The Use of ProteoTuner Technology to Study Nuclear Factor κB Activation by Heavy Ions

Nuclear factor κB (NF-κB) activation might be central to heavy ion-induced detrimental processes such as cancer promotion and progression and sustained inflammatory responses. A sensitive detection system is crucial to better understand its involvement in these processes. Therefore, a DD-tdTomato fluorescent protein-based reporter system was previously constructed with human embryonic kidney (HEK) cells expressing DD-tdTomato as a reporter under the control of a promoter containing NF-κB binding sites (HEK-pNFκB-DD-tdTomato-C8). Using this reporter cell line, NF-κB activation after exposure to different energetic heavy ions (¹⁶O, 95 MeV/n, linear energy transfer—LET 51 keV/µm; ¹²C, 95 MeV/n, LET 73 keV/μm; ³⁶Ar, 95 MeV/n, LET 272 keV/µm) was quantified considering the dose and number of heavy ions hits per cell nucleus that double NF-κB-dependent DD-tdTomato expression. Approximately 44 hits of ¹⁶O ions and ≈45 hits of ¹²C ions per cell nucleus were required to double the NF-κB-dependent DD-tdTomato expression, whereas only ≈3 hits of ³⁶Ar ions were sufficient. In the presence of Shield-1, a synthetic molecule that stabilizes DD-tdTomato, even a single particle hit of ³⁶Ar ions doubled NF-κB-dependent DD-tdTomato expression. In conclusion, stabilization of the reporter protein can increase the sensitivity for NF-κB activation detection by a factor of three, allowing the detection of single particle hits’ effects.     

Kisspeptin-10 Rescues Cholinergic Differentiated SHSY-5Y Cells from α-Synuclein-Induced Toxicity In Vitro

The neuropathological substrate of dementia with Lewy bodies (DLB) is defined by the inextricable cross-seeding accretion of amyloid-β (Aβ) and α-synuclein (α-syn)-laden deposits in cholinergic neurons. The recent revelation that neuropeptide kisspeptin-10 (KP-10) is able to mitigate Aβ toxicity via an extracellular binding mechanism may provide a new horizon for innovative drug design endeavors. Considering the sequence similarities between α-syn’s non-amyloid-β component (NAC) and Aβ’s C-terminus, we hypothesized that KP-10 would enhance cholinergic neuronal resistance against α-syn’s deleterious consequences through preferential binding. Here, human cholinergic SH-SY5Y cells were transiently transformed to upsurge the mRNA expression of α-syn while α-syn-mediated cholinergic toxicity was quantified utilizing a standardized viability-based assay. Remarkably, the E46K mutant α-syn displayed elevated α-syn mRNA levels, which subsequently induced more cellular toxicity compared with the wild-type α-syn in choline acetyltransferase (ChAT)-positive cholinergic neurons. Treatment with a high concentration of KP-10 (10 µM) further decreased cholinergic cell viability, while low concentrations of KP-10 (0.01–1 µM) substantially suppressed wild-type and E46K mutant α-syn-mediated toxicity. Correlating with the in vitro observations are approximations from in silico algorithms, which inferred that KP-10 binds favorably to the C-terminal residues of wild-type and E46K mutant α-syn with CDOCKER energy scores of −118.049 kcal/mol and −114.869 kcal/mol, respectively. Over the course of 50 ns simulation time, explicit-solvent molecular dynamics conjointly revealed that the docked complexes were relatively stable despite small-scale fluctuations upon assembly. Taken together, our findings insinuate that KP-10 may serve as a novel therapeutic scaffold with far-reaching implications for the conceptualization of α-syn-based treatments.     

Inhibitory Effect of Avenanthramides (Avn) on Tyrosinase Activity and Melanogenesis in α-MSH-Activated SK-MEL-2 Cells: In Vitro and In Silico Analysis

Melanin causes melasma, freckles, age spots, and chloasma. Anti-melanogenic agents can prevent disease-related hyperpigmentation. In the present study, the dose-dependent tyrosinase inhibitory activity of Avenanthramide (Avn)-A-B-C was demonstrated, and 100 µM Avn-A-B-C produced the strongest competitive inhibition against inter-cellular tyrosinase and melanin synthesis. Avn-A-B-C inhibits the expression of melanogenesis-related proteins, such as TRP1 and 2. Molecular docking simulation revealed that AvnC (−7.6 kcal/mol) had a higher binding affinity for tyrosinase than AvnA (−7.3 kcal/mol) and AvnB (−6.8 kcal/mol). AvnC was predicted to interact with tyrosinase through two hydrogen bonds at Ser360 (distance: 2.7 Å) and Asn364 (distance: 2.6 Å). In addition, AvnB and AvnC were predicted to be skin non-sensitizers in mammals by the Derek Nexus Quantitative Structure–Activity Relationship system.     

The Pro-Inflammatory Deletion Allele of the NF-κB1 Polymorphism Is Characterized by a Depletion of Subunit p50 in Sepsis

The functionally important NF-κB1 promoter polymorphism (−94ins/delATTG) significantly shapes inflammation and impacts the outcome of sepsis. However, exploratory studies elucidating the molecular link of this genotype-dependent pattern are lacking. Accordingly, we analyzed lipopolysaccharide-stimulated peripheral blood mononuclear cells from both healthy volunteers (n = 20) and septic patients (n = 10). All individuals were genotyped for the −94ins/delATTG NF-κB1 promoter polymorphism. We found a diminished nuclear activity of the NF-κB subunit p50 in ID/DD genotypes after 48 h of lipopolysaccharide stimulation compared to II genotypes (p = 0.025). This was associated with higher TNF-α (p = 0.005) and interleukin 6 concentrations (p = 0.014) and an increased production of mitochondrial radical oxygen species in ID/DD genotypes (p = 0.001). Although ID/DD genotypes showed enhanced activation of mitochondrial biogenesis, they still had a significantly diminished cellular ATP content (p = 0.046) and lower mtDNA copy numbers (p = 0.010) compared to II genotypes. Strikingly, these findings were mirrored in peripheral blood mononuclear cells taken from septic patients. Our results emphasize the crucial aspect of considering NF-κB subunits in sepsis. We showed here that the deletion allele of the NF-κB1 (−94ins/delATTG) polymorphism was associated with the lower nuclear activity of subunit p50, which, in turn, was associated with aggravated inflammation and mitochondrial dysfunction.     

Advanced Glycation End-Products Enhance Lung Cancer Cell Invasion and Migration

Effects of carboxymethyllysine (CML) and pentosidine, two advanced glycation end-products (AGEs), upon invasion and migration in A549 and Calu-6 cells, two non-small cell lung cancer (NSCLC) cell lines were examined. CML or pentosidine at 1, 2, 4, 8 or 16 μmol/L were added into cells. Proliferation, invasion and migration were measured. CML or pentosidine at 4–16 μmol/L promoted invasion and migration in both cell lines, and increased the production of reactive oxygen species, tumor necrosis factor-α, interleukin-6 and transforming growth factor-β1. CML or pentosidine at 2–16 μmol/L up-regulated the protein expression of AGE receptor, p47^phox, intercellular adhesion molecule-1 and fibronectin in test NSCLC cells. Matrix metalloproteinase-2 protein expression in A549 and Calu-6 cells was increased by CML or pentosidine at 4–16 μmol/L. These two AGEs at 2–16 μmol/L enhanced nuclear factor κ-B (NF-κ B) p65 protein expression and p38 phosphorylation in A549 cells. However, CML or pentosidine at 4–16 μmol/L up-regulated NF-κB p65 and p-p38 protein expression in Calu-6 cells. These findings suggest that CML and pentosidine, by promoting the invasion, migration and production of associated factors, benefit NSCLC metastasis.     

GSK343, an Inhibitor of Enhancer of Zeste Homolog 2, Reduces Glioblastoma Progression through Inflammatory Process Modulation: Focus on Canonical and Non-Canonical NF-κB/IκBα Pathways

Glioblastoma (GB) is a tumor of the central nervous system characterized by high proliferation and invasiveness. The standard treatment for GB includes radiotherapy and chemotherapy; however, new therapies are needed. Particular attention was given to the role of histone methyltransferase enhancer of zeste-homolog-2 (EZH2) in GB. Recently, several EZH2-inhibitors have been developed, particularly GSK343 is well-known to regulate apoptosis and autophagy processes; however, its abilities to modulate canonical/non-canonical NF-κB/IκBα pathways or an immune response in GB have not yet been investigated. Therefore, this study investigated for the first time the effect of GSK343 on canonical/non-canonical NF-κB/IκBα pathways and the immune response, by an in vitro, in vivo and ex vivo model of GB. In vitro results demonstrated that GSK343 treatments 1, 10 and 25 μM significantly reduced GB cell viability, showing the modulation of canonical/non-canonical NF-κB/IκBα pathway activation. In vivo GSK343 reduced subcutaneous tumor mass, regulating canonical/non-canonical NF-κB/IκBα pathway activation and the levels of reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD). Ex vivo results confirmed the anti-proliferative effect of GSK343 and also demonstrated its ability to regulate immune response through CXCL9, CXCL10 and CXCL11 expression in GB. Thus, GSK343 could represent a therapeutic strategy to counteract GB progression, thanks to its ability to modulate canonical/non-canonical NF-κB/IκBα pathways and immune response.     

14-3-3γ Regulates Lipopolysaccharide-Induced Inflammatory Responses and Lactation in Dairy Cow Mammary Epithelial Cells by Inhibiting NF-κB and MAPKs and Up-Regulating mTOR Signaling

As a protective factor for lipopolysaccharide (LPS)-induced injury, 14-3-3γ has been the subject of recent research. Nevertheless, whether 14-3-3γ can regulate lactation in dairy cow mammary epithelial cells (DCMECs) induced by LPS remains unknown. Here, the anti-inflammatory effect and lactation regulating ability of 14-3-3γ in LPS-induced DCMECs are investigated for the first time, and the molecular mechanisms responsible for their effects are explored. The results of qRT-PCR showed that 14-3-3γ overexpression significantly inhibited the mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS). Enzyme-linked immunosorbent assay (ELISA) analysis revealed that 14-3-3γ overexpression also suppressed the production of TNF-α and IL-6 in cell culture supernatants. Meanwhile, CASY-TT Analyser System showed that 14-3-3γ overexpression clearly increased the viability and proliferation of cells. The results of kit methods and western blot analysis showed that 14-3-3γ overexpression promoted the secretion of triglycerides and lactose and the synthesis of β-casein. Furthermore, the expression of genes relevant to nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPKs) and lactation-associated proteins were assessed by western blot, and the results suggested that 14-3-3γ overexpression inactivated the NF-κB and MAPK signaling pathways by down-regulating extracellular signal regulated protein kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK) and inhibitor of NF-κB (IκB) phosphorylation levels, as well as by inhibiting NF-κB translocation. Meanwhile, 14-3-3γ overexpression enhanced the expression levels of β-casein, mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), serine/threonine protein kinase Akt 1 (AKT1), sterol regulatory element binding protein 1 (SREBP1) and peroxisome proliferator-activated receptor gamma (PPARγ). These results suggest that 14-3-3γ was able to attenuate the LPS-induced inflammatory responses and promote proliferation and lactation in LPS-induced DCMECs by inhibiting the activation of the NF-κB and MAPK signaling pathways and up-regulating mTOR signaling pathways to protect against LPS-induced injury.     

Protective Role of Andrographolide in Bleomycin-Induced Pulmonary Fibrosis in Mice

Idiopathic pulmonary fibrosis (IPF) is a chronic devastating disease with poor prognosis. Multiple pathological processes, including inflammation, epithelial mesenchymal transition (EMT), apoptosis, and oxidative stress, are involved in the pathogenesis of IPF. Recent findings suggested that nuclear factor-κB (NF-κB) is constitutively activated in IPF and acts as a central regulator in the pathogenesis of IPF. The aim of our study was to reveal the value of andrographolide on bleomycin-induced inflammation and fibrosis in mice. The indicated dosages of andrographolide were administered in mice with bleomycin-induced pulmonary fibrosis. On day 21, cell counts of total cells, macrophages, neutrophils and lymphocytes, alone with TNF-α in bronchoalveolar lavage fluid (BALF) were measured. HE staining and Masson’s trichrome (MT) staining were used to observe the histological alterations of lungs. The Ashcroft score and hydroxyproline content of lungs were also measured. TGF-β1 and α-SMA mRNA and protein were analyzed. Activation of NF-κB was determined by western blotting and electrophoretic mobility shift assay (EMSA). On day 21 after bleomycin stimulation, andrographolide dose-dependently inhibited the inflammatory cells and TNF-α in BALF. Meanwhile, our data demonstrated that the Ashcroft score and hydroxyproline content of the bleomycin-stimulated lung were reduced by andrographolide administration. Furthermore, andrographloide suppressed TGF-β1 and α-SMA mRNA and protein expression in bleomycin-induced pulmonary fibrosis. Meanwhile, andrographolide significantly dose-dependently inhibited the ratio of phospho-NF-κB p65/total NF-κB p65 and NF-κB p65 DNA binding activities. Our findings indicate that andrographolide compromised bleomycin-induced pulmonary inflammation and fibrosis possibly through inactivation of NF-κB. Andrographolide holds promise as a novel drug to treat the devastating disease of pulmonary fibrosis.     

Protective Effect of Resveratrol against IL-1β-Induced Inflammatory Response on Human Osteoarthritic Chondrocytes Partly via the TLR4/MyD88/NF-κB Signaling Pathway: An “in Vitro Study”

Resveratrol is a natural polyphenolic compound that prevents inflammation in chondrocytes and animal models of osteoarthritis (OA) via yet to be defined mechanisms. The purpose of this study was to determine whether the protective effect of resveratrol on IL-1β-induced human articular chondrocytes was associated with the TLR4/MyD88/NF-κB signaling pathway by incubating human articular chondrocytes (harvested from osteoarthritis patients) with IL-1β before treatment with resveratrol. Cell viability was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and TNFα levels in culture supernatants were measured by ELISA(Enzymelinked immunosorbent assay). The levels of TLR4 and its downstream signaling targets (MyD88 and TRAF6) and IL-1β were assessed by measuring the levels of mRNA and protein expression by real-time RT-PCR and western blot analysis, respectively, in addition to assessing NF-κB activation. In addition, TLR4 siRNA was used to block TLR4 expression in chondrocytes further demonstrating that resveratrol prevented IL-1β-mediated inflammation by TLR4 inhibition. We found that resveratrol prevented IL-1β-induced reduction in cell viability. Stimulation of chondrocytes with IL-1β caused a significant up-regulation of TLR4 and its downstream targets MyD88 and TRAF6 resulting in NF-κB activation associated with the synthesis of IL-1β and TNFα. These IL-1β-induced inflammatory responses were all effectively reversed by resveratrol. Furthermore, activation of NF-κB in chondrocytes treated with TLR4 siRNA was significantly attenuated, but not abolished, and exposure to resveratrol further reduced NF-κB translocation. These data suggested that resveratrol prevented IL-1β-induced inflammation in human articular chondrocytes at least in part by inhibiting the TLR4/MyD88/NF-κB signaling pathway suggesting that resveratrol has the potential to be used as a nutritional supplement to counteract OA symptoms.     

Indigo Pulverata Levis (Chung-Dae,Persicaria tinctoria) Alleviates Atopic Dermatitis-like Inflammatory Responses In Vivo and In Vitro

Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with a type 2 T helper cell (Th2) immune response. The Indigo Pulverata Levis extract (CHD) is used in traditional Southeast Asian medicine; however, its beneficial effects on AD remain uninvestigated. Therefore, we investigated the therapeutic effects of CHD in 2,4-dinitrochlorobenzene (DNCB)-induced BALB/c mice and tumor necrosis factor (TNF)-α- and interferon gamma (IFN)-γ-stimulated HaCaT cells. We evaluated immune cell infiltration, skin thickness, and the serum IgE and TNF-α levels in DNCB-induced AD mice. Moreover, we measured the expression levels of pro-inflammatory cytokines, mitogen-activated protein kinase (MAPK), and the nuclear factor-kappa B (NF-κB) in the mice dorsal skin. We also studied the effect of CHD on the translocation of NF-κB p65 and inflammatory chemokines in HaCaT cells. Our in vivo results revealed that CHD reduced the dermis and epidermis thicknesses and inhibited immune cell infiltration. Furthermore, it suppressed the proinflammatory cytokine expression and MAPK and NF-κB phosphorylations in the skin tissue and decreased serum IgE and TNF-α levels. In vitro results indicated that CHD downregulated inflammatory chemokines and blocked NF-κB p65 translocation. Thus, we deduced that CHD is a potential drug candidate for AD treatment.     

Linear Ubiquitination Mediates EGFR-Induced NF-κB Pathway and Tumor Development

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that instigates several signaling cascades, including the NF-κB signaling pathway, to induce cell differentiation and proliferation. Overexpression and mutations of EGFR are found in up to 30% of solid tumors and correlate with a poor prognosis. Although it is known that EGFR-mediated NF-κB activation is involved in tumor development, the signaling axis is not well elucidated. Here, we found that plakophilin 2 (PKP2) and the linear ubiquitin chain assembly complex (LUBAC) were required for EGFR-mediated NF-κB activation. Upon EGF stimulation, EGFR recruited PKP2 to the plasma membrane, and PKP2 bridged HOIP, the catalytic E3 ubiquitin ligase in the LUBAC, to the EGFR complex. The recruitment activated the LUBAC complex and the linear ubiquitination of NEMO, leading to IκB phosphorylation and subsequent NF-κB activation. Furthermore, EGF-induced linear ubiquitination was critical for tumor cell proliferation and tumor development. Knockout of HOIP impaired EGF-induced NF-κB activity and reduced cell proliferation. HOIP knockout also abrogated the growth of A431 epidermal xenograft tumors in nude mice by more than 70%. More importantly, the HOIP inhibitor, HOIPIN-8, inhibited EGFR-mediated NF-κB activation and cell proliferation of A431, MCF-7, and MDA-MB-231 cancer cells. Overall, our study reveals a novel linear ubiquitination signaling axis of EGFR and that perturbation of HOIP E3 ubiquitin ligase activity is potential targeted cancer therapy.     

Human Caspase 12 Enhances NF-κB Activity through Activation of IKK in Nasopharyngeal Carcinoma Cells

Human nasopharyngeal carcinoma (NPC) is a highly invasive cancer associated with proinflammation. Caspase-12 (Casp12), an inflammatory caspase, is implicated in the regulation of NF-κB-mediated cellular invasion via the modulation of the IκBα protein in NPC cells. However, the effect mechanisms of Casp12 need to be elucidated. NPC cells were transfected with the full length of human Casp12 cDNA (pC12) and the effect of human Casp12 (hCasp12) on the NF-κB activity was investigated. We found ectopic expression of hCasp12 increased the NF-κB activity accompanied by an increased p-IκBα expression and a decreased IκBα expression. Treatment of BMS, a specific IKK inhibitor, and pC12-transfected cells markedly decreased the NF-κB activity and ameliorated the expression level of IκBα reduced by hCasp12. Co-immunoprecipitation assays validated the physical interaction of hCasp12 with IKKα/β, but not with NEMO. Furthermore, the NF-κB activity of ΔCasp12-Q (a mutated catalytic of hCasp12) transfected cells was concentration-dependently induced, but lower than that of hCasp12-transfected cells. Importantly, the hCasp12-mediated NF-kB activity was enhanced by TNFα stimulation. That indicated a role of the catalytic motif of hCasp12 in the regulation of the NF-κB activity. This study indicated hCasp12 activated the NF-κB pathway through the activation of IKK in human NPC cells.     

Inhibitory Effects of Saururus chinensis Extract on Receptor for Advanced Glycation End-Products-Dependent Inflammation and Diabetes-Induced Dysregulation of Vasodilation

Advanced glycation end-products (AGEs) and the receptor for AGEs (RAGE) are implicated in inflammatory reactions and vascular complications in diabetes. Signaling pathways downstream of RAGE are involved in NF-κB activation. In this study, we examined whether ethanol extracts of Saururus chinensis (Lour.) Baill. (SE) could affect RAGE signaling and vascular relaxation in streptozotocin (STZ)-induced diabetic rats. Treatment with SE inhibited AGEs-modified bovine serum albumin (AGEs-BSA)-elicited activation of NF-κB and could compete with AGEs-BSA binding to RAGE in a dose-dependent manner. Tumor necrosis factor-α (TNF-α) secretion induced by lipopolysaccharide (LPS)—a RAGE ligand—was also reduced by SE treatment in wild-type Ager^+/+ mice as well as in cultured peritoneal macrophages from Ager^+/+ mice but not in Ager^−/− mice. SE administration significantly ameliorated diabetes-related dysregulation of acetylcholine-mediated vascular relaxation in STZ-induced diabetic rats. These results suggest that SE would inhibit RAGE signaling and would be useful for the improvement of vascular endothelial dysfunction in diabetes.     

Cucurbitacin B Down-Regulates TNF Receptor 1 Expression and Inhibits the TNF-α-Dependent Nuclear Factor κB Signaling Pathway in Human Lung Adenocarcinoma A549 Cells

Pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), induce the expression of intracellular adhesion molecule-1 (ICAM-1) by activating the nuclear factor κB (NF-κB) signaling pathway. In the present study, we found that cucurbitacin B decreased the expression of ICAM-1 in human lung adenocarcinoma A549 cells stimulated with TNF-α or interleukin-1α. We further investigated the mechanisms by which cucurbitacin B down-regulates TNF-α-induced ICAM-1 expression. Cucurbitacin B inhibited the nuclear translocation of the NF-κB subunit RelA and the phosphorylation of IκBα in A549 cells stimulated with TNF-α. Cucurbitacin B selectively down-regulated the expression of TNF receptor 1 (TNF-R1) without affecting three adaptor proteins (i.e., TRADD, RIPK1, and TRAF2). The TNF-α-converting enzyme inhibitor suppressed the down-regulation of TNF-R1 expression by cucurbitacin B. Glutathione, N-acetyl-L-cysteine, and, to a lesser extent, L-cysteine attenuated the inhibitory effects of cucurbitacin B on the TNF-α-induced expression of ICAM-1, suggesting that an α,β-unsaturated carbonyl moiety is essential for anti-inflammatory activity. The present results revealed that cucurbitacin B down-regulated the expression of TNF-R1 at the initial step in the TNF-α-dependent NF-κB signaling pathway.     

Preparation and Characterization of a Chloroperoxidase-like Catalytic Antibody

The small molecule, meso-tetra(α,α,α,α-o-phenylacetamidophenyl) porphyrin (Mr1147.0) was used as complete antigen to elicit MAb through the immunization and cell fusion techniques. The MAb 1F2 obtained was demonstrated to be very pure by MALDI/TOFMS. The subtype of MAb 1F2 is IgG2a, which has a relative molecular weight of 156,678.8 Da.No significant change in the intensity of absorption peaks in UV and CD spectra was observed over a pH range between 6 and 12. The high stability of the abzyme and the tight binding between Fe porphyrin and antibody were also demonstrated. V_max, K_m, κcat, κcat/K_m for abzyme are 5.18 × 10⁻⁸ Ms⁻¹, 1.50 × 10⁻⁸ M, 0.518 s⁻¹, 3.45 × 10⁷ M⁻¹s⁻¹, respectively. The data obtained indicate that catalytic antibody has high catalytic activity. The chloroperoxidase activity of MAb 1F2-Fe porphyrin complex is stable from 10 °C to 60 °C.     

Hydrostatic Pressure Influences HIF-2 Alpha Expression in Chondrocytes

Hypoxia-inducible factor (HIF)-2α is considered to play a major role in the progression of osteoarthritis. Recently, it was reported that pressure amplitude influences HIF-2α expression in murine endothelial cells. We examined whether hydrostatic pressure is involved in expression of HIF-2α in articular chondrocytes. Chondrocytes were cultured and stimulated by inflammation or hydrostatic pressure of 0, 5, 10, or 50 MPa. After stimulation, heat shock protein (HSP) 70, HIF-2α, nuclear factor kappa B (NF-κB), matrix metalloproteinase (MMP)-13, MMP-3, and vascular endothelial growth factor (VEGF) gene expression were evaluated. The levels of all gene expression were increased by inflammatory stress. When chondrocytes were exposed to a hydrostatic pressure of 5 MPa, HIF-2α, MMP-13, and MMP-3 gene expression increased significantly although those of HSP70 and NF-κB were not significantly different from the control group. In contrast, HIF-2α gene expression did not increase under a hydrostatic pressure of 50 MPa although HSP70 and NF-κB expression increased significantly compared to control. We considered that hydrostatic pressure of 5 MPa could regulate HIF-2α independent of NF-κB, because the level of HIF-2α gene expression increased significantly without upregulation of NF-κB expression at 5 MPa. Hydrostatic pressure may influence cartilage degeneration, inducing MMP-13 and MMP-3 expression through HIF-2α.     

Emodin Ameliorates LPS-Induced Acute Lung Injury,Involving the Inactivation of NF-κB in Mice

Acute lung injury (ALI) and its severe manifestation of acute respiratory distress syndrome (ARDS) are well-known illnesses. Uncontrolled and self-amplified pulmonary inflammation lies at the center of the pathology of this disease. Emodin, the bio-active coxund of herb Radix rhizoma Rhei, shows potent anti-inflammatory properties through inactivation of nuclear factor-κB (NF-κB). The aim of this study was to evaluate the effect of emodin on lipopolysaccharide (LPS)-induced ALI in mice, and its potential bio-mechanism. In our study, BALB/c mice were stimulated with LPS to induce ALI. After 72 h of LPS stimulation, pulmonary pathological changes, lung injury scores, pulmonary edema, myeloperoxidase (MPO) activity, total cells, neutrophils, macrophages, TNF-α, IL-6 and IL-1β in bronchoalveolar lavage fluid (BALF), and MCP-1 and E-selectin expression were notably attenuated by emodin in mice. Meanwhile, our data also revealed that emodin significantly inhibited the LPS-enhanced the phosphorylation of NF-κB p65 and NF-κB p65 DNA binding activity in lung. Our data indicates that emodin potently inhibits LPS-induced pulmonary inflammation, pulmonary edema and MCP-1 and E-selectin expression, and that these effects were very likely mediated by inactivation of NF-κB in mice. These results suggest a therapeutic potential of emodin as an anti-inflammatory agent for ALI/ARDS treatment.     

Toward the Discovery of a Novel Class of Leads for High Altitude Disorders by Virtual Screening and Molecular Dynamics Approaches Targeting Carbonic Anhydrase

For decades, carbonic anhydrase (CA) inhibitors, most notably the acetazolamide-bearing 1,3,4-thiadiazole moiety, have been exploited at high altitudes to alleviate acute mountain sickness, a syndrome of symptomatic sensitivity to the altitude characterized by nausea, lethargy, headache, anorexia, and inadequate sleep. Therefore, inhibition of CA may be a promising therapeutic strategy for high-altitude disorders. In this study, co-crystallized inhibitors with 1,3,4-thiadiazole, 1,3-benzothiazole, and 1,2,5-oxadiazole scaffolds were employed for pharmacophore-based virtual screening of the ZINC database, followed by molecular docking and molecular dynamics simulation studies against CA to find possible ligands that may emerge as promising inhibitors. Compared to the co-crystal ligands of PDB-1YDB, 6BCC, and 6IC2, ZINC12336992, ZINC24751284, and ZINC58324738 had the highest docking scores of −9.0, −9.0, and −8.9 kcal/mol, respectively. A molecular dynamics (MD) simulation analysis of 100 ns was conducted to verify the interactions of the top-scoring molecules with CA. The system’s backbone revealed minor fluctuations, indicating that the CA–ligand complex was stable during the simulation period. Simulated trajectories were used for the MM-GBSA analysis, showing free binding energies of −16.00 ± 0.19, −21.04 ± 0.17, and −19.70 ± 0.18 kcal/mol, respectively. In addition, study of the frontier molecular orbitals of these compounds by DFT-based optimization at the level of B3LYP and the 6-311G(d,p) basis set showed negative values of the HOMO and LUMO, indicating that the ligands are energetically stable, which is essential for forming a stable ligand–protein complex. These molecules may prove to be a promising therapy for high-altitude disorders, necessitating further investigations.