3',4'-Dihydroxyflavone mitigates inflammatory responses by inhibiting LPS and TLR4/MD2 interaction.

BACKGROUND: We previously reported the potential inhibitory activity of 3',4'-dihydroxyflavone (DHF) on nitric oxide (NO) and prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-stimulated macrophages. PURPOSE: We investigated the underlying molecular mechanisms of DHF in LPS-activated macrophages and evaluated its effect on LPS-induced septic shock in mice. METHODS: To explore the anti-inflammatory effect of DHF, nitrite, PGE2, and cytokines were measured in vitro and in vivo experiments. In addition, to verify the molecular signaling pathway, quantitative real time-PCR, luciferase assay, nuclear extraction, electrophoretic mobility shift assay, immunocytochemistry, immunoprecipitation, molecular docking analysis, and myeloid differentiation 2 (MD2)-LPS binding assay were conducted. RESULTS: DHF suppressed the LPS-induced expression of proinflammatory mediators through nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and interferon regulatory factor 3 (IRF3) inactivation pathways in RAW 264.7 macrophages. Importantly, molecular docking analysis and in vitro binding assays showed that DHF interacts with the hydrophobic pocket of MD2 and then interferes with the interaction between LPS and toll-like receptor 4 (TLR4). DHF inhibited LPS-induced oxidative stress by upregulating nuclear factor erythroid 2-related factor 2 (Nrf2). Treatment of LPS-induced endotoxemia mice with DHF reduced the expression levels of pro-inflammatory mediators via the inactivation of NF-κB, AP-1, and signal transducer and activator of transcription 1 (STAT1) in the lung tissue, thus increasing the survival rate. CONCLUSION: Taken together, our data first time revealed the underlying mechanism of the DHF-dependent anti-inflammatory effect by preventing LPS from binding to the TLR4/MD2 complex. Therefore, DHF may be a possible anti-inflammatory agent for the treatment of LPS-mediated inflammatory diseases.

Patriscabrin F from the roots of Patrinia scabra attenuates LPS-induced inflammation by downregulating NF-κB, AP-1, IRF3, and STAT1/3 activation in RAW 264.7 macrophages.

BACKGROUND: The roots of Partrinia scabra have been used as a medicinal herb in Asia. We previously reported that the inhibitory effect of patriscabrin F on lipopolysaccharide (LPS)-induced nitric oxide (NO) production was the most potent than that of other isolated iridoids from the roots of P. scabra. PURPOSE: We investigated the anti-inflammatory activity of patriscabrin F as an active compound of P. scabra and related signaling cascade in LPS-activated macrophages. METHOD: The anti-inflammatory activities of patriscabrin F were determined according to its inhibitory effects on NO, prostaglandin E2 (PGE2), and pro-inflammatory cytokines. The molecular mechanisms were revealed by analyzing nuclear factor-κB (NF-κB), activator protein-1 (AP-1), interferon regulatory factor 3 (IRF3), and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. RESULTS: Patriscabrin F inhibited the LPS-induced production of NO, PGE2, tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6 in both bone-marrow derived macrophages (BMDMs) and RAW 264.7 macrophages. Patriscabrin F downregulated LPS-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), TNF-α, IL-1ß, and IL-6 at the transcriptional level. Patriscabrin F suppressed LPS-induced NF-κB activation by decreasing p65 nuclear translocation, inhibitory κBα (IκBα) phosphorylation, and IκB kinase (IKK)α/ß phosphorylation. Patriscabrin F attenuated LPS-induced AP-1 activity by inhibiting c-Fos phosphorylation. Patriscabrin F suppressed the LPS-induced phosphorylation of IRF3, JAK1/JAK2, and STAT1/STAT3. CONCLUSION: Taken together, our findings suggest patriscabrin F may exhibit anti-inflammatory properties via the inhibition of NF-κB, AP-1, IRF3, and JAK-STAT activation in LPS-induced macrophages.

Chemical Constituents from the Aerial Parts of Agastache rugosa and Their Inhibitory Activities on Prostaglandin E2 Production in Lipopolysaccharide-Treated RAW 264.7 Macrophages.

A new flavone glucoside, acacetin-7-O-(3″-O-acetyl-6″-O-malonyl)-ß-d-glucopyranoside (1), two new phenolic glucosides, (3R,7R)-tuberonic acid-12-O-[6'-O-(E)-feruloyl]-ß-d-glucopyranoside (14) and salicylic acid-2-O-[6'-O-(E)-feruloyl]-ß-d-glucopyranoside (15), and two new phenylpropanoid glucosides, chavicol-1-O-(6'-O-methylmalonyl)-ß-d-glucopyranoside (17) and chavicol-1-O-(6'-O-acetyl)-ß-d-glucopyranoside(18), as well as 26 known compounds, 2-13, 16, and 19-31, were isolated from the aerial parts of Agastache rugose. The structures of the new compounds were established by spectroscopic/spectrometric methods such as HRESIMS, NMR, and ECD. The anti-inflammatory effect of the isolated compounds was evaluated by measuring their inhibitory activities on prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. New compounds 1, 15, 17, and 18 inhibited LPS-induced PGE2 production with IC50 values of 16.8 ± 0.8, 33.9 ± 4.8, 14.3 ± 2.1, and 48.8 ± 4.4 µM, respectively. Compounds 5, 7, 9-11, 13, 19, 20, 22, and 27-30 showed potent inhibitory activities with IC50 values of 1.7-8.4 µM.

Hydrangenol Isolated from the Leaves of Hydrangea serrata Attenuates Wrinkle Formation and Repairs Skin Moisture in UVB-Irradiated Hairless Mice.

Our previous study showed that hydrangenol isolated from Hydrangea serrata leaves exerts antiphotoaging activity in vitro. In this study, we determined its antiphotoaging effect in UVB-irradiated HR-1 hairless mice. We evaluated wrinkle formation, skin thickness, histological characteristics, and mRNA and protein expression using qRT-PCR and Western blot analysis in dorsal skins. Hydrangenol mitigated wrinkle formation, dorsal thickness, dehydration, and collagen degradation. Hydrangenol increased the expression of involucrin, filaggrin, and aquaporin-3 (AQP3) as well as hyaluronic acid (HA) production via hyaluronidase (HYAL)-1/-2 downregulation. Consistent with the recovery of collagen composition, the expression of Pro-COL1A1 was increased by hydrangenol. Matrix metalloproteinase (MMP)-1/-3, cyclooxygenase-2 (COX-2), and interleukin-6 (IL-6) expression was reduced by hydrangenol. Hydrangenol attenuated the phosphorylation of mitogen-activated protein kinases (MAPKs) including ERK and p38, activator protein 1 (AP-1) subunit, and signal transduction and activation of transcription 1 (STAT1). Hydrangenol upregulated the expression of nuclear factor-E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO-1), glutamate cysteine ligase modifier subunit (GCLM), and glutamate cysteine ligase catalysis subunit (GCLC). Taken together, our data suggest that hydrangenol can prevent wrinkle formation by reducing MMP and inflammatory cytokine levels and increasing the expression of moisturizing factors and antioxidant genes.

Anti-Inflammatory Mechanisms of Koreanaside A, a Lignan Isolated from the Flower of Forsythia koreana, against LPS-Induced Macrophage Activation and DSS-Induced Colitis Mice: The Crucial Role of AP-1, NF-κB, and JAK/STAT Signaling.

The current treatment options for inflammatory bowel disease (IBD) are unsatisfactory. Therefore, novel and safer therapies are needed. We previously reported that koreanaside A (KA) showed high radical scavenging activity and suppressed vascular cell adhesion molecule 1 (VCAM-1) expression in vascular smooth muscle cells. However, the molecular mechanisms involved in its anti-inflammatory effect have not been reported. KA inhibited pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nitric oxide (NO), and prostaglandin E2 (PGE2). KA inhibited the production and mRNA expression of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) induced by LPS. KA downregulated the myeloid differentiation primary response 88 (MyD88)-dependent inflammatory gene expressions in the MyD88-overexpressed cells. KA suppressed the LPS-induced transcriptional and DNA-binding activities of activator protein-1 (AP-1) and nuclear factor-kappa B (NF-κB). KA was found to inhibit the phosphorylation of Janus kinase 1/2 (JAK1/2) and signal transducers and activators of transcription 1/3 (STAT1/3). In DSS-induced colitis mice, KA relieved the symptoms of colitis by suppressing inflammatory cell infiltration, restoring tight junction (TJ)- and epithelial-mesenchymal transition (EMT)-related protein expression, and inactivating AP-1, NF-κB, and STAT1/3. Therefore, KA reduced inflammatory responses by downregulating AP-1, NF-κB, and JAK/STAT signaling in LPS-induced macrophages and DSS-induced colitis mice.

Chemical Constituents from Leaves of Hydrangea serrata and Their Anti-photoaging Effects on UVB-Irradiated Human Fibroblasts.

Hydrangea serrata (THUNB.) SER. (Hydrangeaceae) leaves have been used as herbal teas in Korea and Japan. The objective of this study was to identify anti-photoaging compounds in aqueous EtOH extract prepared from leaves of H. serrata and their effects on UVB-irradiated Hs68 human foreskin fibroblasts. Phytochemical study on H. serrata leaves led to the isolation and characterization of ten compounds: hydrangenol, thunberginol A, thunberginol C, hydrangenoside A, hydrangenoside C, cudrabibenzyl A, 2,3,4'-trihydroxystilbene, thunberginol F, quercetin 3-O-ß-D-xylopyranosyl (1-2)-ß-D-galactopyranoside, quercetin 3-O-ß-D-xylopyranosyl (1-2)-ß-D-glucopyranoside. Cudrabibenzyl A, 2,3,4'-trihydroxystilbene, quercetin 3-O-ß-D-xylopyranosyl (1-2)-ß-D-galactopyranoside, quercetin 3-O-ß-D-xylopyranosyl (1-2)-ß-D-glucopyranoside were firstly isolated from H. serrata. We estimated the effects of 10 compounds on cell viability and production of pro-collagen Type I, matrix metalloproteinase (MMP)-1, and hyaluronic acid (HA) after UVB irradiation. Of these compounds, hydrangenol showed potent preventive activities against reduced cell viability and degradation of pro-collagen Type I in UVB-irradiated Hs68 fibroblasts. Hydrangenol had outstanding inductive activities on HA production. It suppressed mRNA expression levels of MMP-1, MMP-3, hyaluronidase (HYAL)-1, HYAL-2, cyclooxygenase-2 (COX-2), interleukin (IL)-6, IL-8, and IL-1ß in UVB-irradiated Hs68 fibroblasts. When Hs68 fibroblasts were exposed to hydrangenol after UVB irradiation, UVB-induced reactive oxygen species (ROS) production was suppressed. Hydrangenol also inhibited the activation of activator protein-1 (AP-1) and signal transduction and activation of transcription 1 (STAT-1) by downregulating phosphorylation of p38 and extracellular signal-regulated kinase (ERK). Our data indicate that hydrangenol isolated from H. serrata leaves has potential protective effects on UVB-induced skin photoaging.

Hydrangea serrata (Thunb.) Ser. Extract Attenuate UVB-Induced Photoaging through MAPK/AP-1 Inactivation in Human Skin Fibroblasts and Hairless Mice.

Skin photoaging is mainly caused by exposure to ultraviolet (UV) light, which increases expressions of matrix metalloproteinases (MMPs) and destroys collagen fibers, consequently inducing wrinkle formation. Nutritional factors have received scientific attention for use as agents for normal skin functions. The aim of this study was to investigate the effect of hot water extracts from the leaves of Hydrangea serrata (Thunb.) Ser. (WHS) against ultraviolet B (UVB)-induced skin photoaging and to elucidate the underlying molecular mechanisms in human foreskin fibroblasts (Hs68) and HR-1 hairless mice. WHS recovered UVB-reduced cell viability and ameliorated oxidative stress by inhibiting intracellular reactive oxygen species (ROS) generation in Hs68 cells. WHS rescued UVB-induced collagen degradation by suppressing MMP expression, and reduced the mRNA levels of inflammatory cytokines. These anti-photoaging activities of WHS were associated with inhibition of the activator protein 1 (AP-1), signal transduction and activation of transcription 1 (STAT1), and mitogen-activated protein kinase (MAPK) signaling pathways. Oral administration of WHS effectively alleviated dorsal skin from wrinkle formation, epidermal thickening, collagen degradation, and skin dehydration in HR-1 hairless mice exposed to UVB. Notably, WHS suppressed UVB activation of the AP-1 and MAPK signaling pathways in dorsal mouse skin tissues. Taken together, our data indicate that WHS prevents UVB-induced skin damage due to collagen degradation and MMP activation via inactivation of MAPK/AP-1 signaling pathway.

Anti-Colitic Effects of Ethanol Extract of Persea americana Mill. through Suppression of Pro-Inflammatory Mediators via NF-κB/STAT3 Inactivation in Dextran Sulfate Sodium-Induced Colitis Mice.

Persea americana Mill, cv. Hass, also known as avocado, has been reported to possess hypolipidemic, anti-diabetic, anti-oxidant, cardioprotective, and photoprotective potencies. However, few studies have reported its anti-colitic effects. In this study, we investigated anti-colitic effects of ethanol extract of P. americana (EEP) in dextran sulfate sodium (DSS)-induced colitic mice and the involved molecular mechanisms. EEP effectively improved clinical signs and histological characteristics of DSS-induced colitis mice. In DSS-exposed colonic tissues, EEP reduced expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and pro-inflammatory cytokines such as interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-α. Moreover, EEP suppressed DSS-induced activation of nuclear factor-kappa B (NF-κB) and signal transducer and activator of transcription 3 (STAT3). Consistent with in vivo results, EEP also suppressed protein and mRNA expression levels of iNOS, COX-2, and pro-inflammatory cytokines via NF-κB and STAT3 inactivation in LPS-induced RAW 264.7 macrophages. Taken together, our data indicate that ethanol extract of avocado may be used as a promising therapeutic against inflammatory bowel diseases by suppressing the NF-κB and STAT3 signaling pathway.

Cirsimarin, a flavone glucoside from the aerial part of Cirsium japonicum var. ussuriense (Regel) Kitam. ex Ohwi, suppresses the JAK/STAT and IRF-3 signaling pathway in LPS-stimulated RAW 264.7 macrophages.

Cirsium japonicum var. ussuriense (Regel) Kitam. ex Ohwi (C. ussuriense) is known as "Dae-Gye" or "Korean milk thistle". C. ussuriense have long been used as a folk medicinal plant for inflammatory diseases such as hepatitis, nephritis, and mastitis in Korea, China, and Japan. To reveal the anti-inflammatory components of C. ussuriense, we isolated three flavone glycosides (linarin, cirsimarin, and hispidulin-7-O-neohesperidoside) from the aerial part of C. ussuriense and evaluated their inhibitory effects on LPS-induced pro-inflammatory mediators in macrophages. We also investigated the involving molecular mechanisms of cirsimarin. Among three flavone glycosides, cirsimarin showed vastly superior inhibitory potency in LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production. Cirsimarin concentration-dependently inhibited LPS-induced inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein and mRNA levels in macrophages. Cirsimarin suppressed the production and mRNA expression of tumor necrosis factor- α (TNF-α) and interleukin (IL)-6 in LPS-stimulated RAW 264.7 and bone marrow-derived macrophages. Moreover, molecular data presented that cirsimarin down-regulated the phosphorylation of Janus kinase (JAK)/signal transducer and activator of transcriptions (STATs) and p38 mitogen-activated protein kinase (MAPK), and nuclear translocation of interferon regulatory factor (IRF)-3. Collectively, cirsimarin may be an active ingredient responsible for anti-inflammatory effects of C. ussuriense and it may act as a promising therapeutic against inflammatory diseases by suppressing the JAK/STAT and IRF-3 signaling pathway.

Immunostimulatory effects of cordycepin-enriched WIB-801CE from Cordyceps militaris in splenocytes and cyclophosphamide-induced immunosuppressed mice.

The medicinal mushroom Cordyceps militaris has been reported to possess anticancer and immunomodulatory effects. We investigated the immunostimulatory effects of culture supernatant of C. militaris (WIB-801CE) by examining its in vitro enhancing effects on cell proliferation and cytokine releases in splenocytes and its in vivo effects on cyclophosphamide-induced immunosuppressed mice. WIB-801CE enhanced normal and methotrexate-induced cell proliferation. WIB-801CE significantly ameliorated interleukin (IL)-2, interferon-γ, and tumor necrosis factor-α secretion in methotrexate-induced splenocytes. Oral administration of WIB-801CE effectively increased the cyclophosphamide-suppressed splenocyte proliferation and natural killer cytotoxic activity. WIB-801CE effectively recovered cyclophosphamide-induced decreases in IL-2, interferon-γ, tumor necrosis factor-α, and IL-10 level. The collective data implicate WIB-801CE as a therapeutic candidate in ameliorating the immunosuppression through immunostimulatory properties.

p-Coumaroyl Anthocyanin Mixture Isolated from Tuber Epidermis of Solanum tuberosum Attenuates Reactive Oxygen Species and Pro-inflammatory Mediators by Suppressing NF-κB and STAT1/3 Signaling in LPS-Induced RAW264.7 Macrophages.

Previously, we first reported the identification of four p-coumaroyl anthocyanins (petanin, peonanin, malvanin, and pelanin) from the tuber epidermis of colored potato (Solanum tuberosum L. cv JAYOUNG). In this study, we investigated the anti-oxidative and anti-inflammatory effects of a mixture of peonanin, malvanin, and pelanin (10 : 3 : 3; CAJY). CAJY displayed considerable radical scavenging capacity of 1, 1-diphenyl-2-picryl-hydrazyl (DPPH), increased mRNA levels of the catalytic and modulatory subunit of glutamate cysteine ligase, and subsequent cellular glutathione content. These increases preceded the inhibition of lipopolysaccharide (LPS)-induced intracellular reactive oxygen species (ROS) production. CAJY inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in a concentration-dependent manner at the protein, mRNA, and promoter activity levels. These inhibitions caused attendant decreases in the production of prostaglandin E2 (PGE2). CAJY suppressed the production and mRNA expression of tumor necrosis factor (TNF)-α and interleukin (IL)-6. Molecular data revealed that CAJY inhibited the transcriptional activity and translocation of nuclear factor κB (NF-κB) and phosphorylation of signal transducer and activator of transcription 1 (STAT1) and STAT3. Taken together, these results suggest that the anthocyanin mixture exerts anti-inflammatory effects in macrophages, at least in part by reducing ROS production and inactivating NF-κB and STAT 1/3.

Ethanol Extract of Potentilla supina Linne Suppresses LPS-induced Inflammatory Responses through NF-κB and AP-1 Inactivation in Macrophages and in Endotoxic mice.

In this study, we investigated the antiinflammatory effects of ethanol extracts of Potentilla. supina Linne (EPS) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and septic mice. EPS suppressed LPS-induced nitric oxide, prostaglandin E2 , TNF-α, interleukin-6 and interleukin-1ß at production and mRNA levels in LPS-induced RAW 264.7 macrophages. Consistent with these observations, EPS attenuated the expressions of inducible nitric oxide synthase and cyclooxygenase-2 by downregulation of their promoter activities. Molecularly, EPS reduced the LPS-induced transcriptional activity and DNA-binding activity of nuclear factor-κB (NF-κB), and this was associated with a decrease of translocation and phosphorylation of p65 NF-κB by inhibiting the inhibitory κB-α degradation and IKK-α/ß phosphorylation. Furthermore, EPS inhibited the LPS-induced activation of activator protein-1 by reducing the expression of c-Fos and c-Jun in nuclear. EPS also suppressed the phosphorylation of mitogen-activated protein kinase, such as p38 mitogen-activated protein kinase and c-Jun N-terminal kinase. In an LPS-induced endotoxemia mouse model, pretreatment with EPS reduced the mRNA levels of inducible nitric oxide synthase, cyclooxygenase-2 and proinflammatory cytokines and increased the survival rate of mice. Collectively, these results suggest that the antiinflammatory effects of EPS were associated with the suppression of NF-κB and activator protein-1 activation and support its possible therapeutic role for the treatment of endotoxemia. Copyright © 2017 John Wiley & Sons, Ltd.

Kyungheechunggan-Tang-01, a New Herbal Medication, Suppresses LPS-Induced Inflammatory Responses through JAK/STAT Signaling Pathway in RAW 264.7 Macrophages.

Medicinal plants have been used as alternative therapeutic tools to alleviate inflammatory diseases. The objective of this study was to evaluate anti-inflammatory properties of Kyungheechunggan-tang- (KCT-) 01, KCT-02, and Injinchunggan-tang (IJCGT) as newly developed decoctions containing 3-11 herbs in LPS-induced macrophages. KCT-01 showed the most potent inhibitory effects on LPS-induced NO, PGE2, TNF-α, and IL-6 production among those three herbal formulas. In addition, KCT-01 significantly inhibited LPS-induced iNOS and COX-2 at protein levels and expression of iNOS, COX-2, TNF-α, and IL-6 at mRNA levels. Molecular data revealed that KCT-01 attenuated the activation of JAK/STAT signaling cascade without affecting NF-κB or AP-1 activation. In ear inflammation induced by croton oil, KCT-01 significantly reduced edema, MPO activity, expression levels of iNOS and COX-2, and STAT3 phosphorylation in ear tissues. Taken together, our findings suggest that KCT-01 can downregulate the expression of proinflammatory genes by inhibiting JAK/STAT signaling pathway under inflammatory conditions. This study provides useful data for further exploration and application of KCT-01 as a potential anti-inflammatory medicine.

Berberine Decreased Inducible Nitric Oxide Synthase mRNA Stability through Negative Regulation of Human Antigen R in Lipopolysaccharide-Induced Macrophages.

Berberine, a major isoquinoline alkaloid found in medicinal herbs, has been reported to possess anti-inflammatory effects; however, the underlying mechanisms responsible for its actions are poorly understood. In the present study, we investigated the inhibitory effects of berberine and the molecular mechanisms involved in lipopolysaccharide (LPS)-treated RAW 264.7 and THP-1 macrophages and its effects in LPS-induced septic shock in mice. In both macrophage cell types, berberine inhibited the LPS-induced nitric oxide (NO) production and inducible NO synthase (iNOS) protein expression, but it had no effect on iNOS mRNA transcription. Suppression of LPS-induced iNOS protein expression by berberine occurred via a human antigen R (HuR)-mediated reduction of iNOS mRNA stability. Molecular data revealed that the suppression on the LPS-induced HuR binding to iNOS mRNA by berberine was accompanied by a reduction in nucleocytoplasmic HuR shuttling. Pretreatment with berberine reduced LPS-induced iNOS protein expression and the cytoplasmic translocation of HuR in liver tissues and increased the survival rate of mice with LPS-induced endotoxemia. These results show that the suppression of iNOS protein expression by berberine under LPS-induced inflammatory conditions is associated with a reduction in iNOS mRNA stability resulting from inhibition of the cytoplasmic translocation of HuR.

α-Solanine Isolated From Solanum Tuberosum L. cv Jayoung Abrogates LPS-Induced Inflammatory Responses Via NF-κB Inactivation in RAW 264.7 Macrophages and Endotoxin-Induced Shock Model in Mice.

α-Solanine, a trisaccharide glycoalkaloid, has been reported to possess anti-cancer effects. In this study, we investigated the anti-inflammatory effects of α-solanine isolated from "Jayoung" a dark purple-fleshed potato by examining its in vitro inhibitory effects on inducible nitric-oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and pro-inflammatory cytokines in LPS-induced RAW 264.7 macrophages and its in vivo effects on LPS-induced septic shock in a mouse model. α-Solanine suppressed the expression of iNOS and COX-2 both at protein and mRNA levels and consequently inhibited nitric oxide (NO) and prostaglandin E2 (PGE2 ) production in LPS-induced RAW 264.7 macrophages. α-Solanine also reduced the production and mRNA expression of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) induced by LPS. Furthermore, molecular mechanism studies indicated that α-solanine inhibited LPS-induced activation of nuclear factor-κB (NF-κB) by reducing nuclear translocation of p65, degradation of inhibitory κBα (IκBα), and phosphorylation of IκB kinaseα/ß (IKKα/ß). In an in vivo experiment of LPS-induced endotoxemia, treatment with α-solanine suppressed mRNA expressions of iNOS, COX-2, IL-6, TNF-α, and IL-1ß, and the activation of NF-κB in liver. Importantly, α-solanine increased the survival rate of mice in LPS-induced endotoxemia and polymicrobial sepsis models. Taken together, our data suggest that the α-solanine may be a promising therapeutic against inflammatory diseases by inhibiting the NF-κB signaling pathway. J. Cell. Biochem. 117: 2327-2339, 2016. © 2016 Wiley Periodicals, Inc.

Constituents of PG201 (Layla(®)), a multi-component phytopharmaceutical, with inhibitory activity on LPS-induced nitric oxide and prostaglandin E2 productions in macrophages.

Fourteen compounds, coumarin (1), demethylsuberosin (2), xanthotoxin (3), psoralen (4), decursinol (5), decursin (6), decursinol angelate (7), chikusetsusaponin IVa (8), chikusetsusaponin IVa methyl ester (9), ethyl caffeate (10), syringaresinol (11), cnidilide (12), farnesol (13), and linoleic acid (14), were isolated from phytopharmaceutical PG201 (Layla(®)) by activity-guided fractionation utilizing inhibitory activity on nitric oxide (NO) production in vitro. The isolates 1-14 were evaluated for their inhibitory activity on LPS-induced NO and prostaglandin E2 (PGE2) productions in RAW 264.7 cells. All the compounds except 14 displayed suppressive effects on LPS-induced NO and PGE2 production with IC50 values ranging from 8 to 60 µM. Among these, compound 10 showed the most potent inhibitory effect on NO production from RAW 264.7 cells with an IC50 value of 8.25 µM. Compounds 2, 9, and 10 exhibited high inhibitory effects on PGE2 production with the IC50 values of 9.42, 7.51, and 6.49 µM, respectively. These findings suggest that compounds 2, 9, and 10 are the potential anti-inflammatory active constituents of PG201 and further study may be needed to explain their mechanism of action.

α-Chaconine isolated from a Solanum tuberosum L. cv Jayoung suppresses lipopolysaccharide-induced pro-inflammatory mediators via AP-1 inactivation in RAW 264.7 macrophages and protects mice from endotoxin shock.

In this study, we investigated the molecular mechanisms underlying the anti-inflammatory effects of α-chaconine in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and in LPS-induced septic mice. α-Chaconine inhibited the expressions of cyclooxygenase-2 (COX-2), interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α) at the transcriptional level, and attenuated the transcriptional activity of activator protein-1 (AP-1) by reducing the translocation and phosphorylation of c-Jun. α-Chaconine also suppressed the phosphorylation of TGF-ß-activated kinase-1 (TAK1), which lies upstream of mitogen-activated protein kinase kinase 7 (MKK7)/Jun N-terminal kinase (JNK) signaling. JNK knockdown using siRNA prevented the α-chaconine-mediated inhibition of pro-inflammatory mediators. In a sepsis model, pretreatment with α-chaconine reduced the LPS-induced lethality and the mRNA and production levels of pro-inflammatory mediators by inhibiting c-Jun activation. These results suggest that the anti-inflammatory effects of α-chaconine are associated with the suppression of AP-1, and support its possible therapeutic role for the treatment of sepsis.

Amomum tsao-ko fruit extract suppresses lipopolysaccharide-induced inducible nitric oxide synthase by inducing heme oxygenase-1 in macrophages and in septic mice.

Amomum tsao-ko Crevost et Lemarié (Zingiberaceae) has traditionally been used to treat inflammatory and infectious diseases, such as throat infections, malaria, abdominal pain and diarrhoea. This study was designed to assess the anti-inflammatory effects and the molecular mechanisms of the methanol extract of A. tsao-ko (AOM) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and in a murine model of sepsis. In LPS-induced RAW 264.7 macrophages, AOM reduced the production of nitric oxide (NO) by inhibiting inducible nitric oxide synthase (iNOS) expression, and increased heme oxygenase-1 (HO-1) expression at the protein and mRNA levels. Pretreatment with SnPP (a selective inhibitor of HO-1) and silencing HO-1 using siRNA prevented the AOM-mediated inhibition of NO production and iNOS expression. Furthermore, AOM increased the expression and nuclear accumulation of NF-E2-related factor 2 (Nrf2), which enhanced Nrf2 binding to antioxidant response element (ARE). In addition, AOM induced the phosphorylation of extracellular regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) and generated reactive oxygen species (ROS). Furthermore, pretreatment with N-acetyl-l-cysteine (NAC; a ROS scavenger) diminished the AOM-induced phosphorylation of ERK and JNK and AOM-induced HO-1 expression, suggesting that ERK and JNK are downstream mediators of ROS during the AOM-induced signalling of HO-1 expression. In LPS-induced endotoxaemic mice, pretreatment with AOM reduced NO serum levels and liver iNOS expression and increased HO-1 expression and survival rates. These results indicate that AOM strongly inhibits LPS-induced NO production by activating the ROS/MAPKs/Nrf2-mediated HO-1 signalling pathway, and supports its pharmacological effects on inflammatory diseases.

Chemopreventive effects of standardized ethanol extract from the aerial parts of Artemisia princeps Pampanini cv. Sajabal via NF-κB inactivation on colitis-associated colon tumorigenesis in mice.

Chronic inflammation is an underlying risk factor of colon cancer, and NF-κB plays a critical role in the development of inflammation-associated colon cancer in an AOM/DSS mouse model. The aim of this study was to determine whether the standardized ethanol extract obtained from the aerial parts of Artemisia princeps Pampanini cv. Sajabal (EAPP) is effective at preventing inflammation-associated colon cancer, and if so, to identify the signaling pathways involved. In the present study, protective efficacy of EAPP on tumor formation and the infiltrations of monocytes and macrophages in colons of an AOM/DSS mouse model were evaluated. It was found that colitis and tumor burdens showed statistically meaningful improvements after EAPP administration. Furthermore, these improvements were accompanied by a reduction in NF-κB activity and in the levels of NF-κB-dependent pro-survival proteins, that is, survivin, cFLIP, XIAP, and Bcl-2. In vitro, EAPP significantly reduced NF-κB activation and the levels of IL-1ß and IL-8 mRNA and pro-survival proteins in HT-29 and HCT-116 colon cancer cells. Furthermore, EAPP caused caspase-dependent apoptosis. Based on these results, the authors suggest EAPP suppresses inflammatory responses and induces apoptosis partly via NF-κB inactivation, and that EAPP could be useful for the prevention of colitis-associated tumorigenesis.

Anti-inflammatory effect of a standardized triterpenoid-rich fraction isolated from Rubus coreanus on dextran sodium sulfate-induced acute colitis in mice and LPS-induced macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Rubus coreanus Miquel (Rosaceae), the Korean black raspberry, has traditionally been used to treat inflammatory diseases including diarrhea, asthma, stomach ailment, and cancer. Although previous studies showed that the 19α-hydroxyursane-type triterpenoids isolated from Rubus coreanus exerted anti-inflammatory activities, their effects on ulcerative colitis and mode of action have not been explored. This study was designed to assess the anti-inflammatory effects and the molecular mechanisms involving19α-hydroxyursane-type triterpenoid-rich fraction from Rubus coreanus (TFRC) on a mice model of colitis and lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. MATERIALS AND METHODS: Experimental colitis was induced by DSS for 7 days in ICR mice. Disease activity indices (DAI) took into account body weight, stool consistency, and gross bleeding. Histological changes and macrophage accumulation were observed by immunohistochemical analysis. Pro-inflammatory markers were determined using immunoassays, RT-PCR, and real time PCR. Signaling pathway involving nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) activation was determined by luciferase assay and Western blotting. RESULTS: In DSS-induced colitis mice, TFRC improved DAIs and pathological characteristics including colon shortening and colonic epithelium injury. TFRC suppressed tissue levels of pro-inflammatory cytokines and reduced macrophage infiltration into colonic tissues. In LPS-induced RAW 264.7 macrophages, TFRC inhibited the production of NO, PGE2, and pro-inflammatory cytokines by down-regulating the activation of NF-κB and p38 MAPK signaling. CONCLUSION: The study demonstrates that TFRC has potent anti-inflammatory effects on DSS-induced colonic injury and LPS-induced macrophage activation, and supports its possible therapeutic and preventive roles in colitis.