Peroxisome Proliferator-Activated Receptor-γ Agonist 15d-Prostaglandin J2 Mediates Neuronal Autophagy after Cerebral Ischemia-Reperfusion Injury

Peroxisome proliferator-activated receptor-γ (PPAR-γ) has recently emerged as potential therapeutic agents for cerebral ischemia-reperfusion (I/R) injury because of anti-neuronal apoptotic actions. However, whether PPAR-γ activation mediates neuronal autophagy in such conditions remains unclear. Therefore, in this study, we investigated the role of PPAR-γ agonist 15-PGJ₂ on neuronal autophagy induced by I/R. The expression of autophagic-related protein in ischemic cortex such as LC3-II, Beclin 1, cathepsin-B and LAMP1 increased significantly after cerebral I/R injury. Furthermore, increased punctate LC3 labeling and cathepsin-B staining occurred in neurons. Treatment with PPAR-γ agonist 15d-PGJ₂ decreased not only autophagic-related protein expression in ischemic cortex, but also immunoreactivity of LC3 and cathepsin-B in neurons. Autophagic inhibitor 3-methyladenine (3-MA) decreased LC3-II levels, reduced the infarct volume, and mimicked some protective effect of 15d-PGJ₂ against cerebral I/R injury. These results indicate that PPAR-γ agonist 15d-PGJ₂ exerts neuroprotection by inhibiting neuronal autophagy after cerebral I/R injury. Although the molecular mechanisms underlying PPAR-γ agonist in mediating neuronal autophagy remain to be determined, neuronal autophagy may be a new target for PPAR-γ agonist treatment in cerebral I/R injury.     

Nicotinamide Phosphoribosyltransferase May Be Involved in Age-Related Brain Diseases

Nicotinamide phosphoribosyltransferase (NAMPT) is a key enzyme for nicotinamide adenine dinucleotide (NAD) biosynthesis, and can be found either intracellularly (iNAMPT) or extracellularly (eNAMPT). Studies have shown that both iNAMPT and eNAMPT are implicated in aging and age-related diseases/disorders in the peripheral system. However, their functional roles in aged brain remain to be established. Here we showed that upon aging, NAMPT level increased in serum but decreased in brain, decreased in cortex and hippocampus but remained unchanged in cerebellum and striatum in brain, and increased in microglia but likely decreased in neuron. Accordingly, total NAD (tNAD) level significantly decreased in hippocampus, cerebellum and striatum in aged brain. Application of recombinant NAMPT, mimicking the elevated serum NAMPT level, enhanced the susceptibility of cerebral endothelial cells to ischemic injury, while inhibition of iNAMPT by FK866, a specific inhibitor, reduced intracellular NAD level and induced neuronal death. Taken together, we have revealed a region- and cell-specific change of NAMPT level in brain and serum upon aging, deduced its potential consequences, which suggests that NAMPT is a regulatory factor in aging and age-related brain diseases.     

Acetylbritannilactone Modulates MicroRNA-155-Mediated Inflammatory Response in Ischemic Cerebral Tissues

Inflammatory responses play a critical role in ischemic brain injury. MicroRNA-155 (miR-155) induces the expression of inflammatory cytokines, and acetylbritannilactone (ABL) exerts potent antiinflammatory actions by inhibiting expression of inflammation-related genes. However, the functions of miR-155 and the actual relationship between ABL and miR-155 in ischemia-induced cerebral inflammation remain unclear. In this study, cerebral ischemia of wild-type (WT) and miR-155^−/− mice was induced by permanent middle cerebral artery occlusion (MCAO). pAd-miR-155 was injected into the lateral cerebral ventricle 24 h before MCAO to induce miR-155 overexpression. MCAO mice and oxygen-glucose deprivation (OGD)-treated BV2 cells were used to examine the effects of ABL and miR-155 overexpression or deletion on the expression of proinflammatory cytokines. We demonstrated that ABL treatment significantly reduced neurological deficits and cerebral infarct volume by inhibiting tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) expression in ischemic cerebral tissue and OGD-treated BV2 cells. Mechanistic studies suggested that the observed decrease in TNF-α and IL-1β expression was attributable to the ABL-induced suppression of the expression of nuclear factor-kappa B (NF-κB) and Toll-like receptor 4 (TLR4). We further found that miR-155 promoted TNF-α and IL-1β expression by upregulating TLR4 and downregulating the expression of suppressor of cytokine signaling 1 (SOCS1) and myeloid differentiation primary response gene 88 (MyD88), while ABL exerted an inhibitory effect on miR-155-mediated gene expression. In conclusion, miR-155 mediates inflammatory responses in ischemic cerebral tissue by modulating TLR4/MyD88 and SOCS1 expression, and ABL exerts its antiinflammatory action by suppressing miR-155 expression, suggesting a novel miR-155-based therapy for ischemic stroke.     

Molecular Mechanisms Responsible for Neuron-Derived Conditioned Medium (NCM)-Mediated Protection of Ischemic Brain

The protective value of neuron-derived conditioned medium (NCM) in cerebral ischemia and the underlying mechanism(s) responsible for NCM-mediated brain protection against cerebral ischemia were investigated in the study. NCM was first collected from the neuronal culture growing under the in vitro ischemic condition (glucose-, oxygen- and serum-deprivation or GOSD) for 2, 4 or 6 h. Through the focal cerebral ischemia (bilateral CCAO/unilateral MCAO) animal model, we discovered that ischemia/reperfusion (I/R)-induced brain infarction was significantly reduced by NCM, given directly into the cistern magna at the end of 90 min of CCAO/MCAO. Immunoblocking and chemical blocking strategies were applied in the in vitro ischemic studies to show that NCM supplement could protect microglia, astrocytes and neurons from GOSD-induced cell death, in a growth factor (TGFβ1, NT-3 and GDNF) and p-ERK dependent manner. Brain injection with TGFβ1, NT3, GDNF and ERK agonist (DADS) alone or in combination, therefore also significantly decreased the infarct volume of ischemic brain. Moreover, NCM could inhibit ROS but stimulate IL-1β release from GOSD-treated microglia and limit the infiltration of IL-β-positive microglia into the core area of ischemic brain, revealing the anti-oxidant and anti-inflammatory activities of NCM. In overall, NCM-mediated brain protection against cerebral ischemia has been demonstrated for the first time in S.D. rats, due to its anti-apoptotic, anti-oxidant and potentially anti-glutamate activities (NCM-induced IL-1β can inhibit the glutamate-mediated neurotoxicity) and restriction upon the infiltration of inflammatory microglia into the core area of ischemic brain. The therapeutic potentials of NCM, TGFβ1, GDNF, NT-3 and DADS in the control of cerebral ischemia in human therefore have been suggested and require further investigation.     

African Swine Fever Virus Infection Induces Tumor Necrosis Factor Alpha Production: Implications in Pathogenesis

We have analyzed the production of tumor necrosis factor alpha (TNF-α) induced by in vitro infection with African swine fever (ASF) virus (ASFV) and the systemic and local release of this inflammatory cytokine upon in vivo infection. An early increase in TNF-α mRNA expression was detected in ASFV-infected alveolar macrophages, and high levels of TNF-α protein were detected by ELISA in culture supernatants from these cells. When animals were experimentally infected with a virulent isolate (E-75), enhanced TNF-α expression in mainly affected organs correlated with viral protein expression. Finally, elevated levels of TNF-α were detected in serum, corresponding to the onset of clinical signs. TNF-α has been reported to be critically involved in the pathogenesis of major clinical events in ASF, such as intravascular coagulation, tissue injury, apoptosis, and shock. In the present study, TNF-α containing supernatants from ASFV-infected cultures induced apoptosis in uninfected lymphocytes; this effect was partially abrogated by preincubation with an anti-TNF-α specific antibody. These results suggest a relevant role for TNF-α in the pathogenesis of ASF.     

Propofol Protects Against Focal Cerebral Ischemia via Inhibition of Microglia-Mediated Proinflammatory Cytokines in a Rat Model of Experimental Stroke

Ischemic stroke induces microglial activation and release of proinflammatory cytokines, contributing to the expansion of brain injury and poor clinical outcome. Propofol has been shown to ameliorate neuronal injury in a number of experimental studies, but the precise mechanisms involved in its neuroprotective effects remain unclear. We tested the hypothesis that propofol confers neuroprotection against focal ischemia by inhibiting microglia-mediated inflammatory response in a rat model of ischemic stroke. Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h followed by 24 h of reperfusion. Propofol (50 mg/kg/h) or vehicle was infused intravenously at the onset of reperfusion for 30 minutes. In vehicle-treated rats, MCAO resulted in significant cerebral infarction, higher neurological deficit scores and decreased time on the rotarod compared with sham-operated rats. Propofol treatment reduced infarct volume and improved the neurological functions. In addition, molecular studies demonstrated that mRNA expression of microglial marker Cd68 and Emr1 was significantly increased, and mRNA and protein expressions of proinflammatory cytokines tumor necrosis factor-α, interleukin-1β and interleukin-6 were augmented in the peri-infarct cortical regions of vehicle-treated rats 24 h after MCAO. Immunohistochemical study revealed that number of total microglia and proportion of activated microglia in the peri-infarct cortical regions were markedly elevated. All of these findings were ameliorated in propofol-treated rats. Furthermore, vehicle-treated rats had higher plasma levels of interleukin-6 and C-reactive protein 24 h after MCAO, which were decreased after treatment with propofol. These results suggest that propofol protects against focal cerebral ischemia via inhibition of microglia-mediated proinflammatory cytokines. Propofol may be a promising therapeutic agent for the treatment of ischemic stroke and other neurodegenerative diseases associated with microglial activation.     

Periodic 17β-Estradiol Pretreatment Protects Rat Brain from Cerebral Ischemic Damage via Estrogen Receptor-β

Although chronic 17β-estradiol (E₂) has been shown to be a cognition-preserving and neuroprotective agent in animal brain injury models, concern regarding its safety was raised by the failed translation of this phenomenon to the clinic. Previously, we demonstrated that a single bolus of E₂ 48 hr prior to ischemia protected the hippocampus from damage in ovariectomized rats via phosphorylation of cyclic-AMP response element binding protein, which requires activation of estrogen receptor subtype beta (ER-β). The current study tests the hypothesis that long-term periodic E₂-treatment improves cognition and reduces post-ischemic hippocampal injury by means of ER-β activation. Ovariectomized rats were given ten injections of E₂ at 48 hr intervals for 21 days. Hippocampal-dependent learning, memory and ischemic neuronal loss were monitored. Results demonstrated that periodic E₂ treatments improved spatial learning, memory and ischemic neuronal survival in ovariectomized rats. Additionally, periodic ER-β agonist treatments every 48 hr improved post-ischemic cognition. Silencing of hippocampal ER-β attenuated E₂-mediated ischemic protection suggesting that ER-β plays a key role in mediating the beneficial effects of periodic E₂ treatments. This study emphasizes the need to investigate a periodic estrogen replacement regimen to reduce cognitive decline and cerebral ischemia incidents/impact in post-menopausal women.     

Neuroprotective Effect of Scutellarin on Ischemic Cerebral Injury by Down-Regulating the Expression of Angiotensin-Converting Enzyme and AT1 Receptor

MethodsAdult Sprague–Dawley rats were administrated with different dosages of Scu by oral gavage for 7 days and underwent permanent middle cerebral artery occlusion (pMCAO). Blood pressure was measured 7 days after Scu administration and 24 h after pMCAO surgery by using a noninvasive tail cuff method. Cerebral blood flow (CBF) was determined by Laser Doppler perfusion monitor and the neuronal dysfunction was evaluated by analysis of neurological deficits before being sacrificed at 24 h after pMCAO. Histopathological change, cell apoptosis and infarct area were respectively determined by hematoxylin–eosin staining, terminal deoxynucleotidyl transfer-mediated dUTP nick end labeling (TUNEL) analysis and 2,3,5-triphenyltetrazolium chloride staining. Tissue angiotensin II (Ang II) and ACE activity were detected by enzyme-linked immunosorbent assays. The expression levels of ACE, Ang II type 1 receptor (AT1R), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) were measured by Western blot and real-time PCR. ACE inhibitory activity of Scu in vitro was detected by the photometric determination.ResultsScu treatment dose-dependently decreased neurological deficit score, infarct area, cell apoptosis and morphological changes induced by pMCAO, which were associated with reductions of ACE and AT1R expression and the levels of Ang II, TNF-α, IL-6, and IL-1β in ischemic brains. Scu has a potent ACE inhibiting activity.ConclusionScu protects brain from acute ischemic injury probably through its inhibitory effect on the ACE/Ang II/AT1 axis, CBF preservation and proinflammation inhibition.     

Molecular insights into the interaction between human nicotinamide phosphoribosyltransferase and Toll-like receptor 4

The secreted form of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), which catalyzes a key reaction in intracellular NAD biosynthesis, acts as a damage-associated molecular pattern triggering Toll-like receptor 4 (TLR4)-mediated inflammatory responses. However, the precise mechanism of interaction is unclear. Using an integrated approach combining bioinformatics and functional and structural analyses, we investigated the interaction between NAMPT and TLR4 at the molecular level. Starting from previous evidence that the bacterial ortholog of NAMPT cannot elicit the inflammatory response, despite a high degree of structural conservation, two positively charged areas unique to the human enzyme (the α1-α2 and β1-β2 loops) were identified as likely candidates for TLR4 binding. However, alanine substitution of the positively charged residues within these loops did not affect either the oligomeric state or the catalytic efficiency of the enzyme. The kinetics of the binding of wildtype and mutated NAMPT to biosensor-tethered TLR4 was analyzed. We found that mutations in the α1-α2 loop strongly decreased the association rate, increasing the K_D value from 18 nM, as determined for the wildtype, to 1.3 μM. In addition, mutations in the β1-β2 loop or its deletion increased the dissociation rate, yielding K_D values of 0.63 and 0.22 μM, respectively. Mutations also impaired the ability of NAMPT to trigger the NF-κB inflammatory signaling pathway in human cultured macrophages. Finally, the involvement of the two loops in receptor binding was supported by NAMPT-TLR4 docking simulations. This study paves the way for future development of compounds that selectively target eNAMPT/TLR4 signaling in inflammatory disorders.     

Protection of Ischemic Postconditioning against Neuronal Apoptosis Induced by Transient Focal Ischemia Is Associated with Attenuation of NF-κB/p65 Activation

Background and Purpose

Accumulating evidences have demonstrated that nuclear factor κB/p65 plays a protective role in the protection of ischemic preconditioning and detrimental role in lethal ischemia-induced programmed cell death including apoptosis and autophagic death. However, its role in the protection of ischemic postconditioning is still unclear.

Methods

Rat MCAO model was used to produce transient focal ischemia. The procedure of ischemic postconditioning consisted of three cycles of 30 seconds reperfusion/reocclusion of MCA. The volume of cerebral infarction was measured by TTC staining and neuronal apoptosis was detected by TUNEL staining. Western blotting was used to analyze the changes in protein levels of Caspase-3, NF-κB/p65, phosphor- NF-κB/p65, IκBα, phosphor- IκBα, Noxa, Bim and Bax between rats treated with and without ischemic postconditioning. Laser scanning confocal microscopy was used to examine the distribution of NF-κB/p65 and Noxa.

Results

Ischemic postconditioning made transient focal ischemia-induced infarct volume decrease obviously from 38.6%±5.8% to 23.5%±4.3%, and apoptosis rate reduce significantly from 46.5%±6.2 to 29.6%±5.3% at reperfusion 24 h following 2 h focal cerebral ischemia. Western blotting analysis showed that ischemic postconditioning suppressed markedly the reduction of NF-κB/p65 in cytoplasm, but elevated its content in nucleus either at reperfusion 6 h or 24 h. Moreover, the decrease of IκBα and the increase of phosphorylated IκBα and phosphorylated NF-κB/p65 at indicated reperfusion time were reversed by ischemic postconditioning. Correspondingly, proapoptotic proteins Caspase-3, cleaved Caspase-3, Noxa, Bim and Bax were all mitigated significantly by ischemic postconditioning. Confocal microscopy revealed that ischemic postconditioning not only attenuated ischemia-induced translocation of NF-κB/p65 from neuronal cytoplasm to nucleus, but also inhibited the abnormal expression of proapoptotic protein Noxa within neurons.

Conclusions

We demonstrated in this study that the protection of ischemic postconditioning on neuronal apoptosis caused by transient focal ischemia is associated with attenuation of the activation of NF-κB/p65 in neurons.     

Induction of nerve growth factor expression and release by mechanical and inflammatory stimuli in chondrocytes: possible involvement in osteoarthritis pain

Introduction

Nerve growth factor (NGF) level is increased in osteoarthritis (OA) joints and is involved in pain associated with OA. Stimuli responsible for NGF stimulation in chondrocytes are unknown. We investigated whether mechanical stress and proinflammatory cytokines may influence NGF synthesis by chondrocytes.

Methods

Primary cultures of human OA chondrocytes, newborn mouse articular chondrocytes or cartilage explants were stimulated by increasing amounts of IL-1β, prostaglandin E₂ (PGE₂), visfatin/nicotinamide phosphoribosyltransferase (NAMPT) or by cyclic mechanical compression (0.5 Hz, 1 MPa). Before stimulation, chondrocytes were pretreated with indomethacin, Apo866, a specific inhibitor of NAMPT enzymatic activity, or transfected by siRNA targeting visfatin/NAMPT. mRNA NGF levels were assessed by real-time quantitative PCR and NGF released into media was determined by ELISA.

Results

Unstimulated human and mouse articular chondrocytes expressed low levels of NGF (19.2 ± 8.7 pg/mL, 13.5 ± 1.0 pg/mL and 4.4 ± 0.8 pg/mL/mg tissue for human and mouse articular chondrocytes and costal explants, respectively). Mechanical stress induced NGF release in conditioned media. When stimulated by IL-1β or visfatin/NAMPT, a proinflammatory adipokine produced by chondocytes in response to IL-1β, a dose-dependent increase in NGF mRNA expression and NGF release in both human and mouse chondrocyte conditioned media was observed. Visfatin/NAMPT is also an intracellular enzyme acting as the rate-limiting enzyme of the generation of NAD. The expression of NGF induced by visfatin/NAMPT was inhibited by Apo866, whereas IL-1β-mediated NGF expression was not modified by siRNA targeting visfatin/NAMPT. Interestingly, PGE₂, which is produced by chondrocytes in response to IL-1β and visfatin/NAMPT, did not stimulate NGF production. Consistently, indomethacin, a cyclooxygenase inhibitor, did not counteract IL-1β-induced NGF production.

Conclusions

These results show that mechanical stress, IL-1β and extracellular visfatin/NAMPT, all stimulated the expression and release of NGF by chondrocytes and thus suggest that the overexpression of visfatin/NAMPT and IL-1β in the OA joint and the increased mechanical loading of cartilage may mediate OA pain via the stimulation of NGF expression and release by chondrocytes.     

Neuroprotective Effect of Neuroserpin in Oxygen-Glucose Deprivation- and Reoxygenation-Treated Rat Astrocytes In Vitro

Neuroserpin (NSP) reportedly exerts neuroprotective effects in cerebral ischemic animal models and patients; however, the mechanism of protection is poorly understood. We thus attempted to confirm neuroprotective effects of NSP on astrocytes in the ischemic state and then explored the relative mechanisms. Astrocytes from neonatal rats were treated with oxygen-glucose deprivation (OGD) followed by reoxygenation (OGD/R). To confirm the neuroprotective effects of NSP, we measured the cell survival rate, relative lactate dehydrogenase (LDH) release; we also performed morphological methods, namely Hoechst 33342 staining and Annexin V assay. To explore the potential mechanisms of NSP, the release of nitric oxide (NO) and TNF-α related to NSP administration were measured by enzyme-linked immunosorbent assay. The proteins related to the NF-κB, ERK1/2, and PI3K/Akt pathways were investigated by Western blotting. To verify the cause-and-effect relationship between neuroprotection and the NF-κB pathway, a NF-κB pathway inhibitor sc3060 was employed to observe the effects of NSP-induced neuroprotection. We found that NSP significantly increased the cell survival rate and reduced LDH release in OGD/R-treated astrocytes. It also reduced NO/TNF-α release. Western blotting showed that the protein levels of p-IKKBα/β and P65 were upregulated by the OGD/R treatment and such effects were significantly inhibited by NSP administration. The NSP-induced inhibition could be significantly reversed by administration of the NF-κB pathway inhibitor sc3060, whereas, expressions of p-ERK1, p-ERK2, and p-AKT were upregulated by the OGD/R treatment; however, their levels were unchanged by NSP administration. Our results thus verified the neuroprotective effects of NSP in ischemic astrocytes. The potential mechanisms include inhibition of the release of NO/TNF-α and repression of the NF-κB signaling pathways. Our data also indicated that NSP has little influence on the MAPK and PI3K/Akt pathways.     

ARRB1/β-arrestin-1 mediates neuroprotection through coordination of BECN1-dependent autophagy in cerebral ischemia

Autophagy, a highly conserved process conferring cytoprotection against stress, contributes to the progression of cerebral ischemia. β-arrestins are multifunctional proteins that mediate receptor desensitization and serve as important signaling scaffolds involved in numerous physiopathological processes. Here, we show that both ARRB1 (arrestin, β 1) and ARRB2 (arrestin, β 2) were upregulated by cerebral ischemic stress. Knockout of Arrb1, but not Arrb2, aggravated the mortality, brain infarction, and neurological deficit in a mouse model of cerebral ischemia. Accordingly, Arrb1-deficient neurons exhibited enhanced cell injury upon oxygen-glucose deprivation (OGD), an in vitro model of ischemia. Deletion of Arrb1 did not affect the cerebral ischemia-induced inflammation, oxidative stress, and nicotinamide phosphoribosyltransferase upregulation, but markedly suppressed autophagy and induced neuronal apoptosis/necrosis in vivo and in vitro. Additionally, we found that ARRB1 interacted with BECN1/Beclin 1 and PIK3C3/Vps34, 2 major components of the BECN1 autophagic core complex, under the OGD condition but not normal conditions in neurons. Finally, deletion of Arrb1 impaired the interaction between BECN1 and PIK3C3, which is a critical event for autophagosome formation upon ischemic stress, and markedly reduced the kinase activity of PIK3C3. These findings reveal a neuroprotective role for ARRB1, in the context of cerebral ischemia, centered on the regulation of BECN1-dependent autophagosome formation.     

Ornithine Decarboxylase Activity in In Vivo and In Vitro Models of Cerebral Ischemia

Ornithine decarboxylase (ODC) is considered the rate-limiting enzyme in polyamine biosynthesis, and an increase in putrescine after central nervous system (CNS) injury appears to be involved in neuronal death. Cerebral ischemia and reperfusion trigger an active series of metabolic events, which eventually lead to neuronal death. In the present study, ODC activity was evaluated following transient focal cerebral ischemia and reperfusion in rat. The middle cerebral artery (MCA) was occluded for 2 h in male rats with an intraluminal suture technique. Animals were sacrificed between 3 and 48 h of reperfusion following MCA occlusion, and ODC activity was assayed in cortex and striatum. ODC activity was also estimated in an in vitro ischemia model using primary rat cortical neuron cultures, at 6–24 h reoxygenation following 1 h oxygen-glucose deprivation (OGD). In cortex, following ischemia, ODC activity was increased at 3 h (P < .05), reached peak levels by 6–9 h (P < .001) and returned to sham levels by 48 h reperfusion. In striatum the ODC activity followed a similar time course, but returned to basal levels by 24 h. This suggests that ODC activity is upregulated in rat CNS following transient focal ischemia and its time course of activation is region specific. In vitro, ODC activity showed a significant rise only at 24 h reoxygenation following ischemic insult. The release of lactate dehydrogenase (LDH), an indicator for cell damage, was also significantly elevated after OGD. 0.25 mM -difluoromethylornithine (DFMO) inhibited ischemia-induced ODC activity, whereas a 10-mM dose of DFMO appears to provide some neuroprotection by suppressing both ODC activity and LDH release in neuronal cultures, suggesting the involvement of polyamines in the development of neuronal cell death.     

Ghrelin reduces cerebral ischemic injury in rats by reducing M1 microglia/macrophages

The purpose of this study was to investigate the effect of Ghrelin on the polarization of microglia/ macrophages after cerebral ischemia (CI) in rats. 60 wild-type SD rats were randomly divided into sham group, CI group, CI+Ghrelin group, 20 rats in each group. The modified Longa suture method was used to establish the middle cerebral artery occlusion (MCAO) model in rats. Before surgery, Ghrelin was injected subcutaneously (100 μg/kg, twice a day) for 4 consecutive weeks. After modeling, neurological function scores were performed with three behavioral experiments: mNSS score, Corner test, and Rotarod test, to evaluate the recovery of neurological function after Ghrelin treatment. At the same time, the brain tissues were collected and stained with 2,3,5- triphenyltetrazolium chloride (TTC) to detect the cerebral infarct volume. RT-qPCR was used to detect the expression of TNF-α and IL-1β in the ischemic brain tissue, and the TUNEL staining was used to detect the apoptosis of brain tissue. Flow cytometry was used to detect the percentage of M1 type microglia/macrophages which were isolated by trypsin digestion of fresh cerebral cortex. Then, the Western blotting and immunofluorescence method were used to detect the phosphorylation level of AKT (P-AKT) and AKT. Compared with the CI group, the neurological function of the rats in the CI+Ghrelin group was dramatically improved, and the cerebral infarction area was dramatically reduced. At the same time, the expression of TNF-α and IL-1β in the ischemic brain tissue of rats in the CI+Ghrelin group decreased, and the apoptotic cells in the brain tissue also decreased. Compared with the CI treatment group, the activation of M1 microglia/macrophages in the cortex of the ischemic side of the infarct and the peri-infarct area in the CI+Ghrelin group was dramatically inhibited. At the same time, the ratio of P-AKT/AKT of the brain tissue in the CI+Ghrelin group was dramatically higher than that of the CI group. In the rat cerebral ischemia model, Ghrelin can promote the repair of brain damage and the recovery of neurological function after ischemia. Its mechanism may be related to activating AKT to selectively reduce M1 microglia/macrophages, reducing inflammation and cell apoptosis in brain tissue.Key words: Cerebral ischemia, ghrelin, microglia/macrophage     

Involvement of Rho-kinase in tumor necrosis factor-α-induced interleukin-6 release from C6 glioma cells

Tumor necrosis factor (TNF)-α stimulated interleukin (IL)-6 release and induced the phosphorylation of myosin phosphatase targeting subunit (MYPT)-1, a Rho-kinase substrate. The IL-6 release was significantly suppressed by Y-27632 and fasudil, Rho-kinase inhibitors. Although IκB inhibitor suppressed the TNF-α-induced IL-6 release, the Rho-kinase inhibitors did not affect the TNF-α-induced IκB phosphorylation. TNF-α induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), and p44/p42 MAP kinase. The TNF-α-induced IL-6 release was suppressed by SB203580, a p38 MAPK inhibitor, or SP600125, a SAPK/JNK inhibitor, but not by PD98059, a MAP kinase/extracellular signal-regulated kinase kinase inhibitor. The Rho-kinase inhibitors attenuated the TNF-α-induced phosphorylation of both p38 MAP kinase and SAPK/JNK.Rho-kinase, which has been used for the clinical treatment of cerebral vasospasms, may be involved in other central nervous system (CNS) disorders such as traumatic injury, stroke, neurodegenerative disease and neuropathic pain. TNF-α, a proinflammatory cytokine that affects the CNS through cytokines, such as IL-6, release from neurons, astrocytes and microglia. Therefore, we investigated the involvement of Rho-kinase in the TNF-α-induced IL-6 release from rat C6 glioma cells.These results strongly suggest that Rho-kinase regulates the TNF-α-induced IL-6 release at a point upstream from p38 MAPK and SAPK/JNK in C6 glioma cells. Therefore, Rho-kinase inhibitor may be considered to be a new clinical candidate for the treatment of CNS disorders in addition to cerebral vasospasms.     

The UII/UT System Mediates Upregulation of Proinflammatory Cytokines through p38 MAPK and NF-κB Pathways in LPS-Stimulated Kupffer Cells

The urotensin II (UII)/UII receptor (UT) system is closely related to immune inflammation. In acute liver failure (ALF), the UII/UT system can promote the production and release of proinflammatory cytokines, inducing an inflammatory injury response in liver tissue. However, the mechanism by which the hepatic UII/UT system promotes proinflammatory cytokine production and release is not clear. To solve this problem, we used primary Kupffer cells (KCs) as the model system in the current study. The results showed that after lipopolysaccharide (LPS) stimulation, KCs showed significantly increased expression and release of UII/UT and proinflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β). Pretreatment with urantide, which is a UT receptor antagonist, significantly inhibited the LPS-stimulated expression and release of UII/UT, TNF-α, and IL-1β by KCs. In addition, LPS stimulation induced nuclear p38 mitogen-activated protein kinase (MAPK) protein phosphorylation and expression of the nuclear nuclear factor κB (NF-κB) p65 subunit in KCs and enhanced the binding activity of NF-κB to DNA molecules, whereas urantide pretreatment significantly inhibited the LPS-stimulated nuclear expression and activity of these molecules in KCs. Therefore, our conclusion is that the UII/UT system mediates LPS-stimulated production and release of proinflammatory cytokine by KCs, and this mediating effect at least partially relies on the inflammatory signaling pathway molecules p38 MAPK and NF-κB.     

Docosahexaenoic acid alleviates cell injury and improves barrier function by suppressing necroptosis signalling in TNF-α-challenged porcine intestinal epithelial cells

Long-chain n-3 polyunsaturated fatty acids are known to have beneficial effects on intestinal health. However, the underling mechanisms are largely unknown. The present study was conducted to investigate whether docosahexaenoic acid (DHA) attenuates TNF-α-induced intestinal cell injury and barrier dysfunction by modulating necroptosis signalling. Intestinal porcine epithelial cell line 1 was cultured with or without 12.5 µg/ml DHA, followed by exposure to 50 ng/ml TNF-α for indicated time periods. DHA restored cell viability and cell number triggered by TNF-α. DHA also improved barrier function, which was indicated by increased trans-epithelial electrical resistance, decreased FD4 flux and increased membrane localisation of zonula occludins (ZO-1) and claudin-1. Moreover, DHA suppressed cell necrosis in TNF-α-challenged cells, as shown in the IncuCyte ZOOM™ live cell imaging system and transmission electron microscopy. In addition, DHA decreased protein expression of TNF receptor, receptor interacting protein kinase 1, RIP3 and phosphorylation of mixed lineage kinase-like protein, phosphoglycerate mutase family 5, dynamin-related protein 1 and high mobility group box-1 protein. Furthermore, DHA suppressed protein expression of caspase-3 and caspase-8. Collectively, these results indicate that DHA is capable of alleviating TNF-α-induced cell injury and barrier dysfunction by suppressing the necroptosis signalling pathway.     

Identification of lipocalin-2 as a PKCδ phosphorylation substrate in neutrophils

Background

PKCδ expressed in neutrophils is implicated in promoting reperfusion injury after ischemic stroke. To understand the molecular and cellular actions of PKCδ, we employed a chemical-genetics approach to identify PKCδ substrates in neutrophils.

Results

We recently generated knock-in mice endogenously expressing analog-specific PKCδ (AS-PKCδ) that can utilize ATP analogs as phosphate donors. Using neutrophils isolated from the knock-in mice, we identified several PKCδ substrates, one of which was lipocalin-2 (LCN2), which is an iron-binding protein that can trigger apoptosis by reducing intracellular iron concentrations. We found that PKCδ phosphorylated LCN2 at T115 and this phosphorylation was reduced in Prkcd^−/− mice. PKCδ colocalized with LCN2 in resting and stimulated neutrophils. LCN2 release from neutrophils after cerebral ischemia was reduced in PKCδ null mice.

Conclusions

These findings suggest that PKCδ phosphorylates LCN2 and mediates its release from neutrophils during ischemia-reperfusion injury.