A Synthetic Loop Replacement Peptide That Blocks Canonical NF‐κB Signaling

Aberrant canonical NF‐κB signaling is implicated in diseases from autoimmune disorders to cancer. A major therapeutic challenge is the need for selective inhibition of the canonical pathway without impacting the many non‐canonical NF‐κB functions. Here we show that a selective peptide‐based inhibitor of canonical NF‐κB signaling, in which a hydrogen bond in the NBD peptide is synthetically replaced by a non‐labile bond, shows an about 10‐fold increased potency relative to the original inhibitor. Not only is this molecule, NBD2, a powerful tool for dissection of canonical NF‐κB signaling in disease models and healthy tissues, the success of the synthetic loop replacement suggests that the general strategy could be useful for discovering modulators of the many protein–protein interactions mediated by such structures.     

Plantamajoside Inhibits UVB and Advanced Glycation End Products‐Induced MMP‐1 Expression by Suppressing the MAPK and NF‐κB Pathways in HaCaT Cells

Photoaging and glycation stress are major causes of skin deterioration. Oxidative stress caused by ultraviolet B (UVB) irradiation can upregulate matrix metalloprotease 1 (MMP‐1), a major enzyme responsible for collagen damage in the skin. Advanced glycation end products (AGEs) accumulate via gradual formation from skin proteins, especially from long‐lived proteins such as dermal elastin and collagen. Plantamajoside (PM), isolated from Plantago asiatica, has various biological effects including anti‐inflammatory and antioxidant effects. In this study, we assessed the protective effects of PM on a human keratinocyte cell line (HaCaT) and primary human dermal fibroblasts (HDF) against stress caused by glyceraldehyde‐induced AGEs (glycer‐AGEs) with UVB irradiation. We found that PM attenuated UVB‐ and‐glycer‐AGEs‐induced MMP‐1 expression in HaCaT and HDF cells and proinflammatory cytokines expression by inhibiting the phosphorylation of mitogen‐activated protein kinases (MAPKs) activated by reactive oxygen species. Specific inhibitors of NF‐κB and MAPKs attenuated the induced expression of MMP‐1. PM also inhibited the phosphorylation of IκBα, and reduced nuclear translocation of NF‐κB in these cells. Furthermore, PM attenuated the upregulation of receptor for AGEs (RAGE) by glycer‐AGEs with UVB irradiation. Therefore, our findings strongly suggest that PM is a promising inhibitor of skin photoaging.     

Tabersonine Inhibits the Lipopolysaccharide-Induced Neuroinflammatory Response in BV2 Microglia Cells via the NF-κB Signaling Pathway

The occurrence and development of neurodegenerative diseases is related to a variety of physiological and pathological changes. Neuroinflammation is one of the major factors that induces and aggravates neurodegenerative diseases. The most important manifestation of neuroinflammation is the activation of microglia. Therefore, inhibiting the abnormal activation of microglia is an important way to alleviate the occurrence of neuroinflammatory diseases. In this research, the inhibitory effect of tabersonine (Tab) on neuroinflammation was evaluated by establishing the BV2 neuroinflammation model induced by lipopolysaccharide (LPS). It was found that Tab significantly inhibited the production and expression of nitric oxide (NO), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and reactive oxygen species (ROS) in BV-2 cells stimulated by LPS. In addition, Tab can also inhibit the activation of nuclear factor-κB (NF-κB) induced by LPS, thus regulating inflammatory mediators such as inducible nitric oxide synthase (iNOS). These results indicated that Tab regulated the release of inflammatory mediators such as NO, IL-1β, TNF-α, and IL-6 by inhibiting NF-κB signaling pathway, and exerting its anti-neuroinflammatory effect. This is the first report regarding the inhibition on LPS-induced neuroinflammation in BV2 microglia cells of Tab, which indicated the drug development potential of Tab for the treatment of neurodegenerative diseases.     

Impaired Chaperone Activity of Human Heat Shock Protein Hsp27 Site‐Specifically Modified with Argpyrimidine

Non‐enzymatic posttranslational modifications (nPTMs) affect at least ～30 % of human proteins, but our understanding of their impact on protein structure and function is limited. Studies of nPTMs are difficult because many modifications are not included in common chemical libraries or protein expression systems and should be introduced site‐specifically. Herein, we probed the effect of the nPTM argpyrimidine on the structure and function of human protein Hsp27, which acquires argpyrimidine at residue 188 in vivo. We developed a synthetic approach to an argpyrimidine building block, which we then incorporated at position 188 of Hsp27 through protein semisynthesis. This modification did not affect the protein secondary structure, but perturbed the oligomeric assembly and impaired chaperone activity. Our work demonstrates that protein function can be altered by a single nPTM and opens up a new area of investigation only accessible by methods that allow site‐selective protein modification.     

Resatorvid‐based Pharmacological Antagonism of Cutaneous TLR4 Blocks UV‐induced NF‐κB and AP‐1 Signaling in Keratinocytes and Mouse Skin

Cutaneous exposure to solar ultraviolet (UV) radiation is a major causative factor in skin carcinogenesis, and improved molecular strategies for efficacious chemoprevention of nonmelanoma skin cancer (NMSC) are urgently needed. Toll‐like receptor 4 (TLR4) signaling has been shown to drive skin inflammation, photoimmunosuppression, and chemical carcinogenesis. Here we have examined the feasibility of genetic and pharmacological antagonism targeting cutaneous TLR4 for the suppression of UV‐induced NF‐κB and AP‐1 signaling in keratinocytes and mouse skin. Using immunohistochemical and proteomic microarray analysis of human skin, we demonstrate for the first time that a significant increase in expression of TLR4 occurs in keratinocytes during the progression from normal skin to actinic keratosis, also detectible during further progression to squamous cell carcinoma. Next, we demonstrate that siRNA‐based genetic TLR4 inhibition blocks UV‐induced stress signaling in cultured keratinocytes. Importantly, we observed that resatorvid (TAK‐242), a molecularly targeted clinical TLR4 antagonist, blocks UV‐induced NF‐κB and MAP kinase/AP‐1 activity and cytokine expression (Il‐6, Il‐8, and Il‐10) in cultured keratinocytes and in topically treated murine skin. Taken together, our data reveal that pharmacological TLR4 antagonism can suppress UV‐induced cutaneous signaling, and future experiments will explore the potential of TLR4‐directed strategies for prevention of NMSC.     

Screening and identification of Caulis Sinomenii bioactive ingredients with dual‐target NF‐κB inhibition and β2‐AR agonizing activities

Caulis Sinomenii (CS) is a valuable traditional medicine in China. Its extract can act as an anti‐inflammatory agent and a vascular smooth muscle relaxant. However, the underlying mechanisms remain unknown. In this study, we developed a simple dual‐target method based on ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry combined with a dual‐target bioactive screening assay for anti‐inflammatory and antispasmodic activities to characterize the chemical structure of various bioactive compounds of CS rapidly. Seven potential NF‐κB inhibitors were identified, including laudanosoline‐1‐O‐xylopyranose, 6‐O‐methyl‐laudanosoline‐1‐O‐glucopyranoside, menisperine, sinomenine, laurifoline, magnoflorine and norsinoacutin. Furthermore, IL‐6 and IL‐8 assays confirmed the anti‐inflammatory effects of these potential NF‐κB inhibitors, in which laudanosoline‐1‐O‐d ‐xylopyranose and menisperine were revealed as novel NF‐κB inhibitors. Among the seven identified alkaloids, three potential β₂‐adrenergic receptor agonists, including sinomenine, magnoflorine and laurifoline, were characterized using a luciferase reporter system to measure for the activity of β₂‐adrenergic receptor agonists. Finally, sinomenine, magnoflorine and laurifoline were identified not only as potential NF‐κB inhibitors but also as potential β₂‐adrenegic receptor agonists, which is the first time this has been reported. Molecular dynamic simulation and docking results suggest that the three dual‐bioactive constituents could not only inhibit Pseudomonas aeruginosa PAK strain‐induced inflammatory responses via a negative regulation of the Braf protein that participates in MAPK signaling pathway but also activate the β₂‐adrenegic receptor. These results suggest that CS extract has dual signaling activities with potential clinical application as a novel drug for asthma.     

Fisetin Inhibits UVB‐induced Cutaneous Inflammation and Activation of PI3K/AKT/NFκB Signaling Pathways in SKH‐1 Hairless Mice

Solar ultraviolet B (UVB) radiation has been shown to induce inflammation, DNA damage, p53 mutations and alterations in signaling pathways eventually leading to skin cancer. In this study, we investigated whether fisetin reduces inflammatory responses and modulates PI3K/AKT/NFκB cell survival signaling pathways in UVB‐exposed SKH‐1 hairless mouse skin. Mice were exposed to 180 mJ cm^?2 of UVB radiation on alternate days for a total of seven exposures, and fisetin (250 and 500 nmol) was applied topically after 15 min of each UVB exposure. Fisetin treatment to UVB‐exposed mice resulted in decreased hyperplasia and reduced infiltration of inflammatory cells. Fisetin treatment also reduced inflammatory mediators such as COX‐2, PGE₂ as well as its receptors (EP1–EP4) and MPO activity. Furthermore, fisetin reduced the level of inflammatory cytokines TNFα, IL‐1β and IL‐6 in UVB‐exposed skin. Fisetin treatment also reduced cell proliferation markers as well as DNA damage as evidenced by increased expression of p53 and p21 proteins. Further studies revealed that fisetin inhibited UVB‐induced expression of PI3K, phosphorylation of AKT and activation of the NFκB signaling pathway in mouse skin. Overall, these data suggest that fisetin may be useful against UVB‐induced cutaneous inflammation and DNA damage.     

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.     

Prunetin 4′-O-Phosphate,a Novel Compound,in RAW 264.7 Macrophages Exerts Anti-Inflammatory Activity via Suppression of MAP Kinases and the NFκB Pathway

Biorenovation, a microbial enzyme-assisted degradation process of precursor compounds, is an effective approach to unraveling the potential bioactive properties of the derived compounds. In this study, we obtained a new compound, prunetin 4′-O-phosphate (P4P), through the biorenovation of prunetin (PRN), and investigated its anti-inflammatory effects in lipopolysaccharide (LPS)-treated RAW 264.7 macrophage cells. The anti-inflammatory effect of P4P was evaluated by measuring the production of prostaglandin-E₂ (PGE₂), nitric oxide (NO), which is an inflammation-inducing factor, and related cytokines such as tumor necrosis factor-α (TNFα), interleukin-1β (IL1β), and interleukin-6 (IL6). The findings demonstrated that P4P was non-toxic to cells, and its inhibition of the secretion of NO—as well as pro-inflammatory cytokines—was concentration-dependent. A simultaneous reduction in the protein expression level of pro-inflammatory proteins such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) was observed. Moreover, the phosphorylation of mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase (JNK), p38 MAPK (p38), and nuclear factor kappa B (NFκB) was downregulated. To conclude, we report that biorenovation-based phosphorylation of PRN improved its anti-inflammatory activity. Cell-based in vitro assays further confirmed that P4P could be applied in the development of anti-inflammatory therapeutics.     

Luteolin-3′-O-Phosphate Inhibits Lipopolysaccharide-Induced Inflammatory Responses by Regulating NF-κB/MAPK Cascade Signaling in RAW 264.7 Cells

Luteolin (LT), present in most plants, has potent anti-inflammatory properties both in vitro and in vivo. Furthermore, some of its derivatives, such as luteolin-7-O-glucoside, also exhibit anti-inflammatory activity. However, the molecular mechanisms underlying luteolin-3′-O-phosphate (LTP)-mediated immune regulation are not fully understood. In this paper, we compared the anti-inflammatory properties of LT and LTP and analyzed their molecular mechanisms of action; we obtained LTP via the biorenovation of LT. We investigated the anti-inflammatory activities of LT and LTP in macrophage RAW 264.7 cells. We confirmed from previously reported literature that LT inhibits the production of nitric oxide and prostaglandin E₂, as well as the expression of inducible NO synthetase and cyclooxygenase-2. In addition, expressions of inflammatory genes and mediators, such as tumor necrosis factor-α, interleukin-6, and interleukin-1β, were suppressed. LTP showed anti-inflammatory activity similar to LT, but better anti-inflammatory activity in all the experiments, while also inhibiting mitogen-activated protein kinase and nuclear factor-kappa B more effectively than LT. At a concentration of 10 μM, LTP showed differences of 2.1 to 44.5% in the activity compared to LT; it also showed higher anti-inflammatory activity. Our findings suggest that LTP has stronger anti-inflammatory activity than LT.     

Bioactivity‐based ultra‐performance liquid chromatography‐coupled quadrupole time‐of‐flight mass spectrometry for NF‐κB inhibitors identification in Chinese Medicinal Preparation Bufei Granule

Traditional Chinese medicine (TCM) preparations have become effective treatments for many diseases. However, their active ingredients are still uncertain and difficult to identify. In this study, we propose a strategy that integrates ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry (UPLC/Q‐TOF‐MS) and bioactive (NF‐κB inhibitor) luciferase reporter assay systems for the rapid determination of various anti‐inflammatory compounds of TCM preparations. In this way, Bufei Granule (BFG), a TCM preparation used for the clinical therapy of asthma, was analyzed by the two ways of component identification and activity detection. Potential anti‐inflammatory constituents were screened by NF‐κB activity assay systems and simultaneously identified according to the mass spectrometry data. Three structural types of NF‐κB inhibitors (caffeic acid derivatives, flavonoids and Pentacyclic triterpenes) were characterized. Further cytokine detection confirmed the anti‐inflammatory effects of the potential NF‐κB inhibitors. Compared with conventional chromatographic separation and inhibitory activity detection, integrating UPLC/Q‐TOF‐MS identification and virtual validation was more convenient and more reliable. This strategy clearly demonstrates that MS data‐based fingerprinting is a meaningful tool not only in identifying constituents in complex matrix but also in directly screening for powerful trace ingredients in TCM preparations. Copyright © 2016 John Wiley & Sons, Ltd.     

Dexamethasone Attenuates Oncostatin M Production via Suppressing of PI3K/Akt/NF-κB Signaling in Neutrophil-like Differentiated HL-60 Cells

Oncostatin M (OSM) plays a role in various inflammatory reactions, and neutrophils are the main source of OSM in pulmonary diseases. However, there is no evidence showing the mechanism of OSM production in neutrophils. While dexamethasone (Dex) has been known to exert anti-inflammatory activity in various fields, the precise mechanisms of OSM downregulation by Dex in neutrophils remain to be determined. Here, we examined how OSM is produced in neutrophil-like differentiated HL-60 cells. Enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and Western blot analysis were utilized to assess the potential of Dex. Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation resulted in OSM elevation in neutrophil-like dHL-60 cells. OSM elevation induced by GM-CSF is regulated by phosphatidylinositol 3-kinase (PI3K)/Akt/nuclear factor (NF)-kB signal cascades. GM-CSF stimulation upregulated phosphorylated levels of PI3K or Akt or NF-κB in neutrophil-like dHL-60 cells. Treatment with Dex decreased OSM levels as well as the phosphorylated levels of PI3K or Akt or NF-κB in neutrophil-like dHL-60 cells. Our findings show the potential of Dex in the treatment of inflammatory diseases via blocking of OSM.     

Pharmacokinetics,protein binding and metabolism of a quinoxaline urea analog as an NF‐κB inhibitor in mice and rats by LC‐MS/MS

13–197 is a novel NF‐κB inhibitor that shows promising in vitro efficacy data against pancreatic cancer. In this study, we characterized the pharmacokinetics, tissue distribution, protein binding and metabolism of 13–197 in mice and rats. A valid, sensitive and selective LC‐MS/MS method was developed. This method was validated for the quantification of 13–197, in the range of 0.1 or 0.2‐500 ng/mL in mouse plasma, liver, kidney, lung, heart, spleen, brain, urine and feces. 13–197 has low bioavailability of 3 and 16% in mice and rats, respectively. It has faster absorption in mice with 12‐fold shorter T_max than in rats. Tissue concentrations were 1.3–69.2‐fold higher in mice than in rats at 72 h after intravenous administration. 13–197 is well distributed to the peripheral tissues and has relatively high tissue–plasma concentration ratios, ranging from 1.8 to 3634, in both mice and rats. It also demonstrated more than 99% binding to plasma proteins in both mice and rats. Finally, <1% of 13–197 is excreted unchanged in urine and feces, and metabolite profiling studies detected more than 20 metabolites in mouse and rat plasma, urine and feces, which indicates that 13–197 is extensively metabolized and primarily eliminated by metabolism rather than by excretion. Copyright © 2013 John Wiley & Sons, Ltd.     

Anti-Hair Loss Effect of Adenosine Is Exerted by cAMP Mediated Wnt/β-Catenin Pathway Stimulation via Modulation of Gsk3β Activity in Cultured Human Dermal Papilla Cells

In the present study, we investigated the molecular mechanisms of adenosine for its hair growth promoting effect. Adenosine stimulated the Wnt/β-catenin pathway by modulating the activity of Gsk3β in cultured human dermal papilla cells. It also activated adenosine receptor signaling, increasing intracellular cAMP level, and subsequently stimulating the cAMP mediated cellular energy metabolism. The phosphorylation of CREB, mTOR, and GSK3β was increased. Furthermore, the expression of β-catenin target genes such as Axin2, Lef1, and growth factors (bFGF, FGF7, IGF-1) was also enhanced. The inhibitor study data conducted in Wnt reporter cells and in cultured human dermal papilla cells demonstrated that adenosine stimulates Wnt/β-catenin signaling through the activation of the adenosine receptor and Gsk3β plays a critical role in transmitting the signals from the adenosine receptor to β-catenin, possibly via the Gαs/cAMP/PKA/mTOR signaling cascade.