Evaluation of anti-inflammatory and atrophogenic effects of glucocorticoids on reconstructed human skin

Topical glucocorticoids (GCs) are extensively used in the treatment of inflammatory skin diseases. However, their long-term use is often accompanied by severe and eventually irreversible adverse effects, with atrophy being the most important limitation. Currently, most non-clinical studies involve animal testing, so the results are not always representative of the situation in humans. The aim of this project was to establish an in vitro test protocol for the evaluation of the anti-inflammatory and atrophic potential of topically applied GCs in reconstructed human skin. Initial studies with fibroblasts and keratinocytes confirmed the anti-inflammatory and atrophogenic effects of GCs, as evidenced by decreased cytokine production and collagen mRNA expression. In non-pretreated reconstructed human skin (EpiDermFT?), the topical application of GCs for seven days strongly reduced the secretion of interleukin (IL)-6. GC-induced skin atrophy, known to appear only after prolonged treatment, was not detected by the analysis of epidermal thickness and collagen mRNA expression. However, reproducible epidermal inflammation was established for the first time in reconstructed human skin. Topical treatment with tumour necrosis factor (TNF) increased IL-6 release and strongly reduced epidermal thickness accompanied by severe parakeratosis. GC treatment of reconstructed human skin reduced IL-6 levels and completely resolved parakeratosis, leading to the normalisation of epidermal thickness. These induced inflammatory conditions mimic more closely the clinical situations in which GCs are used, and therefore appear to be more suitable for future investigations for the establishment of a human-based in vitro test protocol for evaluating wanted and unwanted GC effects.     

Interactions between glucocorticoids and anti-inflammatory peptides

Both pro- and anti-inflammatory mediators regulate the anti-inflammatory actions of glucocorticoids, in part by modifying the binding of glucocorticoids to specific receptors. For instance, somatostatin has been shown to increase glucocorticoid binding and signaling in macrophages. The mechanism of this regulation does not require an increased expression of glucocorticoid receptors but, rather, a stabilization of glucocorticoid receptor-associated heat shock protein 90. This is related to a decrease in calpain activity. Thus calpain inhibition may offer a new and exciting possibility for enhancing the anti-inflammatory efficiency of glucocorticoids.     

The anti-inflammatory effects of exercise: mechanisms and implications for the prevention and treatment of disease

Regular exercise reduces the risk of chronic metabolic and cardiorespiratory diseases, in part because exercise exerts anti-inflammatory effects. However, these effects are also likely to be responsible for the suppressed immunity that makes elite athletes more susceptible to infections. The anti-inflammatory effects of regular exercise may be mediated via both a reduction in visceral fat mass (with a subsequent decreased release of adipokines) and the induction of an anti-inflammatory environment with each bout of exercise. In this Review, we focus on the known mechanisms by which exercise - both acute and chronic - exerts its anti-inflammatory effects, and we discuss the implications of these effects for the prevention and treatment of disease.     

New mechanisms of biological effects of electromagnetic fields

ATP production in mitochondria depends on the nuclear spin and magnetic moment of Mg²⁺ ion in creatine kinase and ATPase. Consequently, the enzymatic synthesis of ATP is an ion-radical process and depends on the external magnetic field and microwave fields that control the spin states of ion-radical pairs and influence the ATP synthesis. The chemical mechanism of ATP synthesis and the origin of biological effects of electromagnetic (microwave) fields are discussed.     

New mechanisms of biological effects of electromagnetic fields

The production of ATP in mitochondria depends on the magnesium nuclear spin and magnetic moment of a Mg2+ ion in creatine kinase and ATPase. This suggests that enzymatic synthesis of ATP is an ion-radical process and thus depends on the external magnetic field (magnetobiology originates from this fact) and microwave fields, which control the spin states of ion-radical pairs and affect the ATP synthesis. The chemical mechanism of ATP synthesis and the origin of biological effects of electromagnetic (microwave) fields are discussed.     

Escin exerts synergistic anti-inflammatory effects with low doses of glucocorticoids in vivo and in vitro

Escin, a natural mixture of triterpenoid saponins isolated from the seed of the horse chestnut (Aesculus hippocastanum), had been demonstrated to possess anti-edematous and anti-inflammatory effects. The present study was designed to investigate whether escin exhibits synergistic anti-inflammatory effects when combined with glucocorticoids. The carrageenan-induced paw edema and pleuritis in bilaterally adrenalectomized rats were used to investigate the anti-inflammatory effects of escin and glucocorticoid alone or combined. The carrageenan-induced paw edema was inhibited only when escin and corticosterone (Cort) were administered together. Co-administration of escin with Cort significantly reduced the volume of exudates and the number of white blood cells of exudates in bilaterally adrenalectomized rats with pleuritis, but treatment with escin or Cort alone at a suboptimal concentration did not show any effect on the pleuritis rats. After the murine macrophagic RAW264.7 cells stimulated by lipopolysaccharide (LPS), they were treated with escin, Cort or escin and Cort. Then nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β) of cell culture supernatants were analyzed. Escin or Cort markedly reduced the content of NO, TNF-α and IL-1β secreted by LPS-stimulated RAW264.7 macrophage cells. The combination of suboptimal concentrations of escin with Cort, which alone could not markedly inhibit the release of inflammatory factors, inhibited the secretion of NO, TNF-α and IL-1β in LPS-stimulated RAW264.7 macrophage cells. The findings suggest escin can synergize with glucocorticoids to enhance their anti-inflammatory effect.     

New anti-inflammatory agents

The pyrrole derivatives la, b and 2a, b were used as precursors for the preparation of N-substituted pyrrole derivatives 3a, b-9a, b and pyrrolo[2,3-d]pyrimidines 13-16. Also, all the newly prepared products were tested for anti-inflammatory activity as analogues to fenamates, and some of them revealed moderate anti-inflammatory activity compared to the standard drug indomethacin.     

The anti-inflammatory effect of leptin on experimental colitis: involvement of endogenous glucocorticoids

The present study was designed to compare the effect of leptin on acute colonic inflammation with that of acute stress exposure, which acts via the hypothalamic-pituitary-adrenal (HPA) axis. Sprague-Dawley rats of both sexes were administered intrarectally with acetic acid. Either leptin (10 microg/kg; i.p.) or saline was injected immediately before and 6 h after the induction of colitis. A group of rats was exposed to water avoidance stress (WAS) for 30 min at the 6th h of colitis induction. RU-486 (2 mg/kg; i.p.), a glucocorticoid receptor antagonist, was injected intraperitoneally, at 12 and 1 h before the initial leptin injection, and at 1 h before the second leptin injection or exposure to WAS. Rats were decapitated at 24 h and the distal 8 cm of the colon were removed for macroscopic and microscopic scoring, determination of tissue wet weight index (WI) and tissue myeloperoxidase activity (MPO). Acetic acid-induced colitis significantly increased macroscopic and microscopic damage scores, WI and MPO, compared to control group. Exposure to acute WAS or treatment with leptin reduced the elevations in damage scores, WI and MPO induced by colitis, but no additive inhibitory effect was observed when WAS and leptin were applied together. RU-486 treatment reversed the inhibitory effects of leptin or WAS on colonic inflammation. Our results demonstrate that exogenous leptin mimics the effects of HPA axis activation on colitis-induced inflammatory process. The results also suggest that the anti-inflammatory effect of leptin involves a tissue neutrophil-dependent mechanism and is dependent on the release of glucocorticoids.     

Computer search for molecular mechanisms of ulcerogenic action of non-steroidal anti-inflammatory drugs

Peptic ulcers are the most frequent side effect of therapy with non-steroidal anti-inflammatory drugs (NSAIDs). Good experimental evidence exists that pathogenesis of peptic ulcers cannot be attributed only to inhibition of cyclooxygenases. The knowledge about other molecular mechanisms of drug action associated with development of peptic ulcers could be useful for design of new safer NSAIDs. However, considerable time and material resources are needed for corresponding experimental studies. For simplification of the experimental search, we have developed an approach for in silico identification of putative molecular mechanisms of drug actions associated with their side effects. We have generated a data set of 85 NSAIDs, which includes information about their structures and side effects. Unknown molecular mechanisms of action of these NSAIDs were evaluated by the computer program PASS (Prediction of Activity Spectra for Substances) predicting more than 3000 molecular mechanisms of action based on structural formula of sub-stances. Statistically significant associations have been found between predicted molecular mechanisms of action and development of peptic ulcers. Twenty six molecular mechanisms of action probably associated with development of peptic ulcers have been found: two of them were known previously and 24 were quite new. Analyzing Gene Ontology data, data on signal and metabolic pathways, and available MEDLINE publication data, we proposed hypotheses on the role of 10 molecular mechanisms of action in the pathogenesis of peptic ulcer.     

Ulcerative colitis impairs the acylethanolamide-based anti-inflammatory system reversal by 5-aminosalicylic acid and glucocorticoids

Studies in animal models and humans suggest anti-inflammatory roles on the N-acylethanolamide (NAE)-peroxisome proliferators activated receptor alpha (PPARα) system in inflammatory bowel diseases. However, the presence and function of NAE-PPARα signaling system in the ulcerative colitis (UC) of humans remain unknown as well as its response to active anti-inflammatory therapies such as 5-aminosalicylic acid (5-ASA) and glucocorticoids. Expression of PPARα receptor and PPARα ligands-biosynthetic (NAPE-PLD) and -degrading (FAAH and NAAA) enzymes were analyzed in untreated active and 5-ASA/glucocorticoids/immunomodulators-treated quiescent UC patients compared to healthy human colonic tissue by RT-PCR and immunohistochemical analyses. PPARα, NAAA, NAPE-PLD and FAAH showed differential distributions in the colonic epithelium, lamina propria, smooth muscle and enteric plexus. Gene expression analysis indicated a decrease of PPARα, PPARγ and NAAA, and an increase of FAAH and iNOS in the active colitis mucosa. Immunohistochemical expression in active colitis epithelium confirmed a PPARα decrease, but showed a sharp NAAA increase and a NAPE-PLD decrease, which were partially restored to control levels after treatment. We also characterized the immune cells of the UC mucosa infiltrate. We detected a decreased number of NAAA-positive and an increased number of FAAH-positive immune cells in active UC, which were partially restored to control levels after treatment. NAE-PPARα signaling system is impaired during active UC and 5-ASA/glucocorticoids treatment restored its normal expression. Since 5-ASA actions may work through PPARα and glucocorticoids through NAE-producing/degrading enzymes, the use of PPARα agonists or FAAH/NAAA blockers that increases endogenous PPARα ligands may yield similar therapeutics advantages.     

Glucocorticoid receptor IB: mediator of anti-inflammatory and teratogenic functions of both glucocorticoids and phenytoin

We studied the glucocorticoid receptor complexes of pulmonary and thymic cytosols of female A/J and CD-1 mice and of hepatoma G2 cells by two column-chromatographic systems, using both [3H]dexamethasone (DEX) and [3H]phenytoin (DPH) as ligands. Three DNA-cellulose adsorbable [3H]DEX-receptor complexes were separated in each system. Molecular sieving gave a 7-, a 5.4-, and a 3.5-nm complex (Stokes radii), and DEAE-Sephadex A-50 chromatography gave a complex eluting in the wash, one at 0.14 M KCl, and one at 0.20 M KCl by a KCl gradient. DPH blocked the binding of the 7- and 3.5-nm, wash, and 0.14 M KCl [3H]DEX complexes. Only two DNA-cellulose adsorbable [3H]DPH complexes, each blocked by DEX, were obtained in each system: a 7- and a 3.5-nm, a wash, and a 0.14 M KCl complex. Thus, there is a common receptor for both DPH and DEX. This receptor has two properties which distinguish it from the 5.4-nm DEX-specific receptor: (i) it binds with a variety of steroids other than glucocorticoids and DPH, and (ii) it rebinds new [3H]DEX or [3H]DPH after loss of ligand during chromatographic separation. These results indicate that DPH binds to receptor IB and not to receptor II of Litwack. [G. Litwack, 1976, in Glutathion: Metabolism and Function (Arias, I.M., and Jakoby, W.B., eds.), pp. 285-299, Raven Press, New York]. We have also found that hepatoma G2 cells have only receptor II. DPH affects neither the induction of tyrosine aminotransferase by DEX nor the basal level of this enzyme in these cells. Moreover, neither DEX nor DPH inhibits the release of [3H]arachidonic acid prelabeled in these cells, as they do in thymocytes which have the common receptor. Thus, it appears that glucocorticoid receptor IB binds DEX and DPH as glucocorticoid agonists mediating the anti-inflammatory and teratogenic action of these drugs, while receptor II apparently is responsible for the induction of tyrosine aminotransferase by DEX.     

Postischemic anti-inflammatory effects of bradykinin preconditioning

We sought to determine the mechanisms whereby brief administration of bradykinin (bradykinin preconditioning, BK-PC) before prolonged ischemia followed by reperfusion (I/R) prevents postischemic microvascular dysfunction. Intravital videomicroscopic approaches were used to quantify I/R-induced leukocyte/endothelial cell adhesive interactions and microvascular barrier disruption in single postcapillary venules of the rat mesentery. I/R increased the number of rolling, adherent, and emigrated leukocytes and enhanced venular albumin leakage, effects that were prevented by BK-PC. The anti-inflammatory effects of BK-PC were largely prevented by concomitant administration of a B(2)-receptor antagonist but not by coincident B(1) receptor blockade, nitric oxide (NO) synthase inhibition, or cyclooxygenase blockade. However, NO synthase blockade during reperfusion after prolonged ischemia was effective in attenuating the anti-inflammatory effects of BK-PC. Pan protein kinase C (PKC) inhibition antagonized the beneficial effects of BK-PC but only when administered during prolonged ischemia. In contrast, specific inhibition of the conventional PKC isotypes failed to alter the effectiveness of BK-PC. These results indicate that bradykinin can be used to pharmacologically precondition single mesenteric postcapillary venules to resist I/R-induced leukocyte recruitment and microvascular barrier dysfunction by a mechanism that involves B(2) receptor-dependent activation of nonconventional PKC isotypes and subsequent formation of NO.     

Chondroprotective and anti-inflammatory effects of sesamin

Osteoarthritis (OA) is a major disability of elderly people. Sesamin is the main compound in Sesamun indicum Linn., and it has an anti-inflammatory effect by specifically inhibiting Δ5-desaturase in polyunsaturated fatty acid biosynthesis. The chondroprotective effects of sesamin were thus studied in a porcine cartilage explant induced with interleukin-1beta (IL-1β) and in a papain-induced osteoarthritis rat model. With the porcine cartilage explant, IL-1β induced release of sulfated-glycosaminoglycan (s-GAG) and hydroxyproline release, and this induction was significantly inhibited by sesamin. This ability to inhibit these processes might be due to its ability to decrease expression of MMP-1, -3 and -13, which can degrade both PGs and type II collagen, both at the mRNA and protein levels. Interestingly, activation of MMP-3 might also be inhibited by sesamin. Moreover, in human articular chondrocytes (HACs), some pathways of IL-1β signal transduction were inhibited by sesamin: p38 and JNK. In the papain-induced OA rat model, sesamin treatment reversed the following pathological changes in OA cartilage: reduced disorganization of chondrocytes in cartilage, increased cartilage thickness, and decreased type II collagen and PGs loss. Sesamin alone might increase formation of type II collagen and PGs in the cartilage tissue of control rats. These results demonstrate that sesamin efficiently suppressed the pathological processes in an OA model. Thus, sesamin could be a potential therapeutic strategy for treatment of OA.