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The article discusses hypoxic pathophysiology and high-altitude medicine as of December 2012, with a focus on protein kinase C (PKC) signal transduction pathways implicated in cerebral ischemic/hypoxic preconditioning (I/HPC) development and injuries. Topics include a diagram depicting the molecular mechanisms underling PKC-isoform signaling pathways, mitogen-activated protein kinase (MAPK), and middle cerebral artery occlusion (MCAO). Additional information is presented on terminal deoxynucleotidyl transferase (TdT) and oxygen-glucose deprivation (OGD).

Abstract: Elevated introcular pressure (IOP)-induced retinal neuron ischemic death includes an early phase of necrosis and prolonged phase of apoptosis. We used this ischemic model to observe the changes of sortilin and p75NTR protein expressions in rat retina. The results of Western blot analysis showed the expression of p75NTR at the band of 75 (mature form), 60 (non-glycosylated pieces) and 50kDa (ectodomain shedding pieces), and the expression of sortilin at the 95 and 90kDa (the mature form). The protein expressions of p75NTR (60 and 50kDa pieces) and sortilin (90kDa) increased significantly (p <0.05) at days 3, 5 and 7 after retinal ischemia. This effect was also confirmed by immunofluorescence staining. Sortilin was primarily present in cell membrane of the ganglion cells layer (GCL) and large ganglion cell bodies by immunofluorescence labeling. There was little expression of p75NTR in the normal retina, while expression increased extensively in GCL, inner plexiform layer (IPL) and inner nuclear layer (INL) after retinal ischemia. p75NTR was shown to co-localize with neurofilament in the axons of neuronal cells by double-labeling. These results suggested that the protein expressions of 60 and 50kDa forms of p75NTR, and the 90kDa mature form of sortilin increased in ischemia-induced retinal neuron of rats. [Copyright &y& Elsevier]

Abstract: Studies have demonstrated that optic nerve transection results in apoptotic cell death of retinal ganglion cells (RGCs) and neurons within lateral geniculate nucleus (LGN). Heat shock protein (Hsp) 70 was reported to be involved in protecting cells from injury under various pathological conditions in vivo and in vitro. To determine the involvement of Hsp70 in protecting neurons within LGN against damage or loss induced by optic nerve injuries, we observed the changes in protein expression and distribution of Hsp70 in LGN at days 1, 3, 7, 14 and 28 after unilateral optic nerve transection in the left eye of Sprague–Dawley rats by using Western blot analysis and immunohistochemical staining. We found that the levels of Hsp70 protein expression increased significantly (p <0.05, n =6 for each group) in both right and left LGN of rats following left optic nerve transection 1–7 days. The maximum of Hsp70 expression reached at day 3. However, Hsp70 protein expression levels in both right and left LGN returned to control levels at 14 and 28 days after left optic nerve lesion. In addition, the increased Hsp70 expression, which mainly localized in the intergeniculate leaflet of LGN, was also observed by immunostaining in right LGN at the end of day 3 after the lesion. These results suggest that increased expression of Hsp70 may be involved in protecting neurons within LGN against damage or loss induced by left optic nerve transection at early stage. [Copyright &y& Elsevier]

Background: Perioperative cerebral ischemia/reperfusion injury is a major contributor to postoperative death and cognitive dysfunction in patients. It was reported that morphine preconditioning (MP) can mimic ischemia/hypoxia preconditioning to protect against ischemia/reperfusion injury. However, the mechanism of MP on the ischemia/reperfusion-induced neuronal apoptosis has not been fully clarified., Methods: The middle cerebral artery occlusion/reperfusion (MCAO/R) model of mice and the oxygen-glucose deprivation/reoxygenation (OGD/R) model in primary cortical neurons were used to mimic ischemic stroke. In vivo, the infarct size was measured by using TTC staining; NDSS, Longa score system, and beam balance test were performed to evaluate the neurological deficits of mice; the expression of the protein was detected by using a western blot. In vitro, the viability of neurons was determined by using CCK-8 assay; the expression of protein and mRNA were assessed by using western blot, RT-qPCR, and immunofluorescent staining; the level of apoptosis was detected by using TUNEL staining., Results: MP can improve the neurological functions of mice following MCAO/R (P<0.001, n=10 per group). MP can decrease the infarct size (P<0.001, n=10 per group) and the level of cleaved-caspase-3 of mice following MCAO/R (P<0.01 or 0.001, n=6 per group). MP can increase the levels of cPKCγ membrane translocation, p-p65, and cFLIPL, and decrease the levels of cleaved-caspase-8, 3 in neurons after OGD/R or MCAO/R 1 d (P<0.05, 0.01 or 0.001, n=6 per group). In addition, MP could alleviate OGD/R-induced cell apoptosis (P<0.001, n=6 per group)., Conclusion: MP alleviates ischemia/reperfusion-induced Caspase 8-dependent neuronal apoptosis through the cPKCγ-NF-κB-cFLIPL pathway., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Type 1 diabetes mellitus (T1DM)-induced cognitive dysfunction is common, but its underlying mechanisms are still poorly understood. In this study, we found that knockout of conventional protein kinase C (cPKC)γ significantly increased the phosphorylation of Tau at Ser214 and neurofibrillary tangles, but did not affect the activities of GSK-3β and PP2A in the hippocampal neurons of T1DM mice. cPKCγ deficiency significantly decreased the level of autophagy in the hippocampal neurons of T1DM mice. Activation of autophagy greatly alleviated the cognitive impairment induced by cPKCγ deficiency in T1DM mice. Moreover, cPKCγ deficiency reduced the AMPK phosphorylation levels and increased the phosphorylation levels of mTOR in vivo and in vitro. The high glucose-induced Tau phosphorylation at Ser214 was further increased by the autophagy inhibitor and was significantly decreased by an mTOR inhibitor. In conclusion, these results indicated that cPKCγ promotes autophagy through the AMPK/mTOR signaling pathway, thus reducing the level of phosphorylated Tau at Ser214 and neurofibrillary tangles., (© 2022. Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences.)

Ischemic stroke, which accounts for nearly 80% of all strokes, leads to white matter injury and neurobehavioral dysfunction, but relevant therapies to inhibit demyelination or promote remyelination after white matter injury are still unavailable. In this study, the middle cerebral artery occlusion/reperfusion (MCAO/R) in vivo and oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro were used to establish the ischemic models. We found that Eph receptor A4 (EphA4) had no effect on the apoptosis of oligodendrocytes using TUNEL staining. In contrast, EphA4 promoted proliferation of oligodendrocyte precursor cells (OPCs), but reduced the numbers of mature oligodendrocytes and the levels of myelin-associated proteins (MAG, MOG, and MBP) in the process of remyelination in ischemic models in vivo and in vitro as determined using PDGFRα-EphA4-shRNA and LV-EphA4 treatments. Notably, conditional knockout of EphA4 in OPCs (EphA4 fl/fl  + AAV-PDGFRα-Cre) improved the levels of myelin-associated proteins and functional recovery following ischemic stroke. In addition, regulation of remyelination by EphA4 was mediated by the Ephexin-1/RhoA/ROCK signaling pathway. Therefore, EphA4 did not affect oligodendrocyte (OL) apoptosis but regulated white matter remyelination after ischemic stroke through the Ephexin-1/RhoA/ROCK signaling pathway. EphA4 may provide a novel and effective therapeutic target in clinical practice of ischemic stroke., (© 2022 Wiley Periodicals LLC.)

Synapses serve as the interface for the transmission of information between neurons in the central nervous system. The structural and functional characteristics of synapses are highly dynamic, exhibiting extensive plasticity that is shaped by neural activity and regulated primarily by trans-synaptic cell-adhesion molecules (CAMs). Prototypical trans-synaptic CAMs, such as neurexins (Nrxs) and neuroligins (Nlgs), directly regulate the assembly of presynaptic and postsynaptic molecules, including synaptic vesicles, active zone proteins, and receptors. Therefore, the trans-synaptic adhesion mechanisms mediated by Nrx-Nlg interaction can contribute to a range of synaptopathies in the context of pathological pain and other neurological disorders. The present review provides an overview of the current understanding of the roles of Nrx-Nlg interaction in the regulation of trans-synaptic connections, with a specific focus on Nrx and Nlg structures, the dynamic shaping of synaptic function, and the dysregulation of Nrx-Nlg in pathological pain. Additionally, we discuss a range of proteins capable of modulating Nrx-Nlg interactions at the synaptic cleft, with the objective of providing a foundation to guide the future development of novel therapeutic agents for managing pathological pain.

Herkinorin is a novel opioid receptor agonist. Activation of opioid receptors, a member of G protein coupled receptors (GPCRs), may play an important role in Herkinorin neuroprotection. GPCRs may modulate NOD-like receptor protein 3 (NLRP3)-mediated inflammatory responses in the mechanisms of inflammation-associated disease and pathological processes. In this study, we investigated the effects of Herkinorin on NLRP3 and the underlying receptor and molecular mechanisms in oxygen-glucose deprivation/reperfusion (OGD/R)-treated rat cortex neurons. First, Western blot analysis showed that Herkinorin can inhibit the activation of NLRP3 and Caspase-1, decrease the expression of interleukin (IL)-1β, and decrease the secretion of IL-6 and tumour necrosis factor α detected by enzyme-linked immunosorbent assay in OGD/R-treated neurons. Then we found that Herkinorin downregulated NLRP3 levels by inhibiting the activation of nuclear factor kappa B (NF-κB) pathway, reducing the phosphorylation level of p65 and IκBα in OGD/R-treated neurons (p < .05 or .01, n = 3 per group). Instead, both the mu opioid receptor (MOR) inhibitor, β-funaltrexamine, and MOR knockdown reversed the effects of Herkinorin on NLRP3 (p < .05 or .01, n = 3 per group). Further, we found that the level of β-arrestin2 decreased in the cell membrane and increased in the cytoplasm after Herkinorin pretreatment in OGD/R-treated neurons. In co-immunoprecipitation experiments, Herkinorin increased the binding of IκBα with β-arrestin2, decreased the ubiquitination level of IκBα, and β-arrestin2 knockdown reversed the effects of Herkinorin on IκBα in OGD/R-treated neurons (p < .05 or .01, n = 3 per group). Our data demonstrated that Herkinorin negatively regulated NLRP3 inflammasome to alleviate neuronal ischemic injury through inhibiting NF-κB pathway mediated primarily by MOR activation. Inhibition of the NF-κB pathway by Herkinorin may be achieved by decreasing the ubiquitination level of IκBα, in which β-arrestin2 may play an important role., (© 2021 Wiley Periodicals LLC.)

Prescription opioids are powerful pain-controlling medications that have both benefits and potentially serious risks. Morphine is one of the preferred analgesics that are widely used to treat chronic pain. However, chronic morphine exposure has been found to cause both functional and structural changes in several brain regions, including the medial prefrontal cortex (mPFC), ventral tegmental area (VTA), and hippocampus (HPC), which lead to addictive behavior. Caveolin-1 (Cav-1), a scaffolding protein of membrane lipid rafts (MLRs), has been shown to organize GPCRs and multiple synaptic signaling proteins within the MLRs to regulate synaptic signaling and neuroplasticity. Previously, we showed that in vitro morphine treatment significantly elevates Cav-1 expression and causes neuroplasticity changes. In this study, we confirmed that chronic morphine exposure can significantly increase Cav-1 expression (P < 0.05) and microtubule-associated protein (MAP-2)-positive neuronal dendritic growth in the hippocampus. Moreover, the rewarding effect and dendritic growth in the HPC induced by chronic morphine exposure were significantly inhibited by hippocampal Cav-1 knockdown. Together, these data suggest that Cav-1 in the hippocampus plays an essential role in the neuroplasticity changes that underlie morphine addiction behaviors., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Activin A plays important roles in ischemic injury and white matter remyelination, but its mechanisms are unclear. In this study, the adult male C57BL/6 J mice were used to establish the model of 1 h middle cerebral artery occlusion/reperfusion (MCAO/R) 1 d to 28 d-induced ischemic stroke in vivo. We found that the neurological outcome was positively correlated with the levels of myelin associated proteins (include MAG, CNPase, MOG and MBP, n = 6 per group) both in corpus callosum and internal capsule of mice with ischemic stroke. The dynamic changes of Luxol fast blue (LFB) staining intensity, oligodendrocyte (CC1 + ) and proliferated oligodendrocyte precursor (Ki67 + /PDGFRα + ) cell numbers indicated demyelination and spontaneous remyelination occurred in the corpus callosum of mice after 1 h MCAO/R 1 d-28 d (n = 6 per group). Activin receptor type I (ACVR1) inhibitor SB431542 aggregated neurological deficits, and reduced MAG, MOG and MBP protein levels of mice with ischemic stroke (n = 6 per group). Meanwhile, recombinant mouse (rm) Activin A enhanced the neurological function recovery, MAG, MOG and MBP protein levels of mice with 1 h MCAO/R 28 d. In addition, the injection of AAV-based ACVR1B shRNA with Olig2 promoter could reverse rmActivin A-induced the increases of CC1 + cell number, LFB intensity, MAG, MOG and MBP protein levels in the corpus callosum (n = 6 per group), and neurological function recovery (n = 10 per group) of mice with 1 h MCAO/R 28 d. These results suggested that Activin A improves the neurological outcome through promoting oligodendroglial ACVR1B-mediated white matter remyelination of mice with ischemic stroke, which may provide a potential therapeutic strategy for ischemic stroke., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Pain is a serious clinical challenge, and is associated with a significant reduction in quality of life and high financial costs for affected patients. Research efforts have been made to explore the etiological basis of pain to guide the future treatment of patients suffering from pain conditions. Findings from studies using KA (kainate) receptor agonist, antagonists and receptor knockout mice suggested that KA receptor dysregulation and dysfunction may govern both peripheral and central sensitization in the context of pain. Additional evidence showed that KA receptor dysfunction may disrupt the finely-tuned process of glutamic acid transmission, thereby contributing to the onset of a range of pathological contexts. In the present review, we summarized major findings in recent studies which examined the roles of KA receptor dysregulation in nociceptive transmission and in pain. This timely overview of current knowledge will help to provide a framework for future developing novel therapeutic strategies to manage pain.

Background: Ketamine, a commonly used nonbarbiturate anesthetic drug, possesses antidepressant properties at subanesthetic doses; however, the underlying mechanisms remain unclear., Materials and Methods: The analgesic and antidepressant effects of ketamine were explored using a complete Freund adjuvant (CFA)-induced peripheral inflammatory pain model in vivo. Mice were first divided into sham or CFA injection group randomly, and were observed for mechanical hyperalgesia, depression-like behavior, and mRNA expression of caveolin-1. Then ketamine was administered in CFA-treated mice at day 7., Results: The behavioral testing results revealed mechanical hyperalgesia and depression in mice from days 7 to 21 after CFA injection. Ketamine reversed depression-like behaviors induced by CFA injection. It also restored the brain-regional expression levels of caveolin-1 in CFA-treated mice. In addition, caveolin-1 mRNA and protein expression were increased in the prefrontal cortex and nucleus accumbens of CFA-treated mice. However, ketamine reversed the increase in caveolin-1 expression in the ipsilateral and contralateral prefrontal cortex and nucleus accumbens, supporting the distinct roles of specific brain regions in the regulation of pain and depression-like behaviors., Conclusions: In CFA-treated mice that exhibited pain behavior and depression-like behavior, ketamine reversed depression-like behavior. The prefrontal cortex and nucleus accumbens are the important brain regions in this regulation network. Despite these findings, other molecules and their mechanisms in the signal pathway, as well as other regions of the brain in the pain matrix, require further exploration.

Stroke is an acute cerebro-vascular disease with high incidence and poor prognosis, most commonly ischemic in nature. In recent years, increasing attention has been paid to inflammatory reactions as symptoms of a stroke. However, the role of inflammation in stroke and its underlying mechanisms require exploration. In this study, we evaluated the inflammatory reactions induced by acute ischemia and found that pyroptosis occurred after acute ischemia both in vivo and in vitro, as determined by interleukin-1β, apoptosis-associated speck-like protein, and caspase-1. The early inflammation resulted in irreversible ischemic injury, indicating that it deserves thorough investigation. Meanwhile, acute ischemia decreased the Sirtuin 1 (Sirt1) protein levels, and increased the TRAF6 (TNF receptor associated factor 6) protein and reactive oxygen species (ROS) levels. In further exploration, both Sirt1 suppression and TRAF6 activation were found to contribute to this pyroptosis. Reduced Sirt1 levels were responsible for the production of ROS and increased TRAF6 protein levels after ischemic exposure. Moreover, N-acetyl-L-cysteine, an ROS scavenger, suppressed the TRAF6 accumulation induced by oxygen-glucose deprivation via suppression of ROS bursts. These phenomena indicate that Sirt1 is upstream of ROS, and ROS bursts result in increased TRAF6 levels. Further, the activation of Sirt1 during the period of ischemia reduced ischemia-induced injury after 72 h of reperfusion in mice with middle cerebral artery occlusion. In sum, these results indicate that pyroptosis-dependent machinery contributes to the neural injury during acute ischemia via the Sirt1-ROS-TRAF6 signaling pathway. We propose that inflammatory reactions occur soon after oxidative stress and are detrimental to neuronal survival; this provides a promising therapeutic target against ischemic injuries such as a stroke.

Stroke causes significant morbidity and mortality worldwide, for which no satisfactory preventive option currently exists. Hypoxic preconditioning (HPC) is a protective strategy for cerebral ischemic stroke. To this end, we have identified, Conventional protein kinase C (cPKC)BetaII to play an important role in HPC. Pathway analysis and protein-protein interaction network building and functional proteomic exploration was used to identify 38 proteins in 6 Kyoto Encyclopedia of Genes and Genomes pathways that interact with cPKCBetaII in brains subjected to HPC. The role of the oxidative phosphorylation pathway was confirmed by experimental validation, and the demonstration that the activity of the complex I and complex V and expression and activity of Ndufv2 and ATP5d was increased. Ndufv2 was co-localized with PKCBetaII in neurons rather than glial cells. Together, these data show that Ndufv2 and the oxidative phosphorylation pathway play an important role in cPKCBetaII-related HPC mediated signalling, likely as an adaptive neuroprotective mechanism.

Treatment of postoperative pain remains a challenge in clinic. Botulinum toxin type A (BoNT/A) and gabapentin regulate the release of neurotransmitters from primary afferent neurons, but their effects of on postoperative pain are not clear. In the current study, using pain behavioral tests, Western blot analysis, and immunocytochemistry, we examined whether BoNT/A, alone or in combination with intrathecal gabapentin, inhibited pain hypersensitivity and attenuated the increase in neurokinin 1 (NK1) receptor internalization in dorsal horn neurons after plantar incision. Our data showed that pretreatment of rats with an intraplantar (2 U) 24 h before plantar incision or intrathecal (0.5 U) injection of BoNT/A 48 h before plantar incision induced a prolonged (3-5 days) decrease in pain scores and mechanical hypersensitivity, as compared to those observed with saline pretreatment. Both intraplantar and intrathecal BoNT/A pretreatment reduced synaptosomal-associated protein 25 levels in the ipsilateral lumbar dorsal root ganglia and spinal cord dorsal horn, and attenuated the increase in NK1 receptor internalization in dorsal horn neurons. Intrathecal administration of a sub-effective dose of gabapentin (50 μg) with BoNT/A (0.5 U) induced greater inhibition of pain hypersensitivity and NK1 receptor internalization than BoNT/A alone. These findings suggest that pretreatment with BoNT/A, alone or in combination with intrathecal gabapentin, may present a promising multimodal analgesia regimen for postoperative pain treatment., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Stroke is one of the leading causes of death in the world, but its underlying mechanisms remain unclear. Both conventional protein kinase C (cPKC)γ and ubiquitin C-terminal hydrolase L1 (UCHL1) are neuron-specific proteins. In the models of 1-hr middle cerebral artery occlusion (MCAO)/24-hr reperfusion in mice and 1-hr oxygen-glucose deprivation (OGD)/24-hr reoxygenation in cortical neurons, we found that cPKCγ gene knockout remarkably aggravated ischaemic injuries and simultaneously increased the levels of cleaved (Cl)-caspase-3 and LC3-I proteolysis product LC3-II, and the ratio of TUNEL-positive cells to total neurons. Moreover, cPKCγ gene knockout could increase UCHL1 protein expression via elevating its mRNA level regulated by the nuclear factor κB inhibitor alpha (IκB-α)/nuclear factor κB (NF-κB) pathway in cortical neurons. Both inhibitor and shRNA of UCHL1 significantly reduced the ratio of LC3-II/total LC3, which contributed to neuronal survival after ischaemic stroke, but did not alter the level of Cl-caspase-3. In addition, UCHL1 shRNA reversed the effect of cPKCγ on the phosphorylation levels of mTOR and ERK rather than that of AMPK and GSK-3β. In conclusion, our results suggest that cPKCγ activation alleviates ischaemic injuries of mice and cortical neurons through inhibiting UCHL1 expression, which may negatively regulate autophagy through ERK-mTOR pathway., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Acute acquired comitant esotropia (AACE) is an unusual presentation of esotropia that occurs after infancy. This study was aimed to study the clinical features and the differences between children and adult patients with AACE in the Chinese populations.This was a retrospective analysis of patients diagnosed with AACE over 4 years; 69 patients (25 females and 44 males) were identified. The patients were divided into 3 groups: < 10 year-old (n = 6, 8.7%), 10-18 year-old (n = 23, 33.3%), and ≥18 year-old (n = 40, 58.0%). Patients underwent medical history, brain and orbital computed tomography, and ophthalmological and orthoptic examinations.The refractions of AACE patients varied among age groups: patients < 10 year-old had mild hypermetropia, while older children and adults showed moderate-to-high myopia (P < .001). The mean angles of esotropia were significantly larger in young children compared with older children and adults (P = .005). There was no significant difference in binocularity detected by either synoptophore or TNO stereoscopic testing among different disease durations. Stereopsis detected by synoptophore and TNO testing showed no significant difference at duration within half a year, but the stereopsis measured by TNO was significantly worse than that detected by synoptophore with extending disease duration (P < .05).AACE seems to occur mostly in older children and adults in the Chinese population. Younger children with AACE seem to demonstrate a common trait of mild hypermetropic refractive errors, while myopia can be seen in older children and adult patients. The duration from onset to treatment of esotropia does not affect the preoperative binocularity.

Conventional protein kinase C (cPKC)γ participated in cerebral hypoxic preconditioning-induced neuroprotection and affected the neurological outcome of ischemic stroked mice. As an independent predictor of ischemic stroke, the internal carotid artery occlusion (ICAO)-caused brain-regional ischemic injury may worsen the neurological outcome of patients. However, the brain-regional ischemic vulnerability and its underlying mechanism remain unclear. In this study, the bilateral ICAO (BICAO) model was applied in cPKCγ wild type (WT) and knockout (KO) mice to determine the cPKCγ impact on brain-regional ischemic vulnerability. The arterial spin labeling (ASL) imaging results showed that 7 days BICAO-induced global ischemia could cause significant blood perfusion loss in prefrontal cortex (69.13%), striatum (61.69%), hypothalamus (67.36%), hippocampus (69.82%) and midbrain (40.53%) of WT mice, along with neurological deficits. Nissl staining and Western blot results indicated that hypothalamus and midbrain had more severe neural cell loss than prefrontal cortex, striatum and hippocampus, which negatively coincided with endogenous cPKCγ protein levels but not blood perfusion loss and cPKCγ membrane translocation levels. Furthermore, we found that cPKCγ KO significantly aggravated the neuron loss in prefrontal cortex, striatum and hippocampus and abolish the regional ischemic vulnerability by using immunofluorescent staining with neuron-specific marker NeuN. Similarly, cPKCγ KO also significantly increased Caspase-3, -8 and -9 cleavage levels in prefrontal cortex, striatum, hippocampus, hypothalamus and midbrain of mice with 24 h BICAO. These results suggested that hypothalamus and midbrain are more vulnerable to ischemia, and endogenous cPKCγ affects the regional ischemic vulnerability through modulating Caspase-8 and -9 dependent cell apoptosis.

Galanin is a neuropeptide with a widespread distribution throughout the nervous and endocrine systems, and recent studies have shown an anti-proliferative effect of galanin on several types of tumors. However, whether and how galanin and its receptors are involved in the regulation of cell proliferation in glioma cells remains unclear. In this study, the roles of galanin and its subtype 1 receptor (GAL1) in the proliferation of human U251 and T98G glioma cells were investigated. We found that galanin significantly suppressed the proliferation of U251 and T98G cells as well as tumor growth in nude mice. However, galanin did not exert apoptotic or cytotoxic effects on these two cell lines. In addition, we showed that galanin decreased the proliferation of U251 and T98G cells via its GAL1 receptor. Finally, we found that the GAL1 receptor was involved in the suppressive effects of galanin by activating ERK1/2.

Nitric oxide (NO) is a relatively short-lived inorganic free radical, which can be produced by different types of cells in multi-cellular organisms. This diffusible messenger functions as either an effector or a second messenger in many intercellular communications or intracellular signaling pathways. NO becomes noxious if it is produced in excess. These effects are mainly mediated by the reactivity of NO with various reactive oxygen species, which can be countered by antioxidant enzymes. In addition, NO can directly modify biological molecules via S-nitrosylation and lead to altered signaling responses. Accumulating evidence suggests that NO has a double-edged role in a dose-dependent, cell-type specific, and biological milieu-dependent way. In the present review, we summarized the synthesis and signaling pathway of NO, and especially focused on its involvement in biological processes, such as endoplasmic reticulum stress, apoptosis and autophagy. Besides, we discussed the functions of NO in the nervous system and its potential role in neurodegenerative diseases. We proposed the target on NO may shed light on the treatment of the related diseases.

Herkinorin is an opiate analgesic with limited adverse effects, functioning as a primary selective atypical opioid µ agonist. The present study aimed to identify whether herkinorin has a positive effect on ischemic/reperfusion (I/R) injury. Adult male C57BL/6 mice were randomly divided into five groups: i) Naïve, ii) sham, iii) I/R, iv) I/R with dimethyl sulfoxide (I/R+D) and v) I/R with herkinorin (I/R+H). The I/R injury model was induced by occluding the middle cerebral artery for 1 h followed by 24 h or 7 days of reperfusion. Neurobehavioral scores and sensorimotor functions were examined 24 h and 7 days following reperfusion. In addition, infarct volumes were examined at these time points using a 2,3,5‑triphenyltetrazolium chloride assay. Herkinorin treatment improved neurobehavioral and sensorimotor functional recovery from I/R‑induced brain injury. There was a significant decrease in infarct volume in the I/R+H group at 24 h or 7 days following reperfusion compared with the I/R and I/R+D groups. Western blotting suggested that the decrease in conventional protein kinase C γ (cPKCγ) membrane translocation in the peri‑infarct region may be attenuated by herkinorin pretreatment. These results indicated that herkinorin may be beneficial in I/R‑induced mouse brain injury, and this may be attributed to the membrane translocation of cPKCγ following activation.

We have reported that neuron-specific conventional protein kinase C (cPKC)γ is involved in the development of cerebral hypoxic preconditioning (HPC) and the neuroprotection against ischemic injuries, but its molecular mechanism is unclear. In this study, the adult and postnatal 24 h C57BL/6J wild-type (cPKCγ +/+ ) and cPKCγ knockout (cPKCγ -/- ) mice were respectively used to establish the models of middle cerebral artery occlusion (MCAO)-induced ischemic stroke in vivo and oxygen-glucose deprivation (OGD)-treated primarily cultured cortical neurons as cell ischemia in vitro. The results showed that cPKCγ knockout could increase the infarct volume and neuronal cell loss in the peri-infarct region, and enhance the neurological deficits, the impaired coordination, and the reduced muscle strength of mice following 1 h MCAO/1-7 days reperfusion. Meanwhile, cPKCγ knockout significantly increased the conversion of LC3-I to LC3-II and beclin-1 protein expression, and resulted in more reductions in P-Akt, P-mTOR, and P-S6 phosphorylation levels in the peri-infarct region of mice with ischemic stroke. The autophagy inhibitor BafA1 could enhance or reduce neuronal cell loss in the peri-infarct region of cPKCγ +/+ and cPKCγ -/- mice after ischemic stroke. In addition, cPKCγ knockout and restoration could aggravate or alleviate OGD-induced neuronal ischemic injury in vitro through Akt-mTOR pathway-mediated autophagy. These results suggested that cPKCγ-modulated neuron-specific autophagy improves the neurological outcome of mice following ischemic stroke through the Akt-mTOR pathway, providing a potential therapeutic target for ischemic stroke.

We previously reported that conventional protein kinase C (cPKC)β participated in hypoxic preconditioning-induced neuroprotection against cerebral ischemic injury, and collapsin response-mediated protein 2 (CRMP2) was identified as a cPKCβ interacting protein. In this study, we explored the regulation of CRMP2 phosphorylation and proteolysis by cPKCβ, and their role in ischemic injury of oxygen-glucose deprivation (OGD)-treated cortical neurons and brains of mice with middle cerebral artery occlusion-induced ischemic stroke. The results demonstrated that cPKCβ-mediated CRMP2 phosphorylation via the cPKCβ-selective activator 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) and inhibition of calpain-mediated CRMP2 proteolysis by calpeptin and a fusing peptide containing TAT peptide and the calpain cleavage site of CRMP2 (TAT-CRMP2) protected neurons against OGD-induced cell death through inhibiting CRMP2 proteolysis in cultured cortical neurons. The OGD-induced nuclear translocation of the CRMP2 breakdown product was inhibited by DOPPA, calpeptin, and TAT-CRMP2 in cortical neurons. In addition, both cPKCβ activation and CRMP2 proteolysis inhibition by hypoxic preconditioning and intracerebroventricular injections of DOPPA, calpeptin, and TAT-CRMP2 improved the neurological deficit in addition to reducing the infarct volume and proportions of cells with pyknotic nuclei in the peri-infact region of mice with ischemic stroke. These results suggested that cPKCβ modulates CRMP2 phosphorylation and proteolysis, and cPKCβ activation alleviates ischemic injury in the cultured cortical neurons and brains of mice with ischemic stroke through inhibiting CRMP2 proteolysis by phosphorylation. Focal cerebral ischemia induces a large flux of Ca(2+) to activate calpain which cleaves collapsin response mediator (CRMP) 2 into breakdown product (BDP). Inhibition of CRMP2 cleavage by calpeptin and TAT-CRMP2 alleviates ischemic injury. Conventional protein kinase C (cPKC)β-mediated phosphorylation could inhibit CRMP2 proteolysis and alleviate ischemic injury in cultured cortical neurons and ischemic stroke-induced mice., (© 2016 International Society for Neurochemistry.)

Synapsins as a family of presynaptic terminal phosphoprotein participates in neuronal development, but their role in the synaptic plasticity of visual cortex is unclear. In this study, the impact of monocular deprivation (MD) on dynamic changes of isoform-specific protein expression and site 1 phosphorylation of synapsins in visual cortex of the postnatal mice were observed by using the technique of Western blot analysis. The results showed that the total (T-) protein levels of synapsins including the isoform of Ia/b, IIa/b and IIIa were about 21-26% of adult level in visual cortex of mice at postnatal 7 days (P7), and then the T-synapsin Ia/b and IIb could quickly reach adult level at P35. However, the T-synapsin IIa and IIIa increased more slowly (71-74% at P35), and then kept increasing in the visual cortex of mice at P60. Unlike to the changes of T-synapsins, the level of phosphorylated (P-) synapsin Ia/b (not IIa/b and IIIa) at site 1 increased with development to the highest level at P21, and then decreased rapidly to a low level in visual cortex of mice at P35-60. In addition, we found that the levels of P-synapsin Ia/b increased significantly in left visual cortex of P28 and P35 (not P21 and P42) mice with 1-week MD of right eye; and no significant changes of T-synapsins were observed in both left and right sides of visual cortex in P21-42 mice with MD treatment. These results suggested that the isoform-specific protein expression and site-1 phosphorylation of synapsins might play a different role in the synaptic plasticity of visual cortex, and MD delays the dynamic changes of phosphorylated synapsin Ia/b at site-1 in contralateral visual cortex of juvenile mice.

We and others previously reported that the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 significantly accumulate with age in mouse lung. This is accompanied by elevated phosphorylation of p38. Here, we further investigate whether aging affects activation of p38 signaling and the inflammatory reaction after exposure to lipopolysaccharide (LPS) in the lungs of mice in vivo and humans ex vivo. The data showed that activation of p38 peaked at 0.5h and then rapidly declined in young (2-month-old) mouse lung, after intranasal inhalation challenge with LPS. In contract, activation of p38 peaked at 24h and was sustained longer in aged (20-month-old) mice. As well as altered p38, activations of its upstream activator MKK and downstream substrate NF-κB were also changed in the lungs of aged mice, which corresponded with the absence in the early phase but delayed increases in concentrations of TNF-α, IL-1β and IL-6. Consistent with the above observations in mice, similar patterns of p38 signaling also occurred in human lungs. Compared with younger lungs from adult-middle aged subjects, the activation of p38, MKK and NF-κB, as well as the production of pro-inflammatory cytokines were significantly increased in the lungs of older subjects ex vivo. Exposure of human lung cells to LPS induced rapid activation of p38, MKK and NF-κB in these cells from adult-middle aged subjects, but not older subjects, with increases in the production of the pro-inflammatory cytokines. The LPS-induced rapid activation in the lung cells from adult-middle aged subjects occurred as early as 0.25h after exposure, and then declined. Compared with adult-middle aged subjects, the LPS exposure did not induce marked changes in the early phase, either in the activation of p38, MKK and NF-κB, or in the production of TNF-α, IL-1β or IL-6 in the lung cells from older subjects. In contrast, these changes occurred relatively late, peaked at 16h and were sustained longer in the lungs of older subjects. These data support the hypothesis that the sustained activation of the p38 signaling pathway at baseline and the absence in the early phase but delayed of p38 signaling pathway response to LPS in the elderly may play important roles in increased susceptibility of aged lungs to inflammatory injury., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Purpose: To assess whether a Valsalva manoeuver influences intra-ocular pressure (IOP), cerebrospinal fluid pressure (CSF-P) and, by a change in the trans-laminar cribrosa pressure difference, optic nerve head morphology., Methods: In the first part of the study, 20 neurological patients (study group 'A') underwent measurement of IOP and lumbar CSF-P measurement in a lying position before and during a Valsalva manoeuver. In the second study part, 20 healthy subjects (study group 'B') underwent ocular tonometry and confocal scanning laser tomography of the optic nerve head before and during a Valsalva manoeuver., Results: During the Valsalva manoeuver in study group 'A', the increase in CSF-P by 10.5 ± 2.7 mmHg was significantly (p < 0.001) higher than the increase in IOP by 1.9 ± 2.4 mmHg. The change in CSF-P was not significantly (p = 0.61) correlated with the change in IOP. During the Valsalva manoeuver in study group 'B', IOP increased by 4.5 ± 4.2 mmHg and optic cup volume (p < 0.001), cup/disc area ratio (p = 0.02), cup/disc diameter ratio (p = 0.03) and maximum optic cup depth (p = 0.01) significantly decreased, while neuroretinal rim volume (p = 0.005) and mean retinal nerve fibre layer thickness (p = 0.02) significantly increased., Conclusions: The Valsalva manoeuver-associated short-term increase in CSF-P was significantly larger than a simultaneous short-term increase in IOP. It led to a Valsalva manoeuver-associated decrease or reversal of the trans-laminar cribrosa pressure difference, which was associated with a change in the three-dimensional optic nerve head morphology: optic cup-related parameters decreased and neuroretinal rim-related parameters enlarged. These findings may be of interest for the pathogenesis of glaucomatous optic neuropathy., (© 2013 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

The understanding of molecular mechanism underlying ischemia/reperfusion-induced neuronal death and neurological dysfunction may provide therapeutic targets for ischemic stroke. The up-regulated miRNA-30a among our previous identified 19 MicroRNAs (miRNAs) in mouse brain after 6 h middle cerebral artery occlusion (MCAO) could negatively regulate Beclin 1 messenger RNA (mRNA) resulting in decreased autophagic activity in tumor cells and cardiomyocytes, but its role in ischemic stroke is unclear. In this study, the effects of miRNA-30a on ischemic injury in N2A cells and cultured cortical neurons after oxygen glucose deprivation (OGD), and mouse brain with MCAO-induced ischemic stroke were evaluated. The results showed that miRNA-30a expression levels were up regulated in the brain of mice after 6 h MCAO without reperfusion, but significantly down regulated in the peri-infarct region of mice with 1 h MCAO/24 h reperfusion and in N2A cells after 1 h OGD/6-48 h reoxygenation. Both the conversion ratio of microtubule-associated protein 1 light chain 3 (LC3)-II/LC3-I and Beclin 1 protein level increased in N2A cells and cultured cortical neurons following 1 h OGD/24 h reoxygenation. The down-regulated miRNA-30a could attenuate 1 h OGD/24 h reoxygenation-induced ischemic injury in N2A cells and cultured cortical neurons through enhancing Beclin 1-mediated autophagy, as miRNA-30a recognized the 3'-untranslated region of beclin 1 mRNA to negatively regulate Beclin 1-protein level via promoting beclin 1 messenger RNA (mRNA) degradation, and Beclin 1 siRNA abolished anti-miR-30a-induced neuroprotection in 1 h OGD/24 h reoxygenation treated N2A cells. In addition, anti-miR-30a attenuated the neural cell loss and improved behavioral outcome of mice with ischemic stroke. These results suggested that down-regulation of miRNA-30a alleviates ischemic injury through enhancing beclin 1-mediated autophagy, providing a potential therapeutic target for ischemic stroke.

Objective: To study the expression level of folate receptor α (FR-α) in glioma tissue and its clinical significance., Methods: Forty-eight human glioma specimens were collected from patients who underwent surgery from March 2012 to March 2013. These specimens were as follows:12 cases of glioblastoma (WHO IV), 6 cases of astrocytoma of each malignancy grade(WHO II, III), 6 cases of oligodendroastrocytoma of each malignancy grade (WHO II, III), 6 cases of oligodendroglioma of each malignancy grade (WHO II, III ). In addition, 6 cases of normal brain tissue resected from brain traumatic patients were taken as negative control, and one case of placental tissue (had got the consent of the parents and their families) was taken as positive control. The expression level of FR-α in tumor tissues was evaluated by Western blot analysis. The results of Western blot analysis were analyzed by t-test. The expression level of FR-α and Ki-67 in tumor tissues was evaluated immunohistochemistry, the results were analyzed by Kruskal-Wallis test and Nemenyi test. The correlation between the expression level of FR-α and cell proliferation index Ki-67 was analyzed by Pearson correlation analysis., Results: Western blot analysis showed that the FR-α was not expressed in normal brain tissue and oligodendroglioma tissue, but highly expressed in astrocytoma, oligodendroastrocytoma and gliomablastoma. The expression level in WHO III astrocytoma was significantly higher than in WHO II (t = 4.497, P < 0.05). FR-α was also highly expressed in oligodendroastrocytoma and its expression level in WHO III was also significantly higher than in the WHO II (t = 2.876, P < 0.05). Foremore, immunohistochemistry analysis also showed that FR-α was not expressed in oligodendroglioma, but expressed in astrocytoma, oligodendroastrocytoma and gliomablastoma. The positive rate of FR-α of WHO III was significantly higher than the WHO II astrocytoma(57.8% ± 2.2% vs. 45.7% ± 2.3%,χ(2) = 3.871, P = 0.034). In oligodendroastrocytoma, the positive rate of FR-α of WHO III was significantly higher than the WHO II(56.5% ± 5.4% vs. 37.1% ± 5.2%,χ(2) = 4.454, P = 0.021). Moreover, the expression level of FR-α in gliomablastoma was highest in all histological types of gliomas, the positive rate of FR-α was up to 65.0% ± 4.5%. Pearson correlation analysis showed that the positive rate of FR-α was positively correlated with Ki-67 index (r = 0.903, P < 0.05)., Conclusions: FR-α is expressed in astrocytoma, oligodendroastrocytoma and glioblastoma, and the expression level of FR-α is positively correlated with malignancy grade and Ki-67 index. Therefore, FR-α may be applied as a special target for diagnosis and treatment of glioma.

Collapsin response mediator protein 2 (CRMP2) is a brain-specific multifunctional adaptor protein involved in neuronal polarity and axonal guidance. Our previous results showed CRMP2 may be involved in the hypoxic preconditioning and ischemic injury, but the mechanism was not clear. This study explored whether CRMP2 was involved in NMDA-induced neural death, and the possible mechanism. Western blot analysis demonstrated that NMDA reduced the phosphorylation of CRMP2 and inspired the cleavage of CRMP2. Also, it was detected that NMDA treatment did not affect the phosphorylation of CRMP2 in early stage (<6 h). Over-expression of CRMP2 aggravated the NMDA-induced injury, suggesting the vital role of CRMP2 in excitotoxicity. Tat-CRMP2 was designed to provide the cleavage site of calpain. Thiazolyl blue tetrazolium bromide assay, Hoechst33342/Propidium Iodide staining and Western blot assay showed that Tat-CRMP2 pretreatment increased cell viability compared with the control group against NMDA exposure by decreasing the cleavage of CRMP2. In conclusion, these studies indicated that cleavage of CRMP2 plays an important role involved in the NMDA-induced injury. The cleavage of CRMP2 may be a promising target for excitatory amino acid-related ischemic and hypoxic injury.

Aging is an inevitable process associated with immune imbalance, which is characterized by a progressive functional decline in major organs, including lung. However, effects of altered Th1/Th2 commitment on lung senescence are largely unknown. To examine effects of altered Th1/Th2 balance on lung aging, we measured proportions of Th1 and Th2 cells and expression of cytokines, chemokines, collagen deposition and other relevant physiological and pathological parameters in 2- and 20-months-old (mo) CXCR3-deficient (CXCR3(-/-)) C57BL/6J mice compared with wild-type (WT) mice. There was a significant weight-loss observed in 20-mo CXCR3(-/-) mice compared with the same aged WT group. Although lung function and structure changed with age in both groups, central airway resistance (Rn), tissue elastance (H) and damping (G) were significantly lower in 20-mo CXCR3(-/-) mice than those of WT mice. In contrast, the whole lung volume (V(L)), the mean linear intercept length of alveolar (L(m)), and the total lung collagen content were significantly elevated in 20-mo CXCR3(-/-) mice. With aging, the lungs of WT mice had typical Th1-type status (increased population of Th1 cells and concentrations of cytokine IFN-γ and CXCR3 ligands) while CXCR3(-/-) mice showed Th2-type polarization (decreased proportion of Th1 cells and concentrations of CXCR3 ligands but increased level of IL-4). Our data suggest that Immunosenescence is associated with lung aging, and that altered Th1/Th2 imbalance favors Th2 predominance in CXCR3(-/-) mice, which contributes to the process of accelerated lung aging in this model., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Cerebral hypoxic preconditioning (HPC) provides neuroprotection by intracellular signaling pathways. We previously demonstrated that novel protein kinase Cε (nPKCε) activation participated in cerebral HPC development. In this study, we explore the role of nPKCε in HPC-induced neuroprotection against middle cerebral artery occlusion (MCAO)-induced ischemic injury and identify its possible signaling molecules. A total of 131 adult male BALB/c mice were divided into eight groups: normoxic control (n=9), HPC (n=9), HPC+εV1-2 (n=13), Sham (n=19), HPC+sham (n=6), Ischemia (I, 6h MCAO, n=31), HPC+I (n=25) and HPC+εV1-2+I (n=19). nPKCε specific inhibitor εV1-2 was administered via intracerebroventricular injection. Western blot, 2,3,5-triphenyltetrazolium chloride staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling were applied to determine nPKCε membrane translocation, infarction volume and programmed cell death (PCD), respectively. Two-dimensional gel electrophoresis (2-De) and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used to identify nPKCε-interacting proteins, followed by bioinformatics analysis of genee ontology (GO) to predict nPKCε-specific signaling pathways. Our results showed that HPC attenuates MCAO-induced brain injuries and stabilized nPKCεmembrane translocation in peri-infarct region, which was abolished by nPKCε-speecific inhibitor εV1-2. Proteomics analysis revealed 8 up- and 3 down-regulated nPKCε-interacting proteins both in cytosolic and particulate fractions of HPC mouse brain. GO analysis predicted 25 significant nPKCε-specific signaling pathways among the 16 identified nPKCε-interacting proteins in brain of HPC mice. This study is the first to report multiple nPKCε-interacting proteins and their signaling pathways in HPC mouse brain, suggesting that nPKCε signaling molecules is responsible for HPC-induced neuroprotection against cerebral ischemic injuries of mice., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Members of the tumor necrosis factor (TNF) superfamily have been revealed to be associated with painful bladder syndrome/interstitial cystitis (PBS/IC). TNF ligand-related molecule 1A (TL1A) and its receptor, death receptor 3 (DR3), belong to the TNF superfamily and have been implicated in chronic inflammatory diseases. Bladder biopsies from 8 female patients clinically diagnosed with PBS/IC according to the National Institute for Diabetes and Digestive and Kidney Diseases criteria and 8 female bladder carcinoma control patients were investigated to test the protein and mRNA expression levels of TL1A and DR3 using western blotting and real-time RT-PCR. The protein level ratio of TL1A to β-actin (IC, 0.65±0.03 vs. controls, 0.25±0.02, P<0.001) and of its receptor DR3 to β-actin (IC, 0.66±0.06 vs. controls, 0.27±0.02, P<0.001) were observed to be significantly higher in the patients with IC. The real-time RT-PCR ΔCts of TL1A minus GAPDH (IC, 7.60±0.52 vs. controls, 10.08±0.32, P<0.001) and the DR3 minus GAPDH (IC, 6.68±0.60 vs. controls, 8.99±0.61, P=0.017) were observed to be significantly lower in the patients with IC, suggesting that the mRNA levels of TL1A and DR3 were higher in the PBS/IC patients. The protein and mRNA expression of TL1A and DR3 are upregulated in the bladder tissues of PBS/IC patients and may be involved in inflammation and apoptosis in PBS/IC.

Propofol and ketamine may provide certain degree of neuroprotection, but the underlying mechanism remains unclear to date. The cAMP response element-binding protein (CREB) was proposed that its phosphorylation at Ser133 (P-CREB) constituted a convergence point involved in neuroprotection. The purpose of this study was to determine whether different dosages of propofol and ketamine could provide neuroprotection against permanent middle cerebral artery occlusion (MCAO)-induced ischemic injuries and the involvement of P-CREB. Eighty adult male BALB/c mice that underwent 6 h MCAO were randomly divided into eight groups: Sham-operation; MCAO + saline; MCAO + 25, 50, 100 mg/kg propofol; and MCAO + 25, 50, 100 mg/kg ketamine (intraperitoneal injection 30 min following MCAO). We found that 50, 100 (not 25) mg/kg propofol, and 25 (not 50 and 100) mg/kg ketamine could significantly reduce the infarct volume, edema ratio and neurological deficit (n = 10 per group) as well as inhibit the decrease of P-CREB level in peri-infarct region when compared with that of MCAO + saline group (n = 6 per group). In addition, the results of double-labeled immunofluorescent staining showed that P-CREB co-localized with neuron-specific marker, NeuN, in the peri-infarct region of 50 mg/kg propofol and 25 mg/kg ketamine treated 6 h MCAO mice (n = 4 per group). These results suggested that inhibition of neuron-specific P-CREB dephosphorylation in the peri-infarct region is involved in high dose propofol and low dose ketamine-induced neuroprotection of 6 h MCAO mice.

The p38 mitogen-activated protein kinase (p38 MAPK) pathway is a key regulator of pro-inflammatory cytokine biosynthesis, which may contribute to the chronic low-grade inflammation observed with aging. We hypothesize that aging up-regulates the activation of p38 MAPK as well as the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in mouse lung, and is accompanied by disturbances in oxidant-antioxidant status. In addition, the elevated protein levels of phosphorylated active form of p38 MAPK (phospho-p38 MAPK) with age are tissue-specific. To test this hypothesis, protein levels of phospho-p38 MAPK were determined using Western blot analysis in isolated lung, brain, heart, spleen, kidney and muscle of young (2-month-old) and aged (20-month-old) male C57BL/6J mice. Results show that phospho-p38 MAPK protein levels, not total-p38 MAPK, increased significantly (p<0.01, n=8) in lung and brain of 20-month-old mice. The activation of p38 MAPK in other tissues was not altered with age. Immunostaining showed that epithelial cells and alveolar macrophages in lung parenchyma were the major cellular sources of phospho-p38 MAPK immunity. As measured by enzyme-linked immunosorbent assay (ELISA), TNF-α, IL-1β and IL-6 in lung homogenates were elevated significantly with age, but there were no differences with age in serum levels except for IL-6. In addition, IL-1β and IL-6 were increased notably while TNF-α was not different with age in bronchoalveolar lavage fluid (BALF). Furthermore, the oxidant-antioxidant status was evaluated by measuring pro-oxidant malondialdehyde (MDA) levels and the activity of reactive oxygen species scavenging enzymes (i.e. superoxide dismutase (SOD) and glutathione (GSH)) in lung homogenates. The results showed that SOD and GSH decreased with age, while MDA did not change. In conclusion, our data demonstrate that p38 MAPK is activated during lung aging with a corresponding increase in pro-inflammatory cytokines and decrease in antioxidant capacity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

As of yet, pharmacological treatments of stroke are only met with mediocre results, which are either ineffective or confounded by adverse effects, thus calling for a better understanding of endogenous neuroprotective mechanism. Previously, we have demonstrated that the translocated activation of conventional protein kinase Cγ (cPKCγ) is involved in the development of cerebral hypoxic preconditioning (HPC), one of the most profound neuroprotective strategies. This study was designed to substantiate the role of cPKCγ and its signaling molecules in HPC-induced neuroprotection against subsequent middle cerebral artery occlusion (MCAO)-induced permanent cerebral ischemic injuries. The effects of HPC and cPKCγ on cerebral ischemic injuries were studied by observing the changes in neurological deficits, infarct volume and neural cell apoptosis. cPKCγ membrane translocation (activation) and its interacting protein synapsin in the ischemic brain were examined by Western blot analysis. Proteomic approaches were employed to identify the cPKCγ-interacting proteins. We found that HPC could markedly attenuate MCAO-induced brain injuries and the decrease of cPKCγ membrane translocation, but cPKCγ inhibitor Go6983 could block HPC-induced neuroprotection. Among the 41 identified cPKCγ-interacting proteins, 17 up- and 6 down-regulated proteins were observed in cytosol or particulate fraction during HPC. In addition, the up-regulated synapsin could reciprocally co-precipitate with cPKCγ both in cytosol and particulate fractions, and Go6983 abolished HPC-induced inhibition on synapsin dephosphorylation in ischemic core and peri-infarct region (penumbra). This study is the first to report multiple cPKCγ-interacting proteins in HPC mouse brain and suggested that cPKCγ signaling molecules, especially the cPKCγ-synapsin pathway, might be responsible for HPC-induced neuroprotection against cerebral ischemic injuries of mice., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Previous studies have demonstrated that the enhanced levels of phosphorylated α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor GluR1 subunits at Serine-831 (pGluR1-Ser-831) and Serine-845 (pGluR1-Ser-845) in the spinal cord dorsal horn are involved in central sensitization of inflammatory pain. However, whether the phosphorylatory regulation of AMPA receptor GluR1 subunits is implicated in the development and maintenance of post-operative pain remains unclear. The current study aims to examine the functional regulation of AMPA receptor GluR1 subunit through its phosphorylation mechanism during the period of post-operative painful events in rats. Our data indicated that the expression of pGluR1-Ser-831 in ipsilateral spinal cord dorsal horn increased significantly at 3 h after incision, then decreased gradually, and returned to the normal level 3 day post-incision. Meanwhile, the expression of pGluR1-Ser-845 and GluR1 in ipsilateral spinal cord dorsal horn remained unchanged. The cumulative pain scores increased at 3 h after incision, gradually decreased afterwards and returned to the baseline values at 4 day after incision and the trend was almost parallel to the expression changes of pGluR1-Ser-831 in spinal dorsal horn. Intrathecal injection of a calcium-dependent protein kinase (PKC) inhibitor, Gö6983 (10 μM), significantly reversed the incision-mediated over-expression of pGluR1-Ser-831 in spinal dorsal horn at 3 h after incision and decreased the cumulative pain scores as well. These results indicate that the phosphorylation of GluR1 subunits at Serine-831 and Serine-845 sites might be differentially regulated following surgical procedures and support a neurobiological mechanism of post-operative pain involved in phosphorylation of AMPA subunits GluR1-Ser-831, but not pGluR1-Ser-845. Our study suggests that the therapeutic targeting the phosphorylation regulation of AMPA receptor GluR1 subunit at Serine-831 site would be potentially significant for treating postoperative pain.

Glutamate-induced excitotoxicity has been implicated in the etiology of stroke, epilepsy, and neurodegenerative diseases. NMDA receptors (NMDARs) play a pivotal role in excitotoxic injury; however, clinical trials testing NMDAR antagonists as neuroprotectants have been discouraging. The development of novel neuroprotectant molecules is being vigorously pursued. Here, we report that downstream regulatory element antagonist modulator (DREAM) significantly inhibits surface expression of NMDARs and NMDAR-mediated current. Overexpression of DREAM showed neuroprotection against excitotoxic neuronal injury, whereas knockdown of DREAM enhanced NMDA-induced toxicity. DREAM could directly bind to the C0 domain of the NR1 subunit. Although DREAM contains multiple binding sites for the NR1 subunit, residues 21-40 of the N terminus are the main binding site for the NR1 subunit. Thus, 21-40 residues might relieve the autoinhibition conferred by residues 1-50 and derepress the DREAM core domain by a competitive mechanism. Intriguingly, the cell-permeable TAT-21-40 peptide, constructed according to the critical binding site of DREAM to the NR1 subunit, inhibits NMDAR-mediated currents in primary cultured hippocampal neurons and has a neuroprotective effect on in vitro neuronal excitotoxic injury and in vivo ischemic brain damage. Moreover, both pretreatment and posttreatment of TAT-21-40 is effective against excitotoxicity. In summary, this work reveals a novel, negative regulator of NMDARs and provides an attractive candidate for the treatment of excitotoxicity-related disease.

Ets-like transcription factor-1 (Elk-1) is a target of mitogen activated protein kinase (MAPK) which has been reported to play a key role in the induction of tolerance to ischemic and hypoxic injury, but little is known about the role of Elk-1 in the development of cerebral hypoxic preconditioning (HPC). In this study, we found that Elk-1 phosphorylation at Ser383 (p-Ser383 Elk-1), not protein expression, increased significantly (P < 0.05) in both hippocampus and frontal cortex of mice after repetitive auto-hypoxia exposures from 1 to 6 times (H1-H6, n = 6 for each group) when compared to that of the normoxic control group (H0, n = 6). The enhanced p-Ser383 Elk-1 was also found in the nuclear extract of H3 mice brain. Similarly, increased p-Ser383 Elk-1 was observed by immunostaining in the hippocampus and frontal cortex of mice from H3 and H6 groups. In addition, we found that the increased p-Ser383 Elk-1 co-localized with a neuron-specific protein, neurogranin, in the brain of H3 mice by using double immunofluorescence labeling. These results suggested that the enhanced neuron-specific phosphorylation of Elk-1 at Ser383 is involved in cerebral HPC of mice.

Erythrocyte-derived depressing factor (EDDF) shows significant protective effects on blood vessels from hypertensive rats, by regulating vascular reactivity, calcium homeostasis, DNA synthesis, and cell cycle progression in vascular smooth muscles (VSMCs). Arteries from hypertensive and aging people have high levels of accumulated calcium. However, in the life span of experimental animals commonly used, arterial calcium content does not reach cytotoxic levels observed in human. An overdose of vitamin D(3) results in a rapid arterial calcium overload. Using rats with arterial calcinosis and age- and gender-matched Wistar controls, we investigated whether EDDF has beneficial effect on blood vessels from animals with arterial calcinosis. Blood vessel functions were impaired in rats with arterial calcinosis, as indicated by decreased Ca(2+)-ATPase activity, increased vasoconstrictor responses to alpha1 adrenoceptor agonist phenylephrine and increased ERK1/2 phosphorylation. Arterial calcium overload also impaired the morphological integrity of VSMCs. EDDF restored the abovementioned abnormalities caused by arterial calcinosis, and inhibited cell cycle progression of VSMCs induced by angiotensin II. In conclusion, EDDF may protect blood vessels from animals with arterial calcinosis, which is mediated by regulating calcium homeostasis, vascular reactivity and cell cycle progression as well as by improving morphological integrity of VSMCs.

Activation of p38 mitogen-activated protein kinase (p38 MAPK) has been implicated as a mechanism of ischemia/hypoxia-induced cerebral injury. The current study was designed to explore the involvement of p38 MAPK in the development of cerebral hypoxic preconditioning (HPC) by observing the changes in dual phosphorylation (p-p38 MAPK) at threonine180 and tyrosine182 sites, protein expression, and cellular distribution of p-p38 MAPK in the brain of HPC mice. We found that the p-p38 MAPK levels, not protein expression, increased significantly (p<0.05) in the regions of frontal cortex, hippocampus, and hypothalamus of mice in response to repetitive hypoxic exposure (H1-H6, n=6 for each group) when compared to values of the control normoxic group (H0, n=6) using Western blot analysis. Similar results were also confirmed by an immunostaining study of the p-p38 MAPK location in the frontal cortex, hippocampus, and hypothalamus of mice from HPC groups. To further define the cell type of p-p38 MAPK positive cells, we used a double-labeled immunofluorescent staining method to co-localize p-p38 MAPK with neurofilaments heavy chain (NF-H, neuron-specific marker), S100 (astrocyte-specific marker), and CD11b (microglia-specific maker), respectively. We found that the increased p-p38 MAPK occurred in microglia of cortex and hippocampus, as well as in neurons of hypothalamus of HPC mice. These results suggest that the cell type-specific activation of p38 MAPK in the specific brain regions might contribute to the development of cerebral HPC mechanism in mice.

Our previous studies have demonstrated that hypoxic precondition (HPC) increased membrane translocation of protein kinase C isoforms and decreased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the brain of mice. The goal of this study was to determine the involvement of p90 KD ribosomal S6 kinase (RSK) in cerebral HPC of mice. Using Western-blot analysis, we found that the levels of membrane/nuclear translocation, but not protein expression of RSK increased significantly in the frontal cortex and hippocampus of HPC mice. In addition, we found that the phosphorylation levels of RSK at the Ser227 site (a PDK1 phosphorylation site), but not at the Thr359/Ser363 sites (ERK1/2 phosphorylated sites) increased significantly in the brain of HPC mice. Similar results were confirmed by an immunostaining study of total RSK and phospho-Ser227 RSK. To further define the cellular populations to express phospho-Ser227 RSK, we found that the expression of phospho-Ser227 RSK co-localized with neurogranin, a neuron-specific marker, in cortex and hippocampus of HPC mice by using double-labeled immunofluorescent staining method. These results suggest that increased RSK membrane/nuclear translocation and PDK1 mediated neuron-specific phosphorylation of RSK at Ser227 might be involved in the development of cerebral HPC of mice.