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Morphine (Mor) has exhibited efficacy in safeguarding neurons against ischemic injuries by simulating ischemic/hypoxic preconditioning (I/HPC). Concurrently, autophagy plays a pivotal role in neuronal survival during IPC against ischemic stroke. However, the involvement of autophagy in Mor-induced neuroprotection and the potential mechanisms remain elusive. Our experiments further confirmed the effect of Mor in cellular and animal models of ischemic stroke and explored its potential mechanism. The findings revealed that Mor enhanced cell viability in a dose-dependent manner by augmenting autophagy levels and autophagic flux in neurons subjected to oxygen-glucose deprivation/reoxygenation (OGD/R). Pretreatment of Mor improved neurological outcome and reduced infarct size in mice with middle cerebral artery occlusion/reperfusion (MCAO/R) at 1, 7 and 14 days. Moreover, the use of autophagy inhibitors nullified the protective effects of Mor, leading to reactive oxygen species (ROS) accumulation, increased loss of mitochondrial membrane potential (MMP) and neuronal apoptosis in OGD/R neurons. Results further demonstrated that Mor-induced autophagy activation was regulated by mTOR-independent activation of the c-Jun NH2- terminal kinase (JNK)1/2 Pathway, both in vitro and in vivo. Overall, these findings suggested Mor-induced neuroprotection by activating autophagy, which were regulated by JNK1/2 pathway in ischemic stroke. [ABSTRACT FROM AUTHOR]

Neuron-specific conventional protein kinase C (cPKC)γ mediates cerebral hypoxic preconditioning (HPC). In parallel, autophagy plays a prosurvival role in ischemic preconditioning (IPC) against ischemic stroke. However, the effect of cPKCγ on autophagy in IPC still remains to be addressed. In this study, adult and postnatal 1-day-old C57BL/6 J wild-type (cPKCγ+/+) and knockout (cPKCγ−/−) mice were used to establish in vivo and in vitro IPC models. The results showed that IPC pretreatment alleviated neuronal damage caused by lethal ischemia, which could be suppressed by autophagy inhibitor 3-MA or bafilomycin A1. Meanwhile, cPKCγ knockout blocked IPC-induced neuroprotection, accompanied by significant increase of LC3-I to LC3-II conversion and Beclin 1 protein level, and a significant decrease in p62 protein level. Immunofluorescent staining results showed a decrease of LC3 puncta numbers in IPC-treated cPKCγ+/+ neurons with fatal ischemia, which was reversed in cPKCγ−/− neurons. In addition, cPKCγ-modulated phosphorylation of mTOR at Ser 2448 and ULK1 at Ser 555, rather than p-Thr-172 AMPK, was detected in IPC-pretreated neurons upon lethal ischemic exposure. The present data demonstrated that cPKCγ-modulated autophagy via the mTOR-ULK1 pathway likely modulated IPC-induced neuroprotection. [ABSTRACT FROM AUTHOR]

The quality of CNC (computer numerical control) machine tool is the key to the survival of manufacturing enterprises. How to comprehensively and accurately evaluate its quality is very important for the timely discovery and improvement of machine tool defects. The current research either only evaluates a certain quality characteristic of CNC machine tool, or ignores the dynamic characteristics of CNC machine tool quality, which reduces the accuracy of quality analysis results. To address these problems, firstly, RMS (reliability, maintainability, supportability) was taken as the comprehensive evaluation indexes of CNC machine tool quality characteristics to improve the comprehensiveness of the analysis. Secondly, considering the dynamic characteristics of machine tool reliability, the ADC (availability, dependability, capability) model was improved to obtain a comprehensive quality evaluation index function ERMS with time-varying characteristics. Finally, the characteristics of ERMS were analyzed, and its evaluation criteria were also given. Several CNC machine tools made in China were tracked, and the collected fault data were analyzed by the proposed method. The ERMS of the machine tools was obtained, and its characteristics were analyzed. The results verify the correctness of the proposed method, which provides theoretical basis and technical support for the improvement of the quality of CNC machine tool and the market competitiveness of enterprises. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Introduction: Glioblastoma multiforme (GBM) is one of the most devastating brain malignancies worldwide and is considered to be incurable. However, the mechanisms underlying its aggressiveness remain unclear. Methods: The expression of ADAM17 in tissue samples was detected by immunohistochemistry. Knockdown and rescue experiments were used to demonstrate the regulatory effect of ADAM17 on the invasion ability of GBM cells. Western Blot and qPCR were used to detect the expression of related proteins and RNAs. Moreover, a luciferase reporter assay was performed to verify whether miR-145 directly binds to the 3′-UTR of ADAM17. Results: We revealed that ADAM17 was overexpressed in GBM tissues and correlated positively with poor prognosis. The knockdown of ADAM17 obviously suppressed the invasiveness of GBM cell lines. Furthermore, we found that knockdown of ADAM17 decreased activation of EGFR/Akt/C/EBP-β signaling, and consequently upregulated miR-145 expression in GBM cell lines. Notably, miR-145 directly targeted the ADAM17 3′-UTR and suppressed expression levels of ADAM17. Conclusions: Our findings define an ADAM17/EGFR/miR-145 feedback loop that drives the GBM invasion. Reciprocal regulation between ADAM17 and miR-145 results in aberrant activation of EGFR signaling, suggesting that inhibition of ADAM17 expression can be an ideal therapeutic strategy for the treatment of GBM. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Previous studies have demonstrated that the enhanced levels of phosphorylated α-amino-3-hydroxy-5-methy-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]