Exosomal miR-409-3p secreted from activated mast cells promotes microglial migration, activation and neuroinflammation by targeting Nr4a2 to activate the NF-κB pathway.

OBJECTIVE: Neuroinflammation plays a critical role in central nervous system diseases. Exosomal miRNAs released from various cells are implicated in cell-to-cell communication. Prior studies have placed substantial emphasis on the role of cytokines in mast cell-microglia interactions during neuroinflammation. However, it has never been clearly determined whether exosomal miRNAs participate in the interaction between mast cells and microglia and thus mediate neuroinflammation. METHODS: The characteristics of exosomes isolated from cell culture supernatants were confirmed by transmission electron microscopy (TEM), nanoparticle-tracking analysis (NTA) and Western blot. The transfer of PKH67-labelled exosomes and Cy3-labelled miR-409-3p was observed by fluorescence microscopy. Migration and activation of murine BV-2 microglial cells were evaluated through Transwell assays and immunofluorescence staining for Iba1 and CD68. CD86, IL-1ß, IL-6 and TNF-α were assessed via qRT-PCR and ELISA. MiR-409-3p was detected by qRT-PCR. Nr4a2 and NF-κB levels were measured by western blot. Regulatory effects were identified by luciferase reporter assays. RESULTS: Lipopolysaccharide (LPS)-stimulated murine P815 mast cells secreted exosomes that were efficiently taken up by murine BV-2 cells, which promoted murine BV-2 cell migration and activation. LPS-P815 exosomes increased the CD86, IL-1ß, IL-6 and TNF-α levels in murine BV-2 microglia. Furthermore, activated mast cells delivered exosomal miR-409-3p to murine BV-2 microglia. Upregulated miR-409-3p promoted murine BV-2 microglial migration, activation and neuroinflammation by targeting Nr4a2 to activate the NF-κB pathway. CONCLUSION: Exosomal miR-409-3p secreted from activated mast cells promotes microglial migration, activation and neuroinflammation by targeting Nr4a2 to activate the NF-κB pathway, which provides evidence that not only cytokines but also exosomal miRNAs participate in neuroinflammation. In the future, targeting exosomal miRNAs may provide new insights into neuroinflammation.

Histamine 2/3 receptor agonists alleviate perioperative neurocognitive disorders by inhibiting microglia activation through the PI3K/AKT/FoxO1 pathway in aged rats.

BACKGROUND: Microglia, the principal sentinel immune cells of the central nervous system (CNS), play an extensively vital role in neuroinflammation and perioperative neurocognitive disorders (PND). Histamine, a potent mediator of inflammation, can both promote and prevent microglia-related neuroinflammation by activating different histamine receptors. Rat microglia express four histamine receptors (H1R, H2R, H3R, and H4R), among which the histamine 1 and 4 receptors can promote microglia activation, whereas the role and cellular mechanism of the histamine 2 and 3 receptors have not been elucidated. Therefore, we evaluated the effects and potential cellular mechanisms of histamine 2/3 receptors in microglia-mediated inflammation and PND. METHODS: This study investigated the role of histamine 2/3 receptors in microglia-induced inflammation and PND both in vivo and in vitro. In the in vivo experiments, rats were injected with histamine 2/3 receptor agonists in the right lateral ventricle and were then subjected to exploratory laparotomy. In the in vitro experiments, primary microglia were pretreated with histamine 2/3 receptor agonists before stimulation with lipopolysaccharide (LPS). Cognitive function, microglia activation, proinflammatory cytokine production, NF-κb expression, M1/M2 phenotypes, cell migration, and Toll-like receptor-4 (TLR4) expression were assessed. RESULTS: In our study, the histamine 2/3 receptor agonists inhibited exploratory laparotomy- or LPS-induced cognitive decline, microglia activation, proinflammatory cytokine production, NF-κb expression, M1/M2 phenotype transformation, cell migration, and TLR4 expression through the PI3K/AKT/FoxO1 pathway. CONCLUSION: Based on our findings, we conclude that histamine 2/3 receptors ameliorate PND by inhibiting microglia activation through the PI3K/AKT/FoxO1 pathway. Our results highlight histamine 2/3 receptors as potential therapeutic targets to treat neurological conditions associated with PND.

Correction to: Mild endoplasmic reticulum stress ameliorates lipopolysaccharide-induced neuroinflammation and cognitive impairment via regulation of microglial polarization.

An amendment to this paper has been published and can be accessed via the original article.

Corrigendum to "Protective Effects of Pretreatment with Oleanolic Acid in Rats in the Acute Phase of Hepatic Ischemia-Reperfusion Injury: Role of the PI3K/Akt Pathway".

[This corrects the article DOI: 10.1155/2014/451826.].

The Mast Cell Is an Early Activator of Lipopolysaccharide-Induced Neuroinflammation and Blood-Brain Barrier Dysfunction in the Hippocampus.

Neuroinflammation contributes to or even causes central nervous system (CNS) diseases, and its regulation is thus crucial for brain disorders. Mast cells (MCs) and microglia, two resident immune cells in the brain, together with astrocytes, play critical roles in the progression of neuroinflammation-related diseases. MCs have been demonstrated as one of the fastest responders, and they release prestored and newly synthesized mediators including histamine, ß-tryptase, and heparin. However, temporal changes in MC activation in this inflammation process remain unclear. This study demonstrated that MC activation began at 2 h and peaked at 4 h after lipopolysaccharide (LPS) administration. The number of activated MCs remained elevated until 24 h after LPS administration. In addition, the levels of histamine and ß-tryptase in the hippocampus markedly and rapidly increased within 6 h and remained higher than the baseline level within 24 h after LPS challenge. Furthermore, mast cell-deficient KitW-sh/W-sh mice were used to investigate the effects of MCs on microglial and astrocytic activation and blood-brain barrier (BBB) permeability at 4 h after LPS stimulation. Notably, LPS-induced proinflammatory cytokine secretion, microglial activation, and BBB damage were inhibited in KitW-sh/W-sh mice. However, no detectable astrocytic changes were found in WT and KitW-sh/W-sh mice at 4 h after LPS stimulation. Our findings indicate that MC activation precedes CNS inflammation and suggest that MCs are among the earliest participants in the neuroinflammation-initiating events.

Pro-inflammatory role of high-mobility group box-1 on brain mast cells via the RAGE/NF-κB pathway.

High-mobility group box-1 (HMGB-1) acts as a pro-inflammatory cytokine contributing to the occurrence of many central inflammatory and infectious disorders. Brain mast cells (MCs) are the first responders to peripheral inflammatory stimulation because of their rapid response to external stimuli coupled with their release of preformed and newly synthesized reactive chemicals. Little is known about the involvement of brain MCs in the pro-inflammatory effects of HMGB-1 on the central nervous system (CNS). Thus, we investigated the activation process of MCs by HMGB-1 and explored whether this process is involved in the pro-inflammatory effects of HMGB-1 on the CNS. In this study, we used P815 cells to study the activating role of HMGB-1 on MCs and to explore its potential mechanism in vitro. In an in vivo study, adult male Sprague-Dawley rats received i.c.v. injection of sterile saline or cromoglycate (stabilizer of MCs) 30 min prior to i.p. injection of HMGB-1. Increased levels of tumor necrosis factor and IL-1ß were observed in the P815 cells, as well as in the rats' brains, after HMGB-1 treatment. Pretreatment with the receptor of advanced glycation endproducts (RAGE)-siRNA inhibited the HMGB-1-induced inflammatory process in the P815 cells. Activation of the RAGE/nuclear factor-κB (NF-κB) pathway was observed in both the P815 cells and rats' brains. In addition, HMGB-1 induced the accumulation of brain MCs in the hippocampal CA1 region, and the blood-brain barrier was disrupted. Pretreatment with cromoglycate, a stabilizer of MCs, mitigated these HMGB-1-induced pro-inflammatory processes in rats. These findings indicate that brain MCs are involved in the pro-inflammatory effect of HMGB-1 on the CNS, probably via activating the RAGE/NF-κB pathway.

Preoperative Sleep Disturbance Exaggerates Surgery-Induced Neuroinflammation and Neuronal Damage in Aged Mice.

Postoperative cognitive dysfunction (POCD) is defined as new cognitive impairment (memory impairment and impaired performance) after surgery, especially in aged patients. Sleep disturbance is a common phenomenon before surgery that has been increasingly thought to affect patient recovery. However, little is known about the functional impact of preoperative sleep disturbance on POCD. Here, we showed that tibial fracture surgery induced cognitive deficit and production of proinflammatory cytokines interleukin-6 (IL-6) and IL-1ß, along with microglia and astrocyte activation, neuronal damage, and blood-brain barrier (BBB) disruption. Preoperative sleep disturbance enhanced the surgery-induced neuroinflammation, neuronal damage, BBB disruption, and memory impairment 24 h after surgery. Taken together, these results demonstrated that preoperative sleep disturbance aggravated postoperative cognitive function in aged mice and the mechanism may be related to central nervous system (CNS) inflammation and neuronal damage.

[Propofol protects human cardiac AC16 cells from CoCl2-induced hypoxic injury].

OBJECTIVE: To explore the effect of propofol on human cardiac AC16 cells under CoCl2-induced hypoxic injury and the possible mechanisms.
 Methods: Human AC16 cardiomyocytes were treated with cobalt chloride (CoCl2) to mimic hypoxic condition in cultured cardiomyocytes. The AC16 cells were divided into 3 groups: a control group, a CoCl2 hypoxia group (CoCl2 group), and a propofol+CoCl2 group (propofol+ CoCl2 group). The cell viability was assessed by cell counting kit-8 (CCK-8). Cell apoptosis ratio (AR) and the mitochondrial membrane potential (Δψm) were detected by flow cytometry. The reactive oxygen species (ROS) production in AC16 cells were determined with the ROS-sensitive fluorescent probe. Meanwhile, total intracellular levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in AC16 cells were detected with commercially available kits. Western blot was used to evaluate the activation of c-Jun N-terminal kinase (JNK) and p38 signaling pathways.
 Results: 1) Compared with the control group, AC16 cell viability was decreased significantly in the CoCl2 group following the treatment with 500 µmol/L CoCl2 (P<0.01); 2) Compared with the control group, AR value in AC16 cells was increased significantly in the CoCl2 group, while Δψm was decreased significantly (all P<0.01). Compared with the CoCl2 group, AR value in AC16 cells was decreased significantly in the propofol+CoCl2 group, while Δψm was increased significantly (both P<0.05); 3) Compared with the control group, the levels of ROS and MDA were increased significantly, and the level of SOD was significantly decreased in the CoCl2 group (all P<0.01). Compared with the CoCl2 group, the ROS and MDA levels in the propofol+CoCl2 group were increased significantly and the SOD levels were decreased significantly (all P<0.05); 4) Compared with the control group, the phosphorylation levels of JNK and p38 were increased significantly (both P<0.05) in the CoCl2 group. Compared with the CoCl2 group, the phosphorylation levels of JNK and p38 were decreased significantly in the propofol+CoCl2 group (both P<0.05).
 Conclusion: The pretreatment with propofol may protect human cardiac AC16 cells from the chemical hypoxia-induced injury through regulation of JNK and p38 signaling pathways.

IL-17A contributes to perioperative neurocognitive disorders through blood-brain barrier disruption in aged mice.

BACKGROUND: Perioperative neurocognitive disorders (PND) occur frequently after surgery, especially in aged patients. Surgery-induced neuroinflammation and blood-brain barrier (BBB) dysfunction play a crucial role in the pathogenesis of PND. Interleukin-17A (IL-17A) increases after surgical stress and will be involved in BBB dysfunction. However, the effect of IL-17A on BBB function during PND remains poorly understood. METHODS: Male wild-type C57BL/6J mice (15 months old) received tibial fracture surgery and fixation to establish the PND model. All the mice were injected intraperitoneally with an IL-17A-neutralizing antibody (Abs) or isotype-control Abs 30 min before tibial fracture surgery. Animal behaviour tests conducted 24 h after surgery included the contextual fear conditioning and Y maze tests. Serum and hippocampus IL-17A levels and hippocampus IL-6 and IL-1ß levels were detected by ELISA. BBB function was detected by Evans blue (EB) test. Hippocampus matrix metalloproteinase-2 (MMP-2)- and MMP-9-positive cells were detected by immunohistochemistry. Hippocampus albumin, occludin, claudin-5 and IL-17A receptors were detected by Western blot. For the in vitro experiment, bEnd.3 cells were incubated with IL-17A. Cell IL-17A receptors were detected by immunofluorescence. Cellular MMP-2, MMP-9, occludin, and claudin-5 were detected by Western blot. RESULTS: Tibial fracture surgery promoted memory impairment, increased levels of IL-17A and IL-17A receptors, inflammatory factor production and BBB dysfunction. IL-17A Abs inhibited this effect, including improving memory function, decreasing inflammatory factor production and alleviating BBB disruption, indicated by decreased tight junctions (TJs) and increased MMPs after surgery. The in vitro study suggested that recombinant IL-17A could upregulate the expression of IL-17A receptors, decrease TJs and increase the level of MMPs in bEnd.3 cells. CONCLUSIONS: Our results suggested that IL-17A-promoted BBB disruption might play an important role in the pathogenesis of PND.

Histamine upregulates the expression of histamine receptors and increases the neuroprotective effect of astrocytes.

BACKGROUND: Astrocytes have attracted increasing attention over recent decades for their role in neuroinflammation. Histamine, a major aminergic brain neurotransmitter, has an important influence on the main activities of astrocytes, such as ion homeostasis, energy metabolism, and neurotransmitter clearance. However, little is known about the impact of histamine on astrocyte immunomodulatory function. METHODS: The expression of all known histamine receptor subtypes was examined in primary astrocytes. Then, primary astrocytes were pretreated with selective histamine receptor antagonists and stimulated with histamine. Cellular activation, proinflammatory cytokine production, and expression of neurotrophic factors were assessed. RESULTS: Astrocytes could constitutively express three histamine receptors (H1R, H2R, and H3R), and these three histamine receptors could be selectively upregulated to varying degrees upon histamine treatment. Histamine also dose-dependently stimulated astrocyte activation and subsequent production of glial cell-derived neurotrophic factor (GDNF), whereas it suppressed the secretion of the proinflammatory factors tumor necrosis factor-alpha (TNF-α) and interleukin-1ß (IL-1ß). The effects of histamine were completely abolished by either an H1R or H3R antagonist, while an H2R antagonist attenuated the effects partly. CONCLUSIONS: The present study identified the expression of H1R, H2R, and H3R on astrocytes. We also demonstrated that negative regulation of astrocytic TNF-α and IL-1ß production and the enhancement of astrocytic GDNF stimulated by histamine were receptor-mediated processes in which all three of the expressed histamine receptors (H1R, H2R, and H3R) were involved. These findings may further clarify the involvement and mechanism of astrocyte activation in neuroinflammation.

Effect of tryptase on mouse brain microvascular endothelial cells via protease-activated receptor 2.

BACKGROUND: Mast cells (MCs), the 'first responders' in brain injury, are able to disrupt the blood-brain barrier (BBB), but the underlying mechanism is not well understood. Tryptase is the most abundant MC secretory product. Protease-activated receptor 2 (PAR-2) has been identified as a specific receptor for tryptase, which is abundantly expressed in brain microvascular endothelial cells. The BBB comprises brain microvascular endothelial cells that display specialised molecular properties essential for BBB function and integrity. Therefore, the purpose of the present study was to investigate the effects of tryptase on mouse brain microvascular endothelial cell line bEnd3 and its potential mechanisms of action. METHODS: Induction of mouse brain microvascular endothelial cell activation by tryptase was examined. Then, mouse brain microvascular endothelial cells were pretreated with a PAR-2 antagonist and stimulated with tryptase. Cellular activation, proinflammatory cytokine production, expression of PAR-2, Toll-like receptors (TLRs) and mitogen-activated protein kinases (MAPK), nuclear factor kappa B (NF-kappa B) phosphorylation were assessed. RESULTS: Tryptase upregulated the production of VCAM-1, MMPs (MMP9 and MMP2), TLR4 and TNF-α and downregulated the expression of the tight junction proteins occludin and claudin-5 in mouse brain microvascular endothelial cell. Among the MAPK and NF-kappa B pathway, ERK and NF-kappa B were activated by tryptase. All of these effects could be eliminated by the PAR-2 inhibitor. CONCLUSION: Based on our findings, we conclude that tryptase can trigger brain microvascular endothelial cell activation and proinflammatory mediator release. These findings may further clarify the involvement and mechanism of tryptase in BBB disruption.

Efficacy of a Dexmedetomidine-Remifentanil Combination Compared with a Midazolam-Remifentanil Combination for Conscious Sedation During Therapeutic Endoscopic Retrograde Cholangio-Pancreatography: A Prospective, Randomized, Single-Blinded Preliminary Trial.

BACKGROUND: Dexmedetomidine as a conscious sedative exhibits both analgesia and respiratory sparing effects. AIMS: We evaluated and compared the sedative effect and the safety of a dexmedetomidine-remifentanil (DR) regimen with a midazolam-remifentanil (MR) combination during the endoscopic retrograde cholangio-pancreatography (ERCP) requiring conscious sedation. METHODS: One-hundred and ninety-eight patients were randomized and divided into two groups. A bolus of midazolam (0.05 mg kg-1) was injected intravenously for MR group, and dexmedetomidine (1 µg kg-1) was pumping for 10 min for DR group. Next, an initial loading dose of 1 µg kg-1 and 0.05-0.2 µg kg-1 min-1 of remifentanil was administered in all patients. Hemodynamic and respiratory changes, Ramsay Sedation Scale, Visual Analogue Scale, endoscopist and patient satisfaction were assessed. Furthermore, adverse events as well as recovery time and discharge time were rated. RESULTS: Patient satisfaction scores were significantly higher in the DR group compared with MR group. The occurrence of desaturation was statistically higher, and the operation time was longer in the MR group. Although no statistically significant values could be determined between the two groups about amnesia and need of additional drug, the DR group was found to require a significantly reduced amount of extra midazolam. Furthermore, nausea during catheterization of oropharynx was found to be more pronounced in the DR group. CONCLUSIONS: The dexmedetomidine-remifentanil protocol provided a parallel sedative efficacy and improved respiratory sparing effects. The higher patient satisfaction scores potentially offer a more reproducible ERCP quality. Adding dexmedetomidine to remifentanil can be used safely as a conscious sedation method during ERCP.

Mild endoplasmic reticulum stress ameliorates lipopolysaccharide-induced neuroinflammation and cognitive impairment via regulation of microglial polarization.

BACKGROUND: Neuroinflammation, which ultimately leads to neuronal loss, is considered to play a crucial role in numerous neurodegenerative diseases. The neuroinflammatory process is characterized by the activation of glial cells such as microglia. Endoplasmic reticulum (ER) stress is commonly associated with impairments in neuronal function and cognition, but its relationship and role in neurodegeneration is still controversial. Recently, it was confirmed that nonharmful levels of ER stress protected against experimental Parkinson's disease. Here, we investigated mild ER stress-based regulation of lipopolysaccharide (LPS)-driven neuroinflammation in rats and in primary microglia. METHODS: Male Sprague-Dawley (SD) rats received the intracerebroventricular injection of the ER stress activator tunicamycin (TM) with or without intraperitoneal injection of the ER stress stabilizer sodium 4-phenylbutyrate (4-PBA) 1 h before LPS administration. The levels of neuroinflammation and memory dysfunction were assessed 24 h after treatment. In addition, the effect of mild ER stress on microglia was determined in vitro. RESULTS: Here, we found that low doses of TM led to mild ER stress without cell or organism lethality. We showed that mild ER stress preconditioning reduced microglia activation and neuronal death as well as improved LPS-induced memory impairment in rats. In addition, pre-exposure to nonlethal doses of TM in microglia showed significant protection against LPS-induced proinflammatory cytokine production and M1/2b polarization. However, sodium 4-PBA, a compound that ameliorates ER stress, ablated this protective effect in vivo and in vitro. CONCLUSIONS: Based on our findings, we conclude that the mild ER stress not only limits the accumulation of misfolded proteins but also protects tissues from harmful endotoxemia insults. Therefore, ER stress preconditioning has potential therapeutic value for the treatment of neurodegenerative diseases.

Blocking ATP-sensitive potassium channel alleviates morphine tolerance by inhibiting HSP70-TLR4-NLRP3-mediated neuroinflammation.

BACKGROUND: Long-term use of morphine induces analgesic tolerance, which limits its clinical efficacy. Evidence indicated morphine-evoked neuroinflammation mediated by toll-like receptor 4 (TLR4) - NOD-like receptor protein 3 (NLRP3) inflammasome was important for morphine tolerance. In our study, we investigated whether other existing alternative pathways caused morphine-induced activation of TLR4 in microglia. We focused on heat shock protein 70 (HSP70), a damage-associated molecular pattern (DAMP), which was released from various cells upon stimulations under the control of KATP channel and bound with TLR4-inducing inflammation. Glibenclamide, a classic KATP channel blocker, can improve neuroinflammation by inhibiting the activation of NLRP3 inflammasome. Our present study investigated the effect and possible mechanism of glibenclamide in improving morphine tolerance via its specific inhibition on the release of HSP70 and activation of NLRP3 inflammasome induced by morphine. METHODS: CD-1 mice were used for tail-flick test to evaluate morphine tolerance. The microglial cell line BV-2 and neural cell line SH-SY5Y were used to investigate the pharmacological effects and the mechanism of glibenclamide on morphine-induced neuroinflammation. The activation of microglia was accessed by immunofluorescence staining. Neuroinflammation-related cytokines were measured by western blot and real-time PCR. The level of HSP70 and related signaling pathway were evaluated by western blot and immunofluorescence staining. RESULTS: Morphine induced the release of HSP70 from neurons. The released HSP70 activated microglia and triggered TLR4-mediated inflammatory response, leading to the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) p65 and the activation of NLRP3 inflammasome. Moreover, anti-HSP70 neutralizing antibody partly attenuated chronic morphine tolerance. The secretion of HSP70 was under the control of MOR/AKT/KATP/ERK signal pathway. Glibenclamide as a classic KATP channel blocker markedly inhibited the release of HSP70 induced by morphine and suppressed HSP70-TLR4-NLRP3 inflammasome-mediated neuroinflammation, which consequently attenuated morphine tolerance. CONCLUSIONS: Our study indicated that morphine-induced extracellular HSP70 was an alternative way for the activation of TLR4-NLRP3 in analgesic tolerance. The release of HSP70 was regulated by MOR/AKT/KATP/ERK pathway. Our study suggested a promising target, KATP channel and a new leading compound, glibenclamide, for treating morphine tolerance.

Lidocaine alleviates morphine tolerance via AMPK-SOCS3-dependent neuroinflammation suppression in the spinal cord.

BACKGROUND: Morphine tolerance is a clinical challenge, and its pathogenesis is closely related to the neuroinflammation mediated by Toll-like receptor 4 (TLR4). In Chinese pain clinic, lidocaine is combined with morphine to treat chronic pain. We found that lidocaine sufficiently inhibited neuroinflammation induced by morphine and improved analgesic tolerance on the basis of non-affecting pain threshold. METHODS: CD-1 mice were utilized for tail-flick test to evaluate morphine tolerance. The microglial cell line BV-2 was utilized to investigate the mechanism of lidocaine. Neuroinflammation-related cytokines were measured by western blotting and real-time PCR. The level of suppressor of cytokine signaling 3 (SOCS3) and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)-related signaling pathway was evaluated by western blotting, real-time PCR, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence staining. RESULTS: Lidocaine potentiated an anti-nociceptive effect of morphine and attenuated the chronic analgesic tolerance. Lidocaine suppressed morphine-induced activation of microglia and downregulated inflammatory cytokines, interleukin-1ß (IL-1ß), and tumor necrosis factor-alpha (TNF-α) via upregulating SOCS3 by activating AMPK. Lidocaine enhanced AMPK phosphorylation in a calcium-dependent protein kinase kinase ß (CaMKKß)-dependent manner. Furthermore, lidocaine decreased the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and inhibited the nuclear factor-κB (NF-κB) in accordance with the inhibitory effects to TLR4. CONCLUSIONS: Lidocaine as a prevalent local anesthetic suppresses morphine tolerance efficiently. AMPK-dependent upregulation of SOCS3 by lidocaine plays a crucial role in the improvement of analgesic tolerance.

Aliskiren protects against myocardial ischaemia-reperfusion injury via an endothelial nitric oxide synthase dependent manner.

Aliskiren, a direct renin inhibitor, has shown potent ability to attenuate hypertension. Our previous research has found that aliskiren protected against myocardial ischaemia-reperfusion (I/R) injury and enhanced phosphorylation of endothelial nitric oxide synthase (eNOS) in spontaneously hypertensive rats. However, whether the cardioprotective effect of aliskiren against myocardial I/R injury was eNOS-dependent is unknown. In the present study, 12-week-old male eNOS knockout (eNOS-/- ) and wild-type C57BL/6J mice (WT) were orally administrated with the dose of 50 mg/kg per day of aliskiren. After a 4-week treatment, aliskiren decreased blood pressure in eNOS-/- mice, and reduced renin-angiotension II levels in both eNOS-/- and WT mice. Aliskiren also improved left ventricular ejection fraction (EF) and fractional shortening (FS), decreased myocardial infarct size, reduced creatine kinase (CK) and lactate dehydrogenase (LDH) activity in plasma, attenuated dihydroethidium (DHE) fluorescence and levels of malondialdehyde (MDA), enhanced superoxide dismutase (SOD) activity and total antioxidant capacity (T-AOC) in myocardium, increased SOD and thioredoxin (Trx) proteins expression in WT mice subjected to 30 minutes of ischaemia followed by reperfusion for 24 hours. However, aliskiren failed to restore all of the above indices in eNOS-/- mice subjected to the same I/R injury. Our study indicated that aliskiren protected against myocardial I/R injury via an eNOS dependent manner.

Lithium Ameliorates LPS-Induced Astrocytes Activation Partly via Inhibition of Toll-Like Receptor 4 Expression.

BACKGROUND/AIMS: Astrocytes are critical for the development of postoperative cognitive dysfunction (POCD). In addition, astrocytes express toll-like receptors 4 (TLR4) and build up responses to innate immune triggers by releasing pro-inflammatory molecules. The pathogenesis of neurological disorders often involves the activation of astrocytes and associated inflammatory processes. Lithium, a primary drug for the treatment of bipolar disorder, has recently been suggested to have a role in neuroprotection during neurodegenerative diseases. In this study, we aimed to investigate whether lithium can ameliorate LPS-induced astrocytes activation via inhibition of TLR4 expression. METHODS: Primary astrocytes cells were pretreated with lithium and stimulated with lipopolysaccharide (LPS). Cellular activation, cytokine production, and TLR4 expression, were assessed. RESULTS: Lithium significantly inhibited LPS-induced astrocytes activation and pro-inflammatory cytokine production, as well as LPS-induced TLR4 expression. CONCLUSIONS: Lithium can inhibit LPS-induced TLR4 expression and astrocytes activation. These results indicate that lithium plays an important role in astrocytes activation and neuroinflammation-related diseases, which may open new avenues for neuroscience and biomedical research, and also offers new insight into the treatment of POCD.

Astrocyte-Derived CCL2 is Associated with M1 Activation and Recruitment of Cultured Microglial Cells.

BACKGROUND/AIMS: Microglia are an essential player in central nervous system inflammation. Recent studies have demonstrated that the astrocytic chemokine, CCL2, is associated with microglial activation in vivo. However, CCL2-induced microglial activation has not yet been studied in vitro. The purpose of the current study was to understand the role of astrocyte-derived CCL2 in microglial activation and to elucidate the underlying mechanism(s). METHODS: Primary astrocytes were pre-treated with CCL2 siRNA and stimulated with TNF-α. The culture medium (CM) was collected and added to cultures of microglia, which were incubated with and without CCR2 inhibitor. Microglial cells were analyzed by quantitative RT-PCR to determine whether they polarized to the M1 or M2 state. Microglial migratory ability was assessed by transwell migration assay. RESULTS: TNF-α stimulated the release of CCL2 from astrocytes, even if the culture media containing TNF-α was replaced with fresh media after 3 h. CM from TNF-α-stimulated astrocytes successfully induced microglial activation, which was ascertained by increased activation of M1 and enhanced migration ability. In contrast, CM from astrocytes pretreated with CCL2 siRNA showed no effect on microglial activation, compared to controls. Additionally, microglia pre-treated with RS102895, a CCR2 inhibitor, were resistant to activation by CM from TNF-α-stimulated astrocytes. CONCLUSION: This study demonstrates that the CCL2/CCR2 pathway of astrocyte-induced microglial activation is associated with M1 polarization and enhanced migration ability, indicating that this pathway could be a useful target to ameliorate inflammation in the central nervous system.

Cerebral Mast Cells Participate In Postoperative Cognitive Dysfunction by Promoting Astrocyte Activation.

BACKGROUND: Astrocytes, the major glial cell type that has been increasingly recognized as contributing to neuroinflammation, are critical in the occurrence and development of postoperative cognitive dysfunction (POCD). Although emerging evidence showed that brain mast cells (MCs) are the "first responders" in neuroinflammation, little is known about the functional communication between MCs and astrocytes. METHODS: In this study, we investigated the potential regulation of astrocyte activation by MCs. Rats received an intracerebroventricular injection of Cromolyn (an MC stabilizer) or sterile saline 30 min before undergoing open tibial fracture surgery, and the levels of neuroinflammation and the degree of memory dysfunction were evaluated at 1 day and 3 days after surgery. In the in vitro study, the effect of activated MCs on astrocytes were further clarified. RESULTS: Surgery increased the number of MCs, the astrocyte activation and the production of inflammatory factors, and resulted in cognitive deficits. Site-directed pre-injection of Cromolyn can inhibit this effect. In the vitro study, the conditioned medium from C48/80-stimulated mast cells (P815) could induce primary astrocyte activation and subsequent production of inflammatory cytokines, which could be inhibited by Cromolyn. CONCLUSION: These findings indicate that activated MCs could trigger astrocyte activation, be involved in neuroinflammation and possibly contribute to POCD. Interactions between MCs and astrocytes could provide potential therapeutic targets for POCD.

Activated brain mast cells contribute to postoperative cognitive dysfunction by evoking microglia activation and neuronal apoptosis.

BACKGROUND: Neuroinflammation plays a key role in the occurrence and development of postoperative cognitive dysfunction (POCD). Microglia, the resident immune cells in the brain, has been increasingly recognized to contribute to neuroinflammation. Although brain mast cells (MCs) are the "first responder" in the brain injury rather than microglia, little is known about the functional aspects of MCs-microglia interactions. METHODS: Male Sprague-Dawley (SD) rats were injected intracerebroventricular with MC stabilizer Cromolyn (100 µg/µl), MC stimulator C48/80 (1 µg/µl), or sterile saline 30 min before open tibial fracture surgery, and the levels of neuroinflammation and memory dysfunction were tested 1 and 3 days after surgery. In addition, the effect of activated MCs on microglia and neurons was determined in vitro. RESULTS: Tibial fracture surgery induced MCs degranulation, microglia activation, and inflammatory factors production, which initiated the acute brain inflammatory response and neuronal death and exhibited cognitive deficit. Site-directed preinjection of the "MCs stabilizer" disodium cromoglycate (Cromolyn) inhibited this effect, including decrease of inflammatory cytokines, reduced MCs degranulation, microglia activation, neuronal death, and improved cognitive function 24 h after the surgery. In vitro study, we found that the conditioned medium from lipopolysaccharide (LPS)-stimulated mast cells line (P815) could induce primary microglia activation through mitogen-activated protein kinase (MAPK) pathway signaling and subsequent production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). In addition, the activated P815 could directly induce neuronal apoptosis and synapse injury with microglia independently. Cromolyn could inhibit P815 activation following improved microglia activation and neuronal loss. CONCLUSIONS: These results implicate that activated MCs could trigger microglia activation and neuronal damage, resulting in central nervous system (CNS) inflammation, and communications of MCs with microglia and neuron could constitute a new and unique therapeutic target for CNS immune inflammation-related diseases.