Biological Effects of β-Glucans on Osteoclastogenesis

Although the anti-tumor and anti-infective properties of β-glucans have been well-discussed, their role in bone metabolism has not been reviewed so far. This review discusses the biological effects of β-glucans on bone metabolisms, especially on bone-resorbing osteoclasts, which are differentiated from hematopoietic precursors. Multiple immunoreceptors that can recognize β-glucans were reported to be expressed in osteoclast precursors. Coordinated co-stimulatory signals mediated by these immunoreceptors are important for the regulation of osteoclastogenesis and bone remodeling. Curdlan from the bacterium Alcaligenes faecalis negatively regulates osteoclast differentiation in vitro by affecting both the osteoclast precursors and osteoclast-supporting cells. We also showed that laminarin, lichenan, and glucan from baker’s yeast, as well as β-1,3-glucan from Euglema gracilisas, inhibit the osteoclast formation in bone marrow cells. Consistent with these findings, systemic and local administration of β-glucan derived from Aureobasidium pullulans and Saccharomyces cerevisiae suppressed bone resorption in vivo. However, zymosan derived from S. cerevisiae stimulated the bone resorption activity and is widely used to induce arthritis in animal models. Additional research concerning the relationship between the molecular structure of β-glucan and its effect on osteoclastic bone resorption will be beneficial for the development of novel treatment strategies for bone-related diseases.     

Inhibitory Effect of LGS and ODE Isolated from the Twigs of Syringa oblata subsp. dilatata on RANKL-Induced Osteoclastogenesis in Macrophage Cells

Osteoblasts and osteoclasts play a pivotal role in maintaining bone homeostasis, of which excessive bone resorption by osteoclasts can cause osteoporosis and various bone diseases. However, current osteoporosis treatments have many side effects, and research on new treatments that can replace these treatments is ongoing. Therefore, in this study, the roles of ligustroside (LGS) and oleoside dimethylester (ODE), a natural product-derived compound isolated from Syringa oblata subsp. dilatata as a novel, natural product-derived osteoporosis treatments were investigated. In the results of this study, LGS and ODE inhibited the differentiation of receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced RAW264.7 cells into osteoclasts without cytotoxicity, and down-regulated the activity of TRAP, a specific biomarker of osteoclasts. In addition, it inhibited bone resorption and actin ring formation, which are important functions and features of osteoclasts. Also, the effects of LGS and ODE on the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) and phosphoinositide 3-kinases (PI3K)/ protein kinase B (Akt)/mechanistic target of rapamycin (mTOR) signaling pathways that play important roles in osteoclast differentiation were evaluated. In the results, LGS and ODE downregulated the phosphorylation of RANKL-induced MAPK and PI3K/Akt/mTOR proteins in a concentration-dependent manner, translocation of NF-κB into the nucleus was inhibited. As a result, the compounds LGS and ODE isolated from S. oblate subsp. dilatata effectively regulated the differentiation of RANKL-induced osteoclasts and inhibited the phosphorylation of signaling pathways that play a pivotal role in osteoclast differentiation. Therefore, these results suggest the possibility of LGS and ODE as new natural product treatments for bone diseases caused by excessive osteoclasts.     

Anti-Osteoporotic Effects of n-trans-Hibiscusamide and Its Derivative Alleviate Ovariectomy-Induced Bone Loss in Mice by Regulating RANKL-Induced Signaling

Osteoporosis is characterized by the deterioration of bone structures and decreased bone mass, leading to an increased risk of fracture. Estrogen deficiency in postmenopausal women and aging are major factors of osteoporosis and are some of the reasons for reduced quality of life. In this study, we investigated the effects of n-trans-hibiscusamide (NHA) and its derivative 4-O-(E)-feruloyl-N-(E)-hibiscusamide (HAD) on receptor activator of nuclear factor kappa-Β (NF-κB) ligand (RANKL)-induced osteoclast differentiation and an ovariectomized osteoporosis mouse model. NHA and HAD significantly inhibited the differentiation of osteoclasts from bone marrow-derived macrophages (BMMs) and the expression of osteoclast differentiation-related genes. At the molecular level, NHA and HAD significantly downregulated the phosphorylation of mitogen-activated protein kinase (MAPK) signaling molecules. However, Akt and NF-κB phosphorylation was inhibited only after NHA or HAD treatment. In the ovariectomy (OVX)-induced osteoporosis model, both NHA and HAD effectively improved trabecular bone structure. C-terminal telopeptide (CTX), a bone resorption marker, and RANKL, an osteoclast stimulation factor, were significantly reduced by NHA and HAD. The tartrate-resistant acid phosphatase (TRAP)-stained area, which indicates the osteoclast area, was also decreased by these compounds. These results show the potential of NHA and HAD as therapeutic agents for osteoporosis.     

Hypoxia-inducible factor-2α mediates senescence-associated intrinsic mechanisms of age-related bone loss

Aging is associated with cellular senescence followed by bone loss leading to bone fragility in humans. However, the regulators associated with cellular senescence in aged bones need to be identified. Hypoxia-inducible factor (HIF)−2α regulates bone remodeling via the differentiation of osteoblasts and osteoclasts. Here, we report that HIF-2α expression was highly upregulated in aged bones. HIF-2α depletion in male mice reversed age-induced bone loss, as evidenced by an increase in the number of osteoblasts and a decrease in the number of osteoclasts. In an in vitro model of doxorubicin-mediated senescence, the expression of Hif-2α and p21, a senescence marker gene, was enhanced, and osteoblastic differentiation of primary mouse calvarial preosteoblast cells was inhibited. Inhibition of senescence-induced upregulation of HIF-2α expression during matrix maturation, but not during the proliferation stage of osteoblast differentiation, reversed the age-related decrease in Runx2 and Ocn expression. However, HIF-2α knockdown did not affect p21 expression or senescence progression, indicating that HIF-2α expression upregulation in senescent osteoblasts may be a result of aging rather than a cause of cellular senescence. Osteoclasts are known to induce a senescent phenotype during in vitro osteoclastogenesis. Consistent with increased HIF-2α expression, the expression of p16 and p21 was upregulated during osteoclastogenesis of bone marrow macrophages. ChIP following overexpression or knockdown of HIF-2α using adenovirus revealed that p16 and p21 are direct targets of HIF-2α in osteoclasts. Osteoblast-specific (Hif-2α^fl/fl;Col1a1-Cre) or osteoclast-specific (Hif-2α^fl/fl;Ctsk-Cre) conditional knockout of HIF-2α in male mice reversed age-related bone loss. Collectively, our results suggest that HIF-2α acts as a senescence-related intrinsic factor in age-related dysfunction of bone homeostasis.Subject terms: Cell biology, Diseases     

Isoorientin Inhibits Amyloid β25–35-Induced Neuronal Inflammation in BV2 Cells by Blocking the NF-κB Signaling Pathway

Alzheimer’s disease (AD) is a severe neurodegenerative disorder. AD is pathologically characterized by the formation of intracellular neurofibrillary tangles, and extracellular amyloid plaques which were comprised of amyloid-beta (Aβ) peptides. Aβ induces neurodegeneration by activating microglia, which triggers neurotoxicity by releasing various inflammatory mediators and reactive oxygen species (ROS). Nuclear factor-kappa B (NF-κB) is expressed in human tissues including the brain and plays an important role in Aβ-mediated neuronal inflammation. Thus, the identification of molecules that inhibit the NF-κB pathway is considered an attractive strategy for the treatment and prevention of AD. Isoorientin (3′,4′,5,7-Tetrahydroxy-6-C-glucopyranosyl flavone; ISO), which can be extracted from several plant species, such as Philostachys and Patrinia is known to have various pharmacological activities such as anticancer, antioxidant, and antibacterial activity. However, the effect of ISO on Aβ-mediated inflammation and apoptosis in the brain has yet to be elucidated. In the present study, we investigated whether ISO regulated Aβ-induced neuroinflammation in microglial cells and further explored the underlying mechanisms. Our results showed that ISO inhibited the expression of iNOS and COX-2 induced by Aβ_25–35. And, it inhibited the secretion of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). In addition, ISO reduced the ROS production in Aβ_25–35-induced BV2 cells and inhibited NF-κB activation. Furthermore, ISO blocked Aβ_25–35-induced apoptosis of BV2 cells. Based on these findings, we suggest that ISO represents a promising therapeutic drug candidate for the treatment and prevention of AD.     

Diterpenoid Compounds Isolated from Chloranthus oldhamii Solms Exert Anti-Inflammatory Effects by Inhibiting the IKK/NF-κB Pathway

Chloranthus oldhamii Solms (CO) is a folk medicine for treating infection and arthritis pain but its pharmacological activity and bioactive compounds remain mostly uncharacterized. In this study, the anti-inflammatory compounds of C. oldhamii were identified using an LPS-stimulated, NF-κB-responsive RAW 264.7 macrophage reporter line. Three diterpenoid compounds, 3α-hydroxy-ent-abieta-8,11,13-triene (CO-9), 3α, 7β-dihydroxy-ent-abieta-8,11,13-triene (CO-10), and decandrin B (CO-15) were found to inhibit NF-κB activity at nontoxic concentrations. Moreover, CO-9 and CO-10 suppressed the expression of IL-6 and TNF-α in LPS-stimulated RAW 264.7 cells. The inhibitory effect of CO-9 on TNF-α and IL-6 expression was further demonstrated using LPS-treated bone marrow-derived macrophages. Furthermore, CO-9, CO-10, and CO-15 suppressed LPS-triggered COX-2 expression and downstream PGE2 production in RAW 264.7 cells. CO-9 and CO-10 also reduced LPS-triggered iNOS expression and nitrogen oxide production in RAW 264.7 cells. The anti-inflammatory mechanism of the most effective compound, CO-9, was further investigated. CO-9 attenuated LPS-induced NF-κB activation by reducing the phosphorylation of IKKα/β (Ser176/180), IκBα (Ser32), and p65 (Ser534). Conversely, CO-9 did not affect the LPS-induced activation of MAPK signaling pathways. In summary, this study revealed new anti-inflammatory diterpenoid compounds from C. oldhamii and demonstrated that the IKK-mediated NK-κB pathway is the major target of these compounds.     

Molecular Regulation of Betulinic Acid on α3β4 Nicotinic Acetylcholine Receptors

Betulinic acid (BA) is a major constituent of Zizyphus seeds that have been long used as therapeutic agents for sleep-related issues in Asia. BA is a pentacyclic triterpenoid. It also possesses various anti-cancer and anti-inflammatory effects. Current commercially available sleep aids typically use GABAergic regulation, for which many studies are being actively conducted. However, few studies have focused on acetylcholine receptors that regulate wakefulness. In this study, we utilized BA as an antagonist of α3β4 nicotinic acetylcholine receptors (α3β4 nAChRs) known to regulate rapid-eye-movement (REM) sleep and wakefulness. Effects of BA on α3β4 nAChRs were concentration-dependent, reversible, voltage-independent, and non-competitive. Site-directed mutagenesis and molecular-docking studies confirmed the binding of BA at the molecular level and showed that the α3 subunit L257 and the β4 subunit I263 residues affected BA binding. These data demonstrate that BA can bind to a binding site different from the site for the receptor’s ligand, acetylcholine (ACh). This suggests that BA may be an effective antagonist that is unaffected by large amounts of ACh released during wakefulness and REM sleep. Based on the above experimental results, BA is likely to be a therapeutically useful sleep aid and sedative.     

Decreased Human Platelet Activation and Mouse Pulmonary Thrombosis by Rutaecarpine and Comparison of the Relative Effectiveness with BAY11-7082: Crucial Signals of p38-NF-κB

Platelets play a critical role in arterial thrombosis. Rutaecarpine (RUT) was purified from Tetradium ruticarpum, a well-known Chinese medicine. This study examined the relative activity of RUT with NF-κB inhibitors in human platelets. BAY11-7082 (an inhibitor of IκB kinase [IKK]), Ro106-9920 (an inhibitor of proteasomes), and RUT concentration-dependently (1–6 μM) inhibited platelet aggregation and P-selectin expression. RUT was found to have a similar effect to that of BAY11-7082; however, it exhibits more effectiveness than Ro106-9920. RUT suppresses the NF-κB pathway as it inhibits IKK, IκBα, and p65 phosphorylation and reverses IκBα degradation in activated platelets. This study also investigated the role of p38 and NF-κB in cell signaling events and found that SB203580 (an inhibitor of p38) markedly reduced p38, IKK, and p65 phosphorylation and reversed IκBα degradation as well as p65 activation in a confocal microscope, whereas BAY11-7082 had no effects in p38 phosphorylation. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay shows that RUT and BAY11-7082 did not exhibit free radical scavenging activity. In the in vivo study, compared with BAY11-7082, RUT more effectively reduced mortality in adenosine diphosphate (ADP)-induced acute pulmonary thromboembolism without affecting the bleeding time. In conclusion, a distinctive pathway of p38-mediated NF-κB activation may involve RUT-mediated antiplatelet activation, and RUT could act as a strong prophylactic or therapeutic drug for cardiovascular diseases.     

Soy Isoflavones Inhibit Both GPIb-IX Signaling and αIIbβ3 Outside-In Signaling via 14-3-3ζ in Platelet

Soy diet is thought to help prevent cardiovascular diseases in humans. Isoflavone, which is abundant in soybean and other legumes, has been reported to possess antiplatelet activity and potential antithrombotic effect. Our study aims to elucidate the potential target of soy isoflavone in platelet. The anti-thrombosis formation effect of genistein and daidzein was evaluated in ex vivo perfusion chamber model under low (300 s⁻¹) and high (1800 s⁻¹) shear forces. The effect of genistein and daidzein on platelet aggregation and spreading was evaluated with platelets from both wildtype and GPIbα deficient mice. The interaction of these soy isoflavone with 14-3-3ζ was detected by surface plasmon resonance (SPR) and co-immunoprecipitation, and the effect of αIIbβ3-mediated outside-in signaling transduction was evaluated by western blot. We found both genistein and daidzein showed inhibitory effect on thrombosis formation in perfusion chamber, especially under high shear force (1800 s⁻¹). These soy isoflavone interact with 14-3-3ζ and inhibited both GPIb-IX and αIIbβ3-mediated platelet aggregation, integrin-mediated platelet spreading and outside-in signaling transduction. Our findings indicate that 14-3-3ζ is a novel target of genistein and daidzein. 14-3-3ζ, an adaptor protein that regulates both GPIb-IX and αIIbβ3-mediated platelet activation is involved in soy isoflavone mediated platelet inhibition.     

The Amyloid Fibril-Forming β-Sheet Regions of Amyloid β and α-Synuclein Preferentially Interact with the Molecular Chaperone 14-3-3ζ

14-3-3 proteins are abundant, intramolecular proteins that play a pivotal role in cellular signal transduction by interacting with phosphorylated ligands. In addition, they are molecular chaperones that prevent protein unfolding and aggregation under cellular stress conditions in a similar manner to the unrelated small heat-shock proteins. In vivo, amyloid β (Aβ) and α-synuclein (α-syn) form amyloid fibrils in Alzheimer’s and Parkinson’s diseases, respectively, a process that is intimately linked to the diseases’ progression. The 14-3-3ζ isoform potently inhibited in vitro fibril formation of the 40-amino acid form of Aβ (Aβ₄₀) but had little effect on α-syn aggregation. Solution-phase NMR spectroscopy of ¹⁵N-labeled Aβ₄₀ and A53T α-syn determined that unlabeled 14-3-3ζ interacted preferentially with hydrophobic regions of Aβ₄₀ (L11-H21 and G29-V40) and α-syn (V3-K10 and V40-K60). In both proteins, these regions adopt β-strands within the core of the amyloid fibrils prepared in vitro as well as those isolated from the inclusions of diseased individuals. The interaction with 14-3-3ζ is transient and occurs at the early stages of the fibrillar aggregation pathway to maintain the native, monomeric, and unfolded structure of Aβ₄₀ and α-syn. The N-terminal regions of α-syn interacting with 14-3-3ζ correspond with those that interact with other molecular chaperones as monitored by in-cell NMR spectroscopy.     

α-Amyrin and β-Amyrin Isolated from Celastrus hindsii Leaves and Their Antioxidant,Anti-Xanthine Oxidase,and Anti-Tyrosinase Potentials

Celastrus hindsii is a popular medicinal plant in Vietnam and Southeast Asian countries as well as in South America. In this study, an amount of 12.05 g of an α-amyrin and β-amyrin mixture was isolated from C. hindsii (10.75 g/kg dry weight) by column chromatography applying different solvent systems to obtain maximum efficiency. α-Amyrin and β-amyrin were then confirmed by gas chromatography-mass spectrometry (GC-MS), electrospray ionization-mass spectrometry (ESI-MS), and nuclear magnetic resonance (NMR). The antioxidant activities of the α-amyrin and β-amyrin mixture were determined via 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,20-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays with IC₅₀ of 125.55 and 155.28 µg/mL, respectively. The mixture exhibited a high potential for preventing gout by inhibiting a relevant key enzyme, xanthine oxidase (XO) (IC₅₀ = 258.22 µg/mL). Additionally, an important enzyme in skin hyperpigmentation, tyrosinase, was suppressed by the α-amyrin and β-amyrin mixture (IC₅₀ = 178.85 µg/mL). This study showed that C. hindsii is an abundant source for the isolation of α-amyrin and β-amyrin. Furthermore, this was the first study indicating that α-amyrin and β-amyrin mixture are promising in future therapies for gout and skin hyperpigmentation.     

The Antiviral Effects of Jasminin via Endogenous TNF-α and the Underlying TNF-α-Inducing Action

Previous studies have reported that recombinant tumor necrosis factor (TNF)-α has powerful antiviral activity but severe systematic side effects. Jasminin is a common bioactive component found in Chinese herbal medicine beverage “Jasmine Tea”. Here, we report that jasminin-induced endogenous TNF-α showed antiviral activity in vitro. The underlying TNF-α-inducing action of jasminin was also investigated in RAW264.7 cells. The level of endogenous TNF-α stimulated by jasminin was first analyzed by an enzyme-linked immunosorbent assay (ELISA) from the cell culture supernatant of RAW264.7 cells. The supernatants were then collected to investigate the potential antiviral effect against herpes simplex virus 1 (HSV-1). The antiviral effects of jasminin alone or its supernatants were evaluated by a plaque reduction assay. The potential activation of the PI3K–Akt pathway, three main mitogen-activated protein kinases (MAPKs), and nuclear factor (NF)–κB signaling pathways that induce TNF-α production were also investigated. Jasminin induces TNF-α protein expression in RAW264.7 cells without additional stimuli 10-fold more than the control. No significant up-expression of type I, II, and III interferons; interleukins 2 and 10; nor TNF-β were observed by the jasminin stimuli. The supernatants, containing jasminin-induced-TNF-α, showed antiviral activity against HSV-1. The jasminin-stimulated cells caused the simultaneous activation of the Akt, MAPKs, and NF–κB signal pathways. Furthermore, the pretreatment of the cells with the Akt, MAPKs, and NF–κB inhibitors effectively suppressed jasminin-induced TNF-α production. Our research provides evidence that endogenous TNF-α can be used as a strategy to encounter viral infections. Additionally, the Akt, MAPKs, and NF–κB signaling pathways are involved in the TNF-α synthesis that induced by jasminin.     

Ameliorating Effect on Aβ-Induced Alzheimer’s Mice by Litsea cubeba Persoon Powder

Alzheimer’s disease (AD) is caused by excessive oxidative damage and aging. The objective of this study was to investigate the anti-dementia effect of LCP fruit powder on amyloid β (Aβ)-induced Alzheimer’s mice. The composition of LCP essential oil was determined by gas chromatography/mass spectrometry. In addition, the water maze was used to evaluate the learning and memorizing abilities of the mice. The concentrations of malondialdehyde (MDA), protein carbonyl, phosphorylated τ-protein, and the deposition of Aβ plaques in mouse brains were also assessed. The results showed that the main components of essential oils in LCP and d-limonene, neral, and geranial contents were 14.15%, 30.94%, and 31.74%, respectively. Furthermore, oral administration with different dosages of LCP significantly decreased the escape time (21.25~33.62 s) and distance (3.23~5.07 m) in the reference memory test, and increased the duration time (26.14~28.90 s) and crossing frequency (7.00~7.88 times) in the target zone of probe test (p < 0.05). LCP also inhibited the contents of MDA and the phosphor-τ-protein from oxidative stress, reduced the brain atrophy by about 3~8%, and decreased the percentage of Aβ plaques from 0.44 to 0.05%. Finally, it was observed that the minimum dosage of LCP fruit powder (LLCP, 30.2 mg/day) could prevent oxidative stress induced by Aβ and subsequently facilitate memory and learning deficits in Aβ-induced neurotoxicity and cognitively impaired mice.     

Biochemical and Structural Analysis of a Glucose-Tolerant β-Glucosidase from the Hemicellulose-Degrading Thermoanaerobacterium saccharolyticum

β-Glucosidases (Bgls) convert cellobiose and other soluble cello-oligomers into glucose and play important roles in fundamental biological processes, providing energy sources in living organisms. Bgls are essential terminal enzymes of cellulose degradation systems and attractive targets for lignocellulose-based biotechnological applications. Characterization of novel Bgls is important for broadening our knowledge of this enzyme class and can provide insights into its further applications. In this study, we report the biochemical and structural analysis of a Bgl from the hemicellulose-degrading thermophilic anaerobe Thermoanaerobacterium saccharolyticum (TsaBgl). TsaBgl exhibited its maximum hydrolase activity on p-nitrophenyl-β-d-glucopyranoside at pH 6.0 and 55 °C. The crystal structure of TsaBgl showed a single (β/α)₈ TIM-barrel fold, and a β8-α14 loop, which is located around the substrate-binding pocket entrance, showing a unique conformation compared with other structurally known Bgls. A Tris molecule inhibited enzyme activity and was bound to the active site of TsaBgl coordinated by the catalytic residues Glu163 (proton donor) and Glu351 (nucleophile). Titration experiments showed that TsaBgl belongs to the glucose-tolerant Bgl family. The gatekeeper site of TsaBgl is similar to those of other glucose-tolerant Bgls, whereas Trp323 and Leu170, which are involved in glucose tolerance, show a unique configuration. Our results therefore improve our knowledge about the Tris-mediated inhibition and glucose tolerance of Bgl family members, which is essential for their industrial application.     

Comparative Studies on Regioselectivity of α- and β-Linked Glucan Tosylation

Alpha- and beta-linked 1,3-glucans have been subjected to conversion with p-toluenesulfonic acid (tosyl) chloride and triethylamine under homogeneous reaction conditions in N,N-dimethyl acetamide/LiCl. Samples with a degree of substitution of tosyl groups (DS_Ts) of up to 1.91 were prepared by applying 5 mol reagent per mole repeating unit. Hence, the reactivity of α-1,3-glucan is comparable with cellulose and starch, while the β-1,3-linked glucan curdlan is less reactive. The samples dissolve in aprotic dipolar media independent of the DS_Ts and possess a solubility in less polar solvents that depends on the DS_Ts. NMR studies on the tosyl glucans and of the peracylated derivatives showed a preferred tosylation of position 2 of the repeating unit. However, the selectivity is less pronounced compared with starch. It could be concluded that the α-configurated glycosidic bond directs tosyl groups towards position 2.     

β-Carotene Inhibits Expression of Matrix Metalloproteinase-10 and Invasion in Helicobacter pylori-Infected Gastric Epithelial Cells

Matrix metalloproteinases (MMPs), key molecules of cancer invasion and metastasis, degrade the extracellular matrix and cell–cell adhesion molecules. MMP-10 plays a crucial role in Helicobacter pylori-induced cell-invasion. The mitogen-activated protein kinase (MAPK) signaling pathway, which activates activator protein-1 (AP-1), is known to mediate MMP expression. Infection with H. pylori, a Gram-negative bacterium, is associated with gastric cancer development. A toxic factor induced by H. pylori infection is reactive oxygen species (ROS), which activate MAPK signaling in gastric epithelial cells. Peroxisome proliferator-activated receptor γ (PPAR-γ) mediates the expression of antioxidant enzymes including catalase. β-Carotene, a red-orange pigment, exerts antioxidant and anti-inflammatory properties. We aimed to investigate whether β-carotene inhibits H. pylori-induced MMP expression and cell invasion in gastric epithelial AGS (gastric adenocarcinoma) cells. We found that H. pylori induced MMP-10 expression and increased cell invasion via the activation of MAPKs and AP-1 in gastric epithelial cells. Specific inhibitors of MAPKs suppressed H. pylori-induced MMP-10 expression, suggesting that H. pylori induces MMP-10 expression through MAPKs. β-Carotene inhibited the H. pylori-induced activation of MAPKs and AP-1, expression of MMP-10, and cell invasion. Additionally, it promoted the expression of PPAR-γ and catalase, which reduced ROS levels in H. pylori-infected cells. In conclusion, β-carotene exerts an inhibitory effect on MAPK-mediated MMP-10 expression and cell invasion by increasing PPAR-γ-mediated catalase expression and reducing ROS levels in H. pylori-infected gastric epithelial cells.     

Caragana rosea Turcz Methanol Extract Inhibits Lipopolysaccharide-Induced Inflammatory Responses by Suppressing the TLR4/NF-κB/IRF3 Signaling Pathways

Caragana rosea Turcz, which belongs to the Leguminosae family, is a small shrub found in Northern and Eastern China that is known to possess anti-inflammatory properties and is used to treat fever, asthma, and cough. However, the underlying molecular mechanisms of its anti-inflammatory effects are unknown. Therefore, we used lipopolysaccharide (LPS) in RAW264.7 macrophages to investigate the molecular mechanisms that underlie the anti-inflammatory activities of a methanol extract of Caragana rosea (Cr-ME). We showed that Cr-ME reduced the production of nitric oxide (NO) and mRNA levels of iNOS, TNF-α, and IL-6 in a concentration-dependent manner. We also found that Cr-ME blocked MyD88- and TBK1-induced NF-κB and IRF3 promoter activity, suggesting that it affects multiple targets. Moreover, Cr-ME reduced the phosphorylation levels of IκBα, IKKα/β and IRF3 in a time-dependent manner and regulated the upstream NF-κB proteins Syk and Src, and the IRF3 protein TBK1. Upon overexpression of Src and TBK1, Cr-ME stimulation attenuated the phosphorylation of the NF-κB subunits p50 and p65 and IRF3 signaling. Together, our results suggest that the anti-inflammatory activity of Cr-ME occurs by inhibiting the NF-κB and IRF3 signaling pathways.     

Design and Synthesis of New Benzo[d]oxazole-Based Derivatives and Their Neuroprotective Effects on β-Amyloid-Induced PC12 Cells

A series of novel synthetic substituted benzo[d]oxazole-based derivatives (5a–5v) exerted neuroprotective effects on β-amyloid (Aβ)-induced PC12 cells as a potential approach for the treatment of Alzheimer’s disease (AD). In vitro studies show that most of the synthesized compounds were potent in reducing the neurotoxicity of Aβ_25-35-induced PC12 cells at 5 μg/mL. We found that compound 5c was non-neurotoxic at 30 μg/mL and significantly increased the viability of Aβ_25-35-induced PC12 cells at 1.25, 2.5 and 5 μg/mL. Western blot analysis showed that compound 5c promoted the phosphorylation of Akt and glycogen synthase kinase (GSK-3β) and decreased the expression of nuclear factor-κB (NF-κB) in Aβ_25-35-induced PC12 cells. In addition, our findings demonstrated that compound 5c protected PC12 cells from Aβ_25-35-induced apoptosis and reduced the hyperphosphorylation of tau protein, and decreased the expression of receptor for AGE (RAGE), β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1), inducible nitric oxide synthase (iNOS) and Bcl-2-associated X protein/B-cell lymphoma 2 (Bax/Bcl-2) via Akt/GSK-3β/NF-κB signaling pathway. In vivo studies suggest that compound 5c shows less toxicity than donepezil in the heart and nervous system of zebrafish.     

Polyopes affinis Suppressed IFN-γ- and TNF-α-Induced Inflammation in Human Keratinocytes via Down-Regulation of the NF-κB and STAT1 Pathways

Polyopes affinis is a red algal species commonly found on the South coast and near Jeju Island, Korea. This study aimed to determine whether P. affinis extracts can inhibit the pathogenesis of T-helper-2 (Th2)-mediated inflammation in a human keratinocyte cell line of atopic dermatitis (AD). Cells were incubated with 10 ng/mL of interferon gamma (IFN-γ) and 10 ng/mL of tumor necrosis factor-alpha (TNF-α) at various concentrations of PAB (10, 30, and 60 µg/mL) and PAA (100, 500, and 1000 µg/mL) extracts. A gene-ontology (GO)-enrichment analysis revealed that PAB significantly enriched the genes associated with biological processes such as cell adhesion, immune response, inflammation, and chemokine-mediated pathways. PAB suppressed the expression of the secretory proteins and mRNAs that are associated with the thymus and the production of activation-regulated chemokines (TARC/CCL17) and macrophage-derived chemokines (MDC/CCL22). The effect of the extract on mitogen-activated protein kinases (MAPKs) was related to its inhibition of TARC/CCL17 and MDC/CCL22 production by blocking NF-κB and STAT1 activation. These results suggest that seaweed extract may improve AD by regulating pro-inflammatory chemokines. In conclusion, we first confirmed the existence of phloroglucinol, a polyphenol formed from a precursor called phlorotannin, which is present in PAB, and this result proved the possibility of PAB being used as a treatment for AD.