Anti-inflammatory properties of Salograviolide A purified from Lebanese plant Centaurea ainetensis.

BACKGROUND: Anti-inflammatory activities of medicinal plants have largely been attributed to their content of sesquiterpene lactones (SLs). SLs are predominantly found in the sunflower family Asteraceae and have been isolated from many plants of this family, particularly Centaurea. The anti-inflammatory activities of extract of Centaurea ainetensis, a Lebanese endemic plant, and the isolated active molecule were assessed for their potential ant-inflammatory activities. METHODS: Plant extract from Centaurea ainetensis, and the isolated active ingredient Salograviolide A (SA), a sesquiterpene lactones guaianolide, were used for the study. Western blotting and electrophoretic mobility shift assays were used to test the effects of the plant extract and SA on interleukin-1 (IL-1) induced increase in cyclooxygenase-2 (COX-2) levels and in nuclear factor-kappaB (NF-kappaB) translocation in an intestinal epithelial cell (IEC) of inflammation. Their effects on inflammation score and cytokine levels were also studied in an iodoacetoamide-induced rat model of inflammation. RESULTS: Plant extract and SA were shown to reverse the effects observed by IL-1 on COX-2 levels and NF-kappaB translocation in IEC. SA decreased the level of inflammatory cytokines and the level of inflammation in the animal model. CONCLUSION: These findings suggest that SA may be useful in the development of natural therapies for inflammatory diseases.

Interleukin-6 and Cyclooxygenase-2 downregulation by fatty-acid fractions of Ranunculus constantinopolitanus.

BACKGROUND: Medicinal plants represent alternative means for the treatment of several chronic diseases, including inflammation. The genus Ranunculus, a representative of the Ranunculaceae family, has been reported to possess anti-inflammatory, analgesic, antiviral, antibacterial, antiparasitic and antifungal activities, possibly due to the presence of anemonin and other. Different studies have shown the occurrence of unusual fatty acids (FAs) in Ranunculaceae; however, their therapeutic role has not been investigated. The purpose of this study is to characterize potential anti-inflammatory bioactivities in Ranunculus constantinopolitanus D'Urv., traditionally used in Eastern Mediterranean folk medicine. METHODS: The aerial part of R. constantinopolitanus was subjected to methanol (MeOH) extraction and solvent fractionation. The bioactive fraction (I.2) was further fractionated using column chromatography, and the biologically active subfraction (Y2+3) was identified using infrared (IR) spectroscopy, nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS). The effects of I.2 and Y2+3 on cell viability were studied in mouse mammary epithelial SCp2 cells using trypan blue exclusion method. To study the anti-inflammatory activities of I.2 and Y2+3, their ability to reduce interleukin (IL)-6 levels was assessed in endotoxin (ET)-stimulated SCp2 cells using enzyme-linked immunosorbent assay (ELISA). In addition, the ability of Y2+3 to reduce cyclooxygenase (COX)-2 expression was studied in IL-1-treated mouse intestinal epithelial Mode-K cells via western blotting. Data were analyzed by one-way analysis of variance (ANOVA), Student-Newman-Keuls (SNK), Tukey HSD, two-sample t-test and Dunnett t-tests for multiple comparisons. RESULTS: The chloroform fraction (I.2) derived from crude MeOH extract of the plant, in addition to Y2+3, a FA mix isolated from this fraction and containing palmitic acid, C18:2 and C18:1 isomers and stearic acid (1:5:8:1 ratio), reduced ET-induced IL-6 levels in SCp2 cells without affecting cell viability or morphology. When compared to fish oil, conjugated linoleic acid (CLA) and to individual FAs as palmitic, linoleic, oleic and stearic acid or to a mix of these FAs (1:5:8:1 ratio), Y2+3 exhibited higher potency in reducing ET-induced IL-6 levels within a shorter period of time. Y2+3 also reduced COX-2 expression in IL-1-treated Mode-K cells. CONCLUSION: Our studies demonstrate the existence of potential anti-inflammatory bioactivities in R. constantinopolitanus and attribute them to a FA mix in this plant.

Cross-talk between intestinal epithelial cells and immune cells in inflammatory bowel disease.

Inflammatory bowel disease (IBD) involves functional impairment of intestinal epithelial cells (IECs), concomitant with the infiltration of the lamina propria by inflammatory cells. We explored the reciprocal paracrine and direct interaction between human IECs and macrophages (MΦ) in a co-culture system that mimics some aspects of IBD. We investigated the expression of intercellular junctional proteins in cultured IECs under inflammatory conditions and in tissues from IBD patients. IECs establish functional gap junctions with IECs and MΦ, respectively. Connexin (Cx26) and Cx43 expression in cultured IECs is augmented under inflammatory conditions; while, Cx43-associated junctional complexes partners, E-cadherin, ZO-1, and ß-catenin expression is decreased. The expression of Cx26 and Cx43 in IBD tissues is redistributed to the basal membrane of IEC, which is associated with decrease in junctional complex proteins' expression, collagen type IV expression and infiltration of MΦ. These data support the notion that the combination of paracrine and hetero-cellular communication between IECs and MΦs may regulate epithelial cell function through the establishment of junctional complexes between inflammatory cells and IECs, which ultimately contribute to the dys-regulation of intestinal epithelial barrier.

Protein regulators of eicosanoid synthesis: role in inflammation.

A variety of factors contribute to the complex course of inflammation. Microbiological, immunological and toxic agents can initiate the inflammatory response by activating a variety of humoral and cellular mediators. In the early phase of inflammation, excessive amounts of cytokines and inflammatory mediators are released. These factors activate, in addition to other signaling pathways, the lipid synthesis pathways, which play a crucial role in the pathogenesis of organ dysfunction. Arachidonic acid (AA), the precursor of pro-inflammatory eicosanoids, is released from membrane phospholipids by the action of phospholipase A(2) (PLA(2)), and is metabolized to prostaglandins (PGs) and leukotrienes (LTs) by the action of cyclooxygenase (COX) and lipoxygenase (LO) enzymes, respectively. Disordered activation of PLA(2), LO and COX enzymes have been implicated in many inflammatory diseases. PLA(2) is activated by phospholipase-A(2)-activating protein (PLAP) and LO by 5-lipoxygenase-activating protein (FLAP). The inducible form of COX-2 enzyme, which is usually not present under basal conditions, is induced in inflammation. In this article the function of these enzymes in eicosanoid synthesis, their regulation, and their implication in inflammatory disorders will be reviewed. The properties, function and regulation of the protein activators PLAP and FLAP will also be discussed.

The role of NF-kappaB in mediating the anti-inflammatory effects of IL-10 in intestinal epithelial cells.

In inflammatory bowel disease, cells that infiltrate the mucosa regulate intestinal epithelial cell function partly through release of pro- and anti-inflammatory cytokines. The aim of this study is to evaluate the role of the anti-inflammatory cytokine, IL-10, on normal mouse intestinal epithelial cells (Mode-K) in the absence or presence of IL-1. Western blotting assays and immunocytochemistry were used to identify the presence of IL-1 and IL-10 receptors on Mode-K cells; and electrophoretic mobility shift assays were used to study the activation of NF-kappaB transcription factor. Stimulation of Mode-K cells with IL-1 or IL-10 did not modify IL-1 and IL-10 receptor expression levels. IL-1 induced the synthesis of the enzyme cyclooxygenase-2 (COX-2) through the activation and translocation of p65 subunit of NF-kappaB. Inhibition of translocated p65 binding to DNA, inhibited COX-2 production and induced apoptosis. IL-10 inhibited IL-1-induced effects on IKB-alpha and IKB-beta proteins through stabilizing these proteins; subsequently causing inhibition of NF-kappaB translocation to the nucleus and any subsequent induction of COX-2. These data support a role for IL-10 in the regulation of IEC function under inflammatory conditions and the involvement of COX-2 in inhibiting apoptosis in mouse intestinal epithelial cells.

Regulation of nuclear factor-kappaB in intestinal epithelial cells in a cell model of inflammation.

BACKGROUND: Interleukin-1 (IL-1), an inflammatory cytokine whose levels are elevated in inflamed mucosa, causes part of its effect on intestinal epithelial cells (IEC) through inducing ceramide production. AIM: To study the role of nuclear factor-kappaB (NF-kappaB), a pro-inflammatory and anti-apoptotic factor, in IL-1-treated IEC. METHODS: NF-kappaB activity and levels of apoptotic proteins were assessed by electrophoretic mobility shift assay and RNA-protection assay, respectively. RESULTS: IL-1 and ceramide, which have been shown to partially mediate IL-1 effects on IEC, activated NF-kappaB levels significantly. This activation was due to a decrease in IkappaB-alpha and IkappaB-beta protein levels. Moreover, the ratio of mRNA levels of anti-apoptotic to pro-apoptotic proteins was significantly increased in IL-1-treated IEC. CONCLUSION: NF-kappaB may play a key role in the regulation of the expression of pro-inflammatory and/or apoptotic genes in inflammatory bowel disease, making this protein an attractive target for therapeutic intervention.

Stage-specific effect of N-(4-hydroxyphenyl)retinamide on cell growth in squamous cell carcinogenesis.

Squamous cell carcinoma (SCC) is the most prevalent form of epithelial cancer. SCC results when normal epithelial cells undergo multiple neoplastic changes that culminate in the evolution of an invasive cancer. Retinoids are commonly used as chemopreventive and treatment agents in skin cancer; however, SCC progression is accompanied by a gradual loss of retinoid responsiveness. The synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) has shown promising anti-neoplastic activity in a variety of tumor cells, including those that are resistant to all-trans retinoic acid (t-RA). We investigated the effect of HPR on growth and apoptosis of squamous cells at different stages of carcinogenesis. We then determined if retinoic acid receptor (RAR) overexpression affected the outcome of HPR treatment. To model SCC malignant progression, we used a panel of murine keratinocytes representing different stages of squamous cell carcinogenesis. This panel consisted of primary keratinocytes, SP1 and 308 papilloma cell lines, the PAM-212 squamous carcinoma cell line, and the spindle I7 cell line. With the exception of the primary keratinocytes, all cells were unresponsive to t-RA treatment. Pharmacological concentrations of HPR were non-cytotoxic to all keratinocytes tested and HPR sensitivity was stage-dependent, with the papilloma cell lines being the most sensitive, and the spindle cells being the most resistant. Overexpression of RARgamma in SP1 papilloma cells enhanced growth suppression and apoptosis induction by HPR. HPR-induced growth suppression was accompanied by a simultaneous block in the G(1) phase of the cell cycle in RAR-transduced and control SP1 cells and differential regulation of cell cycle and apoptotic mediators.

IL-1 stimulates ceramide accumulation without inducing apoptosis in intestinal epithelial cells.

BACKGROUND: In inflammatory bowel disease (IBD), cytokine levels (such as interleukin-1 (IL-1)) are elevated. We have shown previously that IL-1 activates phospholipid signaling pathways in intestinal epithelial cells (EEC), leading to increased ceramide levels. AIM: To determine whether ceramide induces apoptosis in IEC. METHODS: Apoptosis was evaluated by annexin-V binding or Hoechst nuclear staining. Levels of bcl-2, bcl-x, bax, p53 and p21 were determined by Western blotting, and celi cycle analysis was determined by flow cytometry. RESULTS: IL-1 increased ceramide accumulation in a time-dependent and concentration-dependent manner with a peak response at 4 h, with [IL-1] = 30 ng/ml. Neither IL-1 nor ceramide induced apoptosis in EEC, but they increased bcl-2 levels and decreased bax and p21 levels without affecting bcl-x and p53 levels. They also caused a slight but significant increase in the G2/M phase. These data suggest a role for ceramide in IBD and suggest a possible mechanism for the enhanced tumorigenic activity in IBD patients.