Anti-inflammatory Effects Of Glucocorticoids Research Articles

Protopanaxadiol Derivative: A Plant Origin of Novel Selective Glucocorticoid Receptor Modulator with Anti-inflammatory Effect

Constant efforts have been made to move towards maintaining the positive anti-inflammatory functions of glucocorticoids (GCs) while minimizing side effects. The anti-inflammatory effect of GCs is mainly attributed to the inhibition of major inflammatory pathways such as NF-κB through GR transrepression, while its side effects are mainly mediated by transactivation. Here, we investigated the selective glucocorticoid receptor modulator (SGRM)-like properties of a plant-derived compound. In this study, glucocorticoid receptor (GR)-mediated alleviation of inflammation by SP-8 was investigated by a combination of in vitro, in silico, and in vivo approaches. Molecular docking and cellular thermal shift assay suggested that SP-8 bound stably to the active site of GR via hydrogen bonding and hydrophobic interactions. SP-8 activated GR, induced GR nuclear translocation, and inhibited NF-κB pathway activation. Furthermore, SP-8 did not up-regulate the gene and protein expression of PEPCK and TAT in HepG2 cells, and it did not induce fat deposition like GC and has little effect on bone metabolism. Interestingly, SP-8 upregulated GR protein expression and did not cause GR phosphorylation at Ser211 in RAW264.7 cells. This work proved that SP-8 dissociated characteristics of transrepression and transactivation can be separated. In addition, the in vitro and in vivo anti-inflammatory effects of SP-8 were confirmed in LPS-induced RAW 264.7 cells and in a mouse model of DSS-induced ulcerative colitis, respectively. In conclusion, SP-8 might serve as a potential SGRM and might hold great potential for therapeutic use in inflammatory diseases.

Stress circuitry: mechanisms behind nervous and immune system communication that influence behavior.

Inflammatory processes are increased by stress and contribute to the pathology of mood disorders. Stress is thought to primarily induce inflammation through peripheral and central noradrenergic neurotransmission. In healthy individuals, these pro-inflammatory effects are countered by glucocorticoid signaling, which is also activated by stress. In chronically stressed individuals, the anti-inflammatory effects of glucocorticoids are impaired, allowing pro-inflammatory effects to go unchecked. Mechanisms underlying this glucocorticoid resistance are well understood, but the precise circuits and molecular mechanisms by which stress increases inflammation are not as well known. In this narrative review, we summarize the mechanisms by which chronic stress increases inflammation and contributes to the onset and development of stress-related mood disorders. We focus on the neural substrates and molecular mechanisms, especially those regulated by noradrenergic signaling, that increase inflammatory processes in stressed individuals. We also discuss key knowledge gaps in our understanding of the communication between nervous and immune systems during stress and considerations for future therapeutic strategies. Here we highlight the mechanisms by which noradrenergic signaling contributes to inflammatory processes during stress and how this inflammation can contribute to the pathology of stress-related mood disorders. Understanding the mechanisms underlying crosstalk between the nervous and immune systems may lead to novel therapeutic strategies for mood disorders and/or provide important considerations for treating immune-related diseases in individuals suffering from stress-related disorders.

4-Octyl Itaconate and Dimethyl Fumarate Induce Secretion of the Anti-Inflammatory Protein Annexin A1 via NRF2.

Annexin A1 is a key anti-inflammatory effector protein that is involved in the anti-inflammatory effects of glucocorticoids. 4-Octyl itaconate (4-OI), a derivative of the endogenous metabolite itaconate, which is abundantly produced by LPS-activated macrophages, has recently been identified as a potent anti-inflammatory agent. The anti-inflammatory effects of 4-OI share a significant overlap with those of dimethyl fumarate (DMF), a derivate of another Krebs cycle metabolite fumarate, which is already in use clinically for the treatment of inflammatory diseases. In this study we show that both 4-OI and DMF induce secretion of the 33-kDa form of annexin A1 from murine bone marrow-derived macrophages, an effect that is much more pronounced in LPS-stimulated cells. We also show that this 4-OI- and DMF-driven annexin A1 secretion is NRF2-dependent and that other means of activating NRF2 give rise to the same response. Lastly, we demonstrate that the cholesterol transporter ABCA1, which has previously been implicated in annexin A1 secretion, is required for this process in macrophages. Our findings contribute to the growing body of knowledge on the anti-inflammatory effects of the Krebs cycle metabolite derivatives 4-OI and DMF.

Combined Statin and Glucocorticoid Therapy for the Safer Treatment of Preterm Birth.

Prematurity is strongly associated with poor respiratory function in the neonate. Rescue therapies include treatment with glucocorticoids due to their anti-inflammatory and maturational effects on the developing lung. However, glucocorticoid treatment in the infant can increase the risk of long-term cardiovascular complications including hypertension, cardiac, and endothelial dysfunction. Accumulating evidence implicates a molecular link between glucocorticoid excess and depletion of nitric oxide (NO) bioavailability as a mechanism underlying the detrimental effects of postnatal steroids on the heart and circulation. Therefore, combined glucocorticoid and statin therapy, by increasing NO bioavailability, may protect the developing cardiovascular system while maintaining beneficial effects on the lung. We investigated combined glucocorticoid and statin therapy using an established rodent model of prematurity and combined experiments of cardiovascular function in vivo, with those in isolated organs as well as measurements at the cellular and molecular levels. We show that neonatal glucocorticoid treatment increases the risk of later cardiovascular dysfunction in the offspring. Underlying mechanisms include decreased circulating NO bioavailability, sympathetic hyper-reactivity, and NO-dependent endothelial dysfunction. Combined neonatal glucocorticoid and statin therapy protects the developing cardiovascular system by normalizing NO and sympathetic signaling, without affecting pulmonary maturational or anti-inflammatory effects of glucocorticoids. Therefore, combined glucocorticoid and statin therapy may be safer than glucocorticoids alone for the treatment of preterm birth.

Glucocorticoid-induced leucine zipper regulates liver fibrosis by suppressing CCL2-mediated leukocyte recruitment

Liver fibrosis (LF) is a dangerous clinical condition with no available treatment. Inflammation plays a critical role in LF progression. Glucocorticoid-induced leucine zipper (GILZ, encoded in mice by the Tsc22d3 gene) mimics many of the anti-inflammatory effects of glucocorticoids, but its role in LF has not been directly addressed. Here, we found that GILZ deficiency in mice was associated with elevated CCL2 production and pro-inflammatory leukocyte infiltration at the early LF stage, resulting in enhanced LF development. RNA interference-mediated in vivo silencing of the CCL2 receptor CCR2 abolished the increased leukocyte recruitment and the associated hepatic stellate cell activation in the livers of GILZ knockout mice. To highlight the clinical relevance of these findings, we found that TSC22D3 mRNA expression was significantly downregulated and was inversely correlated with that of CCL2 in the liver samples of patients with LF. Altogether, these data demonstrate a protective role of GILZ in LF and uncover the mechanism, which can be targeted therapeutically. Therefore, modulating GILZ expression and its downstream targets represents a novel avenue for pharmacological intervention for treating LF and possibly other liver inflammatory disorders.

JTP-117968, a novel selective glucocorticoid receptor modulator, exhibits significant anti-inflammatory effect while maintaining bone mineral density in mice

Classic glucocorticoids have been prescribed for various inflammatory diseases, such as rheumatoid arthritis, due to their outstanding anti-inflammatory effects. However, glucocorticoids cause numerous unwanted side effects, including osteoporosis and diabetes. Hence, selective glucocorticoid receptor modulators (SGRMs), which retain anti-inflammatory effects with minimized side effects, are among the most anticipated drugs in the clinical field.The assumption is that there are two major mechanisms of action via glucocorticoid receptors, transrepression (TR) and transactivation (TA). In general, anti-inflammatory effects of glucocorticoids are largely due to TR, while the side effects associated with glucocorticoids are mostly mediated through TA. We previously reported that JTP-117968, a novel SGRM, maintained partial TR activity while remarkably reducing the TA activity. In this study, we investigated the anti-inflammatory effect of JTP-117968 on a lipopolysaccharide (LPS) challenge model and collagen-induced arthritis (CIA) model in mice. Meanwhile, we tested the effect of JTP-117968 on the bone mineral density (BMD) in mouse femur to evaluate the side effect. Based on the evaluation, JTP-117968 reduced the plasma levels of tumor necrosis factor α induced by LPS challenge in mice significantly. Remarkably, CIA development was suppressed by JTP-117968 comparably with prednisolone and PF-802, an active form of fosdagrocorat that has been developed clinically as an orally available SGRM. Strikingly, the side effect of JTP-117968 on mouse femoral BMD was much lower than those of PF-802 and prednisolone. Therefore, JTP-117968 has attractive potential as a new therapeutic option against inflammatory diseases with minimized side effects compared to classic glucocorticoids.

Efficacy of synthetic glucocorticoids in COVID-19 endothelites.

Since March 2020, the world has been fighting a global pandemic caused by a new coronavirus SARS-CoV-2 (COVID-19). SARS-CoV-2 is responsible for severe acute respiratory syndrome, an airway disease that can be severe and fatal in a percentage of cases. Patients with severe COVID-19 can develop extrapulmonary lesions, with renal, hepatic, cardiac, neurological, and tissue involvement that can cause further severe complications. On December 21, 2021, the European Medicines Agency (EMA) authorized the marketing of the first COVID-19 vaccine. However, several randomized trials are ongoing to find effective, safe, and widely available treatments. The most severe stages of COVID-19 infection are characterized by a multi-system inflammatory state induced by a cytokine storm causing multi-organ injury. Epidemiologic evidence has shown that glucocorticoids (GCs), particularly dexamethasone, are used in severe, hospitalized patients with COVID-19 with good therapeutic benefit. COVID-19 can also damage the endothelial system, causing microcirculatory disturbances and consequently leading to functional organ disorders. The combination of endothelial dysfunction with a generalized inflammatory state may contribute to the general pro-coagulative state described in patients with COVID-19 with increased risk of venous and arterial occlusions. The aim of this article is to describe the therapeutic utility of GCs in stabilizing the vascular endothelial barrier in COVID-19 infection. Indeed, we believe that the stabilization of the endothelial barrier and the anti-inflammatory effect of GCs could be the main effect underlying the therapeutic efficacy in COVID-19 patients.

Overexpression of GILZ in macrophages limits systemic inflammation while increasing bacterial clearance in sepsis in mice.

Studies support the beneficial effects of glucocorticoids (GCs) during septic shock, steering research toward the potential role of GC-induced proteins in controlling excessive inflammatory responses. GILZ is a glucocorticoid-induced protein involved in the anti-inflammatory effects of GCs. We investigated whether the overexpression of GILZ specifically limited to monocytes and macrophages (M/M) alone could control inflammation, thus improving the outcome of septic shock in animal models. We also monitored the expression of GILZ in M/M from septic mice and septic-shock patients. M/M from patients and septic mice displayed significantly lower expression of GILZ than those isolated from controls. Furthermore, transgenic mice (Tg-mice) experiencing sepsis, with increased expression of GILZ restricted to M/M, showed lower frequencies of inflammatory monocytes than their littermates and lower plasma levels of inflammatory cytokines. Tg-mice also had lower blood bacterial counts. We further established that the upregulation of GILZ in M/M enhanced their phagocytic capacity in in vivo assays. The increase of GILZ in M/M was also sufficient to improve the survival rates of septic mice. These results provide evidence for a central role of both GILZ and M/M in the pathophysiology of septic shock and a possible clue for the modulation of inflammation in this disease.

Humanin is a novel regulator of Hedgehog signaling and prevents glucocorticoid-induced bone growth impairment

Glucocorticoids (GCs) are frequently used to treat chronic disorders in children, including inflammation and cancer. Prolonged treatment with GCs is well known to impair bone growth, an effect linked to increased apoptosis and suppressed proliferation in growth plate chondrocytes. We hypothesized that the endogenous antiapoptotic protein humanin (HN) may prevent these effects. Interestingly, GC-induced bone growth impairment and chondrocyte apoptosis was prevented in HN overexpressing mice, HN-treated wild-type mice, and in HN-treated cultured rat metatarsal bones. GC-induced suppression of chondrocyte proliferation was also prevented by HN. Furthermore, GC treatment reduced Indian Hedgehog expression in growth plates of wild-type mice but not in HN overexpressing mice or HN-treated wild-type animals. A Hedgehog (Hh) antagonist, vismodegib, was found to suppress the growth of cultured rat metatarsal bones, and this effect was also prevented by HN. Importantly, HN did not interfere with the desired anti-inflammatory effects of GCs. We conclude that HN is a novel regulator of Hh signaling preventing GC-induced bone growth impairment without interfering with desired effects of GCs. Our data may open for clinical studies exploring a new possible strategy to prevent GC-induced bone growth impairment by cotreating with HN.-Zaman, F., Zhao, Y., Celvin, B., Mehta, H. H., Wan, J., Chrysis, D., Ohlsson, C., Fadeel, B., Cohen, P., Savendahl, L. Humanin is a novel regulator of Hedgehog signaling and prevents glucocorticoid-induced bone growth impairment.

Annexin-A1: Therapeutic Potential in Microvascular Disease.

Annexin-A1 (ANXA1) was first discovered in the early 1980's as a protein, which mediates (some of the) anti-inflammatory effects of glucocorticoids. Subsequently, the role of ANXA1 in inflammation has been extensively studied. The biology of ANXA1 is complex and it has many different roles in both health and disease. Its effects as a potent endogenous anti-inflammatory mediator are well-described in both acute and chronic inflammation and its role in activating the pro-resolution phase receptor, FPR2, has been described and is now being exploited for therapeutic benefit. In the present mini review, we will endeavor to give an overview of ANXA1 biology in relation to inflammation and functions that mediate pro-resolution that are independent of glucocorticoid induction. We will focus on the role of ANXA1 in diseases with a large inflammatory component focusing on diabetes and microvascular disease. Finally, we will explore the possibility of exploiting ANXA1 as a novel therapeutic target in diabetes and the treatment of microvascular disease.

Glucocorticoid-Induced Leucine Zipper: A Novel Anti-inflammatory Molecule.

Glucocorticoids (GCs) are the most commonly used drugs for treatment of autoimmune and inflammatory diseases. Their efficacy is due to their ability to bind cytoplasmic receptors (glucocorticoid receptors, GR) and other cytoplasmic proteins, thus regulating gene expression. Although GCs are potent life-saving drugs, their therapeutic effects are transitory and chronic use of GCs is accompanied by serious side effects. Therefore, new drugs are needed to replace GCs. We have identified a gene, glucocorticoid-induced leucine zipper (GILZ or tsc22d3), that is rapidly and invariably induced by GCs. Human GILZ is a 135-amino acid protein that mediates many GC effects, including inhibition of the NF-κB and MAPK pathways. Similar to GCs, GILZ exerts anti-inflammatory activity in experimental disease models, including inflammatory bowel diseases and arthritis. While transgenic mice that overexpress GILZ are more resistant, GILZ knockout mice develop worse inflammatory diseases. Moreover, the anti-inflammatory effect of GCs is attenuated in GILZ-deficient mice. Importantly, in vivo delivery of recombinant GILZ protein cured colitis and facilitated resolution of lipopolysaccharide-induced inflammation without apparent toxic effects. A synthetic GILZ-derived peptide, corresponding to the GILZ region that interacts with NF-κB, was able to suppress experimental autoimmune encephalomyelitis. Collectively, these findings indicate that GILZ is an anti-inflammatory molecule that may serve as the basis for designing new therapeutic approaches to inflammatory diseases.

Anti-inflammatory actions of Caesalpinin M2 in experimental colitis as a selective glucocoricoid receptor modulator

Although repression of inflammatory gene expression makes glucocorticoids (GCs) powerful anti-inflammatory agents, side effects limit usage and drive the search for improved glucocorticoid receptor (GR) ligands. It has been postulated that the anti-inflammatory effects of GCs are primarily mediated by GR’s activity in transrepressing major inflammation pathways such as NF-κB pathway, whereas their side effects are mostly mediated by GR’s transactivation. In this study, we found that Caesalpinin M2 (C-M2), a cassane furanoditerpene isolated from a Chinese medical plant, exerts an anti-inflammatory potential both in vitro and in vivo. C-M2 inhibited the expression of proinflammatory cytokine IL-1β and IL-6 in LPS-activated bone marrow-derived macrophages. Meanwhile, C-M2 treatment attenuated DSS-induced experimental acute colitis in mice and did not cause side effects, such as spleen involution, like dexamethasone treatment. Molecular docking and cellular thermal shift assay demonstrated that C-M2 could bind to GR in the ligand binding site. We showed that C-M2 mediates gene-inhibitory effects by activating GR. More importantly, C-M2 failed to induce GR binding to glucocorticoid response element-dependent genes and in turn activate their transcription. But it did repress NF-κB-dependent transcription by facilitating the interaction between GR and p65. Taken together, this non-steroidal compound of plant origin may exert anti-inflammatory actions as a selective GR modulator and might hold great potential for therapeutic use in inflammatory diseases.

Glucocorticoids Modulate Th1 and Th2 Responses in Asthmatic Mouse Models by Inhibition of Notch1 Signaling

Background: Notch1 has been linked to the pathogenesis of asthma due to its contribution on Th1/Th2 imbalance. γ-Secretase inhibitor (GSI) acts as an effective blocker of Notch1 signaling. Glucocorticoids (GCs) are the most effective anti-inflammatory drugs for asthma. The present study investigated the involvement of the Notch1 pathway in the anti-inflammatory effect of GCs and its association with Th1/Th2 balance. Methods: The asthma model was established in BALB/c mice by sensitization with ovalbumin (OVA). Dexamethasone (DEX; 1 mg/kg) and/or GSI (0.03 mg/kg) was orally or intranasally administrated. Results: Compared to the OVA-sensitized mice, the administration of DEX and/or GSI significantly ameliorated the airway inflammation infiltration, goblet cell metaplasia, and airway hyper-responsiveness. The expression of IL-4 and IL-13, as well as the ratios of eosinophils and lymphocytes, were significantly decreased, whereas IFN-γ and IL-2 levels were significantly increased in bronchoalveolar lavage fluid after the administration of DEX and GSI. The expressions of the Notch1/NICD1 pathway were decreased after DEX and/or GSI administration in lung tissues, especially in CD4+ T cells. Also, a reduction of GATA3 and elevation of T-bet levels were correlated with the upregulation of Th1/Th2 ratios in lung tissues. Conclusions: Through the inhibition of Notch1 signaling, both GSI and GCs could regulate Th1/Th2 balance involved in allergic airway inflammation in OVA-induced asthma.

Mineralocorticoid receptor blockade improves glucocorticoid-induced skin atrophy but partially ameliorates anti-inflammatory actions in an irritative model in human skin explants.

We recently demonstrated that blockade of the mineralocorticoid receptor (MR) effectively ameliorated GC-induced skin atrophy in healthy human skin explants and epidermal MR knockout mice. However, whether MR blockade improves the therapeutic index of glucocorticoids (GCs) in skin pathology was not investigated. We assessed the effects of GCs, MR antagonists (MRA) or both, in SDS-treated human skin explants. All treatments restored SDS-augmented epidermal thickness but only GC plus MRA restored the expression of COL1A1. However, MRA alone or in combination with GCs may exert a dual role in regulating inflammatory cytokines. Thus, although combined treatment may be beneficial to improve irritative skin, extensive in vivo testing is required to establish whether the anti-inflammatory effects of GCs are maintained in the presence of MRA.

Dexamethasone attenuates methacholine-mediated aquaporin 5 downregulation in human nasal epithelial cells via suppression of NF-κB activation.

Cholinergic stimulation plays a major role in inflammatory airway diseases. However, its role in airway surface liquid homeostasis and aquaporin 5 (AQP5) regulation remains unclear. In this study we sought to determine the effects of methacholine and dexamethasone on AQP5 expression in human nasal epithelial cells (HNEpC). HNEpC were cultured with methacholine or dexamethasone at 4 concentrations in vitro. The subcellular distribution of AQP5 was explored using immunocytochemistry. The pharmacologic effects of methacholine and dexamethasone on the expression of the phosphorylation of cyclic adenosine monophosphate-responsive element binding protein (p-CREB), AQP5, and nuclear factor-kappaB (NF-κB) were examined using Western blotting. AQP5 was found to be located in cell membrane and cytoplasm and present in every group without a statistically significant difference. Methacholine inhibited expression of AQP5 and p-CREB in HNEpC, whereas dexamethasone increased these protein levels dose-dependently in a statistically significant manner. In turn, HNEpC treated with methacholine and dexamethasone showed the same trends as those intervened separately with these 2 drugs. Moreover, dexamethasone had the ability to reverse the inhibitory effect of methacholine. Western blotting revealed that, after incubation with 10-4 mol/L methacholine, NF-κB increased significantly, by 186.67%, compared with the untreated control group. Again, such an increase could be significantly reversed after dexamethasone treatment. NF-κB activation is important for inhibition of p-CREB/AQP5 expression after methacholine intervention, and dexamethasone adjusts it to the opposite side. This observation could provide additional insight into the anti-inflammatory effects of glucocorticoids that contribute to maintaining airway surface liquid and mucosal defense.

JTP-117968, a novel selective glucocorticoid receptor modulator, exhibits improved transrepression/transactivation dissociation

Classic glucocorticoids that have outstanding anti-inflammatory effects are still widely prescribed for the treatment of various inflammatory and autoimmune diseases. Conversely, glucocorticoids cause numerous unwanted side effects, particularly systemically dosed glucocorticoids. Therefore, selective glucocorticoid receptor modulator (SGRM), which maintains beneficial anti-inflammatory effects while reducing the occurrence of side effects, is one of the most anticipated drugs. However, there have been no SGRMs marketed to date.The assumption is that there are two major mechanisms of action of glucocorticoids via glucocorticoid receptors, transrepression (TR) and transactivation (TA). In general, the anti-inflammatory effects of glucocorticoids are mostly mediated through TR, while the side effects associated with glucocorticoids are largely caused by TA.We started to evaluate novel orally available SGRMs that maintain anti-inflammatory effects while minimizing adverse effects by favoring TR over TA. Based on this evaluation, we discovered JTP-117968, (4b'S,7′R,8a'S)-4b'-benzyl-7′-hydroxy-N-(2-methylpyridin-3-yl)-7′-(trifluoromethyl)-4b',6′,7′,8′,8a',10'-hexahydro-5′H-spiro[cyclopropane-1,9′-phenanthrene]-2′-carboxamide, a non-steroidal SGRM. JTP-117968 has partial TR activity, but exhibits extremely low TA activity. The maximum TR efficacy of JTP-117968 was comparable to its structural analogue, PF-802, (4bS,7R,8aR)-4b-Benzyl-7-hydroxy-N-(2-methylpyridin-3-yl)-7-(trifluoromethyl)-4b,5,6,7,8,8a,9,10-octahydrophenanthrene-2-carboxamide, which is the active form of Fosdagrocorat that has been developed clinically as a first-in-class orally available SGRM. Remarkably, the TA activity of JTP-117968 was much weaker than PF-802 not only in in vitro assays, but also in in vivo mice experiments.These findings indicate that JTP-117968 exhibits improved TR/TA dissociation because the compound has significantly lower TA activity compared with an already reported SGRM. Therefore, JTP-117968 is expected to be a useful compound for evaluating ideal SGRMs in the future.

Caffeine modulates glucocorticoid-induced expression of CTGF in lung epithelial cells and fibroblasts

BackgroundAlthough caffeine and glucocorticoids are frequently used to treat chronic lung disease in preterm neonates, potential interactions are largely unknown. While anti-inflammatory effects of glucocorticoids are well defined, their impact on airway remodeling is less characterized. Caffeine has been ascribed to positive effects on airway inflammation as well as remodeling. Connective tissue growth factor (CTGF, CCN2) plays a key role in airway remodeling and has been implicated in the pathogenesis of chronic lung diseases such as bronchopulmonary dysplasia (BPD) in preterm infants. The current study addressed the impact of glucocorticoids on the regulation of CTGF in the presence of caffeine using human lung epithelial and fibroblast cells.MethodsThe human airway epithelial cell line H441 and the fetal lung fibroblast strain IMR-90 were exposed to different glucocorticoids (dexamethasone, budesonide, betamethasone, prednisolone, hydrocortisone) and caffeine. mRNA and protein expression of CTGF, TGF-β1-3, and TNF-α were determined by means of quantitative real-time PCR and immunoblotting. H441 cells were additionally treated with cAMP, the adenylyl cyclase activator forskolin, and the selective phosphodiesterase (PDE)-4 inhibitor cilomilast to mimic caffeine-mediated PDE inhibition.ResultsTreatment with different glucocorticoids (1 μM) significantly increased CTGF mRNA levels in H441 (p < 0.0001) and IMR-90 cells (p < 0.01). Upon simultaneous exposure to caffeine (10 mM), both glucocorticoid-induced mRNA and protein expression were significantly reduced in IMR-90 cells (p < 0.0001). Of note, 24 h exposure to caffeine alone significantly suppressed basal expression of CTGF mRNA and protein in IMR-90 cells. Caffeine-induced reduction of CTGF mRNA expression seemed to be independent of cAMP levels, adenylyl cyclase activation, or PDE-4 inhibition. While dexamethasone or caffeine treatment did not affect TGF-β1 mRNA in H441 cells, increased expression of TGF-β2 and TGF-β3 mRNA was detected upon exposure to dexamethasone or dexamethasone and caffeine, respectively. Moreover, caffeine increased TNF-α mRNA in H441 cells (6.5 ± 2.2-fold, p < 0.05) which has been described as potent inhibitor of CTGF expression.ConclusionsIn addition to well-known anti-inflammatory features, glucocorticoids may have adverse effects on long-term remodeling by TGF-β1-independent induction of CTGF in lung cells. Simultaneous treatment with caffeine may attenuate glucocorticoid-induced expression of CTGF, thereby promoting restoration of lung homeostasis.

COMPARATIVE ANALYSIS OF BIOLOGICAL EFFECTS OF SELECTIVE ACTIVATOR OF THE GLUCOCORTICOID RECEPTOR CPDA ON DIFFERENT SUBTYPES OF BREAST CANCER CELL LINES

Glucocorticoids (GCs) are often used as an adjuvant therapy to reduce the adverse effects of chemotherapy in breast cancer patients. Moreover, GCs can display pro-proliferative or anti-proliferative effects on BC cells depending on their molecular subtype. In addition, long-term use of GCs can induce drug resistance and tumor progression. The biological activity of GCs is mediated by glucocorticoid receptor (GR) via either transrepression or transactivation. The anti-inflammatory effects of GCs are thought to be due to transrepression, while side effects, drug resistance and tumor progression/metastasis are associated with transactivation. We have previously demonstrated that Compound A, a selective GR agonist (SEGRA), has a GR-dependent antitumor effect on blood cancer cells in vitro, not triggering the GR transactivation. This study was focused on the analysis of the CpdA activity in BC models in vitro. We demonstrated the antiproliferative effect of CpdA on BC cells and its ability to induce transrepression of GR-depended genes such as CCND1-3, COX-2, iNOS without the induction of transactivation. A comparative analysis showed that CpdA was an effective and safe alternative to dexamethasone in adjuvant chemotherapy for breast cancer.

P-158 YI Antiinflammatory Effects of Protein Kinase Inhibitor Pyrrole Derivate Having Antitumor Activity

In our previous studies we showed antitumor activity of pyrrole derivate 1-(4-Cl-benzyl)-3-Cl-4-(CF3-fenylamino)-1H-pyrrol-2,5-dione (MI-1) on in vitro (cell lines of human colorectal adenocarcinoma COLO205, SW-620) and in vivo (1,2-dimethylhydrazine-induced rat colon cancer) models. MI-1 is an inhibitor of protein kinases EGFR, VEGFR, IGF1R, Src, PDK1, which are overexpressed in human colorectal tumors. It is known that EGFR and IGF1R can activate PI3K/Akt and following NF-κB signaling pathways. EGFR can also activate Jak/STAT pathway. PDK1 and Src are the key effectors of PI3K/Akt pathway. The consequence of activation of these signaling pathways, in addition to the induction of cell proliferation and survival, are pro-inflammatory effect. Furthermore, the roles of inflammation and oxidative stress in tumor initiation and progression are acknowledged, as well as their close relationship. So antiinflammatory properties of MI-1 as an inhibitor of named kinases were assumed, and the antiinflammatory and antioxidant effects of MI-1 on rat ulcerative colitis model were aimed to be discovered. Acetic acid induced ulcerative colitis model was used. Forty Male Wistar rats were induced by 2 rectal applications of 4% acetic acid (1 mL) weekly. MI-1 (2.7 mg/kg) and a reference drug prednisolone (0.7 mg/kg) administrations were provided daily for 14 days starting simultaneously with the 1st acetic acid application. The internal bowel surface was visually inspected, the integrity of bowel mucosa and inflammatory features were evaluated by 10 and 11 grade scales on macro- (Sehirli A, 2009) and light microscopy levels (Appleyard CB, 1995) respectively, and the grade of total injury (GTI) was calculated. Thiobarbituric acid active products (TBAP), protein carbonyl groups (PCG), intracellular superoxide dismutase (SOD) and catalase (CAT) activities as indicators of colon mucosa redox status were measured spectrophotometrically in bowel mucosa homogenates. Colonic clefts and small ulcers, edema, epithelium desquamation and inflammatory features manifested by submucosa lymphoid and histiocytic infiltration and extravasation appeared under colitis condition. GTI was equal to 10 (GTIcontrol = 0). Increase of TBAP (by 89%) and PCG (by 60%) and decrease of SOD (by 40%) in bowel mucosa were observed. Prednisolone escaped the ulcers, edema and epithelium desquamation, and decreased GTI to 3—some inflammatory features (lymphoid and histiocytic infiltration of submucosa) were persisted. SOD activity was leveled by prednisolone, but TBAP and PCG remained higher than control ones by 52% and 42% respectively. MI-1 restored colon mucosa integrity, decreased mucosa inflammation down to local foci of lymphocytes and histiocytes (GTI = 1), leveled TBAP, PCG and SOD. MI-1 possessed antiinflammatory properties more expressed that prednisolone ones, as well as normalized mucosa redox balance. Since antiinflammatory effects of glucocorticoids are realized through inhibition of phospholipase A2 followed prostaglandins synthesis decrease, and less through COX-2 inhibition, thereat effects of MI-1 through inflammatory signaling pathways are multiple, probably determining MI-1 more expressed and complex impact on inflammatory process. Thus MI-1 has a prospect for correction of inflammatory and carcinogenesis processes through multiple mechanisms of action.

Glucocorticoid Receptor-β Is Downregulated by Vitamin D Treatment in Children with Low Vitamin D Levels but Not in Allergic Asthma

Background: Vitamin D (VitD) enhances the anti-inflammatory effects of glucocorticoids (GC) in vitro. It was hypothesized that VitD3 (colecalciferol) treatment could have an impact on the expression of glucocorticoid receptors (GRs) in a group of children with low VitD levels, with or without asthma. Methods: Asthmatic children and healthy controls, all with low serum VitD levels (25-hydroxyvitamin D [25(OH)D] level <30 ng/mL) were recruited. VitD3 treatment at a dose of 300,000 IU was given orally. Blood samples were obtained at admission and 1 month after the treatment to examine serum 25(OH)D levels and the relative gene expression (RGE) of GR-α and -β in peripheral blood mononuclear cells. Results: Twenty-four children with asthma (Mage 11.1 ± 2.1 years) and 14 healthy controls (Mage 11.5 ± 1.7 years) were studied. The expression of GR-β was significantly higher in the control group at baseline compared with those with asthma (p = 0.006). With VitD3 treatment, there was a decrease in GRβ expression at...