NOX2-mediated reactive oxygen species are double-edged swords in focal cerebral ischemia in mice.

BACKGROUND: Reactive oxygen species (ROS) often promote acute brain injury after stroke, but their roles in the recovery phase have not been well studied. We tested the hypothesis that ROS activity mediated by NADPH oxidase 2 (NOX2) contributes to acute brain injury but promotes functional recovery during the delayed phase, which is linked with neuroinflammation, autophagy, angiogenesis, and the PI3K/Akt signaling pathway. METHODS: We used the NOX2 inhibitor apocynin to study the role of NOX2 in brain injury and functional recovery in a middle cerebral artery occlusion (MCAO) stroke mouse model. Infarct size, neurological deficits and behavior were evaluated on days 3, 7, 10 and 14 after reperfusion. In addition, dynamic NOX2-induced ROS levels were measured by dihydroethidium (DHE) staining. Autophagy, inflammasomes, and angiogenesis were measured by immunofluorescence staining and western blotting. RNA sequencing was performed, and bioinformatics technology was used to analyze differentially expressed genes (DEGs), as well as the enrichment of biological functions and signaling pathways in ischemia penumbra at 7 days after reperfusion. Then, Akt pathway-related proteins were further evaluated by western blotting. RESULTS: Our results showed that apocynin injection attenuated infarct size and mortality 3 days after stroke but promoted mortality and blocked functional recovery from 5 to 14 days after stroke. DHE staining showed that ROS levels were increased at 3 days after reperfusion and then gradually declined in WT mice, and these levels were significantly reduced by the NOX2 inhibitor apocynin. RNA-Seq analysis indicated that apocynin activated the immune response under hypoxic conditions. The immunofluorescence and western blot results demonstrated that apocynin inhibited the NLRP3 inflammasome and promoted angiogenesis at 3 days but promoted the NLRP3 inflammasome and inhibited angiogenesis at 7 and 14 days after stroke, which was mediated by regulating autophagy activation. Furthermore, RNA-Seq and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that apocynin injection resulted in PI3K-Akt signaling pathway enrichment after 7 days of MCAO. We then used an animal model to show that apocynin decreased the protein levels of phosphorylated PI3K and Akt and NF-κB p65, confirming that the PI3K-Akt-NF-κB pathway is involved in apocynin-mediated activation of inflammation and inhibition of angiogenesis. CONCLUSIONS: NOX2-induced ROS production is a double-edged sword that exacerbates brain injury in the acute phase but promotes functional recovery. This effect appears to be achieved by inhibiting NLRP3 inflammasome activation and promoting angiogenesis via autophagy activation.

LncRNA GAS5/miR-137 Is a Hypoxia-Responsive Axis Involved in Cardiac Arrest and Cardiopulmonary Cerebral Resuscitation.

Background: Cardiac arrest/cardiopulmonary resuscitation (CA/CPR) represents one of the devastating medical emergencies and is associated with high mortality and neuro-disability. Post-cardiac arrest syndrome (PCAS) is mechanistically ascribed to acute systemic ischemia/reperfusion(I/R) injury. The lncRNA/microRNA/mRNA networks have been found to play crucial roles in the pathogenesis of the hypoxia-responsive diseases. Nonetheless, the precise molecular mechanisms by which lncRNA/miRNA/mRNA axes are involved in the astrocyte-microglia crosstalk in CA/CPR have not been fully elucidated. Methods: We collected and purified the exosomes from the blood of CA/CPR patients and supernatant of OGD/R-stimulated astrocytes. On the basis of microarray analysis, bioinformatic study, and luciferase activity determination, we speculated that lncRNA GAS5/miR-137 is implicated in the astrocyte-microglia crosstalk under the insult of systemic I/R injury. The regulation of lncRNA GAS5/miR-137 on INPP4B was examined by cellular transfection in OGD/R cell culture and by lateral ventricle injection with miR-137 agomir in CA/CPR mice model. Flow cytometry and immunofluorescence staining were performed to detect the microglial apoptosis, M1/M2 phenotype transformation, and neuroinflammation. Neurological scoring and behavior tests were conducted in CA/CPR group, with miR-137 agomir lateral-ventricle infusion and in their controls. Results: In all the micRNAs, miR-137 was among the top 10 micRNAs that experienced greatest changes, in both the blood of CA/CPR patients and supernatant of OGD/R-stimulated astrocytes. Bioinformatic analysis revealed that miR-137 was sponged by lncRNA GAS5, targeting INPP4B, and the result was confirmed by Luciferase activity assay. qRT-PCR and Western blotting showed that lncRNA GAS5 and INPP4B were over-expressed whereas miR-137 was downregulated in the blood of CA/CPR patients, OGD/R-stimulated astrocytes, and brain tissue of CA/CPR mice. Silencing lncRNA GAS5 suppressed INPP4B expression, but over-expression of miR-137 negatively modulated its expression. Western blotting exhibited that PI3K and Akt phosphorylation was increased when lncRNA GAS5 was silenced or miR-137 was over-expressed. However, PI3K and Akt phosphorylation was notably suppressed in the absence of miR-137, almost reversing their phosphorylation in the silencing lncRNA GAS5 group. Then we found that GAS5 siRNA or miR-137 mimic significantly increased cell viability and alleviated apoptosis after OGD/R injury. Furthermore, over-expression of miR-137 attenuated microglial apoptosis and neuroinflammation in CA/CPR mice model, exhibiting significantly better behavioral tests after CA/CPR. Conclusion: LncRNA GAS5/miR-137 may be involved in the astrocyte-microglia communication that inhibits PI3K/Akt signaling activation via regulation of INPP4B during CA/CPR.

Worsened brain edma and the level of serum HMGB1 and S100B of IL-4 knockout mice after traumatic brain injury / 中华行为医学与脑科学杂志

Objective To investigate the changes of brain edma and expression of blood high mobil-ity group box 1(HMGB1) and calcium binding protein S100B after traumatic brain injury (TBI) in IL-4 knockout (IL-4 KO) mice,and to explore the effects of IL-4 on traumatic brain injury. Methods Twenty male wild type ( WT) or twenty male IL-4 KO BALB/cJ mice were randomly divided into WT sham TBI group,WT TBI group,IL-4 KO sham TBI group and IL-4 KO TBI group(n=10 in each group).The model of traumatic brain injury was established by the free falling body epidural impact method,then the brain water content was measured. The expression of aquaporin-4 ( APQ4) and HMGB1 in injured brain of each group was detected by Western blot,and the concentration of HMGB1 and S100B in serum was detected by ELISA assay. Results ( 1 ) The brain water content of injured lateral brain of BALB/cJ mice with IL-4 gene knockout was significantly higher than that of wild type mice with brain injury model (WT group: (80.03± 0.35)％;IL-4 KO group:(81.93±0.41)％;P<0.05).(2) The Western blot showed that the expression of AQP4 and HMGB1 in brain tissue of BALB/cJ mice with IL-4 gene knockout was significantly higher than those in wild type mice after traumatic brain injury. ( 3) The results of ELISA showed that the levels of HMGB1 and S100B in the serum of IL-4 knockout BALB/cJ mice were significantly higher than those of wild type mice (HMGB1:WT group:(9.21±0.74)ng/ml;IL-4 gene knock-out group:(13.39±1.33)ng/ml,P<0.05;S100B protein:WT group:(11.11±0.84)pg/ml;IL-4 KO group: (18.11±2.02)pg/ml,P<0.05 ). Conclusion The brain tissue water content and the expression of APQ4 are increased in IL-4 KO TBI mice.The expression of HMGB1 in brain issue and serum and S100B in serum are also up-regulated.