Drug-induced anti-inflammatory response in A549 cells, as detected by Raman spectroscopy: a comparative analysis of the actions of dexamethasone and p-coumaric acid.

Corticosteroids are widely used as effective treatments for the control of chronic inflammatory diseases. However, because their long-term administration carries serious consequences, there is a need to investigate alternative therapies to reduce or even replace their use. In this regard, phenolic compounds have been presented as an alternative for the treatment of inflammatory diseases. p-Coumaric acid, a natural phenolic compound found throughout nature, exhibits antioxidative and anti-inflammatory properties. Herein, using a combination of Raman spectroscopy with principal component analysis and hierarchical cluster analysis, the inflammatory process induced by cigarette smoke extract (CSE) in epithelial cells treated with either a corticosteroid or p-coumaric acid was monitored in vitro. Our findings showed that p-coumaric acid had a significant anti-inflammatory effect in CSE-activated epithelial cells, and thus may be a useful alternative to corticosteroids for the treatment of airway inflammation in chronic obstructive pulmonary disease. In addition, multivariate analysis of the cell spectral data indicated that the mechanisms of action of the two drugs occur through different routes.

Effects of the Chinese herb formula Yufeining on stable chronic obstructive pulmonary disease: A randomized, double-blind, placebo-controlled trial.

BACKGROUND: A Chinese herb formula Yufeining (YFN) has showed promise in the treatment of stable chronic obstructive pulmonary disease (COPD), less is known that the impact of YFN in combination with standard Western treatments on lung inflammation. This study evaluated the safety and efficacy of YFN as a treatment for stable COPD and as an anti-inflammatory agent. METHODS: Sixty patients with stable COPD were randomly assigned to two treatment groups (YFN treatment, N = 30; placebo treatment, N = 30). Both groups received inhaled steroids and bronchodilators during an 8-week intervention, and patient status was assessed at 8 weeks later and 4 months after treatment. The primary outcome included clinical efficacy. The secondary outcomes involved CAT score, mMRC grade, six-minute walking distance (6MWD). IL-8, TNF-α, IL-17A, LTB4, TGF-ß1 and CRP were also detection in peripheral serum, as well as adverse reaction conditions. RESULTS: The YFN group demonstrated a significant improvement in clinical efficacy (compare 89.3% to 63.3% in the placebo group; P < 0.05). CAT scores and mMRC grades significantly decreased (P < 0.05, P < 0.01), and 6MWD significantly increased (P<0.05), after YFN treatment. The levels of IL-8, TNF-α, LTB4 and CRP decreased significantly after 8 weeks of treatment compared to baseline levels in both groups. Only in the YFN treatment group, the levels of IL-17A decreased significantly after treatment compared to baseline levels (P < 0.05). No changes were observed inTGF-ß1 from pre-to post-treatment in either group (P > 0.05). Serum levels of IL-8, TNF-α, IL-17A, LTB4 and CRP decreased significantly after YFN treatment compared to the placebo group (P < 0.05). CONCLUSION: A combinatorial treatment approach with YFN, inhaled steroids and bronchodilators produced a clinically effective treatment for stable COPD, leading to a significant decrease in circulating inflammatory mediators. The study appeared YFN was safety. CLINICAL TRIAL REGISTRATION NUMBER: No. ChiCTR-IOR-17013577.

Astragalus polysaccharides enhance the immune response to avian infectious bronchitis virus vaccination in chickens.

Astragalus polysaccharides (APS) are biological macromolecules extracted from Astragalus species that have strong immunoregulatory properties. In this study, APS were employed as an adjuvant for an avian infectious bronchitis virus (IBV) vaccine, and its effects on the cellular immune and humoral immune responses to vaccination in chicken were investigated. One hundred and fifty chicken were randomly divided into five groups (n = 30, each group). The chickens in all groups, except for the unvaccinated control group, were vaccinated with an IBV DNA vaccine. Three of the four vaccinated groups were administered different doses of APS (APSL, 10 mg/kg; APSM, 50 mg/kg; and APSH, 100 mg/kg) after the first vaccination, and the remaining vaccinated group served as a control, without any additional treatment. At 14, 28, and 42 days after the first vaccination, serum anti-IBV antibody titers; peripheral lymphocyte proliferation; and the mRNA expression of IL-1ß, IL-2, IL-8, and TNF-α in the spleen were assessed by enzyme-linked immunosorbent assay (ELISA), the cell counting kit-8 (CCK-8), and real time quantitative RT-PCR (qRT-PCR), respectively. At most time points, the titer of IBV-specific antibodies, lymphocyte proliferation, and IL-1ß, IL-2, IL-8, and TNF-α mRNA expression levels were higher in three APS groups than in the vaccine control group, and these increases were dose-dependent. These data suggest that APS could be used as an adjuvant for IBV vaccination to provide better protection against IBV infection.

Transcriptome profiling of gene expression during immunisation trial against Fasciola hepatica: identification of genes and pathways involved in conferring immunoprotection in a murine model.

BACKGROUND: Fasciolosis remains a significant food-borne trematode disease causing high morbidity around the world and affecting grazing animals and humans. A deeper understanding concerning the molecular mechanisms by which Fasciola hepatica infection occurs, as well as the molecular basis involved in acquiring protection is extremely important when designing and selecting new vaccine candidates. The present study provides a first report of microarray-based technology for describing changes in the splenic gene expression profile for mice immunised with a highly effective, protection-inducing, multi-epitope, subunit-based, chemically-synthesised vaccine candidate against F. hepatica. METHODS: The mice were immunised with synthetic peptides containing B- and T-cell epitopes, which are derived from F. hepatica cathepsin B and amoebapore proteins, as novel vaccine candidates against F. hepatica formulated in an adjuvant adaptation vaccination system; they were experimentally challenged with F. hepatica metacercariae. Spleen RNA from mice immunised with the highest protection-inducing synthetic peptides was isolated, amplified and labelled using Affymetrix standardised protocols. Data was then background corrected, normalised and the expression signal was calculated. The Ingenuity Pathway Analysis tool was then used for analysing differentially expressed gene identifiers for annotating bio-functions and constructing and visualising molecular interaction networks. RESULTS: Mice immunised with a combination of three peptides containing T-cell epitopes induced high protection against experimental challenge according to survival rates and hepatic damage scores. It also induced differential expression of 820 genes, 168 genes being up-regulated and 652 genes being down-regulated, p value <0.05, fold change ranging from -2.944 to 7.632. A functional study of these genes revealed changes in the pathways related to nitric oxide and reactive oxygen species production, Interleukin-12 signalling and production in macrophages and Interleukin-8 signalling with up-regulation of S100 calcium-binding protein A8, Matrix metallopeptidase 9 and CXC chemokine receptor 2 genes. CONCLUSION: The data obtained in the present study provided us with a more comprehensive overview concerning the possible molecular pathways implied in inducing protection against F. hepatica in a murine model, which could be useful for evaluating future vaccine candidates.

Effects of budlein A on human neutrophils and lymphocytes

ABSTRACT Sesquiterpene lactones (SLs) are the active constituents of a variety of medicinal plants used in traditional medicine for the treatment of inflammatory diseases and other ailments. Objective In this study, we evaluated whether budlein A modulates the activation of innate and adaptive immune cells such as neutrophils and lymphocytes. Material and Methods Our research group has investigated several plant species and several compounds have been isolated, identified, and their medical potential evaluated. Budlein A is a SL isolated from the species Aldama buddlejiformis and A. robusta (Asteraceae) and shows anti-inflammatory and anti-nociceptive activities. Advances in understanding how plant-derived substances modulate the activation of innate and adaptive immune cells have led to the development of new therapies for human diseases. Results Budlein A inhibited MPO activity, IL-6, CXCL8, IL-10, and IL-12 production and induces neutrophil apoptosis. In contrast, budlein A inhibited lymphocyte proliferation and IL-2, IL-10, TGF-β, and IFN-γ production, but it did not lead to cell death. Conclusions Collectively, our results indicate that budlein A shows distinct immunomodulatory effects on immune cells.

Shikonin Inhibits Inflammatory Cytokine Production in Human Periodontal Ligament Cells.

Shikonin, which is derived from Lithospermum erythrorhizon, a herb used in traditional medicine, has long been considered to be a useful treatment for various diseases in traditional oriental medicine. Shikonin has recently been reported to have several pharmacological properties, e.g., it has anti-microbial, anti-tumor, and anti-inflammatory effects. The aim of this study was to examine whether shikonin is able to influence the production of interleukin (IL)-6, IL-8, and/or chemokine C-C motif ligand (CCL)20, which contribute to the pathogenesis of periodontal disease, in human periodontal ligament cells (HPDLC). The production levels of IL-6, IL-8, and CCL20 in HPDLC were determined using an ELISA. Western blot analysis was used to detect nuclear factor kappa B (NF-κB) pathway activation in HPDLC. Shikonin prevented IL-1ß- or tumor necrosis factor (TNF)-α-mediated IL-6, IL-8, and CCL20 production in HPDLC. Moreover, we found that shikonin suppressed the phosphorylation and degradation of inhibitor of kappa B-alpha (IκB-α) in IL-1ß- or TNF-α-stimulated HPDLC. These findings suggest that shikonin could have direct beneficial effects against periodontal disease by reducing IL-6, IL-8, and CCL20 production in periodontal lesions.

Regulation of Transforming Growth Factor ß-Activated Kinase Activation by Epigallocatechin-3-Gallate in Rheumatoid Arthritis Synovial Fibroblasts: Suppression of K(63) -Linked Autoubiquitination of Tumor Necrosis Factor Receptor-Associated Factor 6.

OBJECTIVE: Transforming growth factor ß-activated kinase 1 (TAK1) is a key MAPKKK family protein in interleukin-1ß (IL-1ß), tumor necrosis factor (TNF), and Toll-like receptor signaling. This study was undertaken to examine the posttranslational modification of TAK1 and its therapeutic regulation in rheumatoid arthritis (RA). METHODS: The effect of TAK1, IL-1 receptor-associated kinase 1 (IRAK-1), and TNF receptor-associated factor 6 (TRAF6) inhibition was evaluated in IL-1ß-stimulated human RA synovial fibroblasts (RASFs). Western blotting, immunoprecipitation, and 20S proteasome assay were used to study the ubiquitination process in RASFs. The efficacy of epigallocatechin-3-gallate (EGCG), a potent antiinflammatory molecule, in regulating these processes in RASFs was evaluated. Molecular docking was performed to examine the interaction of EGCG with human TAK1, IRAK-1, and TRAF6. These findings were confirmed using a rat model of adjuvant-induced arthritis (AIA). RESULTS: Inhibition of TAK1, but not IRAK-1 or TRAF6, completely abrogated IL-1ß-induced IL-6 and IL-8 synthesis in RASFs. EGCG inhibited TAK1 phosphorylation at Thr(184/187) and occupied the C(174) position, an ATP-binding site, to inhibit its kinase activity. EGCG pretreatment also inhibited K(63) -linked autoubiquitination of TRAF6, a posttranslational modification essential for TAK1 autophosphorylation, by forming a stable H bond at the K(124) position on TRAF6. Furthermore, EGCG enhanced proteasome-associated deubiquitinase expression to rescue proteins from proteasomal degradation. Western blot analyses of joint homogenates from rats with AIA showed a significant increase in K(48) -linked polyubiquitination, TAK1 phosphorylation, and TRAF6 expression when compared to naive rats. Administration of EGCG (50 mg/kg/day) for 10 days ameliorated AIA in rats by reducing TAK1 phosphorylation and K(48) -linked polyubiquitination. CONCLUSION: Our findings provide a rationale for targeting TAK1 for the treatment of RA with EGCG.

Effects of er-miao-san extracts on TNF-alpha-induced MMP-1 expression in human dermal fibroblasts.

BACKGROUND: Various health benefits have been attributed to Er-Miao-San (EMS), a traditional Chinese herbal formulation that contains equal amounts of cortex phellodendri (Phellodendron amurense Ruprecht) and rhizoma atractylodis (Atractylodes lancea D.C). However, its effect on the anti-inflammatory activity in human dermal fibroblasts (HDFs) and the mechanism underlying this effect are unknown. RESULTS: This study investigated the effects of EMS on TNF-α-induced MMP-1 expression in HDFs. Our data show that EMS inhibited TNF-α-induced MMP-1 expression in a concentration-dependent manner. Interestingly, EMS maintained IκB content without inhibiting the phosphorylation of MAPKs, which are well-established upstream kinases of NF-κB. Moreover, EMS reduced the level of nuclear p65 protein in HDFs. Luciferase assay revealed that EMS inhibits the transcriptional activity of NF-κB by stabilizing IκB. Our results show that EMS exerts its anti-inflammatory effect by inhibiting NF-κB-regulated genes such as IL-1ß and IL-8. Moreover, EMS effectively inhibited TNF-α-induced expression of MMP-1 via the NF-κB pathway. CONCLUSIONS: Taken together, our data suggest that EMS could potentially be used as an anti-inflammatory and anti-aging treatment.

Effects of Er-Miao-San extracts on TNF-alpha-indueed MMP-1 expression in human dermal fibroblasts

BACKGROUND: Various health benefits have been attributed to Er-Miao-San (EMS), a traditional Chinese herbal formulation that contains equal amounts of cortex phellodendri (Phellodendron amurense Ruprecht) and rhizoma atractylodis (Atractylodes lancea D.C). However, its effect on the anti-inflammatory activity in human dermal fibroblasts (HDFs) and the mechanism underlying this effect are unknown. RESULTS: This study investigated the effects of EMS on TNF-α-induced MMP-1 expression in HDFs. Our data show that EMS inhibited TNF-α-induced MMP-1 expression in a concentration-dependent manner. Interestingly, EMS maintained IkB content without inhibiting the phosphorylation of MAPKs, which are well-established upstream kinases of NF-kB. Moreover, EMS reduced the level of nuclear p65 protein in HDFs. Luciferase assay revealed that EMS inhibits the transcriptional activity of NF-kBbystabilizing IkB. Our results show that EMS exerts its anti-inflammatory effect by inhibiting NF-kB-regulated genes such as IL-1ß and IL-8. Moreover, EMS effectively inhibited TNF-α-induced expression of MMP-1 via the NF-kBpathway. CONCLUSIONS: Taken together, our data suggest that EMS could potentially be used as an anti-inflammatory and anti-aging treatment.

Anti-inflammatory diterpenoids from the root bark of Acanthopanax gracilistylus.

Five new ent-pimarane (1-3, 7, and 8) and three new ent-kaurane diterpenoids (4-6) and a new oleanane triterpene acid (9), together with 22 known compounds, were isolated from the root bark of the medicinal herb Acanthopanax gracilistylus. The structures of 1-9 were established based on the interpretation of high-resolution MS and 1D- and 2D-NMR data. The absolute configurations of 7 and 11 were determined by single-crystal X-ray diffraction and electronic circular dichroism analysis. Compounds 7 and 8 represent rare naturally occurring structures based on the devinyl ent-pimarane skeleton. Compounds 3, 10, 14, 16, and 17 exhibited potent inhibitory effects on the release of interleukin-1ß (IL-1ß), interleukin-8 (IL-8), and tumor necrosis factor (TNF-α) in lipopolysaccharide-stimulated peripheral blood mononuclear cells.

Chemical constituents from Melastoma dodecandrum and their inhibitory activity on interleukin-8 production in HT-29 cells.

In search of anti-inflammatory lead compounds from traditional Chinese medicines, a bioassay-guided phytochemical study on Melastoma dodecandrum was carried out. As a result, 18 compounds have been isolated. Their chemical structures were determined on the basis of their physicochemical properties and spectral data. Among the isolates, three pentacyclic triterpenoids, ursolic acid (1), asiatic acid (3) and terminolic acid (6), together with one tannin casuarinin (17), were found to significantly decrease interleukin-8 (IL-8) production in human colon cancer cells. The results imply, at least in part, that the anti-inflammatory effect of M. dodecandrum could be due to inhibition of IL-8 production, demonstrated by these naturally occurring compounds described above.

Kaurenoic acid from Sphagneticola trilobata Inhibits Inflammatory Pain: effect on cytokine production and activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway.

Kaurenoic acid [ent-kaur-16-en-19-oic acid (1)] is a diterpene present in several plants including Sphagneticola trilobata. The only documented evidence for its antinociceptive effect is that it inhibits the writhing response induced by acetic acid in mice. Therefore, the analgesic effect of 1 in different models of pain and its mechanisms in mice were investigated further. Intraperitoneal and oral treatment with 1 dose-dependently inhibited inflammatory nociception induced by acetic acid. Oral treatment with 1 also inhibited overt nociception-like behavior induced by phenyl-p-benzoquinone, complete Freund's adjuvant (CFA), and both phases of the formalin test. Compound 1 also inhibited acute carrageenin- and PGE(2)-induced and chronic CFA-induced inflammatory mechanical hyperalgesia. Mechanistically, 1 inhibited the production of the hyperalgesic cytokines TNF-α and IL-1ß. Furthermore, the analgesic effect of 1 was inhibited by l-NAME, ODQ, KT5823, and glybenclamide treatment, demonstrating that such activity also depends on activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway, respectively. These results demonstrate that 1 exhibits an analgesic effect in a consistent manner and that its mechanisms involve the inhibition of cytokine production and activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway.

Antiinflammatory effects of traditional Korean medicine, JinPi-tang and its active ingredient, hesperidin in HaCaT cells.

A traditional Korean medicine, JinPi-tang (JPT), has been used in dermatological therapeutics and cosmeceuticals including antiaging creams and moisturizers. However, it has not been clarified how JPT and its active ingredient, hesperidin (HES), regulates inflammatory reactions in HaCaT cells. Thus, the mechanisms of action of JPT and HES on inflammatory reactions were investigated for the first time in an experimental model. The antiinflammatory effects of JPT and HES in HaCaT cells were investigated by using an enzyme-linked immunosorbent assay and a reverse transcription-polymerase chain reaction as well as western blot analysis. JPT and HES inhibited the H(2)O(2)-induced interleukin-8 and tumor necrosis factor-α production as well as its mRNA expression. In addition, JPT and HES inhibited the activation of the nuclear factor-κB, the phosphorylation of IκBα and the p38 mitogen-activated protein kinase, as well as the activation of cyclooxygenase-2. The findings suggest that JPT and HES would be helpful in the treatment of UV radiation-induced inflammatory skin diseases.

Double blind clinical trial in a series of 115 patients with seborrheic dermatitis: prevention of relapses using a topical modulator of Toll like receptor 2.

AIM: Seborrheic dermatitis is a chronic inflammatory disease aggravated by Malassezia species. Toll-like receptors (TLR) are part of innate immune system that can be activated by yeasts. Previous studies showed that an association of Umbelliferae extract with a lipid (TLR2-Regul™) decreases the IL-8 expression in human skin in contact with M. furfur. The aim of this study was to assess the activity of a topical formulated with TLR2-Regul™ in the prevention of seborrheic dermatitis (SD) relapses. METHODS: Immune-competent SD adult patients were treated for SD (topical imidazoles or steroids). Cleared patients were randomized and received a topical containing TLR2-Regul™ (A) or its vehicle (B). Erythema, scales and pruritus were assessed during two months. RESULTS: The study included 115 patients, mean age 43.4, sex ratio m/f 1.5. At week 4 the relapse rate was 26% (N.=15) in group A and 43% (N.=25) in group B. At W8 the relapse rate was 21% (N.=12) in group A and 40% (N.=23) (P=0.0309). CONCLUSION: In this series of 115 adults with seborrheic dermatitis, patients treated with a topical containing TLR-Regul™ showed a significantly less relapse rate compared with the excipient group (P<0.05). TLR modulation could represent a new therapeutic approach in the prevention of seborrheic dermatitis relapses.

Modulation of matrix metalloproteinase and cytokine production by licorice isolates licoricidin and licorisoflavan A: potential therapeutic approach for periodontitis.

BACKGROUND: Inflammatory cytokines and matrix metalloproteinases (MMPs) produced by resident and inflammatory cells in response to periodontopathogens play a major role in the tissue destruction observed in periodontitis, which is a disease that affects tooth-supporting structures. In the present study, we investigate the effects of licorice-derived licoricidin (LC) and licorisoflavan A (LIA) on the secretion of various cytokines and MMPs by human monocyte-derived macrophages stimulated with Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) lipopolysaccharide (LPS). METHODS: Macrophages were treated with non-toxic concentrations of LC or LIA before being stimulated with A. actinomycetemcomitans LPS. The secretion of cytokines and MMPs and the activation of nuclear factor-kappa B (NF-κB) p65 and activator protein (AP)-1 were assessed by enzyme-linked immunosorbent assays. RESULTS: LC and LIA inhibited the secretion of interleukin (IL)-6 and chemokine (C-C motif) ligand 5 in a concentration-dependent manner but did not affect the secretion of IL-8 by LPS-stimulated macrophages. LC and LIA also inhibited the secretion of MMP-7, -8, and -9 by macrophages. The suppression of cytokine and MMP secretion by LC and LIA was associated with the reduced activation of NF-κB p65 but not that of AP-1. CONCLUSION: The present study suggests that LC and LIA have potential for the development of novel host-modulating strategies for the treatment of cytokine and/or MMP-mediated disorders such as periodontitis.

[Anti-inflammatory and repair activity of two toothpastes in an ex-vivo human gingival mucosa model]. / Comparaison de l'effet anti-inflammatoire et réparateur de deux dentifrices sur un modèle expérimental de muqueuse gingivale humaine.

INTRODUCTION: Our aim was to compare the anti-inflammatory activity of two toothpastes, one including enoxolone 1%, the other including plant extracts and sodium bicarbonate. MATERIAL AND METHODS: Gingival fragments were kept alive ex-vivo. Inflammation, inflammatory mediators (SP and LPS) were applied to culture medium on contact with corium to induce inflammation. The effect of both toothpastes was assessed with histological and biochemical parameters (inflammatory cytokine IL8) of inflammation on the synthesis of collagen and cellular viability. RESULTS: Both toothpaste "A" including enoxolone at 1% and "P" including plant extracts and sodium bicarbonate were effective on edema and vasodilatation. "A" acted on IL8 synthesis, unlike "P". Both toothpastes boosted collagen synthesis by fibroblasts. The percentage of cellular viability for "A" was superior to the currently admitted standard (80%), unlike to "P". DISCUSSION: The mechanisms of action of each toothpaste seem to be different. "A" modulates pro-inflammatory cytokine IL8 expression, unlike "P". The toothpaste "A" seems to be better tolerated.

Expression and regulation of HIF-1alpha in macrophages under inflammatory conditions; significant reduction of VEGF by CaMKII inhibitor.

BACKGROUND: Macrophages expressing the pro-angiogenic transcription factor hypoxia-inducible factor (HIF)-1alpha have been demonstrated in rheumatoid arthritis (RA) in the synovial tissue. Aim of the present study was to investigate intracellular signal transduction regulation of pro-inflammatory HIF-1 alpha expression in macrophages to identify possible new intervention strategies. We investigated the effects of CaMKII-inhibitors amongst other kinase inhibitors, on HIF-1 alpha expression and downstream production of pro-angiogenic factors in macrophages. METHODS: Differentiated THP-1 cells and synovial fluid (SF) macrophages were stimulated with 1 microg/ml LPS with or without pretreatment with specific inhibitors of the ERK pathway (PD98059), the PI3K pathway (LY294002), and the CaMKII pathway (KN93 and SMP-114). mRNA and protein expression of HIF-1 alpha, VEGF, MMP-9, and IL-8 was measured in cell lysates and cell supernatants. RESULTS: HIF-1 alpha protein expression in LPS-stimulated THP-1 macrophages could be blocked by ERK- and PI3K-inhibitors, but also by the CaMKII inhibitor KN93. THP-1 and SF macrophages produced high levels of VEGF, IL-8, and MMP-9, and VEGF protein production was significantly inhibited by PI3K-inhibitor, and by both CaMKII inhibitors. LPS stimulation in an hypoxic environment did not change VEGF levels, suggesting that LPS induced VEGF production in macrophages is more important than the hypoxic induction. CONCLUSIONS: Expression of HIF-1 alpha and downstream effects in macrophages are regulated by ERK-, PI3K, but also by CaMKII pathways. Inhibition of HIF-1alpha protein expression and significant inhibition of VEGF production in macrophages was found using CaMKII inhibitors. This is an unknown but very interesting effect of the CaMKII inhibitor SMP-114, which has been in clinical trial as DMARD for the treatment of RA. This effect may contribute to the anti-arthritic effects of SMP-114.

Interaction of estrogen and tumor necrosis factor alpha in endothelial cell migration and early stage of angiogenesis.

The role of estrogen replacement therapy in postmenopausal women remains controversial. The authors hypothesized that contradictory results with estrogen therapy may be explained by estrogen's potent proangiogenic property, which could be protective in women without atherosclerotic disease but in the presence of chronic inflammation, could lead to destabilization of atherosclerotic plaques. The authors thus examined the interaction between 17beta-estradiol (E2) and the inflammatory cytokine tumor necrosis factor alpha (TNFalpha) in an early stage of angiogenesis. Human umbilical endothelial cells were grown to confluence. Migration was assessed with a wound assay and proliferation was assessed with 5-bromo-2'-deoxyuridine (BrDU). Cells were treated with medium alone, TNFalpha at 0.3, 1, or 20 ng/ml, E2 at 20 nM, or the combination of E2 and TNFalpha. The authors used real-time polymerase chain reaction (PCR) to measure changes in expression of the angiogenesis genes angiopoeitin-2 (Ang-2), vacular endothelial growth factor (VEGF)-A and -C, and interleukin (IL)-8. A large dose of TNFalpha (20 ng/ml) inhibited healing at 24 to 48 h and the addition of E2 preserved some healing. E2 by itself doubled migration, with only a minimal effect on proliferation. A low dose of TNFalpha (0.3 ng/ml) had no effect on migration, 1.0 ng/ml moderately increased it, but the addition of E2 to both doses of TNFalpha increased migration. There was no change in migration when cells were pretreated with E2 and given TNFalpha after wounding, whereas pretreatment with TNFalpha followed by E2 significantly increased wound healing. The nitric oxide synthase (NOS) inhibitor N-nitro-l-arginine-methyl ester (l-NAME) completely blocked the E2 effect on migration. TNFalpha (0.3 and 1.0 ng/ml) increased expression of VEGF-C (2.8 +/- 0.1- and 2.5 +/- 0.2-fold, respectively) and IL-8 (32.8 +/- 1.2- and 42.7 +/- 3.6-fold, respectively) mRNA, but E2 had no significant effect on these molecules. E2 increases the angiogenic activity of TNFalpha. This could potentially worsen the stability of complex atherosclerotic plaques and increase cardiovascular events.

A licorice extract reduces lipopolysaccharide-induced proinflammatory cytokine secretion by macrophages and whole blood.

BACKGROUND: Periodontal diseases are a group of inflammatory disorders initiated by specific Gram-negative periodontopathogenic bacteria that lead to the destruction of tooth-supporting tissues. In this study, we tested whether a carbon dioxide-supercritical extract of Glycyrrhiza uralensis (licorice) can reduce the periodontopathogen-induced inflammatory response. METHODS: Monocyte-derived macrophages were treated with various concentrations of the licorice extract prior to being stimulated with Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) and Porphyromonas gingivalis lipopolysaccharide (LPS). The capacity of the licorice extract to mediate the inflammatory response was also tested in an ex vivo whole blood model stimulated with P. gingivalis LPS. The secretion of interleukin (IL)-1beta, -6, and -8 and tumor necrosis factor-alpha (TNF-alpha) in both models was assessed by enzyme-linked immunosorbent assays. Changes in the phosphorylation state of macrophage intracellular kinases induced by A. actinomycetemcomitans LPS and the licorice extract in the macrophage model were characterized by immunoblotting. RESULTS: The licorice extract exhibited potent anti-inflammatory properties, inhibiting the periodontopathogen LPS-induced IL-1beta, -6, and -8 and TNF-alpha responses of macrophages. The licorice extract inhibited the phosphorylation of important macrophage intracellular signaling proteins, including nuclear factor-kappa B p65 nuclear transcription factor and Jun proto-oncogene-encoded activator protein (AP) 1 transcription factor, which are involved in inflammatory signaling pathways. The licorice extract was also a potent inhibitor of the proinflammatory cytokine response in the ex vivo human whole blood model. CONCLUSION: This CO(2)-supercritical licorice extract is a potential candidate for the development of a new therapy to prevent and/or treat periodontitis-associated tissue destruction.