The edematogenic effect of Micrurus lemniscatus venom is dependent on venom phospholipase A2 activity and modulated by non-neurogenic factors.

Coral snakes mainly cause neurotoxic symptoms in human envenomation, but experimental studies have already demonstrated several pharmacological activities in addition to these effects. This investigation was carried out with the aim of evaluating (1) non-neurogenic mechanisms involved in the inflammatory response induced by Micrurus lemniscatus venom (MLV) in rat hind paws, (2) participation of PLA2 in this response, and (3) neutralizing efficiency of commercial anti-elapid antivenom on edema. MLV promoted a rapid, significant increase in vascular permeability, influx of leukocytes, and disorganization of collagen bundles, as demonstrated by histological analysis. Several pretreatments were applied to establish the involvement of inflammatory mediators in MLV-induced edema (5 µg/paw). Treatment of animals with chlorpromazine reduced MLV-induced edema, indicating participation of TNF-α. However, the inefficiency of other pharmacological treatments suggests that eicosanoids, leukotrienes, and nitric oxide have no role in this type of edema formation. In contrast, PAF negatively modulates this venom-induced effect. MLV was recognized by anti-elapid serum, but this antivenom did not neutralize edema formation. Chemical modification of MLV with p-bromophenacyl bromide abrogated the phospholipase activity and markedly reduced edema, demonstrating PLA2 participation in MLV-induced edema. In conclusion, the non-neurogenic inflammatory profile of MLV is characterized by TNF-α-mediated edema, participation of PLA2 activity, and down-regulation by PAF. MLV induces an influx of leukocytes and destruction of collagen fibers at the site of its injection.

Axonal Protection by Netarsudil, a ROCK Inhibitor, Is Linked to an AMPK-Autophagy Pathway in TNF-Induced Optic Nerve Degeneration.

Purpose: Netarsudil, a Rho kinase inhibitor with norepinephrine transport inhibitory effect, lowers intraocular pressure, however, its effect on axon damage remains to be elucidated. The aim of the current study was to investigate the effect of netarsudil on TNF-induced axon loss and to examine whether it affects phosphorylated-AMP-activated kinase (p-AMPK) and autophagy in the optic nerve. Methods: Intravitreal administration of TNF or TNF with netarsudil was carried out on rats and quantification of axon number was determined. Electron microscopy determined autophagosome numbers. Localization of p-AMPK expression was examined by immunohistochemistry. The changes in p62, LC3-II, and p-AMPK levels were estimated in the optic nerve by immunoblot analysis. The effect of an AMPK activator A769662 or an AMPK inhibitor dorsomorphin on axon number was evaluated. Results: Morphometric analysis revealed apparent protection by netarsudil against TNF-induced axon degeneration. Netarsudil increased autophagosome numbers inside axons. Netarsudil treatment significantly upregulated optic nerve LC3-II levels in both the TNF-treated eyes and the control eyes. Increased p62 protein level induced by TNF was significantly ameliorated by netarsudil. The netarsudil administration alone lessened p62 levels. Netarsudil significantly upregulated the optic nerve p-AMPK levels. A769662 exhibited obvious axonal protection against TNF-induced damage. A769662 treatment upregulated LC3-II levels and the increment of p62 level induced by TNF was significantly ameliorated by A769662. Immunohistochemical analysis revealed that p-AMPK is present in axons. Netarsudil-mediated axonal protection was significantly suppressed by dorsomorphin administration. Conclusions: Netarsudil upregulated p-AMPK and autophagy. Netarsudil-mediated axonal protection may be associated with upregulated p-AMPK.

Celecoxib prevents tumor necrosis factor-α (TNF-α)-induced cellular senescence in human chondrocytes.

Osteoarthritis (OA) is a cartilage degenerative disease commonly observed in the elderly population and significantly impacts the normal life of OA patients. It has been reported that the development of pathological cell senescence in chondrocytes is involved in the pathogenesis of OA. Celecoxib is a common non-steroidal anti-inflammatory drug, and it has been recently reported to exert therapeutic effects on OA. However, its underlying mechanism is still unclear. The present study intends to explore its mechanism and provide fundamental evidence for the application of Celecoxib in the treatment of clinical OA. Tumor necrosis factor-α (TNF-α) was utilized to establish an in vitro model of chondrocytes senescence. The elevated reactive oxygen species (ROS) generation, increased cell cycle arrest in G0/G1 phase, reduced telomerase activity, and upregulated senescence-associatedß-galactosidase (SA-ß-Gal) staining were all observed in TNF-α-treated chondrocytes, which were then dramatically reversed by 10 and 20 µM Celecoxib. In addition, the upregulated DNA damage biomarkers, p-ATM, and p-CHK2, observed in TNF-α-treated chondrocytes were significantly downregulated by 10 and 20 µM Celecoxib. Lastly, the expression level of p21 and p53 was greatly elevated in chondrocytes by stimulation with TNF-α which was then pronouncedly repressed by treatment with Celecoxib. Taken together, our data reveal that Celecoxib ameliorated TNF-α-induced cellular senescence in human chondrocytes.

Acetylcholinesterase inhibition with Pyridostigmine attenuates hypertension and neuroinflammation in the paraventricular nucleus in rat model for Preeclampsia.

Preeclampsia (PE) is characterized by hypertension, autonomic imbalance and inflammation. The subfornical organ (SFO) reportedly relays peripheral inflammatory mediator's signals to the paraventricular nucleus (PVN), a brain autonomic center shown to mediate hypertension in hypertensive rat but not yet in PE rat models. Additionally, we previously showed that Pyridostigmine (PYR), an acetylcholinesterase inhibitor, attenuated placental inflammation and hypertension in PE models. In this study, we investigated the effect of PYR on the activities of these brain regions in PE model. PYR (20 mg/kg/day) was administered to reduced uterine perfusion pressure (RUPP) Sprague-Dawley rat from gestational day (GD) 14 to GD19. On GD19, the mean arterial pressure (MAP) was recorded and samples were collected for analysis. RUPP rats exhibited increased MAP (P = 0.0025), elevated circulating tumor necrosis factor-α (TNF-α, P = 0.0075), reduced baroreflex sensitivity (BRS), increased neuroinflammatory markers including TNF-α, interleukin-1ß (IL-1ß), microglial activation (P = 0.0039), oxidative stress and neuronal excitation within the PVN and the SFO. Changes in MAP, in molecular and cellular expression induced by RUPP intervention were improved by PYR. The ability of PYR to attenuate TNF-α mediated central effect was evaluated in TNF-α-infused pregnant rats. TNF-α infusion-promoted neuroinflammation in the PVN and SFO in dams was abolished by PYR. Collectively, our data suggest that PYR improves PE-like symptoms in rat by dampening placental ischemia and TNF-α-promoted inflammation and pro-hypertensive activity in the PVN. This broadens the therapeutical potential of PYR in PE.

Neuroprotective Effects of Fingolimod in a Cellular Model of Optic Neuritis.

Visual dysfunction resulting from optic neuritis (ON) is one of the most common clinical manifestations of multiple sclerosis (MS), characterized by loss of retinal ganglion cells, thinning of the nerve fiber layer, and inflammation to the optic nerve. Current treatments available for ON or MS are only partially effective, specifically target the inflammatory phase, and have limited effects on long-term disability. Fingolimod (FTY) is an FDA-approved immunomodulatory agent for MS therapy. The objective of the current study was to evaluate the neuroprotective properties of FTY in the cellular model of ON-associated neuronal damage. R28 retinal neuronal cell damage was induced through treatment with tumor necrosis factor-α (TNFα). In our cell viability analysis, FTY treatment showed significantly reduced TNFα-induced neuronal death. Treatment with FTY attenuated the TNFα-induced changes in cell survival and cell stress signaling molecules. Furthermore, immunofluorescence studies performed using various markers indicated that FTY treatment protects the R28 cells against the TNFα-induced neurodegenerative changes by suppressing reactive oxygen species generation and promoting the expression of neuronal markers. In conclusion, our study suggests neuroprotective effects of FTY in an in vitro model of optic neuritis.

In Silico Identification and Evaluation of Natural Products as Potential Tumor Necrosis Factor Function Inhibitors Using Advanced Enalos Asclepios KNIME Nodes.

Tumor necrosis factor (TNF) is a regulator of several chronic inflammatory diseases, such as rheumatoid arthritis. Although anti-TNF biologics have been used in clinic, they render several drawbacks, such as patients' progressive immunodeficiency and loss of response, high cost, and intravenous administration. In order to find new potential anti-TNF small molecule inhibitors, we employed an in silico approach, aiming to find natural products, analogs of Ampelopsin H, a compound that blocks the formation of TNF active trimer. Two out of nine commercially available compounds tested, Nepalensinol B and Miyabenol A, efficiently reduced TNF-induced cytotoxicity in L929 cells and production of chemokines in mice joints' synovial fibroblasts, while Nepalensinol B also abolished TNF-TNFR1 binding in non-toxic concentrations. The binding mode of the compounds was further investigated by molecular dynamics and free energy calculation studies, using and advancing the Enalos Asclepios pipeline. Conclusively, we propose that Nepalensinol B, characterized by the lowest free energy of binding and by a higher number of hydrogen bonds with TNF, qualifies as a potential lead compound for TNF inhibitors' drug development. Finally, the upgraded Enalos Asclepios pipeline can be used for improved identification of new therapeutics against TNF-mediated chronic inflammatory diseases, providing state-of-the-art insight on their binding mode.

Gomisin M2 alleviates psoriasis­like skin inflammation by inhibiting inflammatory signaling pathways.

Psoriasis, a chronic inflammatory skin disease, is characterized by the excessive proliferation and impaired differentiation of epidermal keratinocytes and is accompanied by the increased infiltration of inflammatory cells. The condition requires long­term treatment and has no definitive cure. Hence, supplements and therapeutic agents have been intensely investigated. Gomisin M2 (GM2), a lignan extracted from Schisandra chinensis (Turcz). Baill. (Schisandraceae; S. chinensis), has demonstrated diverse pharmacological properties, including anticancer, anti­inflammatory and antiallergic effects. Based on these findings, the present study examined the effects of GM2 on an imiquimod (IMQ)­induced psoriasis mouse model and on keratinocytes stimulated by tumor necrosis factor (TNF)­α and interferon­Î³. IMQ was topically applied to the back skin of mice for 7 consecutive days, and the mice were orally administered CD. These results showed that the oral administration of GM2 suppressed the symptoms of psoriasis, as evidenced by reductions in skin thickness, psoriasis area severity index scores for psoriasis lesions, transepidermal water loss and myeloperoxidase (MPO)­associated cell infiltration. Furthermore, GM2 reduced the pathologically increased levels of immunoglobulin G2a, MPO and TNF­α in the serum and T helper (Th)1 and Th17 cell populations in the spleen. GM2 decreased the gene expression of inflammatory­related cytokines and chemokines and inhibited the expression of signal transducer and activator of transcription 1 and nuclear factor­κB in the activated keratinocytes. These results suggested that GM2 from S. chinensis is a potential therapeutic candidate to alleviate psoriasis­like skin inflammation.

α-Hydroxyisocaproic Acid Decreases Protein Synthesis but Attenuates TNFα/IFNγ Co-Exposure-Induced Protein Degradation and Myotube Atrophy via Suppression of iNOS and IL-6 in Murine C2C12 Myotube.

There is ongoing debate as to whether or not α-hydroxyisocaproic acid (HICA) positively regulates skeletal muscle protein synthesis resulting in the gain or maintenance of skeletal muscle. We investigated the effects of HICA on mouse C2C12 myotubes under normal conditions and during cachexia induced by co-exposure to TNFα and IFNγ. The phosphorylation of AMPK or ERK1/2 was significantly altered 30 min after HICA treatment under normal conditions. The basal protein synthesis rates measured by a deuterium-labeling method were significantly lowered by the HICA treatment under normal and cachexic conditions. Conversely, myotube atrophy induced by TNFα/IFNγ co-exposure was significantly improved by the HICA pretreatment, and this improvement was accompanied by the inhibition of iNOS expression and IL-6 production. Moreover, HICA also suppressed the TNFα/IFNγ co-exposure-induced secretion of 3-methylhistidine. These results demonstrated that HICA decreases basal protein synthesis under normal or cachexic conditions; however, HICA might attenuate skeletal muscle atrophy via maintaining a low level of protein degradation under cachexic conditions.

A trivalent TNF-R2 as a new tumor necrosis factor alpha-blocking molecule.

The neutralization of tumor necrosis factor alpha (TNFα) with biopharmaceuticals is a successful therapy for inflammatory diseases. Currently, one of the main TNFα-antagonists is Etanercept, a dimeric TNF-R2 ectodomain. Considering that TNFα and its receptors are homotrimers, we proposed that a trimeric TNF-R2 ectodomain could be an innovative TNFα-antagonist. Here, the 3cTNFR2 protein was designed by the fusion of the TNF-R2 ectodomain with the collagen XV trimerization domain. 3cTNFR2 was produced in HEK293 cells and purified by immobilized metal affinity chromatography. Monomers, dimers, and trimers of 3cTNFR2 were detected. The interaction 3cTNFR2-TNFα was assessed. By microscale thermophoresis, the KD value for the interaction was 4.17 ± 0.88 nM, and complexes with different molecular weights were detected by size exclusion chromatography-high performance liquid chromatography. Moreover, 3cTNFR2 neutralized the TNFα-induced cytotoxicity totally in vitro. Although more studies are required to evaluate the anti-inflammatory effect, the results suggest that 3cTNFR2 could be a TNFα-antagonist agent.

Design and characterization of a novel dimeric blood-brain barrier penetrating TNFα inhibitor.

Tumor necrosis factor-alpha (TNFα) inhibitors could prevent neurological disorders systemically, but their design generally relies on molecules unable to cross the blood-brain barrier (BBB). This research was aimed to design and characterize a novel TNFα inhibitor based on the angiopeptide-2 as a BBB shuttle molecule fused to the extracellular domain of human TNFα receptor 2 and a mutated vascular endothelial growth factor (VEGF) dimerization domain. This new chimeric protein (MTV) would be able to trigger receptor-mediated transcytosis across the BBB via low-density lipoprotein receptor-related protein-1 (LRP-1) and inhibit the cytotoxic effect of TNFα more efficiently because of its dimeric structure. Stably transformed CHO cells successfully expressed MTV, and its purification by Immobilized-Metal Affinity Chromatography (IMAC) rendered high purity degree. Mutated VEGF domain included in MTV did not show cell proliferation or angiogenic activities measured by scratch and aortic ring assays, which corroborate that the function of this domain is restricted to dimerization. The pairs MTV-TNFα (Kd 279 ± 40.9 nM) and MTV-LRP1 (Kd 399 ± 50.5 nM) showed high affinity by microscale thermophoresis, and a significant increase in cell survival was observed after blocking TNFα with MTV in a cell cytotoxicity assay. Also, the antibody staining in CHOK1 and bEnd3 cells demonstrated the adhesion of MTV to the LRP1 receptor located in the cell membrane. These results provide compelling evidence for the proper functioning of the three main domains of MTV individually, which encourage us to continue the research with this new molecule as a potential candidate for the systemic treatment of neurological disorders.

Kaempferia parviflora extract inhibits TNF-α-induced release of MCP-1 in ovarian cancer cells through the suppression of NF-κB signaling.

Ovarian clear cell carcinoma (OCCC) is an uncommon subtype of epithelial cell ovarian cancers (EOCs) that has poor response to conventional platinum-based therapy. Therefore, finding new potential therapeutic agents is required. Since inflammatory cytokine, tumor necrosis factor alpha (TNF-α), is strongly expressed in EOCs and associated with the level of tumor grade, disruption of this inflammation pathway may provide another potential target for OCCC treatment. We previously reported that Kaempferia parviflora (KP) extract decreased cell proliferation and induced apoptosis. However, the effects of KP on OCCC, especially the aspects related to inflammatory cytokines, have not been elucidated. Our current study demonstrated the effects of KP extract on cytokine production in TNF-α-induced OCCC TOV-21G cell line. This study showed that KP extract inhibited interleukin 6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) production at both transcription and translation levels via the suppression of nuclear factor-kappa B (NF-κB) signal transduction. In contrast, KP extract increased the expression of inhibitor kappa B (IκB) protein which may delay NF-κB translocation into the nucleus upon TNF-α activation. Moreover, the suppression of cytokines released from KP treated-TOV-21G reduced the migration of monocyte cell (THP-1). KP extract also exhibited the inhibition of IL-6 and MCP-1 production from THP-1 activated by lipopolysaccharides (LPS). Cells treated with KP extract exhibited a decrease in extracellular signal-regulated kinases (ERK1/2) and protein kinase B (AKT) phosphorylation and induced myeloid leukemia cell differentiation protein Mcl-1 (MCL-1) expression. Suppression of inflammatory cytokine and chemokine production and inhibition of tumor-associated macrophage (TAM) migration support the possibility of using KP for OCCC treatment.

The let-7f-5p-Nme4 pathway mediates tumor necrosis factor α-induced impairment in osteogenesis of bone marrow-derived mesenchymal stem cells.

Although tumor necrosis factor α (TNF-α)-mediated inflammation significantly impacts osteoporosis, the mechanisms underlying the osteogenic differentiation defects of bone marrow-derived mesenchymal stem cells (BM-MSCs) caused by TNF-α remain poorly understood. We found that TNF-α stimulation of murine BM-MSCs significantly upregulated the expression levels of several microRNAs (miRNAs), including let-7f-5p, but this increase was significantly reversed by treatment with the kinase inhibitor BAY 11-7082. To study gain- or loss of function, we transfected cells with an miRNA inhibitor or miRNA mimic. We then demonstrated that let-7f-5p impaired osteogenic differentiation of BM-MSCs in the absence and presence of TNF-α, as evidenced by alkaline phosphatase and alizarin red staining as well as quantitative assays of the mRNA levels of bone formation marker genes in differentiated BM-MSCs. Moreover, let-7f-5p targets the 3' untranslated region of Nucleoside diphosphate kinase 4 (Nme4) mRNA and negatively regulates Nme4 expression in mouse BM-MSCs. Ectopic expression of Nme4 completely reversed the inhibitory effects of the let-7f-5p mimic on osteogenic differentiation of mouse BM-MSCs. Furthermore, inhibition of let-7f-5p or overexpression of Nme4 in BM-MSCs restored in-vivo bone formation in an ovariectomized animal model. Collectively, our work indicates that let-7f-5p is involved in TNF-α-mediated reduction of BM-MSC osteogenesis via targeting Nme4.

Penta-acetyl Geniposide Suppresses Migration, Invasion, and Inflammation of TNF-α-Stimulated Rheumatoid Arthritis Fibroblast-Like Synoviocytes Involving Wnt/ß-Catenin Signaling Pathway.

We previously reported that penta-acetyl geniposide ((Ac)5GP, an active derivative of geniposide) showed anti-arthritic effect on adjuvant-induced arthritis (AIA) rats by promoting the apoptosis of AIA fibroblast-like synoviocyte (FLS). This study aimed to demonstrate the effects of (Ac)5GP on migration, invasion, and inflammation of TNF-α-stimulated rheumatoid arthritis (RA) FLS (MH7A cell) and to explore the involved mechanisms. MTT assay was used to determine the applied non-cytotoxic doses of (Ac)5GP (12.5, 25, 50 µM) in vitro. Results of wound-healing, transwell, and phalloidin staining assays indicated that (Ac)5GP reduced the migration, invasion, and F-actin cytoskeletal reorganization of TNF-α-stimulated MH7A. Results of ELISA and western blot assays confirmed that (Ac)5GP reduced TNF-α-induced production of pro-inflammatory cytokines (like IL-1ß, IL-6, IL-8) and matrix metalloproteinases (MMPs, such as MMP-2 and MMP-9). Moreover, (Ac)5GP inhibited TNF-α-induced activation of Wnt/ß-catenin pathway, evidenced by reducing the protein levels of Wnt1, p-GSK-3ß (Ser9), and ß-catenin and preventing ß-catenin nuclear translocation. Importantly, the combination of XAV939 (an inhibitor of Wnt/ß-catenin) promoted the actions of (Ac)5GP on TNF-α-induced migration, invasion, and inflammation, further revealing the involvement of Wnt/ß-catenin pathway underlying the therapeutic effects of (Ac)5GP on TNF-α-stimulated MH7A. In vivo, (Ac)5GP relieved the progression and severity of rat collagen-induced arthritis, related to reducing the levels of IL-1ß, IL-6, IL-8, MMP-2, and MMP-9 as well as inhibiting Wnt/ß-catenin pathway in synovial tissues. Collectively, (Ac)5GP could suppress TNF-α-induced migration, invasion, and inflammation in RA FLS involving Wnt/ß-catenin pathway and (Ac)5GP might be as a candidate agent for RA treatment.

Design and biological evaluation of novel long-acting adalimumab Fab conjugated with the albumin binding domain.

Antigen-binding fragments (Fabs) are preferred alternatives to antibodies for medical application, whereas their short half-lives limit therapeutic effectiveness. Albumin binding domain (ABD) with high affinity for albumin possesses a great potential in enhancing in vivo performance of biotherapeutics. In this study, to mitigate the poor pharmacokinetics of adalimumab Fab targeting tumor necrosis factor-α (TNFα), an ABD fusion strategy was applied innovatively using GA3, ABD035, ABD094 and ABDCon with high affinities for albumin. The prokaryotic expression, bioactivities and half-lives of those novel Fab-ABD fusions were investigated in vitro and in vivo. All Fab-ABD fusions were successfully purified, and they retained similar TNFα-binding activities with the unmodified Fab control, also presented high affinities for human/mouse serum albumin (HSA/MSA). Additionally, the simultaneous binding of the difunctional Fab-ABD fusions to TNFα and albumin was verified, and ABD fused to Fab neither interfered with Fab-TNFα binding nor impaired the association between Fc fragment of IgG receptor and transporter (FcRn) and albumin. Based on the highest binding affinity for HSA and maximal yield, Fab-ABDCon was selected for further evaluation. Fab-ABDCon showed similar thermostability with the Fab control and robust stability in human and mouse plasma. Most notably, the pharmacokinetics of Fab-ABDCon in mice was significantly improved with a 22-fold longer plasma half-life (28.2 h) compared with that of Fab control (1.31 h), which have contributed to its satisfactory therapeutic efficacy in murine TNFα-induced hepatonecrosis model. Thus, Fab-ABDCon could be a promising long-acting candidate suitable for drug development targeting TNFα-mediated inflammatory disease.

Pyropia yezoensis protein protects against TNF­α­induced myotube atrophy in C2C12 myotubes via the NF­κB signaling pathway.

The protein extracted from red algae Pyropia yezoensis has various biological activities, including anti­inflammatory, anticancer, antioxidant, and antiobesity properties. However, the effects of P. yezoensis protein (PYCP) on tumor necrosis factor­α (TNF­α)­induced muscle atrophy are unknown. Therefore, the present study investigated the protective effects and related mechanisms of PYCP against TNF­α­induced myotube atrophy in C2C12 myotubes. Treatment with TNF­α (20 ng/ml) for 48 h significantly reduced myotube viability and diameter and increased intracellular reactive oxygen species levels; these effects were significantly reversed in a dose­dependent manner following treatment with 25­100 µg/ml PYCP. PYCP inhibited the expression of TNF receptor­1 in TNF­α­induced myotubes. In addition, PYCP markedly downregulated the nuclear translocation of nuclear factor­κB (NF­κB) by inhibiting the phosphorylation of inhibitor of κB. Furthermore, PYCP treatment suppressed 20S proteasome activity, IL­6 production, and the expression of the E3 ubiquitin ligases, atrogin­1/muscle atrophy F­box and muscle RING­finger protein­1. Finally, PYCP treatment increased the protein expression levels of myoblast determination protein 1 and myogenin in TNF­α­induced myotubes. The present findings indicate that PYCP may protect against TNF­α­induced myotube atrophy by inhibiting the proinflammatory NF­κB pathway.

Tumour necrosis factor-α (TNF-α) enhances dietary carcinogen-induced DNA damage in colorectal cancer epithelial cells through activation of JNK signaling pathway.

Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer death. Benzo[a]pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazol [4,5-b] pyridine (PhIP) present in cooked meat are pro-carcinogens and considered to be potential risk factors for CRC. Their carcinogenic and mutagenic effects require metabolic activation primarily by cytochrome P450 1 family enzymes (CYPs); the expression of these enzymes can be modulated by aryl hydrocarbon receptor (AhR) activation and the tumour microenvironment, involving mediators of inflammation. In this study, we hypothesized that tumour necrosis factor-α (TNF-α), a key mediator of inflammation, modulates BaP- and PhIP-induced DNA damage in colon cancer epithelial cells. Importantly, we observed that TNF-α alone (0.1-100 pg/ml) induced DNA damage (micronuclei formation) in HCT-116 cells and co-treatment of TNF-α with BaP or PhIP showed higher levels of DNA damage compared to the individual single treatments. TNF-α alone or in combination with BaP or PhIP did not affect the expression levels of CYP1A1 and CYP1B1 (target genes of AhR signaling pathways). The DNA damage induced by TNF-α was elevated in p53 null HTC-116 cells compared to wild type cells, suggesting that TNF-α-induced DNA damage is suppressed by functional p53. In contrast, p53 status failed to affect BaP and PhIP induced micronucleus frequency. Furthermore, JNK and NF-κB signaling pathway were activated by TNF-α treatment but only inhibition of JNK significantly reduced TNF-α-induced DNA damage. Collectively, these findings suggest that TNF-α induced DNA damage involves JNK signaling pathway rather than AhR and NF-κB pathways in colon cancer epithelial cells.

A Novel Peptide Ameliorates TNFα- and LPS-Induced Endothelia Dysfunction in Preeclampsia.

BACKGROUND: To investigate the protective effects of the novel peptide antiendothelial dysfunction peptide in preeclampsia (AEDPPE) on tumor necrosis factor α (TNFα)- and lipopolysaccharide (LPS)-induced injury in the vascular endothelium in preeclampsia. METHODS: The effects of AEDPPE on TNFα-induced vascular endothelial injury were assessed by enzyme-linked immunosorbent assay, quantitative real-time PCR, mitochondrial membrane potential assay, Cell Counting Kit-8 assay, THP-1 monocyte-human umbilical vein endothelial cell (HUVEC) adhesion assay, endothelial tube-forming assay, transcriptomic analysis, preeclamptic symptom analysis, and histological analysis in preeclampsia-like rat models induced by LPS. RESULTS: AEDPPE alleviated the upregulation of antiangiogenic factors including soluble fms-like tyrosine kinase-1, endothelin-1, and tissue plasminogen activator and attenuated the reduction in mitochondrial potential induced by TNFα in HUVECs. In addition, AEDPPE treatment counteracted the decrease in tube formation and decreased the numbers of THP-1 monocytes attached to HUVECs caused by TNFα. Mechanistically, cytokine-cytokine receptor interactions enriched many genes and the TNF signaling pathway may be involved in this phenomenon. Moreover, cotreatment with LPS and AEDPPE significantly reversed the preeclampsia-like phenotype including hypertension and proteinuria and improved the functions of the kidney and placenta. CONCLUSIONS: AEDPPE effectively ameliorated the vascular endothelial injury induced by TNFα and LPS in preeclampsia. We suggest that AEDPPE may be a novel therapeutic candidate for preeclampsia treatment. These findings demonstrate that AEDPPE may play an effective role in ameliorating vascular endothelial dysfunction and be a potential therapeutic agent for preeclampsia.

Compounds DRG and DAG, Two Phenol Glycosides, Inhibit TNF-α-stimulated Inflammatory Response through Blocking NF-kB/AKT/JNK Signaling Pathways in MH7A Cells.

Fourteen constituents were recently isolated from the roots of Dendropanax dentiger with cyclooxygenase-2 (COX-2) inhibitory effects. However, the effect of 14 constituents on rheumatoid arthritis (RA) and their action mechanism remain unclear. The study aimed to explore the anti-RA effect and potential mechanism of these constituents in tumor necrosis factor α (TNF-α)-stimulated human RA fibroblast-like synoviocytes (MH7A cells). The cell viability, nitric oxide (NO) production, inflammatory cytokine levels, and protein expressions were measured by cell counting kit-8 (CCK-8), Griess reagent, ELISA, and Western blot assays, respectively. Results showed that 14 constituents (40 µM) have no cytotoxicity for MH7A cells. Among them, two phenols including 3,4-dimethoxyphenyl-1-O-α-L-rhamnopyranosyl-(1→6)-O-ß-D-glucopyranoside (DRG) and 3,4-dimethoxyphenol-ß-D-apiofuranosyl-(1→6)-ß-D-glucopyranoside (DAG) were shown to significantly inhibit the NO production with IC50 values of 5.25±0.34 and 5.35±0.31 µM, respectively. They also remarkably decreased the release of interleukin (IL)-2, 6, 8, and interferon (IFN)-γ, as well as prominently reduced the phosphorylation protein levels of p65, IkBα, AKT, and JNK at a concentration of 10 µM. Taken together, DRG and DAG could inhibit TNF-α-induced inflammatory response through blocking NF-kB/AKT/JNK signaling pathways in MH7A cells, thus could be promising against RA and other inflammation-related agents.

Quercetin Efficiently Alleviates TNF-α-Stimulated Injury by Signal Transducer and Activator of Transcription 1 and Mitogen-Activated Protein Kinase Pathway in H9c2 Cells: A Protective Role of Quercetin in Myocarditis.

ABSTRACT: This study aimed to evaluate the protective effect of quercetin and its in-depth mechanism in TNF-α-stimulated cardiomyocytes. The differential expression of TNF-alpha (TNF-α) and signal transducer and activator of transcription 1 (STAT1) was analyzed based on the GEO database. H9c2 cells were stimulated with TNF-α to simulate myocarditis. Cell counting kit-8 assay and flow cytometry assay were performed to detect the cell viability and apoptosis. ELISA was used to measure the levels of proinflammatory cytokines (IL-6 and IL-17A) and anti-inflammatory cytokine (IL-10). STAT1 expression was downregulated by transfection with si-STAT1, and its expression was detected using quantitative real-time polymerase chain reaction and Western blot. Western blot was also performed to assess the expression of the mitogen-activated protein kinase (MAPK) pathway-related factors. In this article, TNF-α was highly expressed in patients with myocarditis, and TNF-α (20 µg/mL) declined the viability of H9c2 cells. Quercetin pretreatment partially alleviated the decrease of cell viability, the increase of apoptosis, and the release of inflammatory cytokines (IL-10, IL-6, and IL-17A) induced by TNF-α. In addition, TNF-α increased STAT1 expression, but quercetin prevented the TNF-α-increased STAT1 level. Remarkably, knockdown of STAT1 enhanced the protective effect of quercetin on TNF-α-injured H9c2 cells. Moreover, quercetin restrained the TNF-α-induced activation of the MAPK pathway. Also, the inhibitory effect of quercetin on the pathway was aggravated by STAT1 lacking. In summing, quercetin plays a protective role in TNF-α-stimulated H9c2 cell injury, which may be related to the regulation of STAT1 and MAPK pathway.

Quercetin reverses TNF­α induced osteogenic damage to human periodontal ligament stem cells by suppressing the NF­κB/NLRP3 inflammasome pathway.

Quercetin (Quer) is a typical antioxidant flavonoid from plants that is involved in bone metabolism, as well as in the progression of inflammatory diseases. Elevated levels of tumor necrosis factor­α (TNF­α), a typical pro­inflammatory cytokine, can affect osteogenesis. In the present study, TNF­α was used to establish an in vitro model of periodontitis. The effects of Quer on, as well as its potential role in the osteogenic response of human periodontal ligament stem cells (hPDLSCs) under TNF­α­induced inflammatory conditions and the underlying mechanisms were then investigated. Within the appropriate concentration range, Quer did not exhibit any cytotoxicity. More importantly, Quer significantly attenuated the TNF­α induced the suppression of osteogenesis­related genes and proteins, alkaline phosphatase (ALP) activity and mineralized matrix in the hPDLSCs. These findings were associated with the fact that Quer inhibited the activation of the NF­κB signaling pathway, as well as the expression of NLRP3 inflammation­associated proteins in the inflammatory microenvironment. Moreover, the silencing of NLRP3 by small interfering RNA (siRNA) was found to protect the hPDLSCs against TNF­α­induced osteogenic damage, which was in accordance with the effects of Quer. On the whole, the present study demonstrates that Quer reduces the impaired osteogenesis of hPDLSCs under TNF­α­induced inflammatory conditions by inhibiting the NF­κB/NLRP3 inflammasome pathway. Thus, Quer may prove to be a potential remedy against periodontal bone defects.