Adiponectin Ameliorates GMH-Induced Brain Injury by Regulating Microglia M1/M2 Polarization Via AdipoR1/APPL1/AMPK/PPARγ Signaling Pathway in Neonatal Rats.

Adiponectin (APN), a fat-derived plasma hormone, is a classic anti-inflammatory agent. Multiple studies have demonstrated the beneficial role of APN in acute brain injury, but the effect of APN in germinal matrix hemorrhage (GMH) is unclear, and the underlying molecular mechanisms remain largely undefined. In the current study, we used a GMH rat model with rh-APN treatment, and we observed that APN demonstrated a protective effect on neurological function and an inhibitory effect on neuroinflammation after GMH. To further explore the underlying mechanisms of these effects, we found that the expression of Adiponectin receptor 1 (AdipoR1) primarily colocalized with microglia and neurons in the brain. Moreover, AdiopR1, but not AdipoR2, was largely increased in GMH rats. Meanwhile, further investigation showed that APN treatment promoted AdipoR1/APPL1-mediated AMPK phosphorylation, further increased peroxisome proliferator-activated receptor gamma (PPARγ) expression, and induced microglial M2 polarization to reduce the neuroinflammation and enhance hematoma resolution in GMH rats. Importantly, either knockdown of AdipoR1, APPL1, or LKB1, or specific inhibition of AMPK/PPARγ signaling in microglia abrogated the protective effect of APN after GMH in rats. In all, we propose that APN works as a potential therapeutic agent to ameliorate the inflammatory response following GMH by enhancing the M2 polarization of microglia via AdipoR1/APPL1/AMPK/PPARγ signaling pathway, ultimately attenuating inflammatory brain injury induced by hemorrhage.

BMS-470539 Attenuates Oxidative Stress and Neuronal Apoptosis via MC1R/cAMP/PKA/Nurr1 Signaling Pathway in a Neonatal Hypoxic-Ischemic Rat Model.

Neuronal apoptosis induced by oxidative stress plays an important role in the pathogenesis and progression of hypoxic-ischemic encephalopathy (HIE). Previous studies reported that activation of melanocortin-1 receptor (MC1R) exerts antioxidative stress, antiapoptotic, and neuroprotective effects in various neurological diseases. However, whether MC1R activation can attenuate oxidative stress and neuronal apoptosis after hypoxic-ischemic- (HI-) induced brain injury remains unknown. Herein, we have investigated the role of MC1R activation with BMS-470539 in attenuating oxidative stress and neuronal apoptosis induced by HI and the underlying mechanisms. 159 ten-day-old unsexed Sprague-Dawley rat pups were used. HI was induced by right common carotid artery ligation followed by 2.5 h of hypoxia. The novel-selective MC1R agonist BMS-470539 was administered intranasally at 1 h after HI induction. MC1R CRISPR KO plasmid and Nurr1 CRISPR KO plasmid were administered intracerebroventricularly at 48 h before HI induction. Percent brain infarct area, short-term neurobehavioral tests, Western blot, immunofluorescence staining, Fluoro-Jade C staining, and MitoSox Staining were performed. We found that the expression of MC1R and Nurr1 increased, peaking at 48 h post-HI. MC1R and Nurr1 were expressed on neurons at 48 h post-HI. BMS-470539 administration significantly attenuated short-term neurological deficits and infarct area, accompanied by a reduction in cleaved caspase-3-positive neurons at 48 h post-HI. Moreover, BMS-470539 administration significantly upregulated the expression of MC1R, cAMP, p-PKA, Nurr1, HO-1, and Bcl-2. However, it downregulated the expression of 4-HNE and Bax, as well as reduced FJC-positive cells, MitoSox-positive cells, and 8-OHdG-positive cells at 48 h post-HI. MC1R CRISPR and Nurr1 CRISPR abolished the antioxidative stress, antiapoptotic, and neuroprotective effects of BMS-470539. In conclusion, our findings demonstrated that BMS-470539 administration attenuated oxidative stress and neuronal apoptosis and improved neurological deficits in a neonatal HI rat model, partially via the MC1R/cAMP/PKA/Nurr1 signaling pathway. Early administration of BMS-470539 may be a novel therapeutic strategy for infants with HIE.

Activation of GPR39 with TC-G 1008 attenuates neuroinflammation via SIRT1/PGC-1α/Nrf2 pathway post-neonatal hypoxic-ischemic injury in rats.

BACKGROUND: Hypoxic-ischemic encephalopathy (HIE) is a severe anoxic brain injury that leads to premature mortality or long-term disabilities in infants. Neuroinflammation is a vital contributor to the pathogenic cascade post-HIE and a mediator to secondary neuronal death. As a plasma membrane G-protein-coupled receptor, GPR39, exhibits anti-inflammatory activity in several diseases. This study aimed to explore the neuroprotective function of GPR39 through inhibition of inflammation post-hypoxic-ischemic (HI) injury and to elaborate the contribution of sirtuin 1(SIRT1)/peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α)/nuclear factor, erythroid 2 like 2(Nrf2) in G-protein-coupled receptor 39 (GPR39)-mediated protection. METHODS: A total of 206 10-day-old Sprague Dawley rat pups were subjected to HIE or sham surgery. TC-G 1008 was administered intranasally at 1 h, 25 h, 49 h, and 73 h post-HIE induction. SIRT1 inhibitor EX527, GPR39 CRISPR, and PGC-1α CRISPR were administered to elucidate the underlying mechanisms. Brain infarct area, short-term and long-term neurobehavioral tests, Nissl staining, western blot, and immunofluorescence staining were performed post-HIE. RESULTS: The expression of GPR39 and pathway-related proteins, SIRT1, PGC-1α and Nrf2 were increased in a time-dependent manner, peaking at 24 h or 48-h post-HIE. Intranasal administration of TC-G 1008 reduced the percent infarcted area and improved short-term and long-term neurological deficits. Moreover, TC-G 1008 treatment significantly increased the expression of SIRT1, PGC-1α and Nrf2, but downregulated the expressions of IL-6, IL-1ß, and TNF-α. GPR39 CRISPR EX527 and PGC-1α CRISPR abolished GPR39's neuroprotective effects post-HIE. CONCLUSIONS: TC-G 1008 attenuated neuroinflammation in part via the SIRT1/PGC-1α/Nrf2 pathway in a neonatal rat model of HIE. TC-G 1008 may be a novel therapeutic target for treatment post-neonatal HIE injury.

Establishment of Carotid Artery Dissection and MRI Findings in a Swine Model.

Carotid artery dissection (CAD) is the leading cause of ischemic stroke in young patients; however, the etiology and pathophysiology of CAD remain largely unknown. In our study, two types of dissections (length × width: 1.5 cm × 1/3 circumference of intima, Group I, n = 6; or 1.5 cm × 2/3 circumference of intima, Group II, n = 6) were created between the media and intima. Ultrasound (within 2 h after dissection) showed a dissociated intima in the lumen and obstructed blood flow in the surgical area. Digital subtraction angiography (DSA, 72 h after dissection), magnetic resonance imaging (MRI, 72 h after dissection), and hematoxylin-eosin (H&E, 7 days after dissection) staining confirmed stenosis (33.67 ± 5.66%) in Group I and total occlusion in Group II. In 10 out of 12 swine, the CAD model was established using a detacher and balloon dilation, and morphological outcomes (stenosis or occlusion) after CAD were determined by the size of intimal incision.

Activation of MC1R with BMS-470539 attenuates neuroinflammation via cAMP/PKA/Nurr1 pathway after neonatal hypoxic-ischemic brain injury in rats.

BACKGROUND: Microglia-mediated neuroinflammation plays a crucial role in the pathogenesis of hypoxic-ischemic (HI)-induced brain injury. Activation of melanocortin-1 receptor (MC1R) has been shown to exert anti-inflammatory and neuroprotective effects in several neurological diseases. In the present study, we have explored the role of MC1R activation on neuroinflammation and the potential underlying mechanisms after neonatal hypoxic-ischemic brain injury in rats. METHODS: A total of 169 post-natal day 10 unsexed rat pups were used. HI was induced by right common carotid artery ligation followed by 2.5 h of hypoxia. BMS-470539, a specific selective MC1R agonist, was administered intranasally at 1 h after HI induction. To elucidate the potential underlying mechanism, MC1R CRISPR KO plasmid or Nurr1 CRISPR KO plasmid was administered via intracerebroventricular injection at 48 h before HI induction. Percent brain infarct area, short- and long-term neurobehavioral tests, Nissl staining, immunofluorescence staining, and Western blot were conducted. RESULTS: The expression levels of MC1R and Nurr1 increased over time post-HI. MC1R and Nurr1 were expressed on microglia at 48 h post-HI. Activation of MC1R with BMS-470539 significantly reduced the percent infarct area, brain atrophy, and inflammation, and improved short- and long-term neurological deficits at 48 h and 28 days post-HI. MC1R activation increased the expression of CD206 (a microglial M2 marker) and reduced the expression of MPO. Moreover, activation of MC1R with BMS-470539 significantly increased the expression levels of MC1R, cAMP, p-PKA, and Nurr1, while downregulating the expression of pro-inflammatory cytokines (TNFα, IL-6, and IL-1ß) at 48 h post-HI. However, knockout of MC1R or Nurr1 by specific CRISPR reversed the neuroprotective effects of MC1R activation post-HI. CONCLUSIONS: Our study demonstrated that activation of MC1R with BMS-470539 attenuated neuroinflammation, and improved neurological deficits after neonatal hypoxic-ischemic brain injury in rats. Such anti-inflammatory and neuroprotective effects were mediated, at least in part, via the cAMP/PKA/Nurr1 signaling pathway. Therefore, MC1R activation might be a promising therapeutic target for infants with hypoxic-ischemic encephalopathy (HIE).

GW0742 activates miR-17-5p and inhibits TXNIP/NLRP3-mediated inflammation after hypoxic-ischaemic injury in rats and in PC12 cells.

This study aimed to investigate the effects of PPAR-ß/δ receptor agonist GW0742 on neuroinflammation in a rat model of hypoxia-ischaemia (HI) and in PC12 cells in OGD model. HI was induced by ligating the common carotid artery and inducing hypoxia for 150 minutes. Immunofluorescence was used for quantification of microglia activation and for determining cellular localization of PPAR-ß/δ. Expression of proteins was measured by Western blot. Activation of miR-17-5p by GW0742 was assessed in PC12 cells by Dual-Luciferase Reporter Gene Assay. The endogenous expression of TXNIP, NLRP3, cleaved caspase-1 and IL-1ß was increased after HI. GW0742 treatment significantly reduced the number of activated pro-inflammatory microglia in ipsilateral hemisphere after HI. Mechanistically, GW0742 significantly decreased the expression of TXNIP, NLRP3, IL-6 and TNF-α. Either PPAR-ß/δ antagonist GSK3787, miR-17-5p inhibitor, or TXNIP CRISPR activation abolished the anti-inflammatory effects of GW0742. Activation of PPAR-ß/δ by GW0742 activated miR-17-5p expression in PC12 cells and increased cell viability after OGD, which was accompanied by decreased expression of TXNIP and reduced secretion of IL-1ß and TNF-α. In conclusion, GW0742 may be a promising neurotherapeutic for the management of HI patients.

Rh-CSF1 Attenuates Oxidative Stress and Neuronal Apoptosis via the CSF1R/PLCG2/PKA/UCP2 Signaling Pathway in a Rat Model of Neonatal HIE.

Oxidative stress (OS) and neuronal apoptosis are major pathological processes after hypoxic-ischemic encephalopathy (HIE). Colony stimulating factor 1 (CSF1), binding to CSF1 receptor (CSF1R), has been shown to reduce neuronal loss after hypoxic-ischemia- (HI-) induced brain injury. In the present study, we hypothesized that CSF1 could alleviate OS-induced neuronal degeneration and apoptosis through the CSF1R/PLCG2/PKA/UCP2 signaling pathway in a rat model of HI. A total of 127 ten-day old Sprague Dawley rat pups were used. HI was induced by right common carotid artery ligation with subsequent exposure to hypoxia for 2.5 h. Exogenous recombinant human CSF1 (rh-CSF1) was administered intranasally at 1 h and 24 h after HI. The CSF1R inhibitor, BLZ945, or phospholipase C-gamma 2 (PLCG2) inhibitor, U73122, was injected intraperitoneally at 1 h before HI induction. Brain infarct volume measurement, cliff avoidance test, righting reflex test, double immunofluorescence staining, western blot assessment, 8-OHdG and MitoSOX staining, Fluoro-Jade C staining, and TUNEL staining were used. Our results indicated that the expressions of endogenous CSF1, CSF1R, p-CSF1R, p-PLCG2, p-PKA, and uncoupling protein2 (UCP2) were increased after HI. CSF1 and CSF1R were expressed in neurons and astrocytes. Rh-CSF1 treatment significantly attenuated neurological deficits, infarct volume, OS, neuronal apoptosis, and degeneration at 48 h after HI. Moreover, activation of CSF1R by rh-CSF1 significantly increased the brain tissue expressions of p-PLCG2, p-PKA, UCP2, and Bcl2/Bax ratio, but reduced the expression of cleaved caspase-3. The neuroprotective effects of rh-CSF1 were abolished by BLZ945 or U73122. These results suggested that rh-CSF1 treatment attenuated OS-induced neuronal degeneration and apoptosis after HI, at least in part, through the CSF1R/PLCG2/PKA/UCP2 signaling pathway. Rh-CSF1 may serve as therapeutic strategy against brain damage in patients with HIE.

Rh-CSF1 attenuates neuroinflammation via the CSF1R/PLCG2/PKCε pathway in a rat model of neonatal HIE.

BACKGROUND: Hypoxic-ischemic encephalopathy (HIE) is a life-threatening cerebrovascular disease. Neuroinflammation plays an important role in the pathogenesis of HIE, in which microglia are key cellular mediators in the regulation of neuroinflammatory processes. Colony-stimulating factor 1 (CSF1), a specific endogenous ligand of CSF1 receptor (CSF1R), is crucial in microglial growth, differentiation, and proliferation. Recent studies showed that the activation of CSF1R with CSF1 exerted anti-inflammatory effects in a variety of nervous system diseases. This study aimed to investigate the anti-inflammatory effects of recombinant human CSF1 (rh-CSF1) and the underlying mechanisms in a rat model of HIE. METHODS: A total of 202 10-day old Sprague Dawley rat pups were used. HI was induced by the right common carotid artery ligation with subsequent exposure of 2.5-h hypoxia. At 1 h and 24 h after HI induction, exogenous rh-CSF1 was administered intranasally. To explore the underlying mechanism, CSF1R inhibitor, BLZ945, and phospholipase C-gamma 2 (PLCG2) inhibitor, U73122, were injected intraperitoneally at 1 h before HI induction, respectively. Brain infarct area, brain water content, neurobehavioral tests, western blot, and immunofluorescence staining were performed. RESULTS: The expressions of endogenous CSF1, CSF1R, PLCG2, protein kinase C epsilon type (PKCε), and cAMP response element-binding protein (CREB) were gradually increased after HIE. Rh-CSF1 significantly improved the neurological deficits at 48 h and 4 weeks after HI, which was accompanied by a reduction in the brain infarct area, brain edema, brain atrophy, and neuroinflammation. Moreover, activation of CSF1R by rh-CSF1 significantly increased the expressions of p-PLCG2, p-PKCε, and p-CREB, but inhibited the activation of neutrophil infiltration, and downregulated the expressions of IL-1ß and TNF-α. Inhibition of CSF1R and PLCG2 abolished these neuroprotective effects of rh-CSF1 after HI. CONCLUSIONS: Our findings demonstrated that the activation of CSF1R by rh-CSF1 attenuated neuroinflammation and improved neurological deficits after HI. The anti-inflammatory effects of rh-CSF1 partially acted through activating the CSF1R/PLCG2/PKCε/CREB signaling pathway after HI. These results suggest that rh-CSF1 may serve as a potential therapeutic approach to ameliorate injury in HIE patients.

IRE1α inhibition attenuates neuronal pyroptosis via miR-125/NLRP1 pathway in a neonatal hypoxic-ischemic encephalopathy rat model.

BACKGROUND: Inhibition of inositol-requiring enzyme-1 alpha (IRE1α), one of the sensor signaling proteins associated with endoplasmic reticulum (ER) stress, has been shown to alleviate brain injury and improve neurological behavior in a neonatal hypoxic-ischemic encephalopathy (HIE) rat model. However, there is no information about the role of IRE1α inhibitor as well as its molecular mechanisms in preventing neuronal pyroptosis induced by NLRP1 (NOD-, LRR- and pyrin domain-containing 1) inflammasome. In the present study, we hypothesized that IRE1α can degrade microRNA-125-b-2-3p (miR-125-b-2-3p) and activate NLRP1/caspased-1 pathway, and subsequently promote neuronal pyroptosis in HIE rat model. METHODS: Ten-day old unsexed rat pups were subjected to hypoxia-ischemia (HI) injury, and the inhibitor of IRE1α, STF083010, was administered intranasally at 1 h after HI induction. AntimiR-125 or NLRP1 activation CRISPR was administered by intracerebroventricular (i.c.v) injection at 24 h before HI induction. Immunofluorescence staining, western blot analysis, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), brain infarct volume measurement, neurological function tests, and Fluoro-Jade C staining were performed. RESULTS: Endogenous phosphorylated IRE1α (p-IRE1α), NLRP1, cleaved caspase-1, interleukin-1ß (IL-1ß), and interleukin-18 (IL-18) were increased and miR-125-b-2-3p was decreased in HIE rat model. STF083010 administration significantly upregulated the expression of miR-125-b-2-3p, reduced the infarct volume, improved neurobehavioral outcomes and downregulated the protein expression of NLRP1, cleaved caspase-1, IL-1ß and IL-18. The protective effects of STF083010 were reversed by antimiR-125 or NLRP1 activation CRISPR. CONCLUSIONS: IRE1α inhibitor, STF083010, reduced neuronal pyroptosis at least in part via miR-125/NLRP1/caspase-1 signaling pathway after HI.

cGAS/STING Pathway Activation Contributes to Delayed Neurodegeneration in Neonatal Hypoxia-Ischemia Rat Model: Possible Involvement of LINE-1.

cGAS is a sensor of cytosolic DNA and responds equally to exogenous and endogenous DNA. After recognition of cytosolic dsDNA or ssDNA, cGAS synthesizes the second messenger 2'3'-cGAMP, which then binds to and activates stimulator of interferon genes (STING). STING plays an essential role in responding to pathogenic DNA and self-DNA in the context of autoimmunity. In pathologic conditions, such as stroke or hypoxia-ischemia (HI), DNA can gain access into the cytoplasm of the cell and leak from the dying cells into the extracellular environment, which potentially activates cGAS/STING. Recent in vivo studies of myocardial ischemia, traumatic brain injury, and liver damage models suggest that activation of cGAS/STING is not only a side-effect of the injury, but it can also actively contribute to cell death and apoptosis. We found, for the first time, that cGAS/STING pathway becomes activated between 24 and 48 h after HI in a 10-day-old rat model. Silencing STING with siRNA resulted in decreased infarction area, reduced cortical neurodegeneration, and improved neurobehavior at 48 h, suggesting that STING can contribute to injury progression after HI. STING colocalized with lysosomal marker LAMP-1 and blocking STING reduced the expression of cathepsin B and decreased the expression of Bax and caspase 3 cleavage. We observed similar protective effects after intranasal treatment with cGAS inhibitor RU.521, which were reversed by administration of STING agonist 2'3'-cGAMP. Additionally, we showed that long interspersed element 1 (LINE-1) retrotransposon, a potential upstream activator of cGAS/STING pathway was induced at 48 h after HI, which was evidenced by increased expression of ORF1p and ORF2p proteins and increased LINE-1 DNA content in the cytosol. Blocking LINE-1 with the nucleoside analog reverse-transcriptase inhibitor (NRTI) stavudine reduced infarction area, neuronal degeneration in the cerebral cortex, and reduced the expression of Bax and cleaved caspase 3. Thus, our results identify the cGAS/STING pathway as a potential therapeutic target to inhibit delayed neuronal death after HI.

Ghrelin attenuates oxidative stress and neuronal apoptosis via GHSR-1α/AMPK/Sirt1/PGC-1α/UCP2 pathway in a rat model of neonatal HIE.

Neuronal apoptosis induced by oxidative stress is one of the major pathological processes involved in neurological impairment after hypoxic-ischemic encephalopathy (HIE). Ghrelin, the unique endogenous ligand for the growth hormone secretagogue receptor-1α (GHSR-1α), could take an anti-apoptotic role in the brain. However, whether ghrelin can attenuate neuronal apoptosis by attenuating oxidative stress after hypoxia-ischemia (HI) insult remains unknown. To investigate the beneficial effects of ghrelin on oxidative stress injury and neuronal apoptosis induced by HI, ten-day old unsexed rat pups were subjected to HI injury and exogenous recombinant human ghrelin(rh-Ghrelin) was administered intranasally at 1 h and 24 h after HI induction. [D-Lys3]-GHRP-6, a selective inhibitor of GHSR-1α and Ex527, a selective inhibitor of GHSR-1α were administered intranasally at 1 h before HI induction respectively. Small interfering ribonucleic acid (siRNA) for GHSR-1α were administered by intracerebroventricular (i.c.v) injection at 24 h before HI induction. Neurological tests, immunofluorescence, MitoSox staining, Fluoro-Jade C staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and western blot experiments were performed. Our results indicated that ghrelin significantly improved neurobehavioral outcomes and reduced oxidative stress and neuronal apoptosis. Moreover, ghrelin treatment significantly promoted phosphorylation of AMPK, upregulated the expression of Sirt1, PGC-1α, UCP2 and the ratio of Bcl2/Bax, while it downregulated cleaved caspase-3 levels. The protective effects of ghrelin were reversed by [D-Lys3]-GHRP-6, GHSR-1α siRNA or Ex527. In conclusion, our data demonstrated that ghrelin reduced oxidative stress injury and neuronal apoptosis which was in part via the GHSR-1α/AMPK/Sirt1/PGC-1α/UCP2 signalling pathway after HI. Ghrelin may be a novel therapeutic target for treatment after neonatasl HI injury.

Recombinant Slit2 attenuates neuronal apoptosis via the Robo1-srGAP1 pathway in a rat model of neonatal HIE.

Apoptosis following hypoxic-ischemic injury to the brain plays a major role in neuronal cell death. The neonatal brain is more susceptible to injury as the cortical neurons are immature and there are lower levels of antioxidants. Slit2, an extracellular matrix protein, has been shown to be neuroprotective in various models of neurological diseases. However, there is no information about the role of Slit2 in neonatal hypoxia-ischemia. In this study, we evaluated the effect of Slit2 and its receptor Robo1 in a rat model with neonatal HIE. 10-day old rat pups were used to create the neonatal HIE model. The right common carotid artery was ligated followed by 2.5 h of hypoxia. Recombinant Slit2 was administered intranasally 1 h post HI, recombinant Robo1 was used as a decoy receptor and administered intranasally 1h before HI and srGAP1-siRNA was administered intracerebroventricularly 24 h before HI. Brain infarct area measurement, short-term and long-term neurological function tests, Western blot, immunofluorescence staining, Fluoro-Jade C staining, Nissl staining and TUNEL staining were the assessments done following drug administration. Recombinant Slit2 administration reduced neuronal apoptosis and neurological deficits after neonatal HIE which were reversed by co-administration of recombinant Robo1 and srGAP1-siRNA administration. Recombinant Slit2 showed improved outcomes possibly via the robo1-srGAP1 pathway which mediated the inhibition of RhoA. In this study, the results suggest that Slit2 may help in attenuation of apoptosis and could be a therapeutic agent for treatment of neonatal hypoxic ischemic encephalopathy.

Chemerin reverses neurological impairments and ameliorates neuronal apoptosis through ChemR23/CAMKK2/AMPK pathway in neonatal hypoxic-ischemic encephalopathy.

Hypoxic-ischemic encephalopathy (HIE) is a devastating neurological event that contributes to the prolonged neurodevelopmental consequences in infants. Therapeutic strategies focused on attenuating neuronal apoptosis in the penumbra appears to be promising. Given the increasingly recognized neuroprotective roles of adipokines in HIE, we investigated the potential anti-apoptotic roles of a novel member of adipokines, Chemerin, in an experimental model of HIE. In the present study, 10-day-old rat pups underwent right common carotid artery ligation followed by 2.5 h hypoxia. At 1 h post hypoxia, pups were intranasally administered with human recombinant chemerin (rh-chemerin). Here, we showed that rh-chemerin prevented the neuronal apoptosis and degeneration as evidenced by the decreased expression of the pro-apoptotic markers, cleaved caspase 3 and Bax, as well as the numbers of Fluoro-Jade C and TUNEL-positive neurons. Furthermore, rh-Chemerin reversed neurological and morphological impairments induced by hypoxia-ischemia in neonatal rats at 24 h and 4 weeks after HIE. In addition, chemerin-mediated neuronal survival correlated with the elevation of chemerin receptor 23 (chemR23), phosphorylated calmodulin-dependent protein kinase kinase 2 (CAMKK2), as well as phosphorylated adenosine monophosphate-activated protein kinase (AMPK). Specific inhibition of chemR23, CAMKK2, and AMPK abolished the anti-apoptotic effects of rh-chemerin at 24 h after HIE, demonstrating that rh-chemerin ameliorated neuronal apoptosis partially via activating chemR23/CAMKK2/AMPK signaling pathway. Neuronal apoptosis is a well-established contributing factor of pathological changes and the neurological impairment after HIE. These results revealed mechanisms of neuroprotection by rh-chemerin, and indicated that activation of chemR23 might be harnessed to protect from neuronal apoptosis in HIE.

Long noncoding RNA AC003092.1 promotes temozolomide chemosensitivity through miR-195/TFPI-2 signaling modulation in glioblastoma.

Temozolomide (TMZ) and radiation therapy combination for glioblastoma (GB) patients has been considered as the most effective therapy after surgical procedure. However, the overall clinical prognosis remains unsatisfactory due to intrinsic or developing resistance to TMZ. Recently, increasing evidence suggested that long noncoding RNAs (lncRNAs) play a critical role in various biological processes of tumors, and have been implicated in resistance to various drugs. However, the role of lncRNAs in TMZ resistance is poorly understood. Here, we found that the expression of lncRNA AC003092.1 was markedly decreased in TMZ resistance (TR) of GB cells (U87TR and U251TR) compared with their parental cells (U87 and U251). In patients with glioma, low levels of lncRNA AC003092.1 were correlated with increased TMZ resistance, higher risk of relapse, and poor prognosis. Overexpression of lncRNA AC003092.1 enhances TMZ sensitivity, facilitates cell apoptosis, and inhibits cell proliferation in TMZ-resistant GB cells. In addition, we identified that lncRNA AC003092.1 regulates TMZ chemosensitivity through TFPI-2-mediated cell apoptosis in vitro and in vivo. Mechanistically, further investigation revealed that lncRNA AC003092.1 regulates TFPI-2 expression through miR-195 in GB. Taken together, these data suggest that lncRNA AC003092.1 could inhibit the function of miR-195 by acting as an endogenous CeRNA, leading to increased expression of TFPI-2; this promotes TMZ-induced apoptosis, thereby making GB cells more sensitive to TMZ. Our findings indicate that overexpression of lncRNA AC003092.1 may be a potential therapy to overcome TMZ resistance in GB patients.

Role of PPAR-ß/δ/miR-17/TXNIP pathway in neuronal apoptosis after neonatal hypoxic-ischemic injury in rats.

Activation of peroxisome proliferator-activated receptor beta/delta (PPAR-ß/δ), a nuclear receptor acting as a transcription factor, was shown to be protective in various models of neurological diseases. However, there is no information about the role of PPAR-ß/δ as well as its molecular mechanisms in neonatal hypoxia-ischemia (HI). In the present study, we hypothesized that PPAR-ß/δ agonist GW0742 can activate miR-17-5p, consequently inhibiting TXNIP and ASK1/p38 pathway leading to attenuation of apoptosis. Ten-day-old rat pups were subjected to right common carotid artery ligation followed by 2.5 h hypoxia. GW0742 was administered intranasally 1 and 24 h post HI. PPAR-ß/δ receptor antagonist GSK3787 was administered intranasally 1 h before and 24 h after HI, antimir-17-5p and TXNIP CRISPR activation plasmid were administered intracerebroventricularly 24 and 48 h before HI, respectively. Brain infarct area measurement, neurological function tests, western blot, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), Fluoro-Jade C and immunofluorescence staining were conducted. GW0742 reduced brain infarct area, brain atrophy, apoptosis, and improved neurological function at 72 h and 4 weeks post HI. Furthermore, GW0742 treatment increased PPAR-ß/δ nuclear expression and miR-17-5p level and reduced TXNIP in ipsilateral hemisphere after HI, resulting in inhibition of ASK1/p38 pathway and attenuation of apoptosis. Inhibition of PPAR-ß/δ receptor and miR-17-5p and activation of TXNIP reversed the protective effects. For the first time, we provide evidence that intranasal administration of PPAR-ß/δ agonist GW0742 attenuated neuronal apoptosis at least in part via PPAR-ß/δ/miR-17/TXNIP pathway. GW0742 could represent a therapeutic target for treatment of neonatal hypoxic ischemic encephalopathy (HIE).

Adiponectin attenuates neuronal apoptosis induced by hypoxia-ischemia via the activation of AdipoR1/APPL1/LKB1/AMPK pathway in neonatal rats.

Adiponectin is an important adipocyte-derived plasma protein that has beneficial effects on cardio- and cerebrovascular diseases. A low level of plasma Adiponectin is associated with increased mortality post ischemic stroke; however, little is known about the causal role of Adiponectin as well as its molecular mechanisms in neonatal hypoxia ischemia (HI). In the present study, ten-day-old rat pups were subjected to right common carotid artery ligation followed by 2.5 h hypoxia. Recombinant human Adiponectin (rh-Adiponectin) was administered intranasally 1 h post HI. Adiponectin Receptor 1 (AdipoR1) siRNA, APPL1 siRNA, LKB1 siRNA were administered through intracerebroventricular injection 48 h before HI. Brain infarct area measurement, neurological function test, western blot, Fluoro Jade C (FJC), TUNEL, and immunofluorescence staining were conducted. Results revealed that endogenous Adiponectin, AdipoR1 and APPL1 were increased in a time dependent manner after HI. Administration of rh-Adiponectin reduced brain infarct area, neuronal apoptosis, brain atrophy and improved neurological function at 24 h and 4 weeks post HI. Furthermore, rh-Adiponectin treatment increased Adiponectin, AdipoR1, APPL1, cytosolic LKB1, p-AMPK expression levels and thereby attenuated apoptosis as shown by the decreased expression of the pro-apoptotic marker, Cleaved Caspase 3 (C-Cas3), as well as the number of FJC and TUNEL positively stained neurons. AdipoR1, APPL1 and LKB1 siRNAs abolished the anti-apoptotic effects of rh-Adiponectin at 24 h after HI. Collectively, the data provided evidence that intranasal administration of rh-Adiponectin attenuated neuronal apoptosis at least in part via activating AdipoR1/APPL1/LKB1/AMPK signaling pathway. Adiponectin could represent a therapeutic target for treatment of neonatal hypoxic ischemic encephalopathy.

Sestrin2, as a negative feedback regulator of mTOR, provides neuroprotection by activation AMPK phosphorylation in neonatal hypoxic-ischemic encephalopathy in rat pups.

Hypoxic-ischemic encephalopathy is a condition caused by reduced oxygen and cerebral blood flow to the brain resulting in neurological impairments. Effective therapeutic treatments to ameliorate these disabilities are still lacking. We sought to investigate the role of sestrin2, a highly conserved stress-inducible protein, in a neonatal rat hypoxic-ischemic encephalopathy model. Ten-day-old rat pups underwent right common carotid artery ligation followed by 2.5 h hypoxia. At 1 h post hypoxic-ischemic encephalopathy, rats were intranasally administered with recombinant human sestrin2 and sacrificed for brain infarct area measurement, Fluoro-Jade C, immunofluorescence staining, Western blot, and neurological function testing. rh-sestrin2 reduced brain infarct area, brain atrophy, apoptosis, ventricular area enlargement, and improved neurological function. Western blot showed that sestrin2 expression levels were increased after treatment with rh-sestrin2, and sestrin2 exerts neuroprotective effects via activation of the adenosine monophosphate-activated protein kinase pathway which in turn inhibits mammalian target of rapamycin signaling resulting in the attenuation of apoptosis. In conclusions: Sestrin2 plays an important neuroprotective role after hypoxic-ischemic encephalopathy via adenosine monophosphate-activated protein kinase signaling pathway and serves as a negative feedback regulator of mammalian target of rapamycin. Administration of rh-sestrin2 not only reduced infarct area and brain atrophy, but also significantly improved neurological function.

Sestrin2 induced by hypoxia inducible factor1 alpha protects the blood-brain barrier via inhibiting VEGF after severe hypoxic-ischemic injury in neonatal rats.

OBJECTIVE: Hypoxic ischemic (HI) encephalopathy remains the leading cause of perinatal brain injury resulting in long term disabilities. Stabilization of blood brain barrier (BBB) after HI is an important target, therefore, in this study we aim to determine the role of sestrin2, a stress inducible protein which is elevated after various insults, on BBB stabilization after moderate and severe HI injuries. METHODS: Rat pups underwent common carotid artery ligation followed by either 150min (severe model) or 100min (moderate model) of hypoxia. 1h post HI, rats were intranasally administered with recombinant human sestrin2 (rh-sestrin2) and sacrificed for infarct area, brain water content, righting reflex and geotaxis reflex. Sestrin2 was silenced using siRNA and an activator/inhibitor of hypoxia inducible factor1α (HIF1α) was used to examine their roles on BBB permeability. RESULTS: Rats subjected to severe HI exhibited larger infarct area and higher sestrin2 expression compared to rats in the moderate HI group. rh-sestrin2 attenuated brain infarct and edema, while silencing sestrin2 reversed these protective effects after severe HI. HIF1α induced sestrin2 activation in severe HI but not in moderate HI groups. A HIF1a agonist was shown to increase permeability of the BBB via vascular endothelial growth factor (VEGF) after moderate HI. However, after severe HI, HIF1α activated both VEGF and sestrin2. But HIF1α dependent sestrin2 activation was the predominant pathway after severe HI which inhibited VEGF and attenuated BBB permeability. CONCLUSIONS: rh-sestrin2 attenuated BBB permeability via upregulation of endogenous sestrin2 which was induced by HIF1α after severe HI. However, HIF1α's effects as a prodeath or prosurvival signal were influenced by the severity of HI injury.

Granulocyte-colony stimulating factor activates JAK2/PI3K/PDE3B pathway to inhibit corticosterone synthesis in a neonatal hypoxic-ischemic brain injury rat model.

OBJECTIVE: Our previous study demonstrated that granulocyte-colony stimulating factor (G-CSF)-induced neuroprotection is accompanied by an inhibition of corticosterone production in a neonatal hypoxic-ischemic (HI) rat model. The present study investigates how G-CSF inhibits corticosterone production, using adrenal cortical cells and HI rat pups. METHODS: Cholera toxin was used to induce corticosterone synthesis in a rodent Y1 adrenal cortical cell line by increasing cyclic adenosine monophosphate (cAMP). Both corticosterone and cAMP were quantitatively measured using a commercial enzyme-linked immunosorbent assay (ELISA). The downstream signaling components of the G-CSF receptor, including Janus Kinase 2 (JAK2)/Phosphatidylinositol-3-kinase (PI3K)/Protein kinase B (Akt) and Phosphodiesterase 3B (PDE3B), were detected by western blot. Sprague-Dawley rat pups at the age of 10days (P10) were subjected to unilateral carotid artery ligation followed by hypoxia for 2.5hours. Brain infarction volumes were determined using 2,3,5-triphenyltetrazolium chloride monohydrate (TTC) staining. RESULTS: G-CSF at 30ng/ml inhibited corticosterone synthesis but lost its inhibitory effect at higher doses. The inhibitory effect of G-CSF was conferred by interfering with cAMP signaling via the activation of the JAK2/PI3K/PDE3B signaling pathway. The degradation of cAMP by G-CSF signaling reduced corticosterone production. This mechanism was further verified in the neonatal HI brain injury rat model, in which inhibition of PDE3B reversed the protective effects of G-CSF. CONCLUSION: Our data suggest that the neuroprotective G-CSF reduces corticosterone synthesis at the adrenal level by degrading intracellular cAMP via activation of the JAK2/PI3K/PDE3B pathway.