LPS priming-induced immune tolerance mitigates LPS-stimulated microglial activation and social avoidance behaviors in mice.

In this study, we investigated the regulatory mechanisms underlying the effects of LPS tolerance on the inflammatory homeostasis of immune cells. LPS priming-induced immune tolerance downregulated cyclooxygenase-2, and lowered the production of prostaglandin-E2 in microglial cells. In addition, LPS tolerance downregulated the expression of suppressor of cytokine signaling 3, and inducible nitric oxide synthase/nitric oxide; suppressed the LPS-mediated induction of tumor necrosis factor-α, interleukin (IL)-6, and IL-1; and reduced reactive oxygen species production in microglial cells. LPS stimulation increased the levels of the adaptive response-related proteins heme oxygenase-1 and superoxide dismutase 2, and the levels of heme oxygenase-1 (HO-1) enhanced after LPS priming. Systemic administration of low-dose LPS (0.5 mg/kg) to mice for 4 consecutive days attenuated high-dose LPS (5 mg/kg)-induced inflammatory response, microglial activation, and proinflammatory cytokine expression. Moreover, repeated exposure to low-dose LPS suppressed the recruitment of peripheral monocytes or macrophages to brain regions and downregulated the expression of proinflammatory cytokines. Notably, LPS-induced social avoidance behaviors in mice were mitigated by immune tolerance. In conclusion, immune tolerance may reduce proinflammatory cytokine expression and reactive oxygen species production. Our findings provide insights into the effects of endotoxin tolerance on innate immune cells and social behaviors.

Inhibitory Effects of Urolithins, Bioactive Gut Metabolites from Natural Polyphenols, against Glioblastoma Progression.

We previously reported that proinflammatory cytokines, particularly tumor necrosis factor (TNF)-α, promoted tumor migration, invasion, and proliferation, thus worsening the prognosis of glioblastoma (GBM). Urolithins, the potent metabolites produced by the gut from pomegranate polyphenols, have anticancer properties. To develop an effective therapy for GBM, this study aimed to study the effects of urolithins against GBM. Urolithin A and B significantly reduced GBM migration, reduced epithelial-mesenchymal transition, and inhibited tumor growth. Moreover, urolithin A and B inhibited TNF-α-induced vascular cell adhesion molecule (VCAM)-1 and programmed death ligand 1 (PD-L1) expression, thereby reducing human monocyte (HM) binding to GBM cells. Aryl hydrocarbon receptor (AhR) level had higher expression in patients with glioma than in healthy individuals. Urolithins are considered pharmacological antagonists of AhR. We demonstrated that the inhibition of AhR reduced TNF-α-stimulated VCAM-1 and PD-L1 expression. Furthermore, human macrophage condition medium enhanced expression of PD-L1 in human GBM cells. Administration of the AhR antagonist attenuated the enhancement of PD-L1, indicating the AhR modulation in GBM progression. The modulatory effects of urolithins in GBM involve inhibiting the Akt and epidermal growth factor receptor pathways. The present study suggests that urolithins can inhibit GBM progression and provide valuable information for anti-GBM strategy.

Bradykinin B1 Receptor Affects Tumor-Associated Macrophage Activity and Glioblastoma Progression.

Bradykinin is a small active peptide and is considered an inflammatory mediator in several pathological conditions. Bradykinin exerts its effects by coupling to its receptors, including bradykinin B1 (B1R) and bradykinin B2. B1R has been implicated in the development of various cancers. Our previous study reported that B1R promoted glioblastoma (GBM) development by supporting the migration and invasion of GBM cells. However, the mechanisms underlying the effects of B1R on tumor-associated macrophages (TAMs) and GBM progression remain unknown. Accordingly, to explore the regulatory effects of B1R overexpression (OE) in GBM on tumor-associated immune cells and tumor progression, we constructed a B1R wild-type plasmid and developed a B1R OE model. The results reveal that B1R OE in GBM promoted the expression of ICAM-1 and VCAM-1-cell adhesion molecules-in GBM. Moreover, B1R OE enhanced GBM cell migration ability and monocyte attachment. B1R also regulated the production of the protumorigenic cytokines and chemokines IL-6, IL-8, CXCL11, and CCL5 in GBM, which contributed to tumor progression. We additionally noted that B1R OE in GBM increased the expression of CD68 in TAMs. Furthermore, B1R OE reduced the level of reactive oxygen species in GBM cells by upregulating heme oxygenase-1, an endogenous antioxidant protein, thereby protecting GBM cells from oxidative stress. Notably, B1R OE upregulated the expression of programmed death-ligand 1 in both GBM cells and macrophages, thus providing resistance against T-cell response. B1R OE in GBM also promoted tumor growth and reduced survival rates in an intracranial xenograft mouse model. These results indicate that B1R expression in GBM promotes TAM activity and modulates GBM progression. Therefore, B1R could be an effective target for therapeutic methods in GBM.

Role of Zerumbone, a Phytochemical Sesquiterpenoid from Zingiber zerumbet Smith, in Maintaining Macrophage Polarization and Redox Homeostasis.

Macrophages and microglia are highly versatile cells that can be polarized into M1 and M2 phenotypes in response to diverse environmental stimuli, thus exhibiting different biological functions. In the central nervous system, activated resident macrophages and microglial cells trigger the production of proinflammatory mediators that contribute to neurodegenerative diseases and psychiatric disorders. Therefore, modulating the activation of macrophages and microglia by optimizing the inflammatory environment is beneficial for disease management. Several naturally occurring compounds have been reported to have anti-inflammatory and neuroprotective properties. Zerumbone is a phytochemical sesquiterpenoid and also a cyclic ketone isolated from Zingiber zerumbet Smith. In this study, we found that zerumbone effectively reduced the expression of lipocalin-2 in macrophages and microglial cell lines. Lipocalin-2, also known as neutrophil gelatinase-associated lipocalin (NGAL), has been characterized as an adipokine/cytokine implicated in inflammation. Moreover, supplement with zerumbone inhibited reactive oxygen species production. Phagocytic activity was decreased following the zerumbone supplement. In addition, the zerumbone supplement remarkably reduced the production of M1-polarization-associated chemokines CXC10 and CCL-2, as well as M1-polarization-associated cytokines interleukin (IL)-6, IL-1ß, and tumor necrosis factor-α. Furthermore, the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 and the production of NO were attenuated in macrophages and microglial cells supplemented with zerumbone. Notably, we discovered that zerumbone effectively promoted the production of the endogenous antioxidants heme oxygenase-1, glutamate-cysteine ligase modifier subunit, glutamate-cysteine ligase catalytic subunit, and NAD(P)H quinone oxidoreductase-1 and remarkably enhanced IL-10, a marker of M2 macrophage polarization. Endogenous antioxidant production and M2 macrophage polarization were increased through activation of the AMPK/Akt and Akt/GSK3 signaling pathways. In summary, this study demonstrated the protective role of zerumbone in maintaining M1 and M2 polarization homeostasis by decreasing inflammatory responses and enhancing the production of endogenous antioxidants in both macrophages and microglia cells. This study suggests that zerumbone can be used as a potential therapeutic drug for the supplement of neuroinflammatory diseases.

Study of Baicalin toward COVID-19 Treatment: In silico Target Analysis and in vitro Inhibitory Effects on SARS-CoV-2 Proteases.

Negative impacts of COVID-19 on human health and economic and social activities urge scientists to develop effective treatments. Baicalin is a natural flavonoid, extracted from a traditional medicinal plant, previously reported with anti-inflammatory activity. In this study, we used pharmacophore fitting and molecular docking to screen and determine docking patterns and the binding affinity of baicalin on 3 major targets of SARS-CoV-2 (3-chymotrypsin-like cysteine protease [3CLpro], papain-like protease [PLpro], and RNA-dependent RNA polymerase). The obtained data revealed that baicalin has high pharmacophore fitting on 3CLpro and predicted good binding affinity on PLpro. Moreover, using the enzymatic assay, we examined the inhibitory effect of baicalin in vitro on the screened enzymes. Baicalin also exhibits inhibitory effect on these proteases in vitro. Additionally, we performed pharmacophore-based screening of baicalin on human targets and conducted pathway analysis to explore the potential cytoprotective effects of baicalin in the host cell that may be beneficial for COVID-19 treatment. The result suggested that baicalin has multiple targets in human cell that may induce multiple pharmacological effects. The result of pathway analysis implied that these targets may be associated with baicalin-induced bioactivities that are involved with signals of pro-inflammation factors, such as cytokine and chemokine. Taken together with supportive data from the literature, the bioactivities of bailalin may be beneficial for COVID-19 treatment by reducing cytokine-induced acute inflammation. In conclusion, baicalin is potentially a good candidate for developing new therapeutic to treat COVID-19.

Paliperidone Inhibits Glioblastoma Growth in Mouse Brain Tumor Model and Reduces PD-L1 Expression.

A previous study from our group reported that monocyte adhesion to glioblastoma (GBM) promoted tumor growth and invasion activity and increased tumor-associated macrophages (TAMs) proliferation and inflammatory mediator secretion as well. The present study showed that prescribed psychotropic medicine paliperidone reduced GBM growth and immune checkpoint protein programmed death ligand (PD-L)1 expression and increased survival in an intracranial xenograft mouse model. An analysis of the database of patients with glioma showed that the levels of PD-L1 and dopamine receptor D (DRD)2 were higher in the GBM group than in the low grade astrocytoma and non-tumor groups. In addition, GFP expressing GBM (GBM-GFP) cells co-cultured with monocytes-differentiated macrophage enhanced PD-L1 expression in GBM cells. The enhancement of PD-L1 in GBM was antagonized by paliperidone and risperidone as well as DRD2 selective inhibitor L741426. The expression of CD206 (M2 phenotype marker) was observed to be markedly increased in bone marrow-derived macrophages (BMDMs) co-cultured with GBM. Importantly, treatment with paliperidone effectively decreased CD206 and also dramatically increased CD80 (M1 phenotype marker) in BMDMs. We have previously established a PD-L1 GBM-GFP cell line that stably expresses PD-L1. Experiments showed that the expressions of CD206 was increased and CD80 was mildly decreased in the BMDMs co-cultured with PD-L1 GBM-GFP cells. On the other hands, knockdown of DRD2 expression in GBM cells dramatically decreased the expression of CD206 but markedly increased CD80 expressions in BMDMs. The present study suggests that DRD2 may be involved in regulating the PD-L1 expression in GBM and the microenvironment of GBM. Our results provide a valuable therapeutic strategy and indicate that treatments combining DRD2 antagonist paliperidone with standard immunotherapy may be beneficial for GBM treatment.

Fenofibrate inhibits hypoxia-inducible factor-1 alpha and carbonic anhydrase expression through activation of AMP-activated protein kinase/HO-1/Sirt1 pathway in glioblastoma cells.

Cancer and its associated conditions have significant impacts on public health at many levels worldwide, and cancer is the leading cause of death among adults. Peroxisome proliferator-activated receptor α (PPARα)-specific agonists, fibrates, have been approved by the Food and Drug Administration for managing hyperlipidemia. PPARα-specific agonists exert anti-cancer effects in many human cancer types, including glioblastoma (GBM). Recently, we have reported that the hypoxic state in GBM stabilizes hypoxia-inducible factor-1 alpha (HIF-1α), thus contributing to tumor escape from immune surveillance by activating the expression of the pH-regulating protein carbonic anhydrase IX (CA9). In this study, we aimed to study the regulatory effects of the PPARα agonist fibrate on the regulation of HIF-1α expression and its downstream target protein in GBM. Our findings showed that fenofibrate is the high potency compound among the various fibrates that inhibit hypoxia-induced HIF-1α and CA9 expression in GBM. Moreover, fenofibrate-inhibited HIF-1α expression is mediated by HO-1 activation in GBM cells through the AMP-activated protein kinase (AMPK) pathway. In addition, fenofibrate-enhanced HO-1 upregulation activates SIRT1 and leads to subsequent accumulation of SIRT1 in the nucleus, which further promotes HIF-1α deacetylation and inhibits CA9 expression. Using a protein synthesis inhibitor, cycloheximide, we also observed that fenofibrate inhibited HIF-1α protein synthesis. In addition, the administration of the proteasome inhibitor MG132 showed that fenofibrate promoted HIF-1α protein degradation in GBM. Hence, our results indicate that fenofibrate is a useful anti-GBM agent that modulates hypoxia-induced HIF-1α expression through multiple cellular pathways.

Significant Association of Chitinase 3-like 1 Genotypes to Asthma Risk in Taiwan.

BACKGROUND/AIM: Chitinase 3-like 1 (CHI3L1) is overexpressed in asthma, and negatively associated with forced expiratory volume in the first second. This study aimed at evaluating whether CHI3L1 genotypes affect asthma risk. MATERIALS AND METHODS: The blood samples of 198 asthma patients and 453 control subjects were collected, and the genotypic patterns of CHI3L1 -131C/G (rs4950928) and -247G/A (rs1262491437) were examined. RESULTS: The percentages of CG and GG at CHI3L1 -131C/G were 32.8% and 7.6% among the asthma cases, respectively, significantly higher than the 23.8% and 3.1% among the non-asthmatic healthy subjects (p for trend=0.0009). The allelic frequency distribution analysis showed that the G allele at CHI3L1 - 131C/G conferred a significantly higher asthma risk than the wild-type C allele (p<0.0001). The genotypic and allelic frequency analyses for CHI3L1 -247G/A did not show any significant difference. CONCLUSION: The G allele at CHI3L1-131C/G serves as a biomarker in determining personal susceptibility to asthma in Taiwan.

SIRT1 activation by minocycline on regulation of microglial polarization homeostasis.

Sirtuin 1 (SIRT1) has been reported to be involved in the mechanisms underlying longevity and has also been indicated as a valuable regulator of age-related neurological disorders. Some natural products increase SIRT1 activity and stimulate deacetylation of various proteins. In the present study, SIRT1 overexpression by genetic modification or treatment with SIRT1 activators significantly inhibited the secretion of nitric oxide and expression of inducible nitric oxide synthase, cyclooxygenase 2, and proinflammatory mediator-interleukin 1ß-in microglia. SIRT1 activation also decreased the levels of K379 acetyl-p53 and the protein inhibitor of activated Stat 1 expression in microglial cells. In addition, it dramatically promoted M2 polarization of microglia, which enhanced cell motility and altered phagocytic ability. We also used minocycline, a well-known inhibitor of microglial activation, to study the mechanism of SIRT1 signaling. Minocycline treatment decreased neuroinflammatory responses and promoted M2 polarization of microglia. It also reduced the acetyl-p53 level in the brain tissues in an inflammatory mouse model. Our findings demonstrated that SIRT1 participates in the maintenance of microglial polarization homeostasis and that minocycline exerts regulatory effects on SIRT1 activation. Therefore, our results indicate that SIRT1 activation may be a useful therapeutic target for the treatment of neuroinflammation-associated disorders.

SUMO E3 ligase PIAS1 is a potential biomarker indicating stress susceptibility.

Prior studies suggest that individual differences in stress responses contribute to the pathogenesis of neuropsychiatric disorders. In the present study, we investigated the role of small ubiquitin-like modifier (SUMO) E3 ligase protein inhibitor of activated STAT1 (PIAS1) in mediating stress responses to chronic social defeat stress (CSDS). We found that mRNA and protein levels of PIAS 1 were decreased in the hippocampus of high-susceptibility (HS) mice but not in low-susceptibility (LS) mice after CSDS. Local overexpression of PIAS1 in the hippocampus followed by CSDS exposure promoted stress resilience by attenuating social avoidance and improving anxiety-like behaviors. Viral-mediated gene transfer to generate a conditional knockdown of PIAS1 in the hippocampus promoted social avoidance and stress vulnerability after subthreshold microdefeat. HS mice displayed decreased levels of glucocorticoid receptor (GR) expression, and GR SUMOylation in the hippocampus was associated with stress vulnerability. Furthermore, cytokine/chemokine levels were changed predominantly in the hippocampus of HS mice. These results suggest that hippocampal PIAS1 plays a role in the regulation of stress susceptibility by post-translational modification of GRs.

Peroxisome proliferator-activated receptor alpha accelerates neuronal differentiation and this might involve the mitogen-activated protein kinase pathway.

Activation of peroxisome proliferator-activated receptor alpha (PPARα) has been reported to modulate cell proliferation, migration, and differentiation in astrocytes. In this study, we used a retinoic acid (RA)-induced differentiation model of NTERA-2/clone D1 (NT2) cells to explore the functional significance of PPARα in neuronal differentiation. We found that activating PPARα by Wy14643 accelerated neuronal differentiation via regulating the expression of neuronal markers. RT-PCR assays showed a significant increase in NeuroD expression and a decrease in nestin expression in cells treated concomitantly with RA and Wy14643 for 2 days compared to the levels in cells treated with RA alone. Expression of MAP2 protein, a mature neuronal marker, was markedly upregulated at day 10 of Wy14643 treatment, which was maintained after 21 days of neuronal formation. Corresponding to the changes in MAP2 expression, the expression of Cdk5 was upregulated with Wy14643 exposure from day 10 to day 21. Moreover, cells treated with Wy14643 displayed higher expression levels of phospho-ERK and phospho-p38 in the differentiation process than cell treated with RA alone. These results indicated that activation of PPARα accelerated neuronal differentiation through upregulating the expression of NeuroD, MAP2, and Cdk5 and downregulating the expression of nestin. MAPK signals, ERK and p38, might contribute to the accelerated differentiation process. These findings suggest that PPARα plays a role in regulating neuronal differentiation and may be beneficial for functional recovery from neurological disorders.

Regulatory Effects of Neuroinflammatory Responses Through Brain-Derived Neurotrophic Factor Signaling in Microglial Cells.

Inhibition of microglial over-activation is an important strategy to counter balance neurodegenerative progression. We previously demonstrated that the adenosine monophosphate-activated protein kinase (AMPK) may be a therapeutic target in mediating anti-neuroinflammatory responses in microglia. Brain-derived neurotrophic factor (BDNF) is one of the major neurotrophic factors produced by astrocytes to maintain the development and survival of neurons in the brain, and have recently been shown to modulate homeostasis of neuroinflammation. Therefore, the present study focused on BDNF-mediated neuroinflammatory responses and may provide an endogenous regulation of neuroinflammation. Among the tested neuroinflammation, epigallocatechin gallate (EGCG) and minocycline exerted BDNF upregulation to inhibit COX-2 and proinflammatory mediator expressions. Furthermore, both EGCG and minocycline upregulated BDNF expression in microglia through AMPK signaling. In addition, minocycline and EGCG also increased expressions of erythropoietin (EPO) and sonic hedgehog (Shh). In the endogenous modulation of neuroinflammation, astrocyte-conditioned medium (AgCM) also decreased the expression of COX-2 and upregulated BDNF expression in microglia. The anti-inflammatory effects of BDNF were mediated through EPO/Shh in microglia. Our results indicated that the BDNF-EPO-Shh novel-signaling pathway underlies the regulation of inflammatory responses and may be regarded as a potential therapeutic target in neurodegenerative diseases. This study also reveals a better understanding of an endogenous crosstalk between astrocytes and microglia to regulate anti-inflammatory actions, which could provide a novel strategy for the treatment of neuroinflammation and neurodegenerative diseases.

Differential Characterization of Temozolomide-Resistant Human Glioma Cells.

Glioblastoma multiforme (GBM) is the most common type of primary and malignant tumor occurring in the adult central nervous system. Temozolomide (TMZ) has been considered to be one of the most effective chemotherapeutic agents to prolong the survival of patients with glioblastoma. Many glioma cells develop drug-resistance against TMZ that is mediated by increasing O-6-methylguanine-DNA methyltransferase (MGMT) levels. The expression of connexin 43 was increased in the resistant U251 subline compared with the parental U251 cells. The expression of epithelial-mesenchymal transition (EMT)-associated regulators, including vimentin, N-cadherin, and ß-catenin, was reduced in the resistant U251 subline. In addition, the resistant U251 subline exhibited decreased cell migratory activity and monocyte adhesion ability compared to the parental U251 cells. Furthermore, the resistant U251 subline also expressed lower levels of vascular cell adhesion molecule (VCAM)-1 after treatment with recombinant tumor necrosis factor (TNF)-α. These findings suggest differential characteristics in the drug-resistant GBM from the parental glioma cells.

2-Phenyl-4-quinolone (YT-1) induces G2/M phase arrest and an intrinsic apoptotic mechanism in human leukemia cells.

The present study aimed to investigate the biological effects of the new compound 2­phenyl­4­quinolone (YT­1) on human leukemia cells. Cell viability was determined by propidium iodide (PI) exclusion method followed by flow cytometry. Our results showed that YT­1 inhibited the cell viability and resulted in morphologic changes to the U937, HL­60 and K562 cells, respectively. Among them, U937 cells were the most sensitive cell line. On the contrary, YT­1 had no cytotoxic effects on human fetal skin fibroblast WS1 cells. Flow cytometric analysis indicated that YT­1 induced G2/M phase arrest and apoptosis (sub­G1 population) in U937 cells. The presence of apoptotic bodies evidenced by DAPI staining and DNA fragmentation detected by agarose gel electrophoresis further supported the induction of apoptosis in the YT­1­treated U937 cells. Annexin V/PI staining of U937 cells confirmed that the early apoptotic event occurred after YT­1 exposure. YT­1 disrupted the mitochondrial membrane potential (ΔΨm) in a time­dependent manner. YT­1 increased the protein levels of Bax and Bak but decreased Bcl­2 and Bid protein levels in U937 cells in a time­dependent manner. In addition, YT­1 stimulated the expression of cytochrome c and proteolytic activation of caspase­3 and caspase­9 after exposure to YT­1 in U937 cells. In summary, YT­1 suppressed the viability of U937 leukemia cells through the intrinsic apoptosis pathway. YT­1 is a potential chemotherapeutic candidate for the treatment of leukemia.

YC-1 induces G0/G1 phase arrest and mitochondria-dependent apoptosis in cisplatin-resistant human oral cancer CAR cells.

Oral cancer is a serious and fatal disease. Cisplatin is the first line of chemotherapeutic agent for oral cancer therapy. However, the development of drug resistance and severe side effects cause tremendous problems clinically. In this study, we investigated the pharmacologic mechanisms of YC-1 on cisplatin-resistant human oral cancer cell line, CAR. Our results indicated that YC-1 induced a concentration-dependent and time-dependent decrease in viability of CAR cells analyzed by MTT assay. Real-time image analysis of CAR cells by IncuCyte™ Kinetic Live Cell Imaging System demonstrated that YC-1 inhibited cell proliferation and reduced cell confluence in a time-dependent manner. Results from flow cytometric analysis revealed that YC-1 promoted G0/G1 phase arrest and provoked apoptosis in CAR cells. The effects of cell cycle arrest by YC-1 were further supported by up-regulation of p21 and down-regulation of cyclin A, D, E and CDK2 protein levels. TUNEL staining showed that YC-1 caused DNA fragmentation, a late stage feature of apoptosis. In addition, YC-1 increased the activities of caspase-9 and caspase-3, disrupted the mitochondrial membrane potential (AYm) and stimulated ROS production in CAR cells. The protein levels of cytochrome c, Bax and Bak were elevated while Bcl-2 protein expression was attenuated in YC-1-treated CAR cells. In summary, YC-1 suppressed the viability of cisplatin-resistant CAR cells through inhibiting cell proliferation, arresting cell cycle at G0/G1 phase and triggering mitochondria-mediated apoptosis. Our results provide evidences to support the potentially therapeutic application of YC-1 on fighting against drug resistant oral cancer in the future.

Cholinergic and glutamatergic transmission at synapses between pedunculopotine tegmental nucleus axonal terminals and A7 catecholamine cell group noradrenergic neurons in the rat.

We characterized transmission from the pedunculopotine tegmental nucleus (PPTg), which contains cholinergic and glutamatergic neurons, at synapses with noradrenergic (NAergic) A7 neurons. Injection of an anterograde neuronal tracer, biotinylated-dextran amine, into the PPTg resulted in labeling of axonal terminals making synaptic connection with NAergic A7 neurons. Consistent with this, extracellular stimulation using a train of 10 pulses at 100 Hz evoked both fast and slow excitatory synaptic currents (EPSCs) that were blocked, respectively, by DNQX, a non-N-methyl-d-aspartate receptor blocker, or atropine, a cholinergic muscarinic receptor (mAChR) blocker. Interestingly, many spontaneous-like, but stimulation-dependent, EPSCs, were seen for up to one second after the end of stimulation and were blocked by DNQX and decreased by EGTA-AM, a membrane permeable form of EGTA, showing they are glutamatergic EPSCs causing by asynchronous release of vesicular quanta. Moreover, application of atropine or carbachol, an mAChR agonist, caused, respectively, an increase in the number of asynchronous EPSCs or a decrease in the frequency of miniature EPSCs, showing that mAChRs mediated presynaptic inhibition of glutamatergic transmission of the PPTg onto NAergic A7 neurons. In conclusion, our data show direct synaptic transmission of PPTg afferents onto pontine NAergic neurons that involves cooperation of cholinergic and glutamatergic transmission. This dual-transmitter transmission drives the firing rate of NAergic neurons, which may correlate with axonal and somatic/dendritic release of NA.

CCY-1a-E2 induces G2/M phase arrest and apoptotic cell death in HL-60 leukemia cells through cyclin-dependent kinase 1 signaling and the mitochondria-dependent caspase pathway.

Our previous study demonstrated that 2-[(3-methoxybenzyl)oxy]benzaldehyde (CCY-1a-E2) is a potent compound that acts against multiple human leukemia cell lines. CCY-1a-E2 was also shown to have efficacious anti­leukemic activity in vivo. However, the molecular mechanism of action of CCY­1a­E2 attributed to its anticancer effect remains poorly understood. In the present study, CCY­1a­E2 suppressed cell viability in multiple leukemia cell lines (HL­60, K562, KG­1 and KG­1a) via inhibition of cell proliferation, cell cycle arrest and induction of apoptosis. CCY­1a­E2 exhibited a marked toxic effect on HL­60 cells and displayed low cytotoxicity in normal human peripheral blood mononuclear cells (PBMCs). Results from flow cytometric analysis indicated that CCY­1a­E2 promoted G2/M phase arrest and promoted apoptosis in the HL­60 cells. CCY­1a­E2 treatment upregulated cyclin B, cyclin­dependent kinase 1 (CDK1), cell division cycle 25C (cdc25C) and p21 protein expression. CCY­1a­E2 caused apoptotic cell death and DNA fragmentation as determined by 4',6­diamidino­2­phenylindole (DAPI) staining and DNA gel electrophoresis. Elevated activities of caspase­8, ­9 and ­3 were observed during CCY­1a­E2­induced cell apoptosis; their specific inhibitors were found to block CCY­1a­E2­induced apoptosis, respectively. Moreover, CCY­1a­E2 time­dependently disrupted the mitochondrial membrane potential (ΔΨm), and it enhanced the protein levels of Fas/CD95, cytochrome c, Bax, cleaved PARP, as well as attenuated Bcl­2 expression in the HL­60 cells. Our results provide direct evidence that supports the future potential therapeutic application of CCY-1a-E2 in leukemia.

Interlukin-18 Is a Pivot Regulatory Factor on Matrix Metalloproteinase-13 Expression and Brain Astrocytic Migration.

The expression of matrix metalloproteinase-13 (MMP-13) has been shown to be elevated in some pathophysiological conditions and is involved in the degradation of extracellular matrix in astrocytes. In current study, the function of MMP-13 was further investigated. The conditioned medium (CM) collected from activated microglia increased interleukin (IL)-18 production and enhanced MMP-13 expression in astrocytes. Furthermore, treatment with recombinant IL-18 increased MMP-13 protein and mRNA levels in astrocytes. Recombinant IL-18 stimulation also increased the enzymatic activity of MMP-13 and the migratory activity of astrocytes, while administration of MMP-13 or pan-MMP inhibitors antagonized IL-18-induced migratory activity of astrocytes. In addition, administration of recombinant IL-18 to astrocytes led to the phosphorylation of JNK, Akt, or PKCδ, and treatment of astrocytes with JNK, PI3 kinase/Akt, or PKCδ inhibitors significantly decreased the IL-18-induced migratory activity. Taken together, the results suggest that IL-18-induced MMP-13 expression in astrocytes is regulated by JNK, PI3 kinase/Akt, and PKCδ signaling pathways. These findings also indicate that IL-18 is an important regulator leading to MMP-13 expression and cell migration in astrocytes.

Cobalt Protoporphyrin Upregulates Cyclooxygenase-2 Expression Through a Heme Oxygenase-Independent Mechanism.

Cobalt protoporphyrin (CoPP) is a potent HO-1 inducer and generally known to be an antioxidant in various cell types. Little is known about the CoPP-induced cyclooxygenase-2 (COX-2) expression and its downstream signaling in microglial cells. In current study, CoPP caused concentration- and time-dependent increases in COX-2 expression in microglial cells. Furthermore, activation of apoptosis signal-regulating kinase (ASK) 1/MAP kinase involved in CoPP-induced COX-2 expression in microglia. CoPP also induced P2X7 receptor activation, and treatment of P2X7 inhibitors effectively reduced CoPP-induced COX-2 expression. Protein inhibitor of activated STAT (PIAS) 1 is reported to be involved in modulating anti-inflammatory response through negative regulation of transcription factors. Interestingly, treatment with CoPP markedly induced PIAS1 degradation which is regulated by PI3K, Akt, and glycogen synthase kinase 3α/ß (GSK3α/ß) signaling pathways. These results suggest that CoPP induces COX-2 expression through activating P2X7 receptors and ASK1/MAP kinases as well as PIAS1 degradation signaling pathways. Our study provides a new insight into the regulatory effect of CoPP on neuroinflammation in microglial cells.

Naringenin Suppresses Neuroinflammatory Responses Through Inducing Suppressor of Cytokine Signaling 3 Expression.

Accumulating evidence suggests that neuroinflammation is closely associated with the pathogenesis of neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. The hallmark of neuroinflammation is considered to be microglial activation in the central nervous system (CNS). Activated microglia release pro-inflammatory cytokines which cause neuroinflammation and progressive neuronal cell death. Therefore, inhibition of microglial activation is considered an important strategy in the development of neuroprotective strategy. Naringenin, a flavonoid found in citrus fruits and tomatoes, has been reported to have anti-oxidant, anti-cancer, and anti-inflammatory properties. However, the mechanism of its beneficial anti-inflammatory effects in the CNS is poorly understood. In this study, we demonstrated that naringenin inhibites the release of nitric oxide (NO), the expression of inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), as well as pro-inflammatory cytokines in microglial cells. Treatment of naringenin also induced suppressors of cytokine signaling (SOCS)-3 expression in microglia. The SOCS-3 expression and anti-inflammatory effects of naringenin were found to be regulated by adenosine monophosphate-activated protein kinase α (AMPKα) and protein kinase C δ (PKCδ). Besides, naringenin exerted protective property against neurotoxicity caused by LPS-induced microglial activation. Our findings suggest that naringenin-inhibited iNOS and COX-2 expression is mediated by SOCS-3 activation through AMPKα and PKCδ signaling pathways. In a mouse model, naringenin also showed significant protective effects on microglial activation and improved motor coordination function as well. Therefore, naringenin that involves in anti-neuroinflammatory responses and neuroprotection might be a potential agent for treatment of inflammation-associated disorders.