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.

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.

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.