear SIRT1, deacetylation of PGC-1α, and mitochondrial biogenesis and decreased mortality, behavioral deficits, and brain water content without significant changes in phosphorylated AMP-activated protein kinase (pAMPK) induced by ICH. Activation of SIRT1 with SRT1720 also restored mitochondrial electron transport chain proteins and decreased apoptotic proteins in ICH; however, these changes were reversed after ICH. In contrast, treatment with PGC-1α siRNA yielded opposite effects. To explore the protective effects of SIRT1 after ICH, siRNAs were used to knockdown SIRT1. Treatment with SIRT1 siRNA increased mortality, behavioral deficits, brain water content, mitochondrial dysfunction, and neurocyte apoptosis after ICH. Thus, activation of SIRT1 promotes recovery of mitochondrial protein and function by increasing mitochondrial biogenesis and reduces apoptosis after ICH via the PGC-1α mitochondrial pathway. These data may suggest a new therapeutic approach for ICH injuries.

The NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway plays a critical role in protecting against oxidative stress in brain ischemia and reperfusion injury. Glycogen synthase kinase 3β (GSK-3β) may play a critical role in regulating Nrf2 in a Kelch-like ECH-associated protein 1 (Keap1)-independent manner. However, the relationship between GSK-3β and Nrf2 in brain ischemia and reperfusion injury is not clear. In this study, we explored the mechanisms through which GSK-3β regulates Nrf2 and Nrf-2/ARE pathways in vitro and in vivo. We used oxygen and glucose deprivation/reoxygenation (OGD/R) in primary cultured cortical neurons and a middle cerebral artery occlusion-reperfusion (MCAO/R) rat model to mimic ischemic insult. In this study, GSK-3β siRNA and inhibitors (SB216763 and LiCl) were used to inhibit GSK-3β in vitro and in vivo. After inhibiting GSK-3β, expression of total and nuclear Nrf2, Nrf2-ARE binding activity and expression of Nrf2/ARE pathway-driven genes HO-1 and NQO-1 increased. Overexpression of GSK-3β yielded opposite results. These results suggest that GSK-3β downregulates Nrf2 and the Nrf2/ARE pathway in brain ischemia and reperfusion injury. GSK-3β may be an endogenous antioxidant relevant protein and may represent a new therapeutic target in treatment of ischemia and reperfusion injury.

Background: Nod-like receptor protein 3 (NLRP3) inflammasome is a crucial contributor in the inflammatory process during cerebral ischemia/reperfusion (I/R) injury. ATF4 plays a pivotal role in the pathogenesis of cerebral I/R injury, however, its function and underlying mechanism are not fully characterized yet. In the current study, we examined whether ATF4 ameliorates cerebral I/R injury by inhibiting NLRP3 inflammasome activation and whether mitophagy is involved in this process. In addition, we explored the role of parkin in ATF4-mediated protective effects. Method: To address these issues, healthy male adult Sprague-Dawley rats were exposed to middle cerebral artery occlusion for 1 h followed by 24 h reperfusion. Adeno-associated virus (AAV) and siRNA were injected into rats to overexpress and knockdown ATF4 expression, respectively. After pretreatment with AAV, mdivi-1(mitochondrial division inhibitor-1) was injected into rats to block mitophagy activity. Parkin expression was knockdown using specific siRNA after AAV pretreatment. Result: Data showed that ATF4 overexpression induced by AAV was protective against cerebral I/R injury, as evidenced by reduced cerebral infraction volume, decreased neurological scores and improved outcomes of HE and Nissl staining. In addition, overexpression of ATF4 gene was able to up-regulate Parkin expression, enhance mitophagy activity and inhibit NLRP3 inflammasome-mediated inflammatory response. ATF4 knockdown induced by siRNA resulted in the opposite effects. Furthermore, ATF4-mediated inhibition of NLRP3 inflammasome activation was strongly affected by mitophagy blockage upon mdivi-1 injection. Besides, ATF4-mediated increase of mitophagy activity and inhibition of NLRP3 inflammasome activation were effectively reversed by Parkin knockdown using siRNA. Conclusion: Our study demonstrated that ATF4 is able to alleviate cerebral I/R injury by suppressing NLRP3 inflammasome activation through parkin-dependent mitophagy activity. These results may provide a new strategy to relieve cerebral I/R injury by modulating mitophagy-NLRP3 inflammasome axis.

Nurr1 is a member of the nuclear receptor 4 family of orphan nuclear receptors that is decreased in inflammatory responses and leads to neurons death in Parkinson’s disease. Abnormal expression of Nurr1 have been attributed to various signaling pathways, but little is known about microRNAs (miRNAs) regulation of Nurr1 in ischemia/reperfusion injury. To investigate the post transcriptional regulatory networks of Nurr1, we used a miRNA screening approach and identified miR-145-5p as a putative regulator of Nurr1. By using computer predictions, we identified and confirmed a miRNA recognition element in the 3′UTR of Nurr1 that was responsible for miR-145-5p-mediated suppression. We next demonstrated that overexpression of Nurr1 inhibited TNF-α expression in microglia by trans-repression and finally attenuated ischemia/reperfusion-induced inflammatory and cytotoxic response of neurons. Results of further in vivo study revealed that anti-miR-145-5p administration brought about increasing expression of Nurr1 and reduction of infarct volume in acute cerebral ischemia. Administration of anti-miR-145-5p promotes neurological outcome of rats post MCAO/R. It might be an effective therapeutic strategy to relieve neurons injury upon ischemia/reperfusion of rats through interrupting the axis signaling of miR-145-5p- Nurr1-TNF-α in acute phase.