Safflower Yellow B Protects Brain against Cerebral Ischemia Reperfusion Injury through AMPK/NF-kB Pathway.

Inflammation had showed its important role in the pathogenesis of cerebral ischemia and secondary damage. Safflower yellow B (SYB) had neuroprotective effects against oxidative stress-induced brain injuries, but the mechanisms were still largely unknown to us. In this study, we tried to investigate the anti-inflammation effects of SYB and the possible roles of AMPK/NF-κB signaling pathway on these protective effects. In vivo, brain ischemia/reperfusion (I/R) was induced by transient middle cerebral artery occlusion for 2 h and reperfusion for 20 h. Neurofunctional evaluation, infarction area, and brain water contents were measured. Brain injury markers and inflammatory cytokines levels were measured by ELISA kits. In vitro, cell viability, apoptosis, and LDH leakage were measured after I/R in PC12 cells. The expression and phosphorylation levels of AMPK, NF-κB p65, and P-IκB-α in cytoplasm and nuclear were measured by Western blotting. SiRNA experiment was performed to certify the role of AMPK. The results showed SYB reduced infarct size, improved neurological outcomes, and inhibited brain injury after I/R. In vitro test, SYB treatment alleviated PC12 cells injury and apoptosis and inhibited the inflammatory cytokines (IL-1, IL-6, TNF-α, and COX-2) in a dose-dependent manner. SYB treatment induced AMPK phosphorylation and inhibited NF-κB p65 nuclear translocation both in brain and in PC12 cells. Further studies also showed that the inhibition of NF-κB activity of SYB was through AMPK. In conclusion, SYB protected brain I/R injury through reducing expression of inflammatory cytokines and this effect might be partly due to the inhibition of NF-κB mediated by AMPK.

Effects of microRNA-208a on inflammation and oxidative stress in ketamine-induced cardiotoxicity through Notch/NF-κB signal pathways by CHD9.

Background: MicroRNA can regulate gene expression, and participate in multiple vital activities, such as inflammation, oxidative stress epigenetic modification, cell proliferation, and apoptosis. It plays an important role in the genesis and development of cardiovascular disease.Objective: To assess the role of microRNA-208a in ketamine-induced cardiotoxicity.Methods: All rats were randomly selected into two groups: sham and model groups. After fixed, all rats in the model group was intraperitoneally (IP) injected with 100 mg/kg of ketamine. Heart samples were stained with HE assay. Total RNAs from serum were used to hybridize with the SurePrint G3 Rat Whole Genome GE 8×60 K Microarray G4858A platform.Results: In the rat model with ketamine-induced cardiotoxicity, microRNA-208a expression was increased. Then, over-expression of microRNA-208a increased inflammation and oxidative stress in vitro model. However, down-regulation of microRNA-208a decreased inflammation and oxidative stress in vitro model. Over-expression of microRNA-208a suppressed CHD9 and Notch1, and induced p65 protein expression in vitro model. Overexpression of CHD9 reduced the effects of microRNA-208a on inflammation and oxidative stress in heart cell through Notch/p65 signal pathways. Notch1 activation reduced the effects of microRNA-208a on inflammation and oxidative stress in heart cell through p65 signal pathways.Conclusion: MicroRNA-208a may be a potential biomarker for ketamine-induced cardiotoxicity through inflammation and oxidative stress by Notch/NF-κB signal pathways by CHD9.

The novel estrogenic receptor GPR30 alleviates ischemic injury by inhibiting TLR4-mediated microglial inflammation.

BACKGROUND: The steroid hormone estrogen (17-ß-estradiol, E2) provides neuroprotection against cerebral ischemic injury by activating estrogen receptors. The novel estrogen receptor G protein-coupled receptor 30 (GPR30) is highly expressed in the brain and provides acute neuroprotection against stroke. However, the underlying mechanisms remain unclear. METHODS: In this study, ovariectomized female mice were subjected to middle cerebral artery occlusion (MCAO), and E2, G1, and ICI182780 were administered immediately upon reperfusion. The infarction volume, neurological scores, and neuronal injuries were examined. Primary microglial cells were subjected to oxygen-glucose deprivation (OGD), and the drugs were administered immediately upon reintroduction. The pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6 in penumbra and microglia were assessed by ELISA. The cell viability and lactose dehydrogenase (LDH) release of neurons co-cultured with microglia were analyzed using cell counting kit-8 (CCK8) and LDH release assays. Microglial activation as well as GPR30, Iba1, and Toll-like receptor 4 (TLR4) protein expression and TLR4 mRNA expression were detected. Additionally, NF-κB activity was detected in lipopolysaccharide (LPS)-activated microglia after the activation of GPR30. RESULTS: GPR30 was highly expressed in microglia and significantly increased after ischemic injury. The activation of GPR30 significantly reduced the infarction volume, improved the neurological deficit, and alleviated neuronal injuries. Moreover, GPR30 activation significantly reduced the release of TNF-α, IL-1ß, and IL-6 from ischemic penumbra and microglia subjected to OGD and alleviated neuronal injury as assessed using the CCK8 and LDH assays. Finally, the activation of GPR30 relieved microglial activation, reduced Iba1 and TLR4 protein expression and TLR4 mRNA levels, and inhibited NF-κB activity. CONCLUSIONS: Microglial GPR30 exerts acute neuroprotective effects by inhibiting TLR4-mediated microglial inflammation, which indicates that GPR30 may be a potential target for the treatment of ischemic stroke.

Astrocytic N-Myc Downstream-regulated Gene-2 Is Involved in Nuclear Transcription Factor κB-mediated Inflammation Induced by Global Cerebral Ischemia.

BACKGROUND: Inflammation is a key element in the pathophysiology of cerebral ischemia. This study investigated the role of N-Myc downstream-regulated gene-2 in nuclear transcription factor κB-mediated inflammation in ischemia models. METHODS: Mice (n = 6 to 12) with or without nuclear transcription factor κB inhibitor pyrrolidinedithiocarbamate pretreatment were subjected to global cerebral ischemia for 20 min. Pure astrocyte cultures or astrocyte-neuron cocultures (n = 6) with or without pyrrolidinedithiocarbamate pretreatment were exposed to oxygen-glucose deprivation for 4 h or 2 h. Astrocytic nuclear transcription factor κB and N-Myc downstream-regulated gene-2 expression, proinflammatory cytokine secretion, neuronal apoptosis and survival, and memory function were analyzed at different time points after reperfusion or reoxygenation. Proinflammatory cytokine secretion was also studied in lentivirus-transfected astrocyte lines after reoxygenation. RESULTS: Astrocytic nuclear transcription factor κB and N-Myc downstream-regulated gene-2 expression and proinflammatory cytokine secretion increased after reperfusion or reoxygenation. Pyrrolidinedithiocarbamate pretreatment significantly reduced N-Myc downstream-regulated gene-2 expression and proinflammatory cytokine secretion in vivo and in vitro, reduced neuronal apoptosis induced by global cerebral ischemia/reperfusion (from 65 ± 4% to 47 ± 4%, P = 0.0375) and oxygen-glucose deprivation/reoxygenation (from 45.6 ± 0.2% to 22.0 ± 4.0%, P < 0.001), and improved memory function in comparison to vehicle-treated control animals subjected to global cerebral ischemia/reperfusion. N-Myc downstream-regulated gene-2 lentiviral knockdown reduced the oxygen-glucose deprivation-induced secretion of proinflammatory cytokines. CONCLUSIONS: Astrocytic N-Myc downstream-regulated gene-2 is up-regulated after cerebral ischemia and is involved in nuclear transcription factor κB-mediated inflammation. Pyrrolidinedithiocarbamate alleviates ischemia-induced neuronal injury and hippocampal-dependent cognitive impairment by inhibiting increases in N-Myc downstream-regulated gene-2 expression and N-Myc downstream-regulated gene-2-mediated inflammation.

Alleviation of ischaemia-reperfusion injury by endogenous estrogen involves maintaining Bcl-2 expression via the ERα signalling pathway.

The neuroprotective effects of estrogen against cerebral ischaemia have been confirmed by multiple basic and clinical studies. However, most of these studies used exogenous estrogen administered via different injection methods, and the neuroprotective effects of endogenous estrogen produced by ovaries during different phases of estrous cycle and the underlying mechanisms involved have rarely been explored. In this study, we first identified the stage of estrous cycle via vaginal smears and then measured serum estradiol levels at each phase via radioimmunoassay. We found that the estradiol level was highest in the proestrous and lowest in the diestrous. However, ovariectomy or treatment with the aromatase inhibitor letrozole significantly decreased estradiol levels compared to that of rats in diestrous. Western blotting showed that ovariectomy or letrozole treatment significantly decreased ERα and Bcl-2 protein expression and dramatically increased Bax protein expression compared with the rats in diestrous or proestrous. Rats also underwent 2h of ischaemia via middle cerebral artery occlusion followed by a 24-h reperfusion. Ovariectomy or letrozole treatment significantly decreased the neurological scores and the number of intact neurons detected via Nissl staining and dramatically increased the infarct volume detected via TTC staining and the extent of apoptosis detected via TUNEL staining and Western blotting for cleaved-caspase 3 protein expression. These results demonstrate that endogenous estrogen alleviates ischaemia-reperfusion injury by maintaining Bcl-2 expression via ERα signalling pathway and highlight the neuroprotective effects of endogenous estrogen during different stages of the estrous cycle, providing preliminary information on the underlying mechanism of this process.