Alpinia katsumadai Hayata Volatile Oil Is Effective in Treating 5-Fluorouracil-Induced Mucositis by Regulating Gut Microbiota and Modulating the GC/GR Pathway and the mPGES-1/PGE2/EP4 Pathways.

This study was aimed to investigate the therapeutic effect and mechanism of AKHO on 5-fluorouracil (5-FU)-induced intestinal mucositis in mice. Mouse body weight, diarrhea score, and H&E staining were applied to judge the therapeutic effect of AKHO. 16S rDNA and nontargeted metabolomics have been used to study the mechanism. WB, ELISA, and immunohistochemistry were adopted to validate possible mechanisms. The results demonstrated that AKHO significantly reduced diarrhea scores and intestinal damage induced by 5-FU in mice. AKHO lowered the serum levels of LD and DAO, and upregulated the expressions of ZO-1 and occludin in the ileum. Also, AKHO upregulated the abundance of Lactobacillus in the gut and suppressed KEGG pathways such as cortisol synthesis and secretion and arachidonic acid metabolism. Further validation studies indicated that AKHO downregulated the expressions of prostaglandin E2 (PGE2), microsomal prostaglandin E synthase-1 (mPGES-1), and PGE2 receptor EP4, as well as upregulated the expression of glucocorticoid (GC) receptor (GR), leading to improved intestinal epithelial barrier function. Taken together, AKHO elicited protective effects against 5-FU-induced mucositis by regulating the expressions of tight junction proteins via modulation of GC/GR and mPGES-1/PGE2/EP4 pathway, providing novel insights into the utilization and development of this pharmaceutical/food resource.

Microsomal prostaglandin E2 synthase-1 and its inhibitors: Molecular mechanisms and therapeutic significance.

Inflammation is closely linked to the abnormal phospholipid metabolism chain of cyclooxygenase-2/microsomal prostaglandin E2 synthase-1/prostaglandin E2 (COX-2/mPGES-1/PGE2). In clinical practice, non-steroidal anti-inflammatory drugs (NSAIDs) as upstream COX-2 enzyme activity inhibitors are widely used to block COX-2 cascade to relieve inflammatory response. However, NSAIDs could also cause cardiovascular and gastrointestinal side effects due to its inhibition on other prostaglandins generation. To avoid this, targeting downstream mPGES-1 instead of upstream COX is preferable to selectively block overexpressed PGE2 in inflammatory diseases. Some mPGES-1 inhibitor candidates including synthetic compounds, natural products and existing anti-inflammatory drugs have been proved to be effective in in vitro experiments. After 20 years of in-depth research on mPGES-1 and its inhibitors, ISC 27864 have completed phase II clinical trial. In this review, we intend to summarize mPGES-1 inhibitors focused on their inhibitory specificity with perspectives for future drug development.

Modulation of the PGE2-Mediated Pathway in the Eclosion Blocking Effect of Flumethrin and Terpenoid Subfraction Isolated from Artemesia nilagirica in Rhipicephalus annulatus.

Prostaglandins are a group of important cell-signaling molecules involved in the regulation of ovarian maturation, oocyte development, egg laying and associated behaviors in invertebrates. However, the presence of prostaglandin E2 (PGE2), the key enzymes for PGE2 biosynthesis and its interference by drugs were not investigated previously in the ovary of ticks. The present study was undertaken to assess the modulation of the PGE2-mediated pathway in the eclosion blocking effect of flumethrin and terpenoid subfraction isolated from Artemisia nilagirica in Rhipicephalus annulatus ticks. The acaricidal activities and chemical profiling of the terpenoid subfraction were performed. The localization of the cyclooxygenase1 (COX1) and prostaglandin E synthase (PGES) enzymes and the quantification of PGE2 in the ovaries of the ticks treated with methanol (control), flumethrin and terpenoid subfraction were also undertaken. In addition, the vitellogenin concentration in hemolymph was also assayed. Both flumethrin and the terpenoid subfraction of A. nilagirica elicited a concentration-dependent inhibition of fecundity and blocking of hatching of the eggs. The COX1 could not be detected in the ovaries of treated and control ticks, while there was no significant difference observed in the concentration of vitellogenin (Vg) in them. The presence of PGES in the oocytes of control ticks was confirmed while the immunoreactivities against PGES were absent in the vitellogenic oocytes of ticks treated with flumethrin and terpenoid subfraction. The levels of PGE2 were below the detection limit in the ovaries of the flumethrin-treated ticks, while it was significantly lower in the ovaries of the terpenoid subfraction-treated ticks. Hence, the prostaglandin E synthase and PGE2 were identified as very important mediators for the signaling pathway for ovarian maturation and oviposition in ticks. In addition, the key enzyme for prostaglandin biosynthesis, PGES and the receptors for PGE2 can be exploited as potential drug targets for tick control. The detection of PGES by immunohistochemistry and quantification of PGE2 by LC-MSMS can be employed as valuable tools for screening newer compounds for their eclosion blocking acaricidal effects.

Structure-based screening for the discovery of 1,2,4-oxadiazoles as promising hits for the development of new anti-inflammatory agents interfering with eicosanoid biosynthesis pathways.

The multiple inhibition of biological targets involved in pro-inflammatory eicosanoid biosynthesis represents an innovative strategy for treating inflammatory disorders in light of higher efficacy and safety. Herein, following a multidisciplinary protocol involving virtual combinatorial screening, chemical synthesis, and in vitro and in vivo validation of the biological activities, we report the identification of 1,2,4-oxadiazole-based eicosanoid biosynthesis multi-target inhibitors. The multidisciplinary scientific approach led to the identification of three 1,2,4-oxadiazole hits (compounds 1, 2 and 5), all endowed with IC50 values in the low micromolar range, acting as 5-lipoxygenase-activating protein (FLAP) antagonists (compounds 1 and 2), and as a multi-target inhibitor (compound 5) of arachidonic acid cascade enzymes, namely cyclooxygenase-1 (COX-1), 5-lipoxygenase (5-LO) and microsomal prostaglandin E2 synthase-1 (mPGES-1). Moreover, our in vivo results demonstrate that compound 5 is able to attenuate leukocyte migration in a model of zymosan-induced peritonitis and to modulate the production of IL-1ß and TNF-α. These results are of interest for further expanding the chemical diversity around the 1,2,4-oxadiazole central core, enabling the identification of novel anti-inflammatory agents characterized by a favorable pharmacological profile and considering that moderate interference with multiple targets might have advantages in re-adjusting homeostasis.

Establishment of an in vitro 3D model for neuroblastoma enables preclinical investigation of combined tumor-stroma drug targeting.

The majority of anti-cancer therapies target the proliferating tumor cells, while the tumor stroma, principally unaffected, survives, and provide a niche for surviving tumor cells. Combining tumor cell and stroma-targeting therapies thus have a potential to improve patient outcome. The neuroblastoma stroma contains cancer-associated fibroblasts expressing microsomal prostaglandin E synthase-1 (mPGES-1). mPGES-1-derived prostaglandin E2 (PGE2 ) is known to promote tumor growth through increased proliferation and survival of tumor cells, immune suppression, angiogenesis, and therapy resistance, and we, therefore, hypothesize that mPGES-1 constitutes an interesting stromal target. Here, we aimed to develop a relevant in vitro model to study combination therapies. Co-culturing of neuroblastoma and fibroblast cells in 3D tumor spheroids mimic neuroblastoma tumors with regard to the cyclooxygenase/mPGES-1/PGE2 pathway. Using the spheroid model, we show that the inhibition of fibroblast-derived mPGES-1 enhanced the cytotoxic effect of doxorubicin and vincristine and significantly reduced tumor cell viability and spheroid growth. Cyclic treatment with vincristine in combination with an mPGES-1 inhibitor abrogated cell repopulation. Moreover, inhibition of mPGES-1 potentiated the cytotoxic effect of vincristine on established neuroblastoma allografts in mice. In conclusion, we established a 3D neuroblastoma model, highlighting the potential of combining stromal targeting of mPGES-1 with tumor cell targeting drugs like vincristine.

Anti-osteoarthritic Effects of an Herbal Composition LI73014F2 on Interleukin-1ß-induced Primary Human Articular Chondrocytes.

Osteoarthritis (OA) is one of the most well-characterized joint diseases and is associated with chondrocyte inflammation, metalloproteinase upregulation and apoptosis. LI73014F2 is a novel composition prepared from aqueous extract of Terminalia chebula fruit, alcohol extract of Curcuma longa rhizome, and Boswellia serrata extract at 2:1:2 ratio. Earlier studies have shown that LI73014F2 inhibits cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX) activities, and attenuates clinical symptoms in OA subjects. In the present study, we evaluated the protective anti-inflammatory and anti-apoptotic effects, as well as the underlying mechanisms, of LI73014F2 in interleukin (IL)-1ß-induced inflammation in human primary chondrocytes. Human chondrocytes were treated with LI73014F2 (0, 12.5, 25 and 50 µg/mL) in IL-1ß (10 ng/mL)-containing chondrocyte growth medium for 24 h. Cell viability was assessed using an MTT assay. The pro-inflammatory mediator, inflammatory cytokines, MMPs, apoptosis-related proteins, mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways protein expression levels were detected by western blot analysis. The results demonstrated that LI73014F2 normalized the expressions of COX-2, mPGES-1, PGE2, 5-LOX, LTB4, IL-1ß, TNFα, IL-6, MMP-2, MMP-3, MMP-9, MMP-13, Bax/Bcl-2, cleaved caspase-9 and -3, cleaved PARP, phospho-NF-κB p65 and phospho-p38 MAPK proteins in IL-1ß-induced primary human chondrocytes. Moreover, the data suggested that LI73014F2 reduced IL-1ß-induced inflammation and apoptosis, at least partially via the inhibition of the NF-κB/MAPK signaling pathway. In conclusion, the present findings provide the molecular basis of the anti-OA efficacy of LI73014F2.

The Anti-Inflammatory and Antimicrobial Potential of Selected Ethnomedicinal Plants from Sri Lanka.

Traditional folk medicine in Sri Lanka is mostly based on plants and plant-derived products, however, many of these medicinal plant species are scientifically unexplored. Here, we evaluated the anti-inflammatory and antimicrobial potency of 28 different extracts prepared from seven popular medicinal plant species employed in Sri Lanka. The extracts were subjected to cell-based and cell-free assays of 5-lipoxygenase (5-LO), microsomal prostaglandin E2 synthase (mPGES)-1, and nitric oxide (NO) scavenging activity. Moreover, antibacterial and disinfectant activities were assessed. Characterization of secondary metabolites was achieved by gas chromatography coupled to mass spectrometric (GC-MS) analysis. n-Hexane- and dichloromethane-based extracts of Garcinia cambogia efficiently suppressed 5-LO activity in human neutrophils (IC50 = 0.92 and 1.39 µg/mL), and potently inhibited isolated human 5-LO (IC50 = 0.15 and 0.16 µg/mL) and mPGES-1 (IC50 = 0.29 and 0.49 µg/mL). Lipophilic extracts of Pothos scandens displayed potent inhibition of mPGES-1 only. A methanolic extract of Ophiorrhiza mungos caused significant NO scavenging activity. The lipophilic extracts of G. cambogia exhibited prominent antibacterial and disinfectant activities, and GC-MS analysis revealed the presence of fatty acids, sesquiterpenes and other types of secondary metabolites. Together, our results suggest the prospective utilization of G. cambogia as disinfective agent with potent anti-inflammatory properties.

A Combinatorial Virtual Screening Approach Driving the Synthesis of 2,4-Thiazolidinedione-Based Molecules as New Dual mPGES-1/5-LO Inhibitors.

Dual inhibition of microsomal prostaglandin E2 synthase-1 (mPGES-1) and 5-lipoxygenase (5-LO), two key enzymes involved in pro-inflammatory eicosanoid biosynthesis, represents a new strategy for treating inflammatory disorders. Herein we report the discovery of 2,4-thiazolidinedione-based mPGES-1/5-LO dual inhibitors following a multidisciplinary protocol, involving virtual combinatorial screening, chemical synthesis, and validation of the biological activities for the selected compounds. Following the multicomponent-based chemical route for the decoration of the 2,4-thiazolidinedione core, a large library of virtual compounds was built (∼2.0×104 items) and submitted to virtual screening. Nine selected molecules were synthesized and biologically evaluated, disclosing among them four compounds able to reduce the activity of both enzymes in the mid- and low- micromolar range of activities. These results are of interest for further expanding the chemical diversity around the 2,4-thiazolidinedione central core, facilitating the identification of novel anti-inflammatory agents endowed with a promising and safer pharmacological profile.

Nardosinanone N suppresses LPS-induced macrophage activation by modulating the Nrf2 pathway and mPGES-1.

The side effects of nonsteroidal anti-inflammatory drugs (NSAIDs) in the cardiovascular system mainly result from its inhibitory effect on cyclooxygenase-2 (COX-2). Since NSAIDs are one of the most commonly used anti-inflammatory drugs in the clinic, it is necessary to identify new anti-inflammatory drugs that are safer than NSAIDs. Nardosinanone N (NAN), a compound isolated from the roots and rhizomes of Nardostachys chinensis, was evaluated for its anti-inflammatory effects using the lipopolysaccharide (LPS)-stimulated RAW264.7 cell line and rat peritoneal macrophage models. First, we found that NAN down regulated the levels of nitric oxide (NO), inducible nitric oxide synthase (iNOS) and prostaglandin E2 (PGE2), but not cyclooxygenase-2 (COX-2). Additionally, NAN reduced the M1 macrophage phenotype and increased the M2 macrophage phenotype. Furthermore, mechanistic studies showed that NAN activated the nuclear factor-erythroid 2 -related factor 2 (Nrf2) signaling pathway, which, in turn, increased the expression of antioxidant protein heme oxygenase-1 (HO-1) to achieve its anti-inflammatory effect. Finally, Nrf2 siRNA and the HO-1 inhibitor significantly attenuated the anti-inflammatory effect of NAN. More interestingly, we found that NAN did not affect COX-2 expression and activity but reduced the PGE2 concentration by selective inhibition of microsomal prostaglandin E synthase-1 (mPGES-1). In conclusion, NAN may be a new anti-inflammatory drug that has fewer side effects than NSAIDs and can be a new potential Nrf2 activator and mPGES-1 inhibitor.

A review on mPGES-1 inhibitors: From preclinical studies to clinical applications.

Prostaglandin E2 (PGE2) is a lipid mediator of inflammation and cancer progression. It is mainly formed via metabolism of arachidonic acid by cyclooxygenases (COX) and the terminal enzyme microsomal prostaglandin E synthase-1 (mPGES-1). Widely used non-steroidal anti-inflammatory drugs (NSAIDs) inhibit COX activity, resulting in decreased PGE2 production and symptomatic relief. However, NSAIDs block the production of many other lipid mediators that have important physiological and resolving actions, and these drugs cause gastrointestinal bleeding and/or increase the risk for severe cardiovascular events. Selective inhibition of downstream mPGES-1 for reduction in only PGE2 biosynthesis is suggested as a safer therapeutic strategy. This review covers the recent advances in characterization of new mPGES-1 inhibitors in preclinical models and their future clinical applications.

Inhibition of COX-2, mPGES-1 and CYP4A by isoliquiritigenin blocks the angiogenic Akt signaling in glioma through ceRNA effect of miR-194-5p and lncRNA NEAT1.

BACKGROUND: Arachidonic acid (AA) metabolic enzymes including cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1) and cytochrome P450 (CYP) 4A11 play important roles in glioma angiogenesis. Thus, there is an urgent need to identify the underlying mechanisms and develop strategies to overcome them. METHODS: A homology model of human CYP4A11 was constructed using SYBYL-X 2.0. Structure-based virtual screening against COX-2, mPGES-1 and CYP4A11was performed using the Surflex-Dock of the SYBYL suite. The candidates were further evaluated their antiangiogenic activities in a zebrafish embryo and rabbit corneal angiogenesis model. Laser doppler analysis was used to measure tumor perfusion. The expression of CD31 and α-SMA was measured by immunofluorescence. Western blot was used to measure the expression of HIF-1, Akt and p-Akt. The gene expression of FGF-2, G-CSF, PDGF, TGF-ß, Tie-2, VEGF, lncRNA NEAT1 and miR-194-5p were determined using qPCR. The production of FGF-2, TGF-ß and VEGF were analyzed using ELISA. Bioinformatic analysis and luciferase reporter assays confirmed the interaction between lncRNA NEAT1 and miR-194-5p. RESULTS: The nearly 36,043 compounds from the Traditional Chinese Medicine (TCM) database were screened against COX-2, mPGES-1 and CYP4A11 3D models, and the 17 top flavonoids were identified. In zebrafish screening, isoliquiritigenin (ISL) exhibited the most potent antiangiogenic activities with the EC50 values of 5.9 µM. Conversely, the antiangiogenic effects of ISL in the zebrafish and rabbit corneal models were partly reversed by 20-hydroxyeicosatetraenoic acid (20-HETE) or prostaglandin E2 (PGE2). ISL normalized glioma vasculature and improved the efficacy of temozolomide therapy in the rat C6 glioma model. Inhibition of COX-2, mPGES-1 and CYP4A by ISL decreased FGF-2, TGF-ß and VEGF production in the C6 and U87 glioma cells with p-Akt downregulation, which was reversed by Akt overexpression. Furthermore, ISL downregulated lncRNA NEAT1 but upregulated miR-194-5p in the U87 glioma cell. Importantly, lncRNA NEAT1 overexpression reversed ISL-mediated increase in miR-194-5p expression, and thereby attenuated FGF-2, TGF-ß and VEGF production. CONCLUSIONS: Reprogramming COX-2, mPGES-1 and CYP4A mediated-AA metabolism in glioma by flavonoid ISL inhibits the angiogenic Akt- FGF-2/TGF-ß/VEGF signaling through ceRNA effect of miR-194-5p and lncRNA NEAT1, and may serve as a novel therapeutic strategy for human glioma.

Selenium-rich maize modulates the expression of prostaglandin genes in lipopolysaccharide-stimulated RAW264.7 macrophages.

Cell signaling is necessary for the organs to co-ordinate with the whole body and it includes response to external stimuli, inflammation, hormonal secretions and other various metabolic functions. In the present study, we have focused on the inflammatory signals modulated by the reactive oxygen and nitrogen species (RONS). Under homeostatic conditions, these species turn on the COX-1-dependent arachidonic acid (AA) pathway towards the release of anti-inflammatory enzymes. However, the excess release of these ions induces negative effects in the form of inflammation by turning on the COX-2-dependent AA pathway to release pro-inflammatory enzymes. In the present study, we observed the shunting of the COX-2-dependent AA pathway towards the release of anti-inflammatory enzymes with the supplementation of organic dietary selenium in the form of seleniferous maize extracts. We observed that 500 nM selenium concentration in Se-maize extracts downregulated the COX-2 and mPGES-1 expressions by 3.8- and 3.2-fold and upregulated the GPx-1 and H-PGDS expressions by 5.0- and 5.4-fold, respectively. To facilitate more availability of Se from the dietary matrices, Se-maize extracts were incubated with rMETase. It was observed that the enzyme-treated cells increased the downregulation of COX-2 and mPGES-1 expressions by 24.8- and 21.0-fold and the upregulation of GPx-1 and H-PGDS expressions by 13.2- and 16.5-fold, respectively.

Bavachin attenuates LPS-induced inflammatory response and inhibits the activation of NLRP3 inflammasome in macrophages.

BACKGROUND: Bavachin is a natural product isolated from Psoralea corylifolia L. that has been applied as a traditional medicine in Asian countries. However, the anti-inflammatory effects of bavachin on LPS-induced inflammation and NLRP3 inflammasome activation by macrophages remain unclear. PURPOSE: We investigated the anti-inflammatory effects of bavachin on LPS-activated murine macrophage cell line J774A.1 cells and murine peritoneal macrophages. METHODS: J774A.1 cells and murine peritoneal macrophages were pre-treated with bavachin following LPS treatment. The concentrations of NO, PGE2, IL-6 and IL-12p40 in cell culture supernatant were analyzed. The expressions of iNOS, COX-2, mPGES-1 and MAPKs were analyzed using Western blotting, while NF-κB activity was detected using promoter reporter assay. To examine the activation of NLRP3 inflammasome, J774A.1 cells were incubated with LPS, and then treated with bavachin following treatment with ATP. The concentration of IL-1ß in the cell culture supernatant was measured. The expressions of NLRP3, ASC, caspase-1 and IL-1ß were analyzed using Western blotting. The formation of inflammasome complex was observed by immunofluorescence microscopy. RESULTS: Bavachin suppressed LPS-induced NO and PGE2 production, and decreased iNOS and mPGES-1 expression. Bavachin also reduced LPS-induced IL-6 and IL-12p40 production and decreased the activation of MAPKs and NF-κB. Additionally, bavachin suppressed NLRP3 inflammasome-derived IL-1ß secretion, decreased caspase-1 activation, repressed mature IL-1ß expression, and inhibited inflammasome complex formation. Furthermore, bavachin also suppressed the production of NO, IL-6 and IL-12p40 by LPS-stimulated murine peritoneal macrophages. CONCLUSION: Our experimental results indicated anti-inflammatory effects of bavachin exhibit attenuation of LPS-induced inflammation and inhibit activation of NLRP3 inflammasome in macrophages. These results suggest that bavachin might have potential in treating inflammatory and autoimmune diseases.

Lipophilic extracts of Leucas zeylanica, a multi-purpose medicinal plant in the tropics, inhibit key enzymes involved in inflammation and gout.

ETHNOPHARMACOLOGICAL RELEVANCE: Leucas zeylanica (L.) W.T. Aiton is a popular, multi-purpose medicinal plant in Sri Lanka but the pharmacological potential and the chemical profile have not been systematically investigated to understand and rationalize the reported ethnobotanical significance. AIM OF THE STUDY: The present study was undertaken to scientifically validate the traditional usage of this plant for the treatment of inflammatory conditions, gout and microbial infections. Inhibition of 5-lipoxygenase (5-LO), microsomal prostaglandin E2 synthase (mPGES)-1 and xanthine oxidase (XO) by different extracts of L. zeylanica was investigated to determine the anti-inflammatory and anti-gout activity, respectively. The antibacterial and antifungal activities were also studied and the relevant constituents in the bioactive extracts were tentatively identified. MATERIALS AND METHODS: Cell-free and/or cell-based assays were employed in order to investigate the effects of the extracts against the activity of human 5-LO, mPGES-1 and XO as well as to assess antioxidant properties. The antibacterial activity of the extracts was determined by the broth micro-dilution method against Gram positive and Gram negative bacteria including methicillin-resistant Staphylococcus aureus while the agar dilution method was employed to determine the anti-Candida activity. Gas chromatography coupled to mass spectrometric (GC-MS) analysis enabled the characterization of secondary metabolites in the extracts. RESULTS: The dichloromethane extract of L. zeylanica efficiently inhibited 5-LO activity in stimulated human neutrophils (IC50 = 5.5 µg/mL) and isolated human 5-LO and mPGES-1 (IC50 = 2.2 and 0.4 µg/mL). Potent inhibition of XO was observed by the same extract (IC50 = 47.5 µg/mL), which is the first report of XO-inhibitory activity of a Sri Lankan medicinal plant. Interestingly, significant radical scavenging activity was not observed by this extract. Only the n-hexane extract exhibited antibacterial activity against Staphylococcus aureus and Staphylococcus saprophyticus with a MIC of 250 µg/mL while the anti-Candida activity was moderate. GC-MS analysis revealed the presence of phytosterols, fatty acids, sesquiterpenes, diterpenes and several other types of secondary metabolites. CONCLUSIONS: Potent inhibition of 5-LO, mPGES-1 and XO rationalizes the ethnopharmacological use of L. zeylanica as anti-inflammatory and anti-gout remedy. Interestingly, the antimicrobial activities were not prominent, despite its wide utility as an antimicrobial medication.

The protective role of selenium against cadmium-induced hepatotoxicity in laying hens: Expression of Hsps and inflammation-related genes and modulation of elements homeostasis.

The purpose of this study was to examine the potential role of high selenium (Se) diets in alleviating chronic cadmium (Cd) hepatic toxicity in laying hens. In the present study, 128 healthy 31-week-old laying hens were fed a diet supplemented with Se (Na2SeO3, 2 mg/kg), Cd (CdCl2, 150 mg/kg), or both Se and Cd (150 mg/kg of CdCl2 and 2 mg/kg of Na2SeO3) for 90 days. The expression levels of heat shock proteins (Hsps, including Hsp60, Hsp70 and Hsp90) and inflammation-related factors, including nuclear factor-kappa B p50 (NF-κB), cyclooxygenase-2 (COX-2), prostaglandin E synthases (PTGES), interleukin 1-beta (IL-1ß), and tumor necrosis factor-α (TNF-α) were investigated. The concentrations of 28 elements were also determined. The results indicated that Cd treatment significantly increased the mRNA and protein expression levels of Hsps and significantly improved the expression of inflammation-related genes. Moreover, Cd addition to the diets resulted in disturbances in the systemic balance of 13 elements, leading to decrease in the concentrations of Cr, Mn, Sr, Ba, and Hg and increase in Li, B, Ca, Ti, Fe, Cu, Mo, and Cd concentrations. Treatment with Se significantly alleviated Cd-induced hepatic toxicity, as evidenced by a reduction in Hsp60, Hsp70, Hsp90, NF-κB, COX-2, PTGES, TNF-α, and IL-1ß expression. Additionally, Se and Cd co-treatment alleviated the changes in Li, B, Ca, Fe, Ti, Cu, Mo, Cd, Cr, Se, Sr, Ba, and Hg concentrations, which was in contrast to that upon Cd induction. The study indicated that Se could help against the negative effects of Cd and may be related to the alleviation of Cd-induced Hsps stress and the inflammatory responses along with modulating the element homeostasis.

Common structural and pharmacophoric features of mPGES-1 and LTC4S.

Prostaglandins and leukotrienes are produced in the COX and 5-LOX pathways of the inflammatory process. The current drugs target the upstream enzymes of either of the two pathways, leading to side effects. We have attempted to target the downstream enzymes simultaneously. Two compounds 2 and 3 (10 µM), identified by virtual screening, inhibited mPGES-1 activity by 53.4 ± 4.0 and 53.9 ± 8.1%, respectively. Structural and pharmacophore studies revealed a set of common residues between LTC4S and mPGES-1 as well as four-point pharmacophore mapping onto the inhibitors of both these enzymes as well as 2 and 3. These structural and pharmacophoric features may be exploited for ligand- and structure-based screening of inhibitors and designing of dual inhibitors.

Virtual screening and biological evaluation of novel antipyretic compounds.

Due to the absence of safety of the antipyretics to patients with cardiovascular dysfunction, new targets to treat inflammation have been pursued. mPGES-1 is a promising target because its inhibition would not cause the side-effects related to COX inhibition. To identify novel inhibitors of mPGES-1, we developed a ligand-based pharmacophore model that differentiates true inhibitors from decoys and enlightens the structure-activity relationships for known mPGES-1 inhibitors. The model (four hydrophobic centers, two hydrogen bond acceptor and two hydrogen bond donor points) was employed to select lead-like compounds from ZINC database for in vivo evaluation. Among the 18 compounds selected, five inhibited the fever induced by LPS. The most potent compound (5-(4-fluorophenyl)-3-({6-methylimidazo[1,2-a]pyridin-2-yl}methyl)-2,3dihydro-1,3,4-oxadiazol-2-one) is active peripherally (i.v.) or centrally (i.c.v.) (82.18% and 112% reduction, respectively) and reduces (69.13%) hypothalamic PGE2 production, without significant COX-1/2 inhibition. In conclusion, our in silico approach leads to the selection of a compound that presents the chemical features to inhibit mPGES-1 and reduces fever induced by LPS. Furthermore, the in vivo and in vitro results support the hypothesis that its mechanism of action does not depend on COX inhibition. Hence, it can be considered a promising lead compound for antipyretic development, once it would not have the side-effects of COX-1/2 inhibitors.

Influence of sinomenine upon mesenchymal stem cells in osteoclastogenesis.

With the advent of aging, the morbidity rates of such diseases as osteoarthritis, rheumatoid arthritis, and osteoporosis has witnessed a significant increase. As a common rattan drug, sinomenine (SIN) has been widely applied for the treatment of various arthritic diseases in traditional Chinese medicine (TCM) clinics. Given that SIN has been reported to inhibit the expression of Prostaglandin E2 (PGE2) in several types of cells, in this study, the influence of SIN treatment on PGE2 expression in mesenchymal stem cells (MSCs), thereby changing the osteoprotegerin (OPG) receptor/activator for the nuclear factor-κ B ligand (RANKL) ratio, was investigated. Our results showed that, when compared with the untreated cells, treatment with 0.25mM SIN can down-regulate the mRNA and protein expression levels of the Prostaglandin E synthase 3 (PTGES3) or PGE2 and RANKL, while the OPG was up-regulated. After being cultured with SIN treated MSC-conditioned medium (stMSC-CM), the amount of TRAP-positive multinucleated osteoclasts differentiated from RAW264.7 cells was reduced. Also, the expression levels of specific markers for active osteoclasts were decreased when incubated with stMSC-CM. Moreover, these changes were able to be recovered when the exogenous RANKL was added to the MSC-CM culture. This indicates that the increased OPG/RANKL ratio can reduce the osteoclastogenesis of RAW264.7 cells. Our results demonstrated that SIN has an inhibitory effect on osteoclast differentiation through mechanisms involving the inhibition of the PGE2-induced OPG/RANKL ratio. SIN can also serve as a proinflammatory mediator to regulate the MSC immunosuppressive effects. Our findings suggest that SIN can be useful for the treatment of bone diseases associated with over-activity of osteoclasts.

Effects of 1,25-Dihydroxy Vitamin D3 on Endometriosis.

CONTEXT: Endometriosis is an estrogen-dependent, chronic inflammatory disease. Recent studies have shown that vitamin D (VD) is an effective modulator of the immune system and plays an important role in controlling many inflammatory diseases. OBJECTIVE: The objective of the study was to clarify the in vitro effects of 1,25-dihydroxy vitamin D3 (1,25[OH]2D3) on human endometriotic stromal cells (ESCs) and to determine the serum levels of VD in endometriosis patients. DESIGN, PATIENTS, AND MAIN OUTCOME MEASURES: ESCs were isolated from ovarian endometrioma and cultured with 1,25(OH)2D3. Gene expression of IL-8, cyclooxygenase-2, microsomal prostaglandin E synthase-1, microsomal prostaglandin E synthase-2, cytosolic prostaglandin E synthase, 15-hydroxyprostaglandin dehydrogenase, matrix metalloproteinase (MMP)-2, and MMP-9 was examined using quantitative RT-PCR. The production of IL-8 and prostaglandin E2 was measured using an ELISA and an enzyme immunoassay. Viable cell number was assessed using a cell-counting assay, and DNA synthesis was assessed using the bromodeoxyuridine incorporation assay. Apoptosis was assessed using flow cytometry. The expression of inhibitory-κBα protein was detected using Western blotting. The serum levels of 25-hydroxyvitamin D3 and 1,25(OH)2D3 were measured by a RIA. RESULTS: In vitro studies showed that 1,25(OH)2D3 significantly reduced IL-1ß- or TNF-α-induced inflammatory responses, such as IL-8 expression and prostaglandin activity. 1,25(OH)2D3 also reduced viable ESC numbers and DNA synthesis but did not affect apoptosis. MMP-2 and MMP-9 expressions were reduced by 1,25(OH)2D3. 1,25(OH)2D3 inhibited nuclear factor-κB activation. The serum 25-hydroxyvitamin D3 levels were significantly lower in women with severe endometriosis than in the controls and women with mild endometriosis. Serum 1,25(OH)2D3 levels were not different between groups. CONCLUSIONS: VD modulates inflammation and proliferation in endometriotic cells, and a lower VD status is associated with endometriosis. Taken together, VD supplementation could be a novel therapeutic strategy for managing endometriosis.

Pretreatment of cultured preadipocytes with arachidonic acid during the differentiation phase without a cAMP-elevating agent enhances fat storage after the maturation phase.

Arachidonic acid (AA) and the related prostanoids exert complex effects on the adipocyte differentiation depending on the culture conditions and life stages. Here, we investigated the effect of the pretreatment of cultured 3T3-L1 preadipocytes with exogenous AA during the differentiation phase without 3-isobutyl-1-methylxanthine (IBMX), a cAMP-elevating agent, on the storage of fats after the maturation phase. This pretreatment with AA stimulated appreciably adipogenesis after the maturation phase as evident with the up-regulated gene expression of adipogenic markers. The stimulatory effect of the pretreatment with AA was attenuated by the co-incubation with each of cyclooxygenase (COX) inhibitors. Among exogenous prostanoids and related compounds, the pretreatment with MRE-269, a selective agonist of the IP receptor for prostaglandin (PG) I2, strikingly stimulated the storage of fats in adipocytes. The gene expression analysis of arachidonate COX pathway revealed that the transcript levels of inducible COX-2, membrane-bound PGE synthase-1, and PGF synthase declined more greatly in cultured preadipocytes treated with AA. By contrast, the expression levels of COX-1, cytosolic PGE synthase, and PGI synthase remained constitutive. The treatment of cultured preadipocytes with AA resulted in the decreased synthesis of PGE2 and PGF2α serving as anti-adipogenic PGs although the biosynthesis of pro-adipogenic PGI2 was up-regulated during the differentiation phase. Moreover, the gene expression levels of EP4 and FP, the respective prostanoid receptors for PGE2 and PGF2α, were gradually suppressed by the supplementation with AA, whereas that of IP for PGI2 remained relatively constant. Collectively, these results suggest the predominant role of endogenous PGI2 in the stimulatory effect of the pretreatment of cultured preadipoccytes with AA during the differentiation phase without IBMX on adipogenesis after the maturation phase.