Dihydroquercetin alleviates dopamine neuron loss via regulating TREM2 activation.

BACKGROUND: Parkinson's disease (PD) is a prevalent neurodegenerative disorder, marked by the degeneration of dopamine (DA) neurons in the substantia nigra (SN). Current evidence strongly suggests that neuroinflammation, primarily mediated by microglia, contributes to PD pathogenesis. Triggering receptor expressed on myeloid cells 2 (TREM2) might serve as a promising therapeutic target for PD due to its ability to suppress neuroinflammation. Dihydroquercetin (DHQ) is an important natural dihydroflavone and confers apparent anti-inflammatory, antioxidant and anti-fibrotic effects. Recently, DHQ-mediated neuroprotection was exhibited. However, the specific mechanisms of its neuroprotective effects remain incompletely elucidated. METHODS: In this study, rat models were utilized to induce damage to DA neurons using lipopolysaccharide (LPS) and 6-hydroxydopamine (6-OHDA) to assess the impacts of DHQ on the loss of DA neurons. Furthermore, DA neuronal MN9D cells and microglial BV2 cells were employed to investigate the function of TREM2 in DHQ-mediated DA neuroprotection. Finally, TREM2 knockout mice were used to investigate whether the neuroprotective effects mediated by DHQ through a mechanism dependent on TREM2. RESULTS: The main findings demonstrated that DHQ effectively protected DA neurons against neurotoxicity induced by LPS and 6-OHDA and inhibited microglia-elicited neuroinflammation. Meanwhile, DHQ promoted microglial TREM2 signaling activation. Notably, DHQ failed to reduce inflammatory cytokines release and further present neuroprotection from DA neurotoxicity upon TREM2 silencing. Similarly, DHQ didn't exert DA neuroprotection in TREM2 knockout mice. CONCLUSIONS: These findings suggest that DHQ exerted DA neuroprotection by regulating microglia TREM2 activation.

Activation of neuronal NADPH oxidase NOX2 promotes inflammatory neurodegeneration.

Strong evidence indicates critical roles of NADPH oxidase (a key superoxide-producing enzyme complex during inflammation) in activated microglia for mediating neuroinflammation and neurodegeneration. However, little is known about roles of neuronal NADPH oxidase in neurodegenerative diseases. This study aimed to investigate expression patterns, regulatory mechanisms and pathological roles of neuronal NADPH oxidase in inflammation-associated neurodegeneration. The results showed persistent upregulation of NOX2 (gp91phox; the catalytic subunit of NADPH oxidase) in both microglia and neurons in a chronic mouse model of Parkinson's disease (PD) with intraperitoneal LPS injection and LPS-treated midbrain neuron-glia cultures (a cellular model of PD). Notably, NOX2 was found for the first time to exhibit a progressive and persistent upregulation in neurons during chronic neuroinflammation. While primary neurons and N27 neuronal cells displayed basal expression of NOX1, NOX2 and NOX4, significant upregulation only occurred in NOX2 but not NOX1 or NOX4 under inflammatory conditions. Persistent NOX2 upregulation was associated with functional outcomes of oxidative stress including increased ROS production and lipid peroxidation. Neuronal NOX2 activation displayed membrane translocation of cytosolic p47phox subunit and was inhibited by apocynin and diphenyleneiodonium chloride (two widely-used NADPH oxidase inhibitors). Importantly, neuronal ROS production, mitochondrial dysfunction and degeneration induced by inflammatory mediators in microglia-derived conditional medium were blocked by pharmacological inhibition of neuronal NOX2. Furthermore, specific deletion of neuronal NOX2 prevented LPS-elicited dopaminergic neurodegeneration in neuron-microglia co-cultures separately grown in the transwell system. The attenuation of inflammation-elicited upregulation of NOX2 in neuron-enriched and neuron-glia cultures by ROS scavenger N-acetylcysteine indicated a positive feedback mechanism between excessive ROS production and NOX2 upregulation. Collectively, our findings uncovered crucial contribution of neuronal NOX2 upregulation and activation to chronic neuroinflammation and inflammation-related neurodegeneration. This study reinforced the importance of developing NADPH oxidase-targeting therapeutics for neurodegenerative diseases.

Dysfunction of the noradrenergic system drives inflammation, α-synucleinopathy, and neuronal loss in mouse colon.

Clinical and pathological evidence revealed that α-synuclein (α-syn) pathology seen in PD patients starts in the gut and spreads via anatomically connected structures from the gut to the brain. Our previous study demonstrated that depletion of central norepinephrine (NE) disrupted brain immune homeostasis, producing a spatiotemporal order of neurodegeneration in the mouse brain. The purpose of this study was 1) to determine the role of peripheral noradrenergic system in the maintenance of gut immune homeostasis and in the pathogenesis of PD and 2) to investigate whether NE-depletion induced PD-like α-syn pathological changes starts from the gut. For these purposes, we investigated time-dependent changes of α-synucleinopathy and neuronal loss in the gut following a single injection of DSP-4 (a selective noradrenergic neurotoxin) to A53T-SNCA (human mutant α-syn) over-expression mice. We found DPS-4 significantly reduced the tissue level of NE and increased immune activities in gut, characterized by increased number of phagocytes and proinflammatory gene expression. Furthermore, a rapid-onset of α-syn pathology was observed in enteric neurons after 2 weeks and delayed dopaminergic neurodegeneration in the substantia nigra was detected after 3-5 months, associated with the appearance of constipation and impaired motor function, respectively. The increased α-syn pathology was only observed in large, but not in the small, intestine, which is similar to what was observed in PD patients. Mechanistic studies reveal that DSP-4-elicited upregulation of NADPH oxidase (NOX2) initially occurred only in immune cells during the acute intestinal inflammation stage, and then spread to enteric neurons and mucosal epithelial cells during the chronic inflammation stage. The upregulation of neuronal NOX2 correlated well with the extent of α-syn aggregation and subsequent enteric neuronal loss, suggesting that NOX2-generated reactive oxygen species play a key role in α-synucleinopathy. Moreover, inhibiting NOX2 by diphenyleneiodonium or restoring NE function by salmeterol (a ß2-receptor agonist) significantly attenuated colon inflammation, α-syn aggregation/propagation, and enteric neurodegeneration in the colon and ameliorated subsequent behavioral deficits. Taken together, our model of PD shows a progressive pattern of pathological changes from the gut to the brain and suggests a potential role of the noradrenergic dysfunction in the pathogenesis of PD.

Posttranslational S-nitrosylation modification regulates HMGB1 secretion and promotes its proinflammatory and neurodegenerative effects.

Nuclear protein high-mobility group box 1 (HMGB1) can be actively secreted by activated immune cells and functions as a proinflammatory cytokine. Regulation of HMGB1 secretion is critical for treatment of HMGB1-mediated inflammation and related diseases. This study demonstrates that S-nitrosylation (SNO; the covalent binding of nitric oxide [NO] to cysteine thiols) by inducible nitric oxide synthase (iNOS)-derived NO at Cys106 is essential and sufficient for inflammation-elicited HMGB1 secretion. iNOS deletion or inhibition or Cys106Ser mutation prevents lipopolysaccharide (LPS)- and/or poly(I:C)-elicited HMGB1 secretion. NO donors induce SNO of HMGB1 and reproduce inflammogen-triggered HMGB1 secretion. SNO of HMGB1 promotes its proinflammatory and neurodegenerative effects. Intranigral HMGB1 injection induces chronic microglial activation, dopaminergic neurodegeneration, and locomotor deficits, the key features of Parkinson's disease (PD), in wild-type, but not Mac1 (CD11b/CD18)-deficient, mice. This study indicates pivotal roles for SNO modification in HMGB1 secretion and HMGB1-Mac1 interaction for inflammatory neurodegeneration, identifying a mechanistic basis for PD development.

Macrophages Are Dispensable for Postnatal Pruning of the Cochlear Ribbon Synapses.

Ribbon synapses of cochlear hair cells undergo pruning and maturation before the hearing onset. In the central nervous system (CNS), synaptic pruning was mediated by microglia, the brain-resident macrophages, via activation of the complement system. Whether a similar mechanism regulates ribbon synapse pruning is currently unknown. In this study, we report that the densities of cochlear macrophages surrounding hair cells were highest at around P8, corresponding well to the completion of ribbon synaptic pruning by P8-P9. Surprisingly, using multiple genetic mouse models, we found that postnatal pruning of the ribbon synapses and auditory functions were unaffected by the knockout of the complement receptor 3 (CR3) or by ablations of macrophages expressing either LysM or Cx3cr1. Our results suggest that unlike microglia in the CNS, macrophages in the cochlea do not mediate pruning of the cochlear ribbon synapses.

[Optimization of determination of astragaloside â £ in Astragali Radix by continuous single-factor method].

This paper aims to solve the problems of complicated-unstable test solution preparation process and insufficient extraction of the active ingredient astragaloside Ⅳ in the legal method for the determination of astragaloside Ⅳ in Astragali Radix. The continuous single-factor analysis of seven main factors affecting the content of astragaloside Ⅳ was carried out by HPLC-ELSD, and then the pre-paration method of test solution was optimized. This optimized method exhibited excellent performance in precision, repeatability and stability. The average recovery rate of astragaloside Ⅳ was 99.65% with RSD 2.2%. Astragaloside Ⅳ showed a good linearity between the logarithm of peak area and the logarithm of injection quantity in the range of 0.46-9.1 µg(r=0.999 6). The contents of astragaloside Ⅳ in 29 batches of Astragali Radix were determined by the new and the legal methods. The results showed that the average content of astragaloside Ⅳ in these Astragali Radix samples determined by the former method was 1.458 times than that of the latter one, indicating the new method was simple, reliable and more adequate to extract target compound. According to the results, it is suggested to improve the content standard of astragaloside Ⅳ in Astragali Radix in the new edition of Chinese Pharmacopeia.

Double-stranded RNA-induced dopaminergic neuronal loss in the substantia nigra in the presence of Mac1 receptor.

BACKGROUND: The underlying mechanism of viral infection as a risk factor for Parkinson's disease (PD), the second most common neurodegenerative disease, remains unclear. OBJECTIVE: We used Mac-1-/- and gp91phox-/- transgene animal models to investigate the mechanisms by which poly I:C, a mimic of virus double-stranded RNA, induces PD neurodegeneration. METHOD: Poly I:C was stereotaxically injected into the substantia nigra (SN) of wild-type (WT), Mac-1-knockout (Mac-1-/-) and gp91 phox-knockout (gp91 phox-/-) mice (10 µg/µl), and nigral dopaminergic neurodegeneration, α-synuclein accumulation and neuroinflammation were evaluated. RESULT: Dopaminergic neurons in the nigra and striatum were markedly reduced in WT mice after administration of poly I:C together with abundant microglial activation in the SN, and the expression of α-synuclein was also elevated. However, these pathological changes were greatly dampened in Mac-1-/- and gp91 phox-/- mice. CONCLUSIONS: Our findings demonstrated that viral infection could result in the activation of microglia as well as NADPH oxidase, which may lead to neuron loss and the development of Parkinson's-like symptoms. Mac-1 is a key receptor during this process.

Benzo(a)pyrene exposure induced neuronal loss, plaque deposition, and cognitive decline in APP/PS1 mice.

BACKGROUND: Exposure to benzo(a)pyrene (BaP) was associated with cognitive impairments and some Alzheimer's disease (AD)-like pathological changes. However, it is largely unknown whether BaP exposure participates in the disease progression of AD. OBJECTIVES: To investigate the effect of BaP exposure on AD progression and its underlying mechanisms. METHODS: BaP or vehicle was administered to 4-month-old APPswe/PS1dE9 transgenic (APP/PS1) mice and wildtype (WT) mice for 2 months. Learning and memory ability and exploratory behaviors were evaluated 1 month after the initiation/termination of BaP exposure. AD-like pathological and biochemical alterations were examined 1 month after 2-month BaP exposure. Levels of soluble beta-amyloid (Aß) oligomers and the number of Aß plaques in the cortex and the hippocampus were quantified. Gene expression profiling was used to evaluate alternation of genes/pathways associated with AD onset and progression. Immunohistochemistry and Western blot were used to demonstrate neuronal loss and neuroinflammation in the cortex and the hippocampus. Treatment of primary neuron-glia cultures with aged Aß (a mixture of monomers, oligomers, and fibrils) and/or BaP was used to investigate mechanisms by which BaP enhanced Aß-induced neurodegeneration. RESULTS: BaP exposure induced progressive decline in spatial learning/memory and exploratory behaviors in APP/PS1 mice and WT mice, and APP/PS1 mice showed severer behavioral deficits than WT mice. Moreover, BaP exposure promoted neuronal loss, Aß burden and Aß plaque formation in APP/PS1 mice, but not in WT mice. Gene expression profiling showed most robust alteration in genes and pathways related to inflammation and immunoregulatory process, Aß secretion and degradation, and synaptic formation in WT and APP/PS1 mice after BaP exposure. Consistently, the cortex and the hippocampus of WT and APP/PS1 mice displayed activation of microglia and astroglia and upregulation of inducible nitric oxide synthase (iNOS), glial fibrillary acidic protein (GFAP), and NADPH oxidase (three widely used neuroinflammatory markers) after BaP exposure. Furthermore, BaP exposure aggravated neurodegeneration induced by aged Aß peptide in primary neuron-glia cultures through enhancing NADPH oxidase-derived oxidative stress. CONCLUSION: Our study showed that chronic exposure to environmental pollutant BaP induced, accelerated, and exacerbated the progression of AD, in which elevated neuroinflammation and NADPH oxidase-derived oxidative insults were key pathogenic events.

Through Reducing ROS Production, IL-10 Suppresses Caspase-1-Dependent IL-1ß Maturation, thereby Preventing Chronic Neuroinflammation and Neurodegeneration.

Chronic neuroinflammation contributes to the pathogenesis of Parkinson's disease (PD). However, cellular and molecular mechanisms by which chronic neuroinflammation is formed and maintained remain elusive. This study aimed to explore detailed mechanisms by which anti-inflammatory cytokine interleukin-10 (IL-10) prevented chronic neuroinflammation and neurodegeneration. At 24 h after an intranigral injection of lipopolysaccharide (LPS), levels of NLRP3, pro-caspase-1, pro-IL-1ß, active caspase-1, and mature IL-1ß in the midbrain were much higher in IL-10-/- mice than wildtype mice. Mechanistically, IL-10-/- microglia produced more intracellular reactive oxygen species (iROS) and showed more profound activation of NADPH oxidase (NOX2) than wildtype microglia. Meanwhile, suppression of NOX2-derived iROS production blocked LPS-elicited caspase-1 activation and IL-1ß maturation in IL-10-/- microglia in vitro and in vivo. One month after intranigral LPS injection, IL-10-/- mice revealed more profound microglial activation and dopaminergic neurodegeneration in the substantia nigra than wildtype mice. Importantly, such PD-like pathological changes were prevented by IL-1ß neutralization. Collectively, IL-10 inhibited LPS-elicited production of NOX2-derived iROS thereby suppressing synthesis of NLRP3, pro-caspase-1 and pro-IL-1ß and their activation and cleavage. By this mechanism, IL-10 prevented chronic neuroinflammation and neurodegeneration. This study suggested boosting anti-inflammatory effects of IL-10 and suppressing NLRP3 inflammasome activation could be beneficial for PD treatment.

The pentose phosphate pathway regulates chronic neuroinflammation and dopaminergic neurodegeneration.

BACKGROUND: Metabolic dysfunction and neuroinflammation are increasingly implicated in Parkinson's disease (PD). The pentose phosphate pathway (PPP, a metabolic pathway parallel to glycolysis) converts glucose-6-phosphate into pentoses and generates ribose-5-phosphate and NADPH thereby governing anabolic biosynthesis and redox homeostasis. Brains and immune cells display high activity of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the PPP. A postmortem study reveals dysregulation of G6PD enzyme in brains of PD patients. However, spatial and temporal changes in activity/expression of G6PD in PD remain undetermined. More importantly, it is unclear how dysfunction of G6PD and the PPP affects neuroinflammation and neurodegeneration in PD. METHODS: We examined expression/activity of G6PD and its association with microglial activation and dopaminergic neurodegeneration in multiple chronic PD models generated by an intranigral/intraperitoneal injection of LPS, daily subcutaneous injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 6 days, or transgenic expression of A53T α-synuclein. Primary microglia were transfected with G6PD siRNAs and treated with lipopolysaccharide (LPS) to examine effects of G6PD knockdown on microglial activation and death of co-cultured neurons. LPS alone or with G6PD inhibitor(s) was administrated to mouse substantia nigra or midbrain neuron-glia cultures. While histological and biochemical analyses were conducted to examine microglial activation and dopaminergic neurodegeneration in vitro and in vivo, rotarod behavior test was performed to evaluate locomotor impairment in mice. RESULTS: Expression and activity of G6PD were elevated in LPS-treated midbrain neuron-glia cultures (an in vitro PD model) and the substantia nigra of four in vivo PD models. Such elevation was positively associated with microglial activation and dopaminergic neurodegeneration. Furthermore, inhibition of G6PD by 6-aminonicotinamide and dehydroepiandrosterone and knockdown of microglial G6PD attenuated LPS-elicited chronic dopaminergic neurodegeneration. Mechanistically, microglia with elevated G6PD activity/expression produced excessive NADPH and provided abundant substrate to over-activated NADPH oxidase (NOX2) leading to production of excessive reactive oxygen species (ROS). Knockdown and inhibition of G6PD ameliorated LPS-triggered production of ROS and activation of NF-кB thereby dampening microglial activation. CONCLUSIONS: Our findings indicated that G6PD-mediated PPP dysfunction and neuroinflammation exacerbated each other mediating chronic dopaminergic neurodegeneration and locomotor impairment. Insight into metabolic-inflammatory interface suggests that G6PD and NOX2 are potential therapeutic targets for PD.

Ultrafine carbon particles promote rotenone-induced dopamine neuronal loss through activating microglial NADPH oxidase.

BACKGROUND: Atmospheric ultrafine particles (UFPs) and pesticide rotenone were considered as potential environmental risk factors for Parkinson's disease (PD). However, whether and how UFPs alone and in combination with rotenone affect the pathogenesis of PD remains largely unknown. METHODS: Ultrafine carbon black (ufCB, a surrogate of UFPs) and rotenone were used individually or in combination to determine their roles in chronic dopaminergic (DA) loss in neuron-glia, and neuron-enriched, mix-glia cultures. Immunochemistry using antibody against tyrosine hydroxylase was performed to detect DA neuronal loss. Measurement of extracellular superoxide and intracellular reactive oxygen species (ROS) were performed to examine activation of NADPH oxidase. Genetic deletion and pharmacological inhibition of NADPH oxidase and MAC-1 receptor in microglia were employed to examine their role in DA neuronal loss triggered by ufCB and rotenone. RESULTS: In rodent midbrain neuron-glia cultures, ufCB and rotenone alone caused neuronal death in a dose-dependent manner. In particularly, ufCB at doses of 50 and 100µg/cm2 induced significant loss of DA neurons. More importantly, nontoxic doses of ufCB (10µg/cm2) and rotenone (2nM) induced synergistic toxicity to DA neurons. Microglial activation was essential in this process. Furthermore, superoxide production from microglial NADPH oxidase was critical in ufCB/rotenone-induced neurotoxicity. Studies in mix-glia cultures showed that ufCB treatment activated microglial NADPH oxidase to induce superoxide production. Firstly, ufCB enhanced the expression of NADPH oxidase subunits (gp91phox, p47phox and p40phox); secondly, ufCB was recognized by microglial surface MAC-1 receptor and consequently promoted rotenone-induced p47phox and p67phox translocation assembling active NADPH oxidase. CONCLUSION: ufCB and rotenone worked in synergy to activate NADPH oxidase in microglia, leading to oxidative damage to DA neurons. Our findings delineated the potential role of ultrafine particles alone and in combination with pesticide rotenone in the pathogenesis of PD.

Neurons and astroglia govern microglial endotoxin tolerance through macrophage colony-stimulating factor receptor-mediated ERK1/2 signals.

Endotoxin tolerance (ET) is a reduced responsiveness of innate immune cells like macrophages/monocytes to an endotoxin challenge following a previous encounter with the endotoxin. Although ET in peripheral systems has been well studied, little is known about ET in the brain. The present study showed that brain immune cells, microglia, being different from peripheral macrophages, displayed non-cell autonomous mechanisms in ET formation. Specifically, neurons and astroglia were indispensable for microglial ET. Macrophage colony-stimulating factor (M-CSF) secreted from these non-immune cells was essential for governing microglial ET. Neutralization of M-CSF deprived the neuron-glia conditioned medium of its ability to enable microglia to form ET when microglia encountered two lipopolysaccharide (LPS) treatments. Recombinant M-CSF protein rendered enriched microglia refractory to the second LPS challenge leading to microglial ET. Activation of microglial M-CSF receptor (M-CSFR; also known as CSF1R) and the downstream ERK1/2 signals was responsible for M-CSF-mediated microglial ET. Endotoxin-tolerant microglia in neuron-glia cultures displayed M2-like polarized phenotypes, as shown by upregulation of M2 marker Arg-1, elevated production of anti-inflammatory cytokine interleukin 10, and decreased secretion of pro-inflammatory mediators (tumor necrosis factor α, nitric oxide, prostaglandin E2 and interleukin 1ß). Endotoxin-tolerant microglia protected neurons against LPS-elicited inflammatory insults, as shown by reduced neuronal damages in LPS pre-treatment group compared with the group without LPS pre-treatment. Moreover, while neurons and astroglia became injured during chronic neuroinflammation, microglia failed to form ET. Thus, this study identified a distinct non-cell autonomous mechanism of microglial ET. Interactions of M-CSF secreted by neurons and astroglia with microglial M-CSFR programed microglial ET. Loss of microglial ET could be an important pathogenetic mechanism of inflammation-associated neuronal damages.

The emerging nexus of active DNA demethylation and mitochondrial oxidative metabolism in post-mitotic neurons.

The variable patterns of DNA methylation in mammals have been linked to a number of physiological processes, including normal embryonic development and disease pathogenesis. Active removal of DNA methylation, which potentially regulates neuronal gene expression both globally and gene specifically, has been recently implicated in neuronal plasticity, learning and memory processes. Model pathways of active DNA demethylation involve ten-eleven translocation (TET) methylcytosine dioxygenases that are dependent on oxidative metabolites. In addition, reactive oxygen species (ROS) and oxidizing agents generate oxidative modifications of DNA bases that can be removed by base excision repair proteins. These potentially link the two processes of active DNA demethylation and mitochondrial oxidative metabolism in post-mitotic neurons. We review the current biochemical understanding of the DNA demethylation process and discuss its potential interaction with oxidative metabolism. We then summarise the emerging roles of both processes and their interaction in neural plasticity and memory formation and the pathophysiology of neurodegeneration. Finally, possible therapeutic approaches for neurodegenerative diseases are proposed, including reprogramming therapy by global DNA demethylation and mitohormesis therapy for locus-specific DNA demethylation in post-mitotic neurons.

The effectiveness of problem-based learning on development of nursing students' critical thinking: a systematic review and meta-analysis.

OBJECTIVES: The objective of this systematic review and meta-analysis was to estimate the effectiveness of problem-based learning in developing nursing students' critical thinking. DATA SOURCES: Searches of PubMed, EMBASE, Cumulative Index to Nursing and Allied Health Literature (CINAHL), Proquest, Cochrane Central Register of Controlled Trials (CENTRAL) and China National Knowledge Infrastructure (CNKI) were undertaken to identify randomized controlled trails from 1965 to December 2012, comparing problem-based learning with traditional lectures on the effectiveness of development of nursing students' critical thinking, with no language limitation. The mesh-terms or key words used in the search were problem-based learning, thinking, critical thinking, nursing, nursing education, nurse education, nurse students, nursing students and pupil nurse. REVIEW METHODS: Two reviewers independently assessed eligibility and extracted data. Quality assessment was conducted independently by two reviewers using the Cochrane Collaboration's Risk of Bias Tool. We analyzed critical thinking scores (continuous outcomes) using a standardized mean difference (SMD) or weighted mean difference (WMD) with a 95% confidence intervals (CIs). Heterogeneity was assessed using the Cochran's Q statistic and I(2) statistic. Publication bias was assessed by means of funnel plot and Egger's test of asymmetry. RESULTS: Nine articles representing eight randomized controlled trials were included in the meta-analysis. Most studies were of low risk of bias. The pooled effect size showed problem-based learning was able to improve nursing students' critical thinking (overall critical thinking scores SMD=0.33, 95%CI=0.13-0.52, P=0.0009), compared with traditional lectures. There was low heterogeneity (overall critical thinking scores I(2)=45%, P=0.07) in the meta-analysis. No significant publication bias was observed regarding overall critical thinking scores (P=0.536). Sensitivity analysis showed that the result of our meta-analysis was reliable. Most effect sizes for subscales of the California Critical Thinking Dispositions Inventory (CCTDI) and Bloom's Taxonomy favored problem-based learning, while effect sizes for all subscales of the California Critical Thinking Skills Test (CCTST) and most subscales of the Watson-Glaser Critical Thinking Appraisal (WCGTA) were inconclusive. CONCLUSIONS: The results of the current meta-analysis indicate that problem-based learning might help nursing students to improve their critical thinking. More research with larger sample size and high quality in different nursing educational contexts are required.

CD11b/CD18 (Mac-1) is a novel surface receptor for extracellular double-stranded RNA to mediate cellular inflammatory responses.

During viral infection, extracellular dsRNA is a potent signaling molecule that activates many innate immune cells, including macrophages. TLR3 is a well-known receptor for extracellular dsRNA, and internalization of extracellular dsRNA is required for endosomal TLR3 activation. Preserved inflammatory responses of TLR3-deficient macrophages to extracellular dsRNA strongly support a TLR3-independent mechanism in dsRNA-mediated immune responses. The present study demonstrated that CD11b/CD18 (Mac-1 [macrophage-1 Ag]), a surface integrin receptor, recognized extracellular dsRNA and induced macrophage immune responses. CD11b deficiency reduced inflammatory cytokine induction elicited by polyinosinic:polycytidylic acid (poly I:C; a synthetic dsRNA) in mouse sera and livers, as well as in cultured peritoneal macrophages. dsRNA-binding assay and confocal immunofluorescence showed that Mac-1, especially the CD11b subunit, interacted and colocalized with poly I:C on the surface of macrophages. Further mechanistic studies revealed two distinct signaling events following dsRNA recognition by Mac-1. First, Mac-1 facilitated poly I:C internalization through the activation of PI3K signaling and enhanced TLR3-dependent activation of IRF3 in macrophages. Second, poly I:C induced activation of phagocyte NADPH oxidase in a TLR3-independent, but Mac-1-dependent, manner. Subsequently, phagocyte NADPH oxidase-derived intracellular reactive oxygen species activated MAPK and NF-κB pathways. Our results indicate that extracellular dsRNA activates Mac-1 to enhance TLR3-dependent signaling and to trigger TLR3-independent, but Mac-1-dependent, inflammatory oxidative signaling, identifying a novel mechanistic basis for macrophages to recognize extracellular dsRNA to regulate innate immune responses. This study identifies Mac-1 as a novel surface receptor for extracellular dsRNA and implicates it as a potential therapeutic target for virus-related inflammatory diseases.

NADPH oxidases: novel therapeutic targets for neurodegenerative diseases.

Oxidative stress is a key pathologic factor in neurodegenerative diseases such as Alzheimer and Parkinson diseases (AD, PD). The failure of free-radical-scavenging antioxidants in clinical trials pinpoints an urgent need to identify and to block major sources of oxidative stress in neurodegenerative diseases. As a major superoxide-producing enzyme complex in activated phagocytes, phagocyte NADPH oxidase (PHOX) is essential for host defense. However, recent preclinical evidence has underscored a pivotal role of overactivated PHOX in chronic neuroinflammation and progressive neurodegeneration. Deficiency in PHOX subunits mitigates neuronal damage induced by diverse insults/stresses relevant to neurodegenerative diseases. More importantly, suppression of PHOX activity correlates with reduced neuronal impairment in models of neurodegenerative diseases. The discovery of PHOX and non-phagocyte NADPH oxidases in astroglia and neurons further reinforces the crucial role of NADPH oxidases in oxidative stress-mediated chronic neurodegeneration. Thus, proper modulation of NADPH oxidase activity might hold therapeutic potential for currently incurable neurodegenerative diseases.

Clozapine protects dopaminergic neurons from inflammation-induced damage by inhibiting microglial overactivation.

Increasing evidence suggests a possible involvement of neuroinflammation in some psychiatric disorders, and also pharmacological reports indicate that anti-inflammatory effects are associated with therapeutic actions of psychoactive drugs, such as anti-depressants and antipsychotics. The purpose of this study was to explore whether clozapine, a widely used antipsychotic drugs, displays anti-inflammatory and neuroprotective effects. Using primary cortical and mesencephalic neuron-glia cultures, we found that clozapine was protective against inflammation-related neurodegeneration induced by lipopolysaccharide (LPS). Pretreatment of cortical or mesencephalic neuron-glia cultures with clozapine (0.1 or 1 µM) for 24 h attenuated LPS-induced neurotoxicity. Clozapine also protected neurons against 1-methyl-4-phenylpyridinium(+) (MPP(+))-induced neurotoxicity, but only in cultures containing microglia, indicating an indispensable role of microglia in clozapine-afforded neuroprotection. Further observation revealed attenuated LPS-induced microglial activation in primary neuron-glia cultures and in HAPI microglial cell line with clozapine pretreatment. Clozapine ameliorated the production of microglia-derived superoxide and intracellular reactive oxygen species (ROS), as well as the production of nitric oxide and TNF-α following LPS. In addition, the protective effect of clozapine was not observed in neuron-glia cultures from mice lacking functional NADPH oxidase (PHOX), a key enzyme for superoxide production in immune cells. Further mechanistic studies demonstrated that clozapine pretreatment inhibited LPS-induced translocation of cytosolic subunit p47(phox) to the membrane in microglia, which was most likely through inhibiting the phosphoinositide 3-kinase (PI3K) pathway. Taken together, this study demonstrates that clozapine exerts neuroprotective effect via the attenuation of microglia activation through inhibition of PHOX-generated ROS production and suggests potential use of antipsychotic drugs for neuroprotection.

Rotenone activates phagocyte NADPH oxidase by binding to its membrane subunit gp91phox.

Rotenone, a widely used pesticide, reproduces parkinsonism in rodents and associates with increased risk for Parkinson disease. We previously reported that rotenone increased superoxide production by stimulating the microglial phagocyte NADPH oxidase (PHOX). This study identified a novel mechanism by which rotenone activates PHOX. Ligand-binding assay revealed that rotenone directly bound to membrane gp91(phox), the catalytic subunit of PHOX; such binding was inhibited by diphenyleneiodonium, a PHOX inhibitor with a binding site on gp91(phox). Functional studies showed that both membrane and cytosolic subunits were required for rotenone-induced superoxide production in cell-free systems, intact phagocytes, and COS7 cells transfected with membrane subunits (gp91(phox)/p22(phox)) and cytosolic subunits (p67(phox) and p47(phox)). Rotenone-elicited extracellular superoxide release in p47(phox)-deficient macrophages suggested that rotenone enabled activation of PHOX through a p47(phox)-independent mechanism. Increased membrane translocation of p67(phox), elevated binding of p67(phox) to rotenone-treated membrane fractions, and coimmunoprecipitation of p67(phox) and gp91(phox) in rotenone-treated wild-type and p47(phox)-deficient macrophages indicated that p67(phox) played a critical role in rotenone-induced PHOX activation via its direct interaction with gp91(phox). Rac1, a Rho-like small GTPase, enhanced p67(phox)-gp91(phox) interaction; Rac1 inhibition decreased rotenone-elicited superoxide release. In conclusion, rotenone directly interacted with gp91(phox); such an interaction triggered membrane translocation of p67(phox), leading to PHOX activation and superoxide production.

Fluoxetine protects neurons against microglial activation-mediated neurotoxicity.

Neuroinflammation is closely associated with the pathogenesis of Parkinson's disease (PD) and other neurological disorders. Increasing evidence suggests that inhibition of microglia-mediated neuroinflammation might represent a promising therapeutic potential for PD and related disorders. Fluoxetine, a selective serotonin reuptake inhibitor, is commonly used for the treatment of major depression due to its tolerability and safety profiles. Recent studies have shown that fluoxetine affords robust neuroprotection in a series of neurological disease models. However, the mechanism underlying fluoxetine-mediated neuroprotection remains unclear. Here, by using rat primary midbrain neuronglia cultures, we report that both R and S enantiomers of fluoxetine attenuated chronic neurodegeneration induced by a common inflammogen lipopolysaccharide (LPS) and a neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)). Reconstituted cell culture studies further revealed that microglia were required for fluoxetine-mediated neuroprotection. Fluoxetine significantly inhibited LPS-induced activation of microglia and subsequent release of multiple pro-inflammatory and cytotoxic factors including tumor necrosis factor-α, interleukin-1ß, nitric oxide, and reactive oxygen species. Furthermore, inhibition of microglial NF-κB signaling pathway participated in fluoxetine-mediated neuroprotection. Collectively, fluoxetine exerted neuroprotection against microglia-mediated neurotoxicity. Thus, fluoxetine might hold a potential to retard inflammation-mediated chronic neurodegenerative process of PD.

Transcriptional Factor NF-κB as a Target for Therapy in Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative condition characterized by chronic inflammation. Nuclear factor κB (NF-κB) is a family of inducible transcription factors that are expressed in a wide variety of cells and tissues, including microglia, astrocytes, and neurons, and the classical NF-κB pathway plays a key role in the activation and regulation of inflammatory mediator production during inflammation. Activation of the classical NF-κB pathway is mediated through the activity of the IKK kinase complex, which consists of a heterotrimer of IKKα, IKKß, and IKKγ subunits. Targeting NF-κB has been proposed as an approach to the treatment of acute and chronic inflammatory conditions, and the use of inhibitors specific for either IKKß or IKKγ has now been found to inhibit neurodegeneration of TH+ DA-producing neurons in murine and primate models of Parkinson's disease. These studies suggest that targeting the classical pathway of NF-κB through the inhibition of the IKK complex can serve as a useful therapeutic approach to the treatment of PD.