Sulconazole induces pyroptosis promoted by interferon-γ in monocyte/macrophage lineage cells.

Imidazole derivatives are commonly used as antifungal agents. Here, we aimed to investigate the functions of imidazole derivatives on macrophage lineage cells. We assessed the expression levels of inflammatory cytokines in mouse monocyte/macrophage lineage (RAW264.7) cells. All six imidazole derivatives examined, namely ketoconazole, sulconazole, isoconazole, luliconazole, clotrimazole, and bifonazole, reduced the expression levels of inflammatory cytokines, such as interleukin (IL)-6 and tumor necrosis factor-α, after induction by lipopolysaccharide (LPS) in RAW264.7 cells. These imidazole derivatives also induced cell death in RAW264.7 cells, regardless of the presence or absence of LPS. Since the cell death was characteristic in morphology, we investigated the mode of the cell death. An imidazole derivative, sulconazole, induced gasdermin D degradation together with caspase-11 activation, namely, pyroptosis in RAW264.7 cells and peritoneal macrophages. Furthermore, priming with interferon-γ promoted sulconazole-induced pyroptosis in RAW264.7 cells and macrophages and reduced the secretion of the inflammatory cytokine, IL-1ß, from sulconazole-treated macrophages. Our results suggest that imidazole derivatives suppress inflammation by inducing macrophage pyroptosis, highlighting their modulatory potential for inflammatory diseases.

Ibudilast Reduces IL-6 Levels and Ameliorates Symptoms in Lipopolysaccharide-Induced Sepsis Mice.

In Japan, ibudilast (IBD) is a therapeutic agent used to treat asthma, allergic conjunctivitis, and dizziness caused by cerebrovascular disease. Previously, we have reported that IBD could reduce the secretion of proinflammatory cytokines, including interleukin (IL)-6 and tumor necrosis factor (TNF)-α, in lipopolysaccharide (LPS)-treated RAW264.7 monocyte-linage cells in vitro. In the present study, we examined the anti-inflammatory effects of IBD in vivo. As IL-6 is a biomarker for sepsis and has been suggested to exacerbate symptoms, we determined whether IBD reduces IL-6 levels in vivo and improves sepsis symptoms in animal models. We observed that IBD treatment reduced IL-6 levels in the lungs of LPS-treated mice and improved LPS-induced hypothermia, one of the symptoms of sepsis. In addition, IBD reduced IL-6 and attenuated plasminogen activator inhibitor-1 (PAI-1) and alanine aminotransferase (ALT) levels in the serum of LPS-treated mice. Elevated PAI-1 levels exacerbate sepsis-induced disseminated intravascular coagulation (DIC), and ALT is a biomarker for liver dysfunction. IBD improved the survival of mice administered a lethal dose of LPS. IBD administration ameliorated kidney pathology of model mice. Overall, these results suggest that IBD exerts anti-inflammatory functions in vivo and could be a drug candidate for treating endotoxemia, including sepsis.

Cynandione A causes a dynamic change in SIRT1 nuclear trafficking via PKA signaling and beige adipocyte differentiation in 3T3-L1 cells.

Inducible brown-like adipocytes, also known as beige adipocytes, dissipate energy through thermogenesis. Although recent reports suggest that silent information regulator 2 homolog 1 (SIRT1) promotes beige adipocyte differentiation (beiging), the activation mechanism of SIRT1 remains unknown. Here, we report that cynandione A (CA), a major component of Cynanchum wilfordii, causes dynamic changes in SIRT1 nuclear trafficking via protein kinase cAMP-dependent (PKA) signaling and induces the beiging process in adipocyte lineage cells. SIRT1 is located in both the cytoplasm and the nucleus of 3T3-L1 cells. Using cell fractionation and RNA interference experiments, we found that the translocation of SIRT1 from the cytoplasm to the nucleus was enhanced after CA treatment and was followed by upregulation of beige adipocyte-related gene expression. Moreover, we found that CA-induced SIRT1 nuclear trafficking is dependent on the PKA signaling pathway. These results suggest a novel mechanism of CA by which PKA signaling promotes SIRT1 nuclear trafficking, which permits the docking of SIRT1 to its nuclear substrates, leading to beiging in 3T3-L1 cells.

Oenothein B, a Bioactive Ellagitannin, Activates the Extracellular Signal-Regulated Kinase 2 Signaling Pathway in the Mouse Brain.

(1) Background: Oenothein B, a cyclic dimeric ellagitannin present in various medicinal plants, has been reported to exert diverse effects that are beneficial for the treatment and prevention of diseases, including cancer and infections. We recently showed that oenothein B also functions in the brain because its oral administration to systemic inflammatory model mice reduced inflammatory responses in the brain and suppressed abnormal behavior. (2) Results: The present in vivo results demonstrated that oenothein B activated extracellular signal-regulated kinase 2 and cAMP response element-binding protein in the brain, both of which play important roles in synaptic transmission and learning/memory in the central nervous system (CNS). (3) Conclusions: These results suggest that oenothein B exerts neuroprotective effects on the CNS by not only its anti-inflammatory activity but also by enhancing neuronal signaling pathways.

T-Cell Activation-Inhibitory Assay to Screen Caloric Restriction Mimetics Drugs for Drug Repositioning.

We previously reported a screening method for caloric restriction mimetics (CRM), a group of plant-derived compounds capable of inducing good health and longevity. In the present study, we explored the possibility of using this method to screen CRM drugs for drug repositioning. The method, T-cell activation-inhibitory assay, is based on inductive logic. Most of CRM such as resveratrol have been reported to suppress T-cell activation and have anti-inflammatory functions. Here, we assessed the activity of 12 antiallergic drugs through T-cell activation-inhibitory assay and selected four that showed the lowest IC50 values-ibudilast (IC50 0.97 µM), azelastine (IC50 7.2 µM), epinastine (IC50 16 µM), and amlexanox (IC50 33 µM)-for further investigation. Because azelastine showed high cytotoxicity, we selected only the remaining three drugs to study their biological functions. We found that all the three drugs suppressed the expression of interleukin (IL)-6, an inflammatory cytokine, in lipopolysaccharide-treated macrophage cells, with ibudilast being the strongest suppressor. Ibudilast also suppressed the secretion of another inflammatory cytokine, tumor necrosis factor (TNF)-α, and the expression of an inflammatory enzyme, cyclooxygenase-2, in the cells. These results suggest that T-cell activation-inhibitory assay can be used to screen potential CRM drugs having anti-inflammatory functions for the purpose of drug repositioning.

Citrus Auraptene Induces Expression of Brain-Derived Neurotrophic Factor in Neuro2a Cells.

(1) Background: Our published data have indicated that 1) auraptene (AUR), a citrus ingredient, has neuroprotective effects on the mouse brain, owing to its ability to suppress inflammation, such as causing a reduction in hyperactivation of microglia and astrocytes; 2) AUR has the ability to trigger phosphorylation (activation) of extracellular signal-related kinase (ERK) and cAMP response element-binding protein (CREB) in neuronal cells; 3) AUR has the ability to induce glial cell line-derived neurotrophic factor (GDNF) synthesis/secretion in rat C6 glioma cells. The well-established fact that the ERK-CREB pathway plays an important role in the production of neurotrophic factors, including GDNF and brain-derived neurotrophic factor (BDNF), prompted us to investigate whether AUR would also have the ability to induce BDNF expression in neuronal cells. (2) Methods: Mouse neuroblastoma neuro2a cells were cultured and the effects of AUR on BDNF mRNA expression and protein content were evaluated by RT-PCR and ELISA, respectively. (3) Results: The levels of BDNF mRNA and secreted BDNF were significantly increased by AUR in a dose- and time-dependent manner in neuro2a cells. (4) Conclusion: The induction of BDNF in neuronal cells might be, in part, one of the mechanisms accounting for the neuroprotective effects of AUR.

Heptamethoxyflavone inhibits adipogenesis via enhancing PKA signaling.

3,5,6,7,8,3',4'-heptamethoxyflavone (HMF), a naturally occurring polymethoxyflavone found in citrus peel, is known to have neuroprotective, anti-inflammatory, and immunomodulatory effects. However, the effects of HMF on adipogenesis remain unclear. Here, we demonstrate that HMF inhibits the early stage of adipogenesis and maturation in 3T3-L1 adipocytes. HMF treatment during preadipocyte differentiation for 8 days reduced lipid accumulation in a dose-dependent manner, and the expression levels of key adipogenic transcription factors (peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα)) and the lipogenic transcription factor, sterol regulatory element-binding protein (SREBP1), were lower after the initial 4 days of the differentiation. Moreover, PPARγ expression level was lower even after the initial 2 days, but C/EBPα and SREBP1 expression was not. HMF upregulated the phosphorylation of protein kinase A catalytic subunit α (PKACα), AMP-activated protein kinase (AMPK), and acetyl-CoA carboxylase (ACC) in 3T3-L1 cells. The phosphorylation of ACC leads to the inhibition of adipogenesis. Furthermore, the induction of phosphorylation of AMPK and ACC by HMF was abolished by RNA interference targeting PKACα. Taken together, our results suggest that HMF might inhibit the early stage of adipogenesis via the activation of PKA signaling in 3T3-L1 cells.

Citrus flavonoid 3,5,6,7,8,3',4'-heptamethoxyflavone induces BDNF via cAMP/ERK/CREB signaling and reduces phosphodiesterase activity in C6 cells.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) is associated with onset of several central nervous system disorders, e.g., Parkinson's disease, Alzheimer's disease, depression, epilepsy, and chronic pain. In our previous in vivo studies using ischemic and depression mouse models, we revealed that citrus flavonoid 3,5,6,7,8,3',4'-heptamethoxyflavone (HMF) exerts neuroprotective effects by enhancing the expression of BDNF in astrocytes within the hippocampus. Therefore, in the present study, we examined the mechanism of BDNF induction by HMF in vitro using rat C6 glioma cells. METHODS: C6 glioma cells were treated with HMF (10 µM) or HMF + U0126 (10 µM), HMF + H89 (1 µM), or HMF + K252a (200 nM) for 48 h. The protein level of mature BDNF (m-BDNF), phosphorylated-ERK (p-ERK) and phosphorylated-cAMP-response element binding protein (p-CREB) were measured using western blot analysis. To clarify the mechanism of HMF for increasing m-BDNF, the inhibitory effect of phosphodiesterase 4B (PDE4B) and PDE4D, and intracellular cAMP levels were examined by ELISA. RESULTS: Our findings revealed that the m-BDNF-inducing activity of HMF was abolished by U0126 but not by H89 or K252a. HMF was found to phosphorylate (activate) ERK and cAMP-response element binding protein (CREB), a BDNF transcription factor. HMF inhibited PDE4B and PDE4D activity. Moreover, 10 µM HMF elevated intracellular cAMP levels in C6 cells. CONCLUSIONS: These findings suggest that HMF might exert its neuroprotective effects by inducing m-BDNF expression in C6 cells, model cell line of astrocytes, via the activation of cAMP/ERK/CREB signaling and inhibiting PDE4B or PDE4D.

Citrus Auraptene Induces Glial Cell Line-Derived Neurotrophic Factor in C6 Cells.

We previously demonstrated that auraptene (AUR), a natural coumarin derived from citrus plants, exerts anti-inflammatory effects in the brain, resulting in neuroprotection in some mouse models of brain disorders. The present study showed that treatment with AUR significantly increased the release of glial cell line-derived neurotrophic factor (GDNF), in a dose- and time-dependent manner, by rat C6 glioma cells, which release was associated with increased expression of GDNF mRNA. These results suggest that AUR acted as a neuroprotective agent in the brain via not only its anti-inflammatory action but also its induction of neurotrophic factor. We also showed that (1) the AUR-induced GDNF production was inhibited by U0126, a specific inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) 1/2, and by H89, a specific inhibitor of protein kinase A (PKA); and (2) AUR induced the phosphorylation of cAMP response element-binding protein (CREB), a transcription factor located within the nucleus. These results suggest that AUR-stimulated gdnf gene expression was up-regulated through the PKA/ERK/CREB pathway in C6 cells.

Neuroprotective effect of Citrus kawachiensis (Kawachi Bankan) peels, a rich source of naringin, against global cerebral ischemia/reperfusion injury in mice.

Cerebral ischemia/reperfusion is known to induce the generation of reactive oxygen species and inflammatory responses. Numerous studies have demonstrated that naringin (NGIN) has anti-oxidant and anti-inflammatory properties. We previously reported that Citrus kawachiensis contains a large quantity of NGIN in its peel. In the present study, we orally (p.o.) administered dried peel powder of C. kawachiensis to mice of a transient global ischemia model and found in the hippocampus region that it 1) suppressed neuronal cell death, 2) reversed the reduction in the level of phosphorylated calcium-calmodulin-dependent protein kinase II, 3) had the tendency to reverse the reduction in the level of glutathione, and 4) blocked excessive activation of microglia and astrocytes. These results suggested that the dried peel powder of C. kawachiensis had a neuroprotective effect against ischemic brain via anti-oxidative and anti-inflammatory effects. We also showed that these effects of the dried peel powder were more powerful than those obtained with a comparable amount of NGIN alone.

Heptamethoxyflavone Reduces Phosphodiesterase Activity and T-Cell Growth in vitro.

In a previous study, we reported that interleukin-4 production was reduced in spleen cells of mice administered 3,5,6,7,8,3',4'-heptamethoxyflavone (HMF), which is a polymethoxyflavone found at high concentrations in the peel of various citrus fruits. In this study, we investigated the function of HMF on the growth of T cells cultured from the spleens of mice. HMF decreased the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by anti-CD3/CD28 antibody-stimulated mouse spleen cells. HMF inhibited the activities of phosphodiesterase (PDE) enzymes prepared from bovine brain and human PDE4B and PDE3B enzymes. The cyclic AMP (cAMP) content in anti-CD3/CD28 antibody-stimulated spleen cells increased after HMF treatment in vitro. These results suggest that HMF inhibits T-cell growth and affects immune function via reduced PDE activity and increased cAMP content.

Auraptene Acts as an Anti-Inflammatory Agent in the Mouse Brain.

The anti-inflammatory activity of auraptene (AUR), a citrus coumarin, in peripheral tissues is well-known, and we previously demonstrated that AUR exerts anti-inflammatory effects in the ischemic brain; the treatment of mice with AUR for eight days immediately after ischemic surgery suppressed demise and neuronal cell death in the hippocampus, possibly through its anti-inflammatory effects in the brain. We suggested that these effects were at least partly mediated by the suppression of inflammatory mediators derived from astrocytes. The present study showed that (1) AUR, as a pretreatment for five days before and another three days after ischemic surgery, suppressed microglial activation, cyclooxygenase (COX)-2 expression in astrocytes, and COX-2 mRNA expression in the hippocampus; (2) AUR suppressed the lipopolysaccharide-induced expression of COX-2 mRNA and the mRNA of pro-inflammatory cytokines in cultured astrocytes; (3) AUR was still detectable in the brain 60 min after its intraperitoneal administration. These results support our previous suggestion that AUR directly exerts anti-inflammatory effects on the brain.

3,5,6,7,8,3',4'-Heptamethoxyflavone, a citrus flavonoid, on protection against memory impairment and neuronal cell death in a global cerebral ischemia mouse model.

The present study evaluated the effects of treatment with the citrus flavonoid, 3,5,6,7,8,3',4'-heptamethoxyflavone (HMF) on protection against memory impairment and neuronal death in a global cerebral ischemia mouse model. The results showed that HMF, administrated for three days immediately after ischemic surgery, protected against ischemia-induced memory dysfunction, rescued neuronal cell death in the CA1 cell layer, increased the production of BDNF, stimulated the autophosphorylation of CaMK II and suppressed microglial activation in the hippocampus. These results suggest that HMF has a neuroprotective effect after brain ischemia by inducing BDNF production and anti-inflammatory effects.

Dilation of the inferior colliculus and hypersensitivity to sound in Wnt1-cre and Wnt1-GAL4 double-transgenic mice.

The processing of sound information is mediated by the cochlea and the central auditory system. Among the central auditory system, the inferior colliculus (IC) has leading roles in the acoustic processing. In a previous study, we demonstrated psychiatric disorder-related behavioral abnormalities in a genetically modified animal of Wnt1-cre and Wnt1-GAL4 double-transgenic (dTg) mouse. Here we report an abnormal morphology of the IC and dysacusis in the dTg mice. The IC in the brain of the dTg mice is dilated in appearance and histologic analysis revealed a high cell-density in the IC. Also, the dTg mice showed high scores in a startle response test using a click box that emits a 20-kHz sound. Auditory brainstem response (ABR) test revealed lower ABR thresholds of the dTg mice at a test-stimulus frequency of 32kHz, but not at 4-16kHz. These findings suggest that the dTg mice could be a useful animal model for studying the physiologic function of the IC and the pathophysiology of psychiatric disorder-related dysacusis.

A comparative study of axon-surrounding cells in the two nasal nerve tracts from mouse olfactory epithelium and vomeronasal organ.

The olfactory and vomeronasal systems are the two nasal chemical detectors in mammals. While glial cells in the olfactory nerve tracts have been well-investigated, little is known about cells in the vomeronasal nerve tracts. In the present study, we compared the expression patterns of marker proteins in the cells comprising the two nasal nerve tracts in mice. Neural crest-derived cells surrounded the olfactory nerve axons in the lamina propria of the olfactory epithelium. These cells expressed glial fibrillary acidic protein (GFAP) and p75 glycoprotein, which are markers of olfactory ensheathing cells. Neural crest-derived cells also surrounded the vomeronasal nerve axons in the lamina propria of the vomeronasal epithelium. These nerve axon-surrounding cells, however, did not express GFAP or p75. Rather, the vomeronasal nerve axons expressed GFAP and p75. These results suggest that axon-surrounding cells functionally differ between the olfactory and vomeronasal nerve tracts.

Psychiatric disorder-related abnormal behavior and habenulointerpeduncular pathway defects in Wnt1-cre and Wnt1-GAL4 double transgenic mice.

The neural crest is a unique structure in vertebrates. Wnt1-cre and Wnt1-GAL4 double transgenic (dTg) mice have been used in a variety of studies concerning neural crest cell lineages in which the Cre/loxP or GAL4/UAS system was applied. Here, we show psychiatric disorder-related behavioral abnormalities and histologic alterations in a neural crest-derived brain region in dTg mice. The dTg mice exhibited increased locomotor activity, decreased social interaction, and impaired short-term spatial memory and nesting behavior. The choline acetyltransferase- and vesicular glutamate transporter 2-immunoreactive habenulointerpeduncular fiber tracts that project from the medial habenular nucleus of the epithalamus to the interpeduncular nucleus of the midbrain tegmentum appeared irregular in the dTg mice. Both the medial habenula nucleus and the interpeduncular nucleus were confirmed to be derived from the neural crest. The findings of this study suggest that neural crest-derived cells have pathogenic roles in the development of psychiatric disorders and that the dTg mouse could be a useful animal model for studying the pathophysiology of mental illness such as autism and schizophrenia. Scientists that use the dTg mice as a cre-transgenic deleter line should be cautious in its possible toxicity, especially if behavioral analyses are to be performed.

Neurotrophic effect of citrus auraptene: neuritogenic activity in PC12 cells.

The activation of extracellular signal-regulated kinases (ERK) leads to a number of cellular changes associated with the development of long-term memory. Using cultured cortical neurons, we previously showed that the n-hexane extract prepared from the peels of Citrus grandis (Kawachi bankan) induces the activation of ERK1/2 and that one of the compounds with this ability in the extract is 3,5,6,7,8,3',4'-heptamethoxyflavone (HMF), a Citrus polymethoxyflavone. In fact, we found that HMF has the ability to rescue mice from drug-induced learning impairment. This hexane extract contains auraptene (AUR), a coumarin derivative with a monoterpene unit, together with HMF. The present study was designed to investigate the effect of AUR in vitro. Our results show that 1) AUR had the ability to induce the activation of ERK1/2 in not only cortical neurons but also the rat pheochromocytoma cell line (PC12 cells), which is a model system for studies on neuronal proliferation and differentiation; and 2) AUR had the ability to promote neurite outgrowth from PC12 cells.

Effect of the edible mushroom Mycoleptodonoides aitchisonii on transient global ischemia-induced monoamine metabolism changes in rat cerebral cortex.

We performed a transient bilateral common carotid artery occlusion on rats and investigated whether feeding an aqueous extract of Mycoleptodonoides aitchisonii, an edible mushroom, affected metabolism of monoamines in the cerebral cortex, possibly protecting against ischemic damage. Seventeen days after the surgery, concentrations of the dopamine (DA) metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) and of homovanillic acid (HVA) in the cerebral cortex of the M. aitchisonii-fed group (MV) were higher than in the control ischemia (CV) group. The turnover rate of DA, which was indicated by (DOPAC+HVA)/DA, for the CV group was significantly lower than for the MV group, and the MV group value was the same rate as the sham-operated group. These data indicate that M. aitchisonii affects the dopaminergic neuronal system following brain ischemia damage in the cerebral cortex.

Hydrocephalus and abnormal subcommissural organ in mice lacking presenilin-1 in Wnt1 cell lineages.

Presenilin-1 (PS1) is a transmembrane protein that is in many cases responsible for the development of familial Alzheimer's disease. PS1 is widely expressed in embryogenesis and is essential for neurogenesis, somitogenesis, angiogenesis, and cardiac morphogenesis. To further investigate the role of PS1 in the brain, we inactivated the PS1 gene in Wnt1 cell lineages using the Cre-loxP recombination system. Here we show that conditional inactivation of PS1 in Wnt1 cell lineages results in congenital hydrocephalus and subcommissural organ abnormalities, suggesting a possible role of PS1 in the regulation of cerebrospinal fluid homeostasis.

Estrogen stimulates proliferation and differentiation of neural stem/progenitor cells through different signal transduction pathways.

Our previous study indicated that both 17ß-estradiol (E2), known to be an endogenous estrogen, and bisphenol A (BPA), known to be a xenoestrogen, could positively influence the proliferation or differentiation of neural stem/progenitor cells (NS/PCs). The aim of the present study was to identify the signal transduction pathways for estrogenic activities promoting proliferation and differentiation of NS/PCs via well known nuclear estrogen receptors (ERs) or putative membrane-associated ERs. NS/PCs were cultured from the telencephalon of 15-day-old rat embryos. In order to confirm the involvement of nuclear ERs for estrogenic activities, their specific antagonist, ICI-182,780, was used. The presence of putative membrane-associated ER was functionally examined as to whether E2 can activate rapid intracellular signaling mechanism. In order to confirm the involvement of membrane-associated ERs for estrogenic activities, a cell-impermeable E2, bovine serum albumin-conjugated E2 (E2-BSA) was used. We showed that E2 could rapidly activate extracellular signal-regulated kinases 1/2 (ERK 1/2), which was not inhibited by ICI-182,780. ICI-182,780 abrogated the stimulatory effect of these estrogens (E2 and BPA) on the proliferation of NS/PCs, but not their effect on the differentiation of the NS/PCs into oligodendroglia. Furthermore, E2-BSA mimicked the activity of differentiation from NS/PCs into oligodendroglia, but not the activity of proliferation. Our study suggests that (1) the estrogen induced proliferation of NS/PCs is mediated via nuclear ERs; (2) the oligodendroglial generation from NS/PCs is likely to be stimulated via putative membrane-associated ERs.