Activation of the Akt/FoxO3 signaling pathway enhances oxidative stress-induced autophagy and alleviates brain damage in a rat model of ischemic stroke.

Autophagy has been implicated in stroke. Our previous study showed that the FoxO3 transcription factor promotes autophagy after transient cerebral ischemia/reperfusion (I/R). However, whether the Akt/FoxO3 signaling pathway plays a regulatory role in autophagy in cerebral I/R-induced oxidative stress injury is still unclear. The present study aims to investigate the effects of the Akt/FoxO3 signaling pathway on autophagy activation and neuronal injury in vitro and in vivo. By employing LY294002 or insulin to regulate the Akt/FoxO3 signaling pathway, we found that insulin pretreatment increased cell viability, decreased reactive oxygen species production, and enhanced the expression of antiapoptotic and autophagy-related proteins following H2O2 injury in HT22 cells. In addition, insulin significantly decreased neurological deficit scores and infarct volume and increased the expression of antiapoptotic and autophagy-related proteins following I/R injury in rats. However, LY294002 showed the opposite effects under these conditions. Altogether, these results indicate that Akt/FoxO3 signaling pathway activation inhibited oxidative stress-mediated cell death through activation of autophagy. Our study supports a critical role for the Akt/FoxO3 signaling pathway in autophagy activation in stroke.

ErbB4 processing is involved in OGD/R induced neuron injury.

OBJECTIVE: Our previous study found that ErbB4 gene expression was changed after oxygen-glucose deprivation/reperfusion (OGD/R). However, the exact role and mechanism of ErbB4 in brain ischemia are largely unknown. In this study, we explored the protective effects of ErbB4 and its possible mechanism after OGD/R. METHODS: Cerebral ischemia/reperfusion (I/R) injury model was established in vitro and in vivo. Cell viability, apoptosis, and ROS production were measured by MTT, TUNEL, and fluorescent probe 2', 7'-dichlorofluorescein diacetate (DCFH-DA). Infarct size was evaluated by TTC. We performed bioinformatics analyses to screen for novel key genes involved in ErbB4 changes. RNA-Seq was used to transcriptome analysis. RNA and protein expression were detected by quantitative RT‒PCR and western bloting. RESULTS: The expression of 80-kDa ErbB4 decreased after cerebral I/R injury in vitro and in vivo. Co-expression network analysis revealed that ErbB4 expression was correlated with the changes in Adrb1, Adrb2, Ldlr, and Dab2. Quantitative RT‒PCR revealed that the mRNA expression levels of Adrb1, Adrb2, and Dab2 were upregulated, and that of Ldlr was decreased after OGD/R. Activation of ErbB4 expression by neuregulin 1 (NRG1) significantly promoted cell survival, attenuated hippocampal apoptosis, and decreased ROS production after OGD/R. Furthermore, the elimination of ErbB4 using a specific siRNA reversed these beneficial effects. CONCLUSION: Our data revealed the neuroprotective effects of ErbB4 against OGD/R injury, and the action could be related to changes in the ErbB4 membrane-associated fragment and the expression of Adrb1, Adrb2, Ldlr, and Dab2.

FoxO3 transcription factor promotes autophagy after oxidative stress injury in HT22 cells.

Autophagy has been implicated in neurodegenerative diseases. Forkhead box O3 (FoxO3) transcription factors promote autophagy in heart and inhibit oxidative damage. Here we investigate the role of FoxO3 transcription factors in regulating autophagy after oxidative stress injury in immortalized mouse hippocampal cell line (HT22). The present study confirms that hydrogen peroxide (H2O2) injury could induce autophagy and FoxO3 activation in HT22 cells. In addition, overexpression of FoxO3 enhanced H2O2-induced autophagy activation and suppressed neuronal cell damage, while knockdown of FoxO3 reduced H2O2-induced autophagy activation and exacerbated neuronal cell injury. Inhibition of autophagy by 3-methyladenine (3-MA) resulted in reduced cell viability, increased production of reactive oxygen species (ROS), promoted nuclear condensation, and decreased expression of antiapoptotic and autophagy-related proteins, indicating that autophagy may have protective effects on H2O2-induced injury in HT22 cells. Moreover, overexpression of FoxO3 prevented exacerbation of brain damage induced by 3-MA. Taken together, these results show that activation of FoxO3 could induce autophagy and inhibit H2O2-induced damage in HT22 cells. Our study demonstrates the critical role of FoxO3 in regulating autophagy in brain.

FoxO3 transcription factor promotes autophagy after transient cerebral ischemia/reperfusion.

Aim: Autophagy was activated after cerebral ischemia reperfusion (I/R) injury. However, the molecular mechanisms underlying regulation of autophagy in cerebral I/R injury were not completely understood. Studies reported that Forked-box class O (FoxO) transcription factors involved in autophagy and might be the regulator of autophagy in multiple cells. In this study, we investigated the effects of FoxO3 on regulating autophagy after cerebral I/R injury. Materials and methods: Rats were subjected to MCAO for 2 h and reperfusion for different times, western blot was used to examine the expression of p-FoxO3, FoxO3 and the autophagic marker LC3 and Beclin-1 in penumbral region. Then rats were injected with WT-FoxO3 or TM-FoxO3 adenovirus by lateral cerebral ventricle to increase the function of FoxO3, western blot was used to examine the expression of LC3 and Beclin-1 in penumbral region. TTC and HE staining were used to evaluate the effects of increased FoxO3 activation on I/R induced brain damage. Results: Our studies showed that I/R injury resulted in induction of autophagy in penumbral brain tissue with concomitant dephosphorylation of FoxO3, consistent with increased activity of nuclear FoxO3 transcription factor. Increased FoxO3 activation led to autophagy significantly increased and had a protective effects on I/R injury. Conclusion: These data revealed an important role of FoxO3 in regulating autophagy in brain, and provided a new approach for further prevention and treatment of cerebral ischemia.

Neuroprotective effect of 2-(4-methoxyphenyl)ethyl-2-acetamido-2-deoxy-ß-D-pyranoside against sodium nitroprusside-induced neurotoxicity in HT22 cells.

2-(4-Methoxyphenyl) ethyl-2-acetamido-2-deoxy-ß-D-pyranoside (GlcNAc-Sal), the salidroside analog was synthesized and shown to inhibit hypoglycemia and serum limitation induced apoptosis in PC12 cells. This study investigated the protective effects of GlcNAc-Sal on sodium nitroprusside (SNP)-induced cytotoxicity in HT22 cells. Cell viability tests and Hoechst 33342 staining comfirmed that GlcNAc-Sal pretreatment attenuated SNP-stimulated apoptotic cell death in HT22 cells in a concentration-dependent manner. The measurements of reactive oxygen species (ROS), nitric oxide (NO) production and apoptosis-related gene and protein expression suggest that the protection of GlcNAc-Sal, shown in this study, might be mediated by inhibiting intracellular ROS and NO production, and regulating apoptosis-related gene and protein expression during SNP stimulation. Perhaps, this study might contribute to the development of GlcNAc-Sal as an agent for preventing and/or treating a variety of NO-induced brain diseases.

NLRP3 Inflammasome Activation Leads to Epileptic Neuronal Apoptosis.

BACKGROUND: While the NOD-Like Receptor Protein-3 (NLRP3) inflammasome is involved in a variety of nervous system diseases, its role in epilepsy still needs to be further investigated. METHODS: The expressions of NLRP3 inflammasome and apoptosis related proteins were examined by Western blot. MTT was used to assess cell viability. The role of NLRP3 inflammasome in epileptic neuronal apoptosis was further validated in NLRP3 knockout (KO) mice by Nissl staining. RESULTS: Exposure of SH-SY5Y cells to free-Mg2+ solutions increased the expression of NLRP3 inflammasome with a concomitant increase in neuronal apoptosis. This effect was inhibited in cells treated with MCC950 as a common NLRP3 inhibitor, thereby implicating the role of NLRP3 inflammasome in epileptic neuronal apoptosis. In vivo relevance of this finding was further corroborated in the NLRP3 KO mice. Compared with the wild type mice, neuronal loss induced by pentylenetetrazole was significantly inhibited in the NLRP3 KO mice. CONCLUSION: The study presented herein demonstrates the interaction between NLRP3 inflammasome and epilepsy progression. In addition, MCC950 might represent an important therapeutic drug for the treatment of NLRP3 inflammasome driven epileptogenic activity.

Pretreatment with 2-(4-methoxyphenyl)ethyl-2-acetamido-2-deoxy-ß-D-pyranoside attenuates cerebral ischemia/reperfusion-induced injury in vitro and in vivo.

Salidroside, extracted from the root of Rhodiola rosea L, is known for its pharmacological properties, in particular its neuroprotective effects. 2-(4-Methoxyphenyl) ethyl-2-acetamido-2-deoxy-ß-D-pyranoside (GlcNAc-Sal), an analog of salidroside, was recently synthesized and shown to possess neuroprotective properties. The purpose of the current study was to investigate the neuroprotective effects of GlcNAc-Sal against oxygen-glucose deprivation-reperfusion (OGD-R)-induced neurotoxicity in vitro and global cerebral ischemia-reperfusion (GCI-R) injury in vivo. Cell viability tests and Hoechst 33342 staining confirmed that GlcNAc-Sal pretreatment markedly attenuated OGD-R induced apoptotic cell death in immortalized mouse hippocampal HT22 cells. Western blot, immunofluorescence and PCR analyses revealed that GlcNAc-Sal pretreatment restored the balance of pro- and anti-apoptotic proteins and inhibited the activation of caspase-3 and PARP induced by OGD-R treatment. Further analyses showed that GlcNAc-Sal pretreatment antagonized reactive oxygen species (ROS) generation, iNOS-derived NO production and NO-related apoptotic cell death during OGD-R stimulation. GCI-R was induced by bilateral common carotid artery occlusion (BCCAO) and reperfusion in mice in vivo. Western blot analysis showed that GlcNAc-Sal pretreatment decreased the expression of caspase-3 and increased the expression of Bcl-2 (B-cell lymphoma 2)/Bax (Bcl-2-associated X protein) induced by GCI-R treatment. Our findings suggest that GlcNAc-Sal pretreatment prevents brain ischemia reperfusion injury by the direct or indirect suppression of cell apoptosis and GlcNAc-Sal could be developed as a broad-spectrum agent for the prevention and/or treatment of cerebral ischemic injury.