RU.521 mitigates subarachnoid hemorrhage-induced brain injury via regulating microglial polarization and neuroinflammation mediated by the cGAS/STING/NF-κB pathway.

BACKGROUND: The poor prognosis of subarachnoid hemorrhage (SAH) is often attributed to neuroinflammation. The cGAS-STING axis, a cytoplasmic pathway responsible for detecting dsDNA, plays a significant role in mediating neuroinflammation in neurological diseases. However, the effects of inhibiting cGAS with the selective small molecule inhibitor RU.521 on brain injury and the underlying mechanisms after SAH are still unclear. METHODS: The expression and microglial localization of cGAS following SAH were investigated with western blot analysis and immunofluorescent double-staining, respectively. RU.521 was administered after SAH. 2'3'-cGAMP, a second messenger converted by activated cGAS, was used to activate cGAS-STING. The assessments were carried out by adopting various techniques including neurological function scores, brain water content, blood-brain barrier permeability, western blot analysis, TUNEL staining, Nissl staining, immunofluorescence, morphological analysis, Morris water maze test, Golgi staining, CCK8, flow cytometry in the in vivo and in vitro settings. RESULTS: Following SAH, there was an observed increase in the expression levels of cGAS in rat brain tissue, with peak levels observed at 24 h post-SAH. RU.521 resulted in a reduction of brain water content and blood-brain barrier permeability, leading to an improvement in neurological deficits after SAH. RU.521 had beneficial effects on neuronal apoptosis and microglia activation, as well as improvements in microglial morphology. Additionally, RU.521 prompted a shift in microglial phenotype from M1 to M2. We also noted a decrease in the production of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6, and an increase in the level of the anti-inflammatory cytokine IL-10. Finally, RU.521 treatment was associated with improvements in cognitive function and an increase in the number of dendritic spines in the hippocampus. The therapeutic effects were mediated by the cGAS/STING/NF-κB pathway and were found to be abolished by 2'3'-cGAMP. In vitro, RU.521 significantly reduced apoptosis and neuroinflammation. CONCLUSION: The study showed that SAH leads to neuroinflammation caused by microglial activation, which contributes to early brain injury. RU.521 improved neurological outcomes and reduced neuroinflammation by regulating microglial polarization through the cGAS/STING/NF-κB pathway in early brain injury after SAH. RU.521 may be a promising candidate for the treatment of neuroinflammatory pathology after SAH. Video Abstract.

HACE1 exerts a neuroprotective role against oxidative stress in cerebral ischemia-reperfusion injury by activating the PI3K/AKT/Nrf2 pathway.

HECT domain and Ankyrin repeat-containing E3 ubiquitin protein ligase 1 (HACE1) is an E3 ubiquitin ligase involving oxidative stress, an important contributor in cerebral ischemia-reperfusion injury (CIRI). It was proposed to be associated with the PI3K/AKT pathway and Nrf2 nuclear translocation, which are important players of oxidative stress. Therefore, we supposed that HACE1 might affect CIRI by regulating the PI3K/AKT/Nrf2 pathway. Here, we used the transient middle cerebral artery occlusion-reperfusion (tMCAO/R) model to induce CIRI in rats and found lower HACE1 expression in ischemic rats compared with the control. To explore the exact role of HACE1, the lentivirus vector carrying the HACE1 sequence was administrated to rats by intracerebroventricular injection (1 × 109 TU/mL, 9 µL) one week before tMCAO/R operation. HACE1 overexpression alleviated tMCAO/R-induced brain damage in rats. Further studies revealed that it reduced oxidative stress via activating the PI3K/AKT/Nrf2 pathway, thereby inhibiting neuronal apoptosis in the ischemic penumbra of rats with CIRI. Then, differentiated PC12 cells were cultured in oxygen-glucose deprivation-reoxygenation (OGD/R) conditions (OGD: 1 % O2, 94 % N2, and 5 % CO2; R: normal atmosphere) to simulate CIRI in vitro. Similarly, HACE1 overexpression inhibited neuronal apoptosis caused by OGD/R treatment. The PI3K inhibitor LY294002 reversed the inhibitory effects of HACE1 overexpression on oxidative stress in OGD/R-injured cells, accompanied by the inactivated AKT/Nrf2 pathway. Altogether, our results suggest that HACE1 protects against oxidative stress-induced neuronal apoptosis in CIRI by activating the PI3K/AKT/Nrf2 pathway, providing a new insight into the CIRI treatment.

ACEA Attenuates Oxidative Stress by Promoting Mitophagy via CB1R/Nrf1/PINK1 Pathway after Subarachnoid Hemorrhage in Rats.

METHOD: Endovascular perforation was performed to establish a SAH model of rats. ACEA was administered intraperitoneally 1 h after SAH. The CB1R antagonist AM251 was injected intraperitoneally 1 h before SAH induction. Adenoassociated virus- (AAV-) Nrf1 shRNA was infused into the lateral ventricle 3 weeks before SAH induction. Neurological tests, immunofluorescence, DHE, TUNEL, Nissl staining, transmission electron microscopy (TEM), and Western blot were performed. RESULTS: The expression of CB1R, Nrf1, PINK1, Parkin, and LC3II increased and peaked at 24 h after SAH. ACEA treatment exhibited the antioxidative stress and antiapoptosis effects after SAH. In addition, ACEA treatment increased the expression of Nrf1, PINK1, Parkin, LC3II, and Bcl-xl but repressed the expression of Romo-1, Bax, and cleaved caspase-3. Moreover, the TEM results demonstrated that ACEA promoted the formation of mitophagosome and maintained the normal mitochondrial morphology of neurons. The protective effect of ACEA was reversed by AM251 and Nrf1 shRNA, respectively. CONCLUSIONS: This study demonstrated that ACEA alleviated oxidative stress and neurological dysfunction by promoting mitophagy after SAH, at least in part via the CB1R/Nrf1/PINK1 signaling pathway.

c-myc protects mice from ischemia stroke through elevating microRNA-200b-5p-regulated SIRT1 expression.

OBJECTIVE: c-myc has been reported to attenuate ischemia stroke (IS). We initiated the research to uncover the molecular mechanism of c-myc with regard to microRNA (miR)-200b-5p/Sirtuin1 (SIRT1) axis. METHODS: An IS mouse model was prepared by middle cerebral artery occlusion (MCAO). Measurements of c-myc, miR-200b-5p and SIRT1 levels in MCAO mice were conducted. c-myc, miR-200b-5p and SIRT1 expression levels in MCAO mice were detected. The neurological function, production of inflammatory cytokines, neuronal apoptosis, brain tissue pathology and neuronal survival of MCAO mice were observed. RESULTS: c-myc and SIRT1 levels went downward while miR-200b-5p expression went upward in MCAO mice. Elevation of c-myc or suppression of miR-200b-5p improved neurological function, reduced inflammation and neuronal apoptosis, and attenuated brain tissue pathology and neuronal survival of MCAO mice. Enhancement of miR-200b-5p or knockdown of SIRT1 weakened c-myc-induced protection against MCAO-induced brain injury in mice. CONCLUSION: Overall, c-myc protects mice from IS through elevating miR-200b-5p-targeted SIRT1 expression.

Neuroprotective effects of methylcobalamin in cerebral ischemia/reperfusion injury through activation of the ERK1/2 signaling pathway.

Despite advances in the understanding of the pathophysiology of ischemic stroke, therapeutic options remain limited. Methylcobalamin is an endogenous vitamin B12 that exhibits anti-inflammatory and antiapoptotic activities in a variety of diseases. In this study, we aimed to explore the neuroprotective effects and mechanism of action of methylcobalamin on cerebral ischemic injury in vitro and in vivo. The oxygen and glucose deprivation/reperfusion model and middle cerebral artery occlusion model were used to simulate cerebral ischemic injury in vitro and in vivo. Cell viability, inflammatory factors, cell apoptosis, and protein expression levels were determined. Further, autophagy flux and the cerebral infarction volume were measured. The modified neurological severity score, Longa score, Rotarod assay, and foot-fault test were used to evaluate behavioral changes and neurological deficits in rats. In vitro, methylcobalamin significantly increased cell viability, decreased lactate dehydrogenase release, attenuated inflammatory cytokine expression, reduced the apoptotic proportion, and enhanced autophagy flux after OGD treatment. In addition, Bcl-2 and Beclin1 expression levels and the LC3 II/I ratio were increased, whereas levels of Bax and cleaved caspase-3 were decreased. In vivo, methylcobalamin significantly reduced the cerebral infarction volume and neurological deficits in the rats. Furthermore, methylcobalamin activated the ERK1/2 pathway, whereas ERK1/2 inhibitors diminished its effects in the in vitro and in vivo models. In conclusion, methylcobalamin may exert a neuroprotective effect on cerebral ischemia and is a promising drug candidate for developing novel neuroprotective therapies.

Intranasal delivery of 9-cis retinoic acid reduces beta-amyloid deposition via inhibiting astrocyte-mediated inflammation.

Alzheimer's disease (AD) is associated with the accumulation and deposition of a beta-amyloid (Αß) peptide in the brain, resulting in increased neuroinflammation and synaptic dysfunction. Intranasal delivery of targeted drugs to the brain represents a noninvasive pathway that bypasses the blood-brain barrier and minimizes systemic exposure. The aim of this study was to evaluate the therapeutic effect of intranasally delivered 9-cis retinoic acid (RA) on the neuropathology of an AD mouse model. Herein, we observed dramatically decreased Αß deposition in the brains of amyloid precursor protein (APP) and presenilin 1 (PS1) double-transgenic mice (APP/PS1) treated intranasally with 9-cis RA for 4 weeks compared to that in the brains of vehicle-treated mice. Importantly, intranasal delivery of 9-cis RA suppressed Αß-associated astrocyte activation and neuroinflammation and ultimately restored synaptic deficits in APP/PS1 transgenic mice. These results support the critical roles of Αß-associated neuroinflammation responses to synaptic deficits, particularly during the deposition of Αß. Our findings provide strong evidence that intranasally delivered 9-cis RA attenuates neuronal dysfunction in an AD mouse model and is a promising therapeutic strategy for the prevention and treatment of AD.

Long non-coding RNA AGAP2-AS1 promotes the proliferation of glioma cells by sponging miR-15a/b-5p to upregulate the expression of HDGF and activating Wnt/ß-catenin signaling pathway.

Glioma is a kind of malignant brain tumor which damages the central nervous system of adults. Recent years, the molecular mechanism involved in the initiation and progression of glioma has been widely reported. Long non-coding RNAs (lncRNAs) have been proved to be significant modulators in the biological processes of glioma. In this study, we found that lncRNA AGAP2-AS1 was differentially expressed in glioma tissue samples and cell lines. Kaplan-Meier method was used to analyze the correlation between AGAP2-AS1 expression and the overall survival of glioma patients. Higher expression of AGAP2-AS1 was correlated with the lower overall survival of glioma patients. Functionally, AGAP2-AS1 knockdown inhibited glioma cell proliferation and accelerated glioma cell apoptosis. Mechanistically, AGAP2-AS1 upregulated HDGF by sponging miR-15a/b-5p. The function of AGAP2-AS1-miR-15a/b-5p-HDGF axis was confirmed by performing rescue assays. Experimental results suggested that miR-15a/b-5p and HDGF involved in AGAP2-AS1-mediated glioma cell proliferation. Moreover, AGAP2-AS1 and HDGF were found to activate Wnt/ß-catenin signaling pathway in glioma cell lines. In summary, this study demonstrated that AGAP2-AS1 promoted glioma cell proliferation by sponging miR-15a/b-5p to upregulate the expression of HDGF.

Fluoride related changes in behavioral outcomes may relate to increased serotonin.

Fluoride ingestion has been linked to changes in behavior in mice and rats, related to dose, sex of the animal, and the timing of exposure. Previous studies have shown the behavior of female rats to be most affected by postnatal fluoride exposure, and in this study we determined the effects of postnatal fluoride exposure on anxiety related behavior and serotonin. Mice given 50 ppm fluoride in drinking water had increased entries in the open arms of the elevated plus maze, suggesting reduced anxiety. Both peripheral and central serotonin was increased in the fluoride treated mice. In a cohort of children drinking water containing 2.5 ppm fluoride, serum serotonin was also increased as compared to controls. The mechanisms by which fluoride results in an increase peripheral and central serotonin are not well understood, but warrant further study, as these effects may also be relevant to prenatal fluoride related changes in behavior in both mice and humans.

MiR-760 suppresses non-small cell lung cancer proliferation and metastasis by targeting ROS1.

MicroRNAs (miRNAs) have been shown to be critical regulators in many types of tumors. The aim of our study was to investigate the role of miR-760 in non-small cell lung cancer (NSCLC). We demonstrated that the expression of miR-760 was downregulated in NSCLC tissues compared with the adjacent normal tissues. We also demonstrated that the expression of miR-760 was downregulated in the NSCLC cell lines. Overexpression of miR-760 suppressed the NSCLC cell proliferation, cell cycle, and migration. Moreover, we identified that ROS1 was a direct target of miR-760 in the NSCLC cell. Elevated expression of miR-760 suppressed ROS1 expression in the NSCLC cell. We also demonstrated that the expression of ROS1 was higher in the NSCLC tissues than in the adjacent lung tissues. MiR-760 expression level was reversely associated with the expression level of ROS1 in the NSCLC tissues. In summary, we showed that miR-760 suppressed the NSCLC cell proliferation, cell cycle, and migration through regulating the ROS1 expression. These data suggested that miR-760 may act as a tumor suppressor gene in the NSCLC partly through regulating ROS1 expression.

Correction to: MiR-760 suppresses non-small cell lung cancer proliferation and metastasis by targeting ROS1.

In the original article the authors list and the credit to the corresponding authors were not complete.

miR-1297 regulates neural stem cell differentiation and viability through controlling Hes1 expression.

OBJECTIVES: Neural stem cells (NSCs) are self-renewing, undifferentiated and multipotent precursors that can generate neuronal and glial lineages. MicroRNAs (miRNAs) are small non-coding RNAs that act crucial roles in cell proliferation, differentiation and migration. However, the role of miR-1297 in the development of NSCs is still unknown. MATERIALS AND METHODS: Primary NSCs were isolated from rat's embryos. The expression of miR-1297 and Hes1 were measured by qRT-PCR. Western blot was performed to detect the protein expression of Hes1, ß-tubulin-III and GFAP. RESULTS: We showed that miR-1297 expression was upregulated during NSC differentiation, while the expression of Hes1 was decreased during NSC differentiation. Elevated expression of miR-1297 promoted the NSCs viability and increased the formation of NSCs to neurospheres. Ecoptic expression of miR-1297 promoted ß-tubulin-III expression in the NSCs. Overexpression of miR-1297 decreased GFAP expression in the NSCs. Furthermore, we demonstrated that miR-1297 regulated NSCs viability and differentiation by directly targeting Hes1. Overexpression of miR-1297 suppressed Hes1 expression in the NSCs. CONCLUSIONS: These results suggested that miR-1297 played an important role in NSCs viability and differentiation through inhibiting Hes1 expression.

Long nonding RNA UCA1 regulates neural stem cell differentiation by controlling miR-1/Hes1 expression.

Neural stem cells are able to self-renew and generate glial and neuronal lineages. Neural stem cell may serve as therapeutic method for neurological disorders including spinal cord injuries, Parkinson's disease, Huntington's disease and Alzheimer's disease. Long noncoding RNAs (lncRNAs) are longer than 200 nucleotides with limited protein-coding capacity. Recent studies have demonstreated that lncRNAs play an important role in several cellular processes including cell differentiation, cell development, proliferation, apoptosis, invasion and migration. However, the role of lncRNA human urothelial carcinoma associated 1 (UCA1) in the development of neural stem cells remains unknown. In this study, we showed that the expression of UCA1 was upregulated in the neural stem cell in a time-dependent manner. Knockdown of UCA1 suppressed the neural stem cell proliferation. Inhibition of UCA1 decreased the expression of nestin and the formation of neurosphere. Moreover, knockdown of UCA1 suppressed the neural stem cell differentiation to astrocyte and promoted the neural stem cell differentiation to neuron. Furthermore, we demonstrated that knockdown of UCA1 increased the expression of miR-1 in the neural stem cell and suppressed the expresion of Hes1, which is one target gene of miR-1. In addition, ectopic expression of Hes1 could impair siUCA1-induced neural stem cells proliferation. Overexpression of Hes1 suppressed siUCA1-induced ß-tubulin expression and promoted siUCA1-inhibited GFAP expression in the neural stem cell. These results suggested that UCA1 regulated the neural stem cell proliferation and differentiation through regulating Hes1 expression.

miR-381 Regulates Neural Stem Cell Proliferation and Differentiation via Regulating Hes1 Expression.

Neural stem cells are self-renewing, multipotent and undifferentiated precursors that retain the capacity for differentiation into both glial (astrocytes and oligodendrocytes) and neuronal lineages. Neural stem cells offer cell-based therapies for neurological disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease and spinal cord injuries. However, their cellular behavior is poorly understood. MicroRNAs (miRNAs) are a class of small noncoding RNAs involved in cell development, proliferation and differentiation through regulating gene expression at post-transcriptional level. The role of miR-381 in the development of neural stem cells remains unknown. In this study, we showed that overexpression of miR-381 promoted neural stem cells proliferation. It induced the neural stem cells differentiation to neurons and inhibited their differentiation to astrocytes. Furthermore, we identified HES1 as a direct target of miR-381 in neural stem cells. Moreover, re-expression of HES1 impaired miR-381-induced promotion of neural stem cells proliferation and induce neural stem cells differentiation to neurons. In conclusion, miR-381 played important role in neural stem cells proliferation and differentiation.

MicroRNA-575 targets BLID to promote growth and invasion of non-small cell lung cancer cells.

This study was designed to detect miR-575 expression and function in non-small cell lung cancer (NSCLC). A higher expression of miR-575 in NSCLC tissues was observed compared with adjacent non-neoplastic tissues. Furthermore, re-introduction of miR-575 significantly promoted cell proliferation, migration, and invasion in the NSCLC line. Moreover, we showed that BLID is negatively regulated by miR-575 at the posttranscriptional level, via a specific target site within the 3'UTR. Overexpression of BLID counteracted miR-575-induced proliferation and invasion in NSCLC cells. The expression of BLID is frequently downregulated in NSCLC tumors and cell lines and inversely correlates with miR-575 expression. The findings of this study contribute to the current understanding of the functions of miR-575 in NSCLC.

Angiotensin converting enzyme 2/Ang-(1-7)/mas axis protects brain from ischemic injury with a tendency of age-dependence.

BACKGROUND: The angiotensin (Ang) converting enzyme 2 (ACE2)/Ang-(1-7)/Mas receptor pathway is an important component of the renin-angiotensin system and has been suggested to exert beneficial effects in ischemic stroke. AIMS: This study explored whether the ACE2/Ang-(1-7)/Mas pathway has a protective effect on cerebral ischemic injury and whether this effect is affected by age. METHODS: We used three-month and eight-month transgenic mice with neural over-expression of ACE2 (SA) and their age-matched nontransgenic (NT) controls. Neurological deficits and ischemic stroke volume were determined following middle cerebral artery occlusion (MCAO). In oxygen and glucose deprivation (OGD) experiments on brain slices, the effects of the Mas receptor agonist (Ang1-7) or antagonist (A779) on tissue swelling, Nox2/Nox4 expression reactive oxygen species (ROS) production and cell death were measured. RESULTS: (1) Middle cerebral artery occlusion -induced ischemic injury and neurological deficit were reduced in SA mice, especially in eight-month animals; (2) OGD-induced tissue swelling and cell death were decreased in SA mice with a greater reduction seen in eight-month mice; (3) Ang-(1-7) and A779 had opposite effects on OGD-induced responses, which correlated with changes in Nox2/Nox4 expression and ROS production. CONCLUSIONS: Angiotensin converting enzyme 2/Ang-(1-7)/Mas axis protects brain from ischemic injury via the Nox/ROS signaling pathway, with a greater effect in older animals.