esculetin and Inflammation

Encapsulation of esculetin into DSPE-MPEG2000 carrier was performed to improve its water solubility and oral bioavailability, as well as enhance its anti-inflammatory effect on a mouse model of ulcerative colitis that was induced with dextran sulphate sodium (DSS).. We determined the. The PS of Esc-NLC was 102.29 ± 0.63 nm with relative standard deviation (RSD) of 1.08% (with poly-dispersity index-PDI of 0.197 ± 0.023), while the ZP was -15.67 ± 1.39 mV with RSD of 1.24%. Solubility of esculetin was improved coupled with prolonged release time. Its pharmacokinetic parameters were compared with that of free esculetin, wherein the maximum concentration of the drug in plasma was increased by 5.5 times. Of note, bioavailability of the drug was increased by 1.7 times, while the half-life was prolonged by 2.4 times. In the anti-colitis efficacy experiment, the mice in Esc and Esc-NLC groups exhibited significantly reduced levels of TNF-α, IL-1β, and IL-6 in their sera comparable to the DSS group. Colon histopathological examination revealed that mice with ulcerative colitis in both Esc and Esc-NLC groups displayed improved inflammation, amid the Esc-NLC groups having the best prophylactic treatment effect.. Esc-NLC could ameliorate DSS-induced ulcerative colitis by improving bioavailability, prolonging drug release time and regulating cytokine release. This observation confirmed the potential of Esc-NLC to reduce inflammation in ulcerative colitis, albeit the need for follow-up research to verify the application of this strategy to clinical treatment of ulcerative colitis.

Topics: Animals; Colitis, Ulcerative; Excipients; Inflammation; Interleukin-6; Lipids; Mice; Tumor Necrosis Factor-alpha

Esculetin is a bioactive compound of medicinal herb Hydrangea paniculata, and has showed anti-oxidation and anti-inflammation bioactivities. Renal local oxidative stress and inflammation are import contributors for progression of lupus nephritis (LN).. In the present study, the renal protective effect of esculetin against LN was evaluated using MRL/lpr mice.. MRL/lpr mice were orally administrated with esculetin (20 mg/kg and 40 mg/kg) from 10 to 20 weeks and then renal function and kidney pathology were analyzed.. Esculetin significantly attenuated renal impairment in MRL/lpr mice by reducing blood urea nitrogen (BUN), serum creatinine (Scr) and albuminuria, and ameliorated the glomerular hypertrophy, tubular interstitial fibrosis and mononuclear cell infiltration into interstitium. mRNA microarray suggested that esculetin could significantly down-regulate complement cascade, inflammation and fibrosis pathway, and up-regulate Nrf2-related anti-oxidation genes. Most surprising finding in the current study was that esculetin could inhibit the complement activation both in classical and alternative pathway using in vitro hemolysis assay, further enzyme assay suggested that esculetin blocked the C3 convertase (C4b2a) to exert this inhibitory capability. Molecular docking predicted that esculetin had four conventional hydrogen bonds interacting with C4b2a, and CDOCKER energy is relatively lower. Luciferase reporter gene demonstrated that esculetin could activate Nrf2 signaling pathway, and further flow cytometry confirmed that anti-oxidation bioactivity of esculetin was dependent on Nrf2 activation. On the other hand, esculetin could inhibit NFκB nuclear translocation and TGFβ-smad3 profibrosis pathway.. Esculetin shows beneficial effect on LN progression, and it may be a good natural leading compound for design of chemical compounds to treat LN.

Topics: Animals; Blood Urea Nitrogen; Complement Activation; Creatinine; Disease Progression; Dose-Response Relationship, Drug; Female; Hydrangea; Inflammation; Lupus Nephritis; Mice; Mice, Inbred MRL lpr; Molecular Docking Simulation; NF-E2-Related Factor 2; NF-kappa B; Signal Transduction; Umbelliferones

Esculetin, a natural derivative from the traditional and widely-used Chinese medicinal herb Cortex Fraxini, has a variety of pharmacological effects, especially in anti-inflammation. However, it is not clear whether esculetin has a therapeutic effect on sepsis. This study aimed to investigate the anti-inflammatory and protective effects of esculetin on early sepsis. The results showed that the lung injury was significantly relieved with the treatment of esculetin, accompanied with the restrained production of inflammatory factors including IL-1β, IL-6, TNF-α, CCL2 and iNOS during the early phase of E.coli-induced sepsis. Of note, activation of NF-κB and STAT1/STAT3 signals, the main upstream signals of many inflammatory factors, were attenuated by esculetin in both lung tissues from septic mice and LPS-stimulated macrophage. These findings suggested that the protection of esculetin against early sepsis should be related to its anti-inflammatory effect, which was at least partly due to its inhibition on NF-κB and STAT1/STAT3 signaling pathway in macrophage. Thus, esculetin could serve as a potential therapeutic agent by rebalancing innate immune response in macrophage for the treatment of early sepsis.

Topics: Animals; Inflammation; Lipopolysaccharides; Mice; NF-kappa B; Sepsis; Signal Transduction; STAT1 Transcription Factor; STAT3 Transcription Factor; Umbelliferones

Allergic rhinitis (AR) is a type of respiratory disease closely associated with chronic inflammation. Esculetin is a natural coumarin derivative and has been reported to possess anti-allergic and anti-inflammatory effects. However, the roles of esculetin in AR have not been studied. In this study, we aimed to examine the effect of esculetin on AR using an in vitro model. The human nasal epithelial cells (HNEpC) were stimulated by histamine for 24 hours with or without the pretreatment of esculetin. The mRNA levels and production of inflammatory cytokines including IL-6 and IL-8, as well as mucin 5AC (MUC5AC) were measured using qRT-PCR and ELISA, respectively. The results showed that esculetin suppressed histamine-induced expression and secretion of IL-6, IL-8, and MUC5AC in HNEpCs. Furthermore, we examined the effect of esculetin on NF-κB pathway by detecting the expression levels of NF-κB p65, p-p65 and IκBα using western blot analysis. Esculetin treatment suppressed the histamine-induced p-p65 expression and p-IκBα degradation. Inhibiting NF-κB pathway suppressed histamine-induced production of IL-6, IL-8, and MUC5AC in HNEpCs. These findings suggested that esculetin suppressed histamine-induced production of inflammatory cytokines and mucin in HNEpCs, which were partly mediated by the inhibition of NF-κB pathway.

Topics: Cell Line; Cytokines; Epithelial Cells; Gene Expression Regulation; Histamine; Humans; Inflammation; Interleukin-6; Interleukin-8; Mucin 5AC; NF-kappa B; Nose; Umbelliferones

Age-related macular degeneration (AMD) is the most common cause of visual loss. The dry AMD is characterized by retinal pigment epithelium (RPE) death and changes in AMD lead to severe loss of vision. Coumarin-derived esculetin has a number of therapeutic and pharmacological effects such as anti-inflammatory and antioxidant with various mechanisms. The purpose of this study was to investigate the effects of esculetin treatment on lipopolysaccharide (LPS)-induced inflammation, oxidative stress, and cell survival.. Human RPE cells (ARPE-19) were incubated for 24-72 h with 5 μg/ml LPS to induce inflammation and oxidative stress. Esculetin (5 μM) was used to protect the cells from LPS-induced damage. The cell viability was evaluated by quantitative 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test. Interleukin 6 (IL-6), IL-12, and vascular endothelial growth factor (VEGF) levels were determined by enzyme-linked immunosorbent assay (ELISA). IL-1β, tumor necrosis factor receptor (TNFR), TNF-related apoptosis-inducing ligand (TRAIL), catalase, glutathione peroxidase (GPx), superoxide dismutase 1 (CuZnSOD) and SOD2 (MnSOD) mRNA expressions were analyzed by RT-quantitative polymerase chain reaction. Apoptosis was monitored by cell-based cytometer. NF-kappa B (NF-κB) p65/RelA levels were determined by ELISA, and NF-κB protein expression and extracellular signal-regulated kinase (ERK1/2) phosphorylation were evaluated by Western blot analysis.. Esculetin treatment significantly suppressed LPS-induced cell death mediated by apoptosis and necrosis in a concentration-dependent manner. While LPS caused significant inflammation with cytokine increase in cells, esculetin reduced the expression of LPS-induced cytokines, VEGF, TNFR, and TRAIL. Furthermore, exposure to LPS increased the expression of GPx and mitochondrial MnSOD, leading to oxidative stress in the cells. Esculetin treatment attenuated phosphorylation of ERK1/2 and NF-κB expression mediated by LPS.. These results suggest that esculetin may be an alternative treatment option for endotoxin-induced inflammation and oxidative stress, which therefore may inhibit the development of LPS-mediated AMD.

Topics: Antioxidants; Cell Death; Cell Survival; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Gene Expression Regulation; Humans; Inflammation; Lipopolysaccharides; Macular Degeneration; NF-kappa B; Oxidative Stress; Polymerase Chain Reaction; Retinal Pigment Epithelium; RNA; Umbelliferones

This study investigated the effects and mechanism of esculetin (6,7-dihydroxycoumarin) on non-alcoholic fatty liver in diabetic mice fed high-fat diet (HFD). The diabetic mice model was induced by injection of streptozotocin, after which they were fed HFD diet with or without esculetin for 11 weeks. Non-diabetic mice were provided a normal diet. Diabetes induced hepatic hypertrophy, lipid accumulation and droplets; however, esculetin reversed these changes. Esculetin treatment in diabetic mice fed HFD significantly down-regulated expression of lipid synthesis genes (Fasn, Dgat2 and Plpp2) and inflammation genes (Tlr4, Myd88, Nfkb, Tnfα and Il6). Moreover, the activities of hepatic lipid synthesis enzymes (fatty acid synthase and phosphatidate phosphohydrolase) and gluconeogenesis enzyme (glucose-6-phosphatase) in the esculetin group were decreased compared with the diabetic group. In addition, esculetin significantly reduced blood HbA

Topics: Animals; Blood Glucose; Body Weight; Chemokines; Diabetes Mellitus, Experimental; Diet, High-Fat; Gene Expression Regulation; Inflammation; Insulin; Lipid Metabolism; Lipid Peroxidation; Liver; Male; Mice, Inbred C57BL; Non-alcoholic Fatty Liver Disease; Superoxide Dismutase; Umbelliferones

Some studies suggest that 5-lipoxygenase (5-LOX) inhibition or leukotriene receptor antagonism may effectively attenuate different kinds of pain. In the present study, we investigated whether esculetin (which, among other actions, potently inhibits 5-LOX) possesses analgesic activity in acute non-inflammatory pain and acute inflammatory pain models in rats. We also examined the effects of zileuton, a selective 5-LOX inhibitor, on esculetin activity. Plasma concentrations of leukotriene B4 (LTB4 ) after administration of esculetin were also determined. Esculetin (1.25-20 mg/kg, i.p.) dose-dependently alleviated hyperalgesia and exhibited antinociceptive effects in both experimental models. The greatest effect of esculetin was observed with a dose of 20 mg/kg. In carrageenan-induced inflammatory pain in rats, 20 mg/kg esculetin reversed or mitigated hyperalgesia, increasing the threshold to mechanical stimuli from a control value of -23.8 ± 1.8% to 15.2 ± 2.2% (P < 0.01) and that to thermal stimuli from -52.5 ± 6.1% to -9.5 ± 3.9% (P < 0.01). In non-inflammatory pain, after esculetin (20 mg/kg) administration the threshold values to mechanical and thermal stimuli increased to 75.9 ± 4.2% and 59.2 ± 4.3%, respectively (P < 0.01 for both). Zileuton (30 mg/kg, p.o.) alone slightly but significantly increased the pain threshold in the non-inflammatory and inflammatory acute pain models. Pretreatment with 30 mg/kg, p.o., zileuton significantly enhanced the analgesic activity of 5 mg/kg, i.p., esculetin in both pain models. Moreover, esculetin (10 mg/kg, i.p.) decreased LTB4 concentrations in the blood from 244 ± 29 pg/mL in the control group to 185 ± 11 pg/mL (P < 0.005). The results of the present study suggest the involvement of the 5-LOX pathway in esculetin analgesia.

Topics: Analgesia; Analgesics; Animals; Arachidonate 5-Lipoxygenase; Disease Models, Animal; Hydroxyurea; Hyperalgesia; Inflammation; Leukotriene B4; Lipoxygenase Inhibitors; Male; Pain; Pain Threshold; Rats; Umbelliferones

Although previous studies have demonstrated that the natural coumarin compound esculetin possesses various pharmacological properties, the molecular mechanism of esculetin-mediated anti-inflammatory potential is not fully understood. In this study, we determined the effects of esculetin on lipopolysaccharide (LPS)-induced inflammatory responses of murine RAW 264.7 macrophages. The results indicate that esculetin inhibits LPS-induced nitric oxide and prostaglandin E2 production in a concentration-dependent manner, and inhibits inducible nitric oxide synthase and cyclooxygenase-2 expression in RAW 264.7 cells. Esculetin also significantly suppresses the production of inflammatory cytokines, including tumor necrosis factor-α and interleukin-1β, which was concomitant with a decrease in their expression levels. Furthermore, it was observed that esculetin attenuated the LPS-mediated nuclear factor-kappa B (NF-κB) translocation associated with the blocking of inhibitor of NF-κB (IκB)-α degradation as well as reactive oxygen species (ROS) production, without any significant cytotoxicity. These data suggest that, by blocking NF-κB activation, esculetin suppresses LPS-elicited inflammatory events, and this is mediated, at least in part, by inhibiting the generation of ROS. Collectively, these findings provide mechanistic insights into the anti-inflammatory action of esculetin in macrophages.

Topics: Animals; Anti-Inflammatory Agents; Cell Line; Cyclooxygenase 2; Dinoprostone; I-kappa B Proteins; Inflammation; Inflammation Mediators; Interleukin-1beta; Lipopolysaccharides; Macrophages; Mice; NF-KappaB Inhibitor alpha; Nitric Oxide; Reactive Oxygen Species; Umbelliferones

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature

Four coumarins were isolated from the EtOAc extract of the flower-tops of Santolina oblongifolia Boiss. (Compositae). They were identified as 7-methoxycoumarin (herniarin) (1), 6,7-dihydroxycoumarin (aesculetin) (2), 6-methoxy-7-glucosidylcoumarin (scopolin) (3), and 6-hydroxy-7-methoxycoumarin (scopoletin) (4). This is the first report of the isolation of aesculetin and scopolin from the genus Santolina. The isolated coumarins showed marked activity as inhibitors of eicosanoid-release from ionophore-stimulated mouse peritoneal macrophages.

Topics: Animals; Anti-Inflammatory Agents; Calcimycin; Carrageenan; Chromatography, Thin Layer; Coumarins; Dinoprostone; Inflammation; Ionophores; Leukotrienes; Macrophages, Peritoneal; Magnetic Resonance Spectroscopy; Mice; Plant Extracts; Plants, Medicinal; Rats; Rats, Wistar; Spain; Spectrophotometry, Ultraviolet