The anti-herpetic activity of trichosanthin via the nuclear factor-κB and p53 pathways

AimsTrichosanthin (TCS) is a type I ribosome-inactivating protein. We have previously shown that TCS induces a more potent apoptosis in infected cells over uninfected cells, but the mechanism underlying it is unclear. In this study, we explored the anti-HSV-1 mechanism of TCS through the nuclear factor-κB (NF-κB) and p53 pathways in human epithelial carcinoma (HEp-2) cells with wild type p53.Main methodsThe western blot, electrophoretic mobility shift assay, chromatin immunoprecipitation assay, enzyme-linked immunosorbent assay and cytokinesis-block micronucleus were applied in this study.Key findingsIt was shown that TCS inhibited the HSV-1-induced NF-κB activation. Meanwhile, in HSV-1 infected cells, TCS treatment activated significantly more p53 and BAX, with no DNA damage and less S phase arrest compared with uninfected cells. The activation of BAX in infected cells correlated with the cell death signaling of p53.SignificanceTaken together, these results suggest that the anti-HSV-1 effect of TCS is related to its suppression of NF-κB activation and regulation of p53-dependent cell death in infected cells.     

Cepharanthine suppresses osteoclast formation by modulating the nuclear factor-κB and nuclear factor of activated T-cell signaling pathways

The increased activation of osteoclasts is the major manifestation of several lytic bone diseases, including osteoporosis, rheumatoid arthritis, aseptic loosening of orthopedic implants, Paget disease and malignant bone diseases. One important bone-protective therapy in these diseases focuses on the inhibition of osteoclast differentiation and resorptive function. Given that the deleterious side-effects of currently available drugs, it is beneficial to search for effective and safe medications from natural compounds. Cepharanthine (CEP) is a compound extracted from Stephania japonica and has been found to have antioxidant and anti-inflammatory effects. In this study, we found that CEP inhibited receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast formation and bone-resorbing activities using osteoclastogenesis and bone resorption assay. By polymerase chain reaction, we also found that CEP inhibited the expression of osteoclast-differentiation marker genes including Ctsk, Calcr, Atp6v0d2, Mmp9 and Nfatc1. Mechanistic analyses including Western blot and luciferase reporter assay revealed that CEP inhibited RANKL-induced activation of NF-κB and nuclear factor of activated T-cell, which are essential for the formation of osteoclast. Collectively, these data suggested that CEP can potentially be used as an alternative therapy for preventing or treating osteolytic diseases.     

YTHDF2 mediates LPS-induced osteoclastogenesis and inflammatory response via the NF-κB and MAPK signaling pathways

Aberrant elevation of osteoclast differentiation and function is responsible for disrupting bone homeostasis in various inflammatory bone diseases. YTH domain family 2 (YTHDF2) is a well-known m⁶A-binding protein that plays an essential role in regulating cell differentiation and inflammatory processes by mediating mRNA degradation. However, the regulatory role of YTHDF2 in inflammatory osteoclast differentiation remains unelucidated. Here, we detected the expression of m⁶A-related genes and found that YTHDF2 was upregulated in RANKL-primed osteoclast precursors stimulated with lipopolysaccharide (LPS). Ythdf2 knockdown in RAW264.7 cells and primary bone marrow-derived macrophages (BMMs) enhanced osteoclast formation and bone resorption, which was assessed by TRAP staining assay and pit formation assay. Ythdf2 depletion upregulated osteoclast-related gene expression and proinflammatory cytokine secretion. In contrast, overexpression of Ythdf2 produced the reverse effect. Furthermore, Ythdf2 knockdown enhanced the phosphorylation of IKKα/β, IκBα, ERK, P38 and JNK. NF-κB and MAPK signaling pathway inhibitors effectively abrogated the enhanced expression of Nfact1, c-Fos, IL-1β and TNF-α caused by Ythdf2 knockdown. Mechanistically, the mRNA stability assay revealed that Ythdf2 depletion led to stabilization of Tnfrsf11a, Traf6, Map4k4, Map2k3, Map2k4 and Nfatc1 mRNA. In summary, our findings demonstrated that YTHDF2 has a negative regulatory role in LPS-induced osteoclast differentiation and the inflammatory response via the NF-κB and MAPK signaling pathways.     

LPS-induced NF-κB expression in THP-1Blue cells correlates with neopterin production and activity of indoleamine 2,3-dioxygenase

Neopterin production is induced in human monocyte-derived macrophages and dendritic cells upon stimulation with Th1-type cytokine interferon-γ (IFN-γ). In parallel, IFN-γ induces the tryptophan-(trp)-degrading enzyme indoleamine 2,3-dioxygenase (IDO) and triggers the formation of reactive oxygen species (ROS). Translocation of the signal transduction element nuclear factor-κB (NF-κB) is induced by ROS and accelerates the pro-inflammatory response by activation of other pro-inflammatory pathways. Therefore, a close relationship between NF-κB expression, the production of neopterin and the degradation of trp can be assumed, although this has not been demonstrated so far. In the present in vitro study we compared the influence of lipopolysaccharide (LPS) on NF-κB activation, neopterin formation and the degradation of trp in THP-1Blue cells, which represent the human myelomonocytic cell line THP-1 stably transfected with an NF-κB inducible reporter system.In cells stimulated with LPS, a significant induction of NF-κB was observed, and this was paralleled by an increase of kynureunine (kyn) and neopterin concentrations and a decline of trp. The increase of the kyn to trp quotient indicates accelerated IDO activity. Higher LPS concentrations and longer incubation of cells were associated with higher activities of all three biochemical pathways and significant correlations existed between NF-κB activation, neopterin release and trp degradation (all p < 0.001). We conclude that there is a parallel induction of NF-κB, neopterin formation and trp degradation in monocytic THP-1 cells, which is elicited by pro-inflammatory triggers like LPS during innate immune responses.     

Indoxyl sulfate upregulates renal expression of MCP-1 via production of ROS and activation of NF-κB, p53, ERK, and JNK in proximal tubular cells

AimsMonocyte chemotactic protein-1 (MCP-1) plays an important role in recruiting monocytes/macrophages to injured tubulointerstitial tissue. The present study examined whether indoxyl sulfate, a uremic toxin, regulates renal expression of MCP-1.Main methodsThe effect of indoxyl sulfate on the expression of MCP-1 was determined using human proximal tubular cells (HK-2 cells) and following animals: (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive indoxyl sulfate-administered rats (DN + IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive indoxyl sulfate-administered rats (DH + IS).Key findingsDN + IS, DH, and DH + IS rats showed significantly increased mRNA expression of MCP-1 in the kidneys compared with DN rats. DH + IS rats tended to show increased mRNA expression of MCP-1 in the kidneys compared with DH rats. Immunohistochemistry demonstrated the stimulatory effects of indoxyl sulfate on MCP-1 expression and monocyte/macrophage infiltration in the kidneys. Indoxyl sulfate upregulated mRNA and protein expression of MCP-1 in HK-2 cells. Indoxyl sulfate induced activation of ERK, p38, and JNK as well as of NF-κB and p53 in HK-2 cells. An antioxidant, and inhibitors of NF-κB, p53, ERK pathway (MEK1/2), and JNK suppressed indoxyl sulfate-induced mRNA expression of MCP-1 in HK-2 cells.SignificanceIndoxyl sulfate upregulates renal expression of MCP-1 through production of reactive oxygen species (ROS), and activation of NF-κB, p53, ERK, and JNK in proximal tubular cells. Thus, accumulation of indoxyl sulfate in chronic kidney disease might be involved in the pathogenesis of tubulointerstitial injury through induction of MCP-1 in the kidneys.     

Transforming growth factor-β1 suppresses serum deprivation-induced apop-tosis via activation of ERK1/ERK2 and the inhibition of p38 activity and ceramide production

In this report we studied the effects and mechanism of transforming growth factor-β1 (TGF-β1) on serum deprivation-induced cell apoptosis. Serum deprivation induces apoptosis, which is associated with an increase in intracellular ceramide level and with the activation of p38 mitogen-activated protein (MAP) kinase. Inhibition of p38 MAP kinase by SB203580 significantly reduced apoptosis induced by serum-deprivation. Treatment of cells with TGF-β1 stimulated cell proliferation and suppressed the serum deprivation-induced apoptotic response. The anti-apoptotic effect of TGF-β1 is correlated with its ability to inhibit the serum deprivation-induced activation of p38 MAP kinase and the increase in intracellular ceramide level. In     

Tumor necrosis factor alpha promotes the proliferation of human nucleus pulposus cells via nuclear factor-κB,c-Jun N-terminal kinase,and p38 mitogen-activated protein kinase

Although tumor necrosis factor alpha (TNF-α) is known to play a critical role in intervertebral disc (IVD) degeneration, the effect of TNF-α on nucleus pulposus (NP) cells has not yet been elucidated. The aim of this study was to explore the effect of TNF-α on proliferation of human NP cells. NP cells were treated with different concentrations of TNF-α. Cell proliferation was determined by cell counting kit-8 (CCK-8) analysis and Ki67 immunofluorescence staining, and expression of cyclin B1 was studied by quantitative real-time RT-PCR. Cell cycle was measured by flow cytometry and cell apoptosis was analyzed using an Annexin V–fluorescein isothiocyanate (FITC) & propidium iodide (PI) apoptosis detection kit. To identify the mechanism by which TNF-α induced proliferation of NP cells, selective inhibitors of major signaling pathways were used and Western blotting was carried out. Treatment with TNF-α increased cell viability (as determined by CCK-8 analysis) and expression of cyclin B1 and the number of Ki67-positive and S-phase NP cells, indicating enhancement of proliferation. Consistent with this, NP cell apoptosis was suppressed by TNF-α treatment. Moreover, inhibition of NF-κB, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) blocked TNF-α-stimulated proliferation of NP cells. In conclusion, the current findings suggest that the effect of TNF-α on IVD degeneration involves promotion of the proliferation of human NP cells via the NF-κB, JNK, and p38 MAPK pathways.     

Effect of thymopentin on the production of cytokines, heat shock proteins, and NF-κB signaling proteins

In vivo effects of thymopentin, an active fragment of the naturally occurring thymic hormone thymopoietin, on the production of cytokines, nitric oxide, heat shock proteins, and signaling proteins NF-κB, phNF-κB, and IκB-α in lymphoid cells of male NMRI mice was studied. Activation of production of several cytokines (IL-1α, IL-2, IL-6, IL-10, and IFN-γ), nitric oxide, and heat shock proteins (HSP70 and HSP90) was observed in peritoneal macrophages and spleen lymphocytes of mice that received intraperitoneal injections of thymopentin (15μg per 100 g body weight). Thymopentin apparently produces stress-like rather than damaging effects. A probable action mechanism of this hormone is activation of the NF-κB signaling pathway, which is most pronounced at the NF-κB phosphorylation stage.     

Ursolic acid inhibits nuclear factor-κB signaling in intestinal epithelial cells and macrophages,and attenuates experimental colitis in mice

Aims

Ursolic acid (UA), a natural pentacyclic triterpenoid acid, has been reported to show immunomodulatory activity. This study investigated the effects of UA on nuclear factor-kappa B (NF-κB) signaling in cells and experimental murine colitis.

Main methods

Human intestinal epithelial cells (IECs) COLO 205 and peritoneal macrophages from IL-10-deficient (IL-10^−/−) mice were pretreated with UA and then stimulated with tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS), respectively. The expression of pro-inflammatory cytokines was determined by real-time RT-PCR and ELISA. The effect of UA on NF-κB signaling was examined by immunoblot analysis to detect IκBα phosphorylation/degradation and electrophoretic mobility shift assay to assess the DNA binding activity of NF-κB. For in vivo studies, dextran sulfate sodium (DSS)-induced acute colitis in C57BL/6 wild-type mice and chronic colitis in IL-10^−/− mice were treated with or without UA. Colitis was quantified by histopathologic evaluation. Immunohistochemical staining for phosphorylated IκBα was performed in the colonic tissue.

Key findings

UA significantly inhibited the production of pro-inflammatory cytokines, IκBα phosphorylation/degradation and NF-κB DNA binding activity in both IEC and IL-10^−/− peritoneal macrophages stimulated with TNF-α and LPS, respectively. UA significantly reduced the severity of DSS-induced murine colitis, as assessed by the disease activity index, colon length, and histopathology. UA also significantly ameliorated the severity of colitis in IL-10^−/− mice. Furthermore, UA suppressed IκBα phosphorylation in the colonic tissue.

Significance

UA inhibits NF-κB activation in both IECs and macrophages, and attenuates experimental murine colitis. These results suggest that UA is a potential therapeutic agent for inflammatory bowel disease.     

C-type natriuretic peptide attenuates LPS-induced endothelial activation: involvement of p38, Akt,and NF-κB pathways

Endothelial activation elicited by inflammatory agents is regarded as a key event in the pathogenesis of several vascular inflammatory diseases. In the present study, the inhibitory effects and underlying mechanism of C-type natriuretic peptide (CNP) on LPS-induced endothelial activation were examined in human umbilical vein endothelial cells (HUVECs). The effect of CNP on adhesion molecule expression was assessed using quantitative real-time RT-PCR and western blotting analyses. The nuclear factor-κB (NF-κB), MAPK, and PI3K/Akt signaling pathways in LPS-stimulated HUVECs were investigated using western blotting analyses, and the production of intracellular reactive oxygen species (ROS) was measured using a fluorescence method. Pretreatment with CNP inhibited LPS-induced expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, E-selectin, and P-selectin in a concentration-dependent manner. CNP suppressed the phosphorylation of p65 and NF-κB activation in LPS-stimulated cells. Moreover, CNP reduced ERK1/2 and p38 phosphorylation induced by LPS but not JNK. Furthermore, CNP induced Akt phosphorylation and activation of hemeoxygenase-1 (HO-1) expression. CNP significantly inhibited the production of intracellular ROS. These results suggest that CNP effectively attenuated LPS-induced endothelial activation by inhibiting the NF-κB and p38 signaling pathways, eliminating LPS-induced intracellular ROS production, and activating the PI3K/Akt/HO-1 pathway in HUVECs; thereby, demonstrating that CNP may be a potential therapeutic target for the treatment of sepsis and inflammatory vascular diseases.     

Different effects of antisense RelA p65 and NF-κB1 p50 oligonucleotides on the nuclear factor-κB mediated expression of ICAM-1 in human coronary endothelial and smooth muscle cells

Background  

Activation of nuclear factor-κB (NF-κB) is one of the key events in early atherosclerosis and restenosis. We hypothesized that tumor necrosis factor-α (TNF-α) induced and NF-κB mediated expression of intercellular adhesion molecule-1 (ICAM-1) can be inhibited by antisense RelA p65 and NF-κB1 p50 oligonucleotides (RelA p65 and NF-κB1 p50).     

Retigeric acid B exhibits antitumor activity through suppression of nuclear factor-κB signaling in prostate cancer cells in vitro and in vivo

Previously, we reported that retigeric acid B (RB), a natural pentacyclic triterpenic acid isolated from lichen, inhibited cell growth and induced apoptosis in androgen-independent prostate cancer (PCa) cells. However, the mechanism of action of RB remains unclear. In this study, we found that using PC3 and DU145 cells as models, RB inhibited phosphorylation levels of IκBα and p65 subunit of NF-κB in a time- and dosage-dependent manner. Detailed study revealed that RB blocked the nuclear translocation of p65 and its DNA binding activity, which correlated with suppression of NF-κB-regulated proteins including Bcl-2, Bcl-x(L), cyclin D1 and survivin. NF-κB reporter assay suggested that RB was able to inhibit both constitutive activated-NF-κB and LPS (lipopolysaccharide)-induced activation of NF-κB. Overexpression of RelA/p65 rescued RB-induced cell death, while knockdown of RelA/p65 significantly promoted RB-mediated inhibitory effect on cell proliferation, suggesting the crucial involvement of NF-κB pathway in this event. We further analyzed antitumor activity of RB in in vivo study. In C57BL/6 mice carrying RM-1 homografts, RB inhibited tumor growth and triggered apoptosis mainly through suppressing NF-κB activity in tumor tissues. Additionally, DNA microarray data revealed global changes in the gene expression associated with cell proliferation, apoptosis, invasion and metastasis in response to RB treatment. Therefore, our findings suggested that RB exerted its anti-tumor effect by targeting the NF-κB pathway in PCa cells, and this could be a general mechanism for the anti-tumor effect of RB in other types of cancers as well.     

Enalapril inhibits nuclear factor-κB signaling in intestinal epithelial cells and peritoneal macrophages and attenuates experimental colitis in mice

Aims

Enalapril, an angiotensin-converting enzyme (ACE) inhibitor, has pleiotropic effects such as anti-inflammatory effects. This study investigated the effect of enalapril on the nuclear factor-kappa B (NF-κB) pathway and on experimental colitis.

Main methods

The human intestinal epithelial cell (IEC) line COLO 205 and peritoneal macrophages from C57BL/6 wild-type mice and IL-10-deficient (IL-10^−/−) mice were prepared and subsequently stimulated with lipopolysaccharide (LPS) alone or LPS plus enalapril. The effect of enalapril on NF-κB signaling was examined by western blotting to detect IκBα phosphorylation/degradation; an electrophoretic mobility shift assay (EMSA) to assess the DNA binding activity of NF-κB; and ELISAs to qualify IL-8, TNF-α, IL-6, and IL-12 production. In in vivo studies, dextran sulfate sodium (DSS)-induced acute colitis in wild-type mice and chronic colitis in IL-10^−/− mice were treated with or without enalapril. Colitis was quantified by histologic scoring, and the phosphorylation of IκBα in the colonic mucosa was assessed using immunohistochemistry.

Key findings

Enalapril significantly inhibited LPS-induced IκBα phosphorylation/degradation, NF-κB binding activity, and pro-inflammatory cytokine production in both IEC and peritoneal macrophages. The administration of enalapril significantly reduced the severity of colitis, as assessed based on histology in both murine colitis models. Furthermore, in colon tissue, the up-regulation of IκBα phosphorylation with colitis induction was attenuated in enalapril-treated mice.

Significance

Enalapril may block the NF-κB signaling pathway, inhibit the activation of IECs and macrophages, and attenuate experimental murine colitis by down-regulating IκBα phosphorylation. These findings suggest that enalapril is a potential therapeutic agent for inflammatory bowel disease.     

Icariin inhibits RANKL-induced osteoclastogenesis via modulation of the NF-κB and MAPK signaling pathways

The receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-RANK regulatory axis is a major regulator of osteoclast differentiation and activation. Icariin, a flavonol glycoside isolated from the Epimedium herb, has been reported to prevents bone loss in ovariectomized mice and inhibits wear particle-induced osteolysis. However, the molecular mechanism through which icariin inhibits RANKL-induced osteoclastogenesis has not been fully understood. Therefore, we aimed to investigate the effects of icariin on RANKL-induced osteoclastogenesis and to elucidate the mechanism underlying this effect. Our results showed that RANKL-induced osteoclastogenesis was inhibited by icariin in bone marrow macrophages (BMMs) and RAW264.7?cells, and that this effect was due to suppression of NF-κB and mitogen-activated protein kinase (MAPK) activation. In addition, icariin inhibited F-actin ring formation and attenuated the bone resorption ability of mature osteoclasts. Collectively, our results indicate that icariin may be a promising potential candidate for the treatment of osteolytic diseases such as osteoporosis. Moreover, our findings lay the foundation for understanding and intervening in osteoclast-related diseases at the molecular level.