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Background: Excessive inflammation poses significant risks to human physical and mental health. Astilbe grandis, a traditional Miao medicine, is renowned for its anti-inflammatory properties. However, the specific anti-inflammatory effects and mechanisms of many compounds within this plant remain unclear. This study aims to investigate the anti-inflammatory effects and mechanisms of two characteristic oleanane triterpenoids, 3α-acetoxyolean-12-en-27-oic acid (1) and 3β-acetoxyolean-12-en-27-oic acid (2), isolated from Astilbe grandis, using lipopolysaccharide (LPS)-induced Macrophages. Methods: The anti-inflammatory effects and mechanisms of compounds 1 and 2 were investigated by establishing an LPS-induced inflammation model in RAW 264.7 cells and THP-1 cells. Nitric oxide (NO) levels were assessed using the Griess method. The concentrations of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1beta (IL-1β) were measured via enzymelinked immunosorbent assay (ELISA). The expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) was determined using western blotting and quantitative real-time PCR (qRT-PCR). Additionally, the phosphorylation level of p65 in nuclear factor-kappa B (NF-κB) was assessed through western blotting. The nuclear translocation of NF-κB p65 was assessed through immunofluorescence staining. Finally, the binding affinity of the compounds to NF-κB p65 target was validated through molecular docking. Results: Compounds 1 and 2 significantly inhibited the expression of NO, TNF-α, IL-6, IL-1β, COX-2, and iNOS in LPS-induced Macrophages. Mechanistically, they attenuated the activation of the NF-κB signaling pathway by downregulating the phosphorylation level and nuclear translocation of p65. Conclusion: This study elucidates the anti-inflammatory activities and potential mechanism of the characteristic oleanane triterpenoids with C-14 carboxyl group, compounds 1 and 2, in LPS-induced Macrophages by inhibiting the NF-κB signaling pathway for the first time. These findings suggest that these two compounds hold promise as potential candidates for anti-inflammatory interventions in the future. [ABSTRACT FROM AUTHOR]

Cannabichromene (CBC), a non-psychoactive cannabinoid found in Cannabis sativa, has recently been shown to possess several medicinal properties. However, how CBC produces anti-inflammatory effects and the mechanisms of this remain poorly studied. Therefore, we extracted and purified the CBC from the Cannabis sativa cv. pink pepper (hemp cultivar). The efficacy of CBC in reducing inflammation in RAW 264.7 macrophages and a λ-carrageenan-induced mouse model was then evaluated. CBC had no cytotoxicity up to a concentration of 20 μM and inhibited nitric oxide production by approximately 50% at a concentration of 20 μM. In addition, CBC treatment significantly inhibited causes of inflammation such as inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) at both the mRNA and protein levels. Moreover, CBC suppressed LPS-stimulated inflammation in RAW 264.7 cells by downregulating the nuclear factor kappa B (NF-kB) and mitogen-activated protein kinase pathways (MAPK). Furthermore, our in vivo experiments confirmed that the λ-carrageenan-induced increase in the levels of the cytokines iNOS, IL-1β, and IL-6 was abrogated following treatment with CBC. Therefore, CBC has potential anti-inflammatory effects and may be useful for preventing or treating inflammation. [ABSTRACT FROM AUTHOR]

Malaria is a parasitic infection responsible for high morbidity and mortality rates worldwide. During the disease, phagocytosis of infected red blood cells by the macrophages induces the production of reactive oxygen (ROS) and nitrogen species (RNS), culminating in parasite death. Curcumin (CUR) is a bioactive compound that has been demonstrated to reduce the production of pro-inflammatory cytokines and chemokines produced by macrophages but to reduce parasitemia in infected mice. Hence, the main purpose of this study is to investigate whether curcumin may interfere with macrophage function and polarization after Plasmodium berghei infection in vitro. In our findings, non-polarized macrophage (M0), classically activated (M1), and alternatively activated (M2) phenotypes showed significantly increased phagocytosis of infected red blood cells (iRBCs) when compared to phagocytosis of uninfected red blood cells (RBCs) 3 h after infection. After 24 h, M1 macrophages exposed to RBCs + CUR showed greater elimination capacity when compared to macrophages exposed to iRBCs + CUR, suggesting the interference of curcumin with the microbicidal activity. Additionally, curcumin increased the phagocytic activity of macrophages when used in non-inflammatory conditions (M0) and reduced the inducible nitric oxide synthase (iNOS) and arginase activities in all macrophage phenotypes infected (M0, M1, and M2), suggesting interference in arginine availability by curcumin and balance promotion in macrophage polarization in neutral phenotype (M0). These results support the view of curcumin treatment in malaria as an adjuvant, promoting a balance between pro- and anti-inflammatory responses for a better clinical outcome. [ABSTRACT FROM AUTHOR]

During a search for natural inflammatory inhibitors, 1-O-acetylbritannilactone (ABL), a sesquiterpene lactone, was isolated from the flowers of Inula britannica. ABL significantly inhibited human neutrophil elastase (HNE) with a half-maximal inhibitory concentration (IC50) of 3.2 ± 0.3 µM, thus did so more effectively than the positive control material (epigallocatechin gallate) (IC50 7.2 ± 0.5 µM). An enzyme kinetic study was performed. ABL noncompetitively inhibited HNE with an inhibition constant Ki of 2.4 µM. ABL inhibited lipopolysaccharide-induced nitric oxide and prostaglandin E2 production by RAW 264.7 cells in a dose-dependent manner, as well as the protein-level expression of inducible nitric oxide synthase and cyclooxygenase-2. The anti-inflammatory effect of ABL was confirmed using a transgenic Tg(mpx:EGFP) zebrafish larval model. The exposure of the larvae to ABL inhibited neutrophil recruitment to the site of injury after tail fin amputation. [ABSTRACT FROM AUTHOR]

Neocinnamomum caudatum (Lauraceae) plant is used in the traditional system of medicine and is considered a potential source of edible fruits, spices, flavoring agents and biodiesel. The leaves, bark and roots of the species are used by local communities for the treatment of inflammatory responses, such as allergies, sinusitis and urinary tract infections. However, there is no scientific evidence to support the molecular mechanism through which this plant exerts its anti-inflammatory effect. The aim of the current research was to characterize the chemical constituents of bark (NCB) and leaf (NCL) essential oil of N. caudatum and to elucidate its anti-inflammatory action in lipopolysaccharide (LPS)-treated RAW 264.7 cells. Essential oils extracted by hydrodistillation were further subjected to gas chromatography mass spectrometry (GC-MS) analysis. The major constituents in bark essential oil identified as β-pinene (13.11%), α-cadinol (11.18%) and α-pinene (10.99%), whereas leaf essential oil was found to be rich in β-pinene (45.21%), myrcene (9.97%) and α-pinene (9.27%). Treatment with NCB and NCL at a concentration of 25 µg/mL exerted significant anti-inflammatory activity by significantly reducing LPS-triggered nitric oxide (NO) production to 45.86% and 61.64%, respectively, compared to the LPS-treated group. In the LPS-treated group, the production of proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β, decreased after treatment with essential oil, alleviating the mRNA levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2. The essential oil also inhibited the production of intracellular ROS and attenuated the depletion of mitochondrial membrane potential in a concentration-dependent manner. Pretreatment with NCB also reduced nuclear factor kappa-B (NF-κB)/p65 translocation and elevated the levels of endogenous antioxidant enzymes in LPS-induced macrophages. The present findings, for the first time, demonstrate the anti-inflammatory potential of both bark and leaf essential oils of N. caudatum. The bark essential oil exhibited a significantly more important anti-inflammatory effect than the leaf essential oil and could be used as a potential therapeutic agent for the treatment of inflammatory diseases. [ABSTRACT FROM AUTHOR]

In this study, the anti-inflammatory effects of nonyl 8-acetoxy-6-methyloctanoate (NAMO), isolated from the cultured marine diatom, Phaeodactylum tricornutum Bohlin, against LPS-induced RAW 264.7 macrophages were evaluated. NAMO has indicated the strongest inhibitory effects against nitric oxide (NO) and prostaglandin E2 (PGE2) production, dosedependently, on lipopolysaccharide (LPS)-induced RAW 264.7 cells. The 50% NO production inhibitory concentration (IC50) of NAMO was 24.8 µM with the least cytotoxic effect in both LDH and MTT assays. Inflammatory stimulators such as LPS, which induce cytokines in the process of macrophage activation and mediate tissue response in different phases of inflammation, were studied. NAMO showed significant and strong suppression of pro-inflammatory cytokine and interleukin-1ß (IL-1ß) production, but no significant inhibitory effect on the production of cytokines including tumor necrosis factor-a (THF-a) and interleukin-6 (IL-6) at the tested concentrations against LPS treatment on RAW macrophages. Western blot analysis was carried out to determine the protein expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins which mediate the suppression effect of NAMO on NO and PGE2 production. The Western blot assay confirmed the suppression of iNOS and COX-2 protein expressions against LPS-stimulated RAW264.7 cells. Collectively, NAMO isolated from the cultured marine diatom, P. tricornutum exhibited a profound anti-inflammatory effect in vitro, suggesting that the compound might have a beneficial effect during the treatment of inflammatory diseases and can be used in functional food applications. [ABSTRACT FROM AUTHOR]

Exogenous ganglioside GM1 has been reported to exert an immunomodulatory effect. We investigated the anti-inflammatory effect of GM1 ganglioside on endotoxin-induced uveitis (EIU) in rats and RAW 264.7 macrophages. Methods: EIU was induced in Lewis rats by administering a subcutaneous injection of lipopolysaccharide (LPS). GM1 was injected intraperitoneally for three consecutive days prior to the LPS injection. Twenty-four hours after the LPS injection, the integrity of the blood-aqueous barrier was evaluated by determining the protein concentration and number of infiltrating cells in the aqueous humor (AqH). Immunohistochemical and Western blot analyses of the iris-ciliary body (ICB) were performed to evaluate the effect of GM1 on the LPS-induced expression of cyclooxygenase-2 (COX-2) and intercellular adhesion molecule-1 (ICAM-1). The effect of GM1 on proinflammatory mediators and signaling cascades was examined in LPS-stimulated RAW 264.7 cells using Western blotting and immunofluorescence staining to further clarify the underlying anti-inflammatory mechanism. Results: GM1 significantly reduced the protein concentration and number of infiltrating cells in the AqH of rats with EIU. GM1 also decreased the LPS-induced expression of the ICAM-1 and COX-2 proteins in the ICB. In RAW 264.7 cells, GM1 inhibited the proinflammatory mediators induced by LPS, including inducible nitric oxide synthase (iNOS), COX-2, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), and this inhibitory effect was potentially mediated by suppressing transforming growth factor-β-activated kinase 1 (TAK1) and reactive oxygen species (ROS)-mediated activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs). Conclusions: Based on this study, GM1 may be a potential anti-inflammatory agent for ocular inflammatory diseases. [ABSTRACT FROM AUTHOR]

It has been reported that 4,5-dihydropyrazole and thiazole derivatives have many biological functions, especially in the aspect of anti-inflammation. According to the strategy of pharmacophore combination, we introduced thiazolinone and dihydropyrazole moiety into steroid skeleton to design and synthesize a novel series of D-ring substituted steroidal 4,5-dihydropyrazole thiazolinone derivatives, and assessed their in vitro anti-inflammatory profiles against Lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophage cells. The anti-inflammatory activities assay demonstrated that compound 12e was considered as the most effective anti-inflammatory drug, which suppressed the expression of pro-inflammatory mediators including nitric oxide (NO), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), it also dose-dependently inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-induced RAW 264.7 macrophage cells. Furthermore, the results of the Western blot analysis showed a correlation between the inhibition of the Nuclear factor-kappa B (NF-κB) and Mitogen-activated protein kinases (MAPKs) signaling pathways and the suppressive effects of compound 12e on pro-inflammatory cytokines. Molecular docking studies of compound 12e into the COX-2 protein receptor (PDB ID: 5IKQ) active site was performed to rationalize their COX-2 inhibitory potency. The results were found to be in line with the biological findings as they exerted more favorable interactions compared to that of dexamethasone (DXM), explaining their remarkable COX-2 inhibitory activity. The findings revealed that these candidates could be identified as potent anti-inflammatory agents, compound 12e could be a promising drug for the treatment of inflammatory diseases. [Display omitted] • This study provided a novel, efficient and simple synthesis of steroidal 4,5-dihydropyrazole thiazolinone derivatives. • In vitro activity assay screened compound 12e with excellent anti-inflammatory biological activity and low cytotoxicity. • 12e inhibited LPS-induced expression of the pro-inflammatory cytokines iNOS, COX-2, TNF-α and IL-6 on RAW 264.7 cells. • 12e exerted anti-inflammatory effects through down-regulation of NF-κB/MAPKs pathway. [ABSTRACT FROM AUTHOR]

Inflammation, an important biological protective response to tissue damage or microbial invasion, is considered to be an alarming signal for the progress of varied biological complications. Based on the previous reports in the literature that proved the noticeable efficacy of pyrazole and thiazole scaffold as well as nitrogen heterocyclic based compounds against acute and chronic inflammatory disease, a new set of novel D-ring substituted steroidal 4,5-dihydropyrazole thiazole derivatives were synthesized and evaluated their anti-inflammatory activities in vitro. Preliminary structure-activity relationship (SAR) analysis was conducted by their inhibitory activities against nitric oxide (NO) release in lipopolysaccharide (LPS)-induced RAW 264.7 cells, and the optimal compound 12b [3β-hydroxy-pregn-5-en-17β-yl-5′- (o- chlorophenyl)− 1′-(4′′- phenyl -[1′′, 3′′]- thiazol-2′′- yl) − 4′,5′-dihydro − 1′H-pyrazol − 3′- yl] exhibited more potent anti-inflammatory activity than the positive control treatment methylprednisolone (MPS), with an IC 50 value of 2.59 μM on NO production and low cytotoxicity against RAW 264.7 cells. In further mechanism study, our results showed that compound 12b significantly suppressed the production of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and inhibited the expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) through blocking NF-κB p65 nuclear translocation and phosphorylation of IκBα. Compound 12b also attenuated LPS-induced activation of c-Jun amino-terminal kinase (JNK) and p38 phosphorylation in RAW 264.7 cells. Molecular docking study revealed the strong binding affinity of compound 12b to the active site of the COX-2 proteins, which confirmed that compound 12b acted as an anti-inflammatory mediator. These results indicate that steroidal derivatives bearing 4,5-dihydropyrazole thiazole structure might be considered for further research and scaffold optimization in designing anti-inflammatory drugs and compound 12b might be a promising therapeutic anti-inflammatory drug candidate. [Display omitted] • This study provided a novel series of D-ring substituted steroidal 4,5-dihydropyrazole thiazole derivatives. • The structures of all targeted synthesized compounds had been characterized. • The anti-inflammatory biological activity of targeted synthesized compounds was assayed. • In vitro activity assay screened compound 12b with excellent anti-inflammatory biological activity in LPS-induced RAW 264.7 cells. [ABSTRACT FROM AUTHOR]

[Display omitted] • Non-starch polysaccharide (ECPs-I) was extracted from finger millet. • Deproteinated non-starch polysaccharide (ECPs-II) prepared by proteinase K. • The molecular weight of polysaccharides were found to range from 43 to 120 kDa. • The extracted polysaccharides did not show any toxic effects on RAW 264.7 cells. • ECPs-II has significantly enhance the RAW 264.7 cells activation. The structural elucidation and investigation of the immunostimulatory effects of crude polysaccharides (ECPs), non-starch polysaccharides (ECPs-I), and deproteinated non-starch polysaccharides (ECPs-II) derived from finger millet (Eleusine coracana) were performed. The extracted ECPs, ECPs-I, and ECPs-II primarily comprised different levels of carbohydrate (82.9, 41.9 and 62.0%), protein (5.3, 38.4 and 22.0%) and uronic acid (0.5, 2.4 and 0.7%). Monosaccharide composition analysis showed that glucose (88.2 and 40.6) was the main sugar unit present in ECPs and ECPs-I, followed by galactose, arabinose, xylose, rhamnose, and mannose; however, ECPs-II contained galactose (42.2%) as a major unit. The average molecular weights of ECPs, ECPs-I, and ECPs-II were found to be 120, 78 and 43 kDa, respectively. The polysaccharides as well as non-starch polysaccharides did not show any toxic effects on RAW 264.7 cells. ECPs-II treatment significantly enhanced the activation of RAW 264.7 cells by inducing nitric oxide (NO) production, upregulating inducible nitric oxide synthase (iNOS) and various pro-inflammatory cytokines including interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor alpha (TNF-α). Furthermore, the RAW 264.7 cells were activated after ECPs-II treatment via the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and mitogen-activated protein kinase signaling (MAPK) pathways induced by a cluster of differentiation 40 (CD40) expression. The structure of ECPs-II mainly consists of (1→3,4)-linked galactopyranosyl and (1→3,5) arabinopyranosyl. [ABSTRACT FROM AUTHOR]

Objective: To demonstrate the anti-inflammatory activity of Brassica napus L. hydrosols (BNH) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Methods: Composition analysis of BNH was conducted via gas chromatography-mass spectrometry after BNH were extracted. The nitric oxide (NO) production was measured using the Griess assay. Prostaglandin E2 (PGE2) production was evaluated with enzyme-linked immunosorbent assay. The effects of BNH on LPS-induced pro-inflammatory enzymes including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were evaluated using Western blot analysis. Furthermore, phosphorylation of nuclear factor-kappa B (NF-κB) and nuclear translocation of NF-κB p65 were evaluated with Western blot analysis and immunofluorescence staining, respectively. Results: Compared with LPS-stimulated cells, BNH markedly decreased the generation of NO and PGE2 in LPS-stimulated RAW 264.7 cells (P<0.01 or P<0.05). Moreover, BNH inhibited protein levels of iNOS and COX-2 (P<0.01). Phosphorylation of NF-κB and nuclear translocation of NF-κB p65 was significantly inhibited by BNH (P<0.01 or P<0.05). Conclusion: The anti-inflammatory activities of BNH were mediated via blockage of the NF-κB signaling pathways in LPS-stimulated RAW 264.7 cells. [ABSTRACT FROM AUTHOR]

Background. The macrophage lineage is characterized by plasticity due to the acquisition of distinct functional phenotypes, and two major subsets are evaluated; classical M1 activation (strong microbicidal activity) and alternative M2 activation (immunoregula-tory functions). The M1 subset expresses inducible nitric oxide synthase (iNOS), which is a primary marker to identify these cells, whereas M2 macrophages are characterized by expression of Arginase-1, found in inflammatory zone 1 (Fizz1), chitinase-like molecule (Ym-1), and CD206. The micro-environmental stimuli and signals in tissues are critical in the macrophage polarization. Toll-like receptors (TLR) ligands, such as lipopolysaccharide (LPS), palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK4), and ArtinM (mannose-binding lectin) are inductors of M1 subset. The impact of TLR2 and TLR4 signals to fight against Cryptococcus gattii infection is unknown, which is a fungal pathogen that preferentially infects the lung of immunocompetent individuals. The macrophages initiate an immune response to combat the C. gattii, then we evaluated in RAW 264.7 cell the effect of TLR2 and TLR4 agonists on the macrophage polarization dynamic and the impact on the growth of C. gattii. Methods and Results. We demonstrated that P3C4, LPS, and ArtinM induced an increase in the levels of iNOS transcripts in RAW 264.7 cells, whereas the relative expression of arginase-1, Ym-1, and Fizz1 was significantly increased in the presence of IL-4 alone. The effects of TLR2 and TLR4 agonists on repolarization from the M2 to M1 subset was evaluated, and the first stimulus was composed of IL-4 and, after 24 h of incubation, the cells were submitted to a second stimulus of P3C4, LPS, ArtinM, or Medium. These TLR agonists induced the production of TNF-a in polarized RAW 264.7 cells to the M2 subset, moreover the measurement of M1/M2 markers using qRT-PCR demonstrated that a second stimulus with LPS for 24 h induced a significant augmentation of levels of iNOS mRNA. This impact of TLR2 and TLR4 agonists in the activation of the RAW 264.7 macrophage was assayed in the presence of C. gattii, the macrophages stimulated with TLR2 and TLR4 agonists for 24 h and co-cultured with C. gattii, as a second stimulus, reached high levels of TNF-a even after incubation with different concentrations of C. gattii. The activation of RAW 264.7 cells induced by TLR2 and TLR4 agonists favored the phagocytosis of C. gattii and inhibited the growth of yeast in the early period of infection. However, RAW 264.7 cells incubated with C. gattii in the presence of TLR2 and TLR4 agonists did not result a significant difference in the colony forming unit (CFU) assay in the early period of C. gattii infection, compared to negative control. Conclusion. Polarized RAW 264.7 cells to the M1 subset with TLR2 and TLR4 agonists did not inhibit the growth of C. gattii, whereas robust immunity was identified that could dysregulate host tolerance to this pathogen. [ABSTRACT FROM AUTHOR]

Background: Zanthoxylum rhetsa is a plant used in traditional medicine and is known to possess health benefits such as antibacterial, antidiabetic, and anti-diarrheal activities. Objectives: The objective of this study was to explore and demonstrate the anti-inflammatory activity of various solvent fractions of Z. rhetsa bark. Materials and Methods: The effect of crude methanolic extract and its fractions (hexane, chloroform, ethyl acetate, and butanol) on the levels of pro-inflammatory cytokines (interleukin-1β, tumor necrosis factor-alpha, and interleukin-6) and inflammatory factors was tested via targeting nuclear factor-kappa B (NF-κB) signaling pathways in lipopolysaccharide (LPS)-stimulated mouse RAW 264.7 macrophages. Results: Treatment with all the solvent fractions at various concentrations (50, 100, and 200 μg/ml) suppressed pro-inflammatory cytokine levels in a dose-dependent manner. Among the fractions, the chloroform fractions exhibited a significant inhibition of LPS-induced inflammation. Moreover, these fractions effectively suppressed the expression of various NF-κB signaling targets, including NF-κB, nitric oxide synthase, and cyclooxygenase-2 as well as inhibited the degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor, alpha (inhibitor of kappa B alpha [IκBα]). This anti-inflammatory effect was mediated by the prevention of IκBα degradation, as this protein prevents the translocation of NF-κB from the cytoplasm to the nucleus, initiating the transcription of pro-inflammatory genes. Conclusion: Thus, the Z. rhetsa chloroform fraction may be an effective natural anti-inflammatory agent against inflammation-associated diseases. [ABSTRACT FROM AUTHOR]

Danshensu, isolated from Salvia miltiorrhiza (Danshen), is known to have anti-inflammatory properties. Therefore danshen is extensively used in many nutraceutical formulations. Reactive oxygen species are essential for the development of hypoxia-induced inflammation. Generation of reactive oxygen species by infiltrating macrophages is common in various diseases such as cardiovascular disease, neurodegenerative disease, tumor, and aging. To explore the mechanism underlying the attenuation of inflammation, we used RAW 264.7 cells as a model and hypoxia as an inducer of inflammation. The results showed the protective mechanism of danshensu on reactive oxygen species production, hypoxia-inducible factor 1-alpha expression, c-Jun N-terminal kinase phosphorylation, nuclear translocation of nuclear factor kappa B, and inducible nitric oxide synthase expression following hypoxia in RAW 264.7 cells. [ABSTRACT FROM AUTHOR]

Ganoderma lucidum immunomodulatory protein (FIP‐glu) is an active ingredient with potential immunoregulatory functions. The study was conducted to explore the immunomodulatory activities of recombinant FIP‐glu (rFIP‐glu) and its possible mechanism in macrophage RAW264.7 cells. In vitro assays of biological activity indicated that rFIP‐glu significantly activated RAW264.7 cells and possessed proinflammatory and anti‐inflammatory abilities. RNA sequencing analysis and Western blot analysis showed that macrophage activation involved PI3K/Akt and MAPK pathways. Furthermore, real‐time quantitative polymerase chain reaction indicated that the PI3K inhibitor LY294002 blocked the messenger RNA (mRNA) levels of MCP‐1 (CCL‐2), the MEK1/2 inhibitor U0126 reduced the mRNA levels of TNF‐α and MCP‐1 (CCL‐2), and the JNK1/2/3 inhibitor SP600125 prevented the upregulation of inducible nitric oxide synthase mRNA in rFIP‐glu‐induced cells. rFIP‐glu did not mediate these inflammatory effects through a general pathway but rather through a different pathway for a different inflammatory mediator. These data imply that rFIP‐glu possessed immunomodulatory activity in macrophages, which was mediated through PI3K/Akt and MAPK pathways. [ABSTRACT FROM AUTHOR]

Inflammatory bowel diseases involve chronic intestinal inflammation which is mostly caused by Crohn's disease and ulcerative colitis (UC) conditions. Patients having UC are prone to colorectal cancer and dysplastic polyps, and also have sporadic adenomas. Syringic acid (SA) possesses many health benefits including antioxidant, anti‐bacterial, and anti‐cancer. This study was aimed to identify the effects of SA on UC, using a murine experimental model. Clinical symptoms and weight loss were significantly reduced in mice induced with dextran sulfate sodium (DSS) and treated with SA, compared to untreated mice. The effects of SA exhibited in DSS‐induced mice were associated with significant decrease in the expressions levels of inflammatory mediators such as inducible nitric oxide synthase (iNOS), cyclooxygenase‐2 (COX‐2), pro‐inflammatory cytokines (tumor necrosis factor [TNF‐α], interleukin [IL‐1β and IL‐6]), remarkable amelioration of colonic architectural disruption, and a significant reduction in the activity of colonic myeloperoxidase. To further confirm the anti‐inflammatory property of SA, RAW 264.7 cells were stimulated with lipopolysaccharides. SA dose‐dependently inhibited the cytokines TNF‐α, IL‐1β, and IL‐6. It also decreased the expressions of p65‐NF‐κB and IκB, thus reducing the activation and nuclear accumulation of p‐STAT‐3Y705. This prohibited the degradation of inhibitory protein, IκB, as well as inhibited the nuclear translocation of p65‐NF‐κB in colonic tissue. It was concluded that SA has a potential to limit inflammation via inhibiting the p65‐NF‐κB and STAT3 signaling; hence it can be used for therapeutic purposes. [ABSTRACT FROM AUTHOR]

The present study was designed to investigate the inhibitory effect of 2,4 bis-[(4-ethoxyphenyl)azo] 5-(3-hydroxybenzylidene) thiazolidine-2,4-dione (TZD-OCH2CH3) on the cyclo-oxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in RAW 264.7 cells. The effects of TZD-OCH2CH3 on COX-2 and iNOS mRNA expression in LPS-activated RAW 264.7 cells were detected by real time PCR. Also, to understand structure and substrate specificity, we have utilized molecular docking simulations (AutoDock Vina) and the active residues in the binding pocket were determined from COX-2 and iNOS. The treatment of RAW 264.7 cells with TZD-OCH2CH3 significantly inhibited LPS-induced COX-2 mRNA expression, corresponding to 46.1% and 61.06% at 30 and 60 µg/mL, respectively. The present study revealed that the TZD-OCH2CH3 had a little effect on iNOS mRNA expression. Meanwhile, the TZD-OCH2CH3 also could inhibit the production of NO compared to single LPS-stimulated cell. According to the results obtained, TZD-OCH2CH3 dramatically suppressed lipopolysaccharide (LPS) induced nitric oxide (NO) production after 24 h, in a concentration-dependent manner with an IC50 of 65 µg/mL. Our data suggest that TZD-OCH2CH3, as a functionally novel agent, inhibits the inflammatory pathway via suppression of COX-2 mRNA expression and also by the inhibition of the iNOS activity. Therefore, this compound could be suggested as a novel therapeutic strategy for inflammation-associated disorders. [ABSTRACT FROM AUTHOR]

• A new family of D-ring pyrazoline derivatives of pregn-5-enes were synthesized. • The structures of all targeted synthesized compounds had been characterized. • The anti-inflammatory biological activity of synthesized compounds was assayed. • Compound 4g was with excellent anti-inflammatory activity and low cytotoxicity. To identify new potential anti-inflammatory agents, a number of novel steroidal derivatives with nitrogen heterocyclic side chains 4a-4l were synthesized and evaluated for their anti-inflammatory effects in activated RAW 264.7 macrophage cells. The synthesis scheme involves two steps, Claisen-Schmidt condensation with the corresponding pregnenolone and aromatic aldehydes as the first step followed by nucleophilic addition of thiosemicarbazide across an α, β-unsaturated carbonyl as a later step. Compound structures were confirmed by 1H NMR, 13C NMR, HRMS, and IR. The compounds were assayed to test their anti-inflammatory effects in activated RAW 264.7 cells. Compound 4g , 3β-hydroxy-pregn-5-en-17β-yl-5′-(m-fluorophenyl)-4′, 5′-dihydro-1′-carbothioic acid amido pyrazole, was identified as the most potent anti-inflammatory agent of the analysed compounds, with an IC 50 value of 0.86 µM on nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW 264.7 cells for 24 h compared to dexamethasone (IC 50 = 0.62 µM) and low cytotoxicity against RAW 264.7 cells. Compound 4g significantly inhibited NO produced by LPS-induced RAW 264.7 cells. Further studies showed that compound 4g markedly inhibited the expression of pro-inflammatory factors, including inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2) in LPS-induced RAW 264.7 cells. These results indicate that derivatives bearing pyrazoline structure might be considered for further research and scaffold optimization in designing anti-inflammatory drugs and compound 4g might be a promising therapeutic anti-inflammatory drug candidate. [ABSTRACT FROM AUTHOR]

Geraniin, a major polyphenolic compound of Geranium sibiricum L, has long been used as an important Chinese herbal medicine for the treatment of a variety of inflammatory pathologies. However, the underlying anti-inflammatory molecular mechanisms of this compound are not clear. The aim of the present study was to investigate the anti-inflammatory activities of geraniin and elucidate the underlying mechanisms. The anti-inflammatory effects of geraniin were studied by using lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Geraniin suppressed the inducible nitric oxide synthase (iNOS) expression, and inhibited reactive oxygen species (ROS) production. Subsequent studies demonstrated that geraniin effectively reduced production of NO and pro-inflammatory cytokines. These effects were mediated by impaired translocation of nuclear factor (NF)-κB and inhibition of the phosphorylation of Akt in LPS-stimulated RAW 264.7 cells. Furthermore, geraniin induced heme oxygenase-1 (HO-1) expression via activation of transcription factor Nrf2. This study gives scientific evidence that geraniin inhibits the LPS-induced expression of inflammatory mediators via suppression of Akt-mediated NF-κB pathway as well as up-regulation of Nrf2/HO-1 pathway, indicating that geraniin has a potential application in inflammatory conditions. [ABSTRACT FROM AUTHOR]

Kaempferol 3- O -(2G-glucosylrutinoside)-7- O -glucoside (KGG) has isolated from Hosta plantaginea flowers and possessed an inhibitory effect on cyclooxygenase 2 (COX-2), could be effective in inhibiting inflammation. However, the anti-inflammatory activity and mechanism of KGG remain unknown. In this study, for the first time, the anti-inflammatory effect of KGG and its potential molecular mechanisms were explored in cells. KGG had no cytotoxicity at concentrations of 1.25, 2.5, 5, 10, 20, and 40 μM by Cell Counting kit-8 assay in RAW 264.7 cells. Besides, KGG concentration-dependently (1.25, 2.5, and 5 μM) inhibited secretions of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-6 in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Western blot showed that the phosphorylation of nuclear factor kappa-B (NF-κB) p65, inhibitor of NF-κB (IκB), p38 MAPK, c-Jun N-terminal kinases (JNK), extracellular signal-regulated kinase (Erk), and protein kinase B (Akt), together with inducible nitric oxide synthase (iNOS) and COX-2 were significantly attenuated by KGG (1.25, 2.5, and 5 μM) in a concentration-dependent relationship. Meanwhile, KGG remarkably enhanced the protein expression of IκB. Taken together, KGG may be one of bioactive phytochemicals from H. plantaginea flowers, and be an anti-inflammatory agent via inhibiting NF-κB, mitogen-activated protein kinases (MAPKs), and Akt signaling pathways. [Display omitted] • Kaempferol 3- O -(2G-glucosylrutinoside)-7- O -glucoside (KGG) exerts anti-inflammatory effect through blocking NF-κB, MAPKS, and Akt pathways. • KGG might be a promising candidate against inflammation. [ABSTRACT FROM AUTHOR]

The purpose of this study was to evaluate the effects of polydatin (PD) on cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expressions at protein and transcriptional levels, as well as the production of prostaglandin E (PGE) and nitric oxide (NO) in lipopolysaccharide (LPS)-induced macrophage RAW 264.7 cells. To elucidate the underlying mechanism responsible for these symptoms, we investigated the phosphorylation of mitogen-activated protein kinase (MAPK) pathway and nuclear factor-κB (NF-κB) expression. NO was analyzed with the Griess method. PGE was measured by enzyme-linked immunosorbent assay (ELISA). iNOS and COX-2 messenger RNA (mRNA) were identified by qPCR assay. iNOS, COX-2, NF-κB, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 protein expressions were detected with Western blot. The results revealed that PD effectively inhibited NO and PGE production, and it is not surprising that PD reduced iNOS and COX-2 expression at protein and transcriptional levels. Additionally, PD significantly ameliorated the activation of NF-κB and the phosphorylation of MAPKs in LPS-induced RAW 264.7 macrophages. These findings suggested that PD exerted potent anti-inflammatory activity in macrophages. [ABSTRACT FROM AUTHOR]

Amber is a type of fossil tree resin with several bioactive properties and has been traced in traditional medicines used in Russia and China. However, its anti-inflammatory activities are poorly characterized. Here, the anti-inflammatory effects of the extract of amber mined from Kaliningrad, Russia was investigated in lipopolysaccharide (LPS)-induced RAW 264.7 cells. The effect of the amber extract on cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Further, its effects on the production of intracellular reactive oxygen species (ROS), NO, and inflammatory cytokines were assessed by 2′,7′-dichlorodihydrofluorescein diacetate staining, Griess test, and cytokine enzyme-linked immunosorbent assays, respectively. Western blotting and real-time reverse transcription-polymerase chain reaction analysis were performed to assess the mRNA and protein expression levels of the inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS). The translocation of the nuclear factor-kappa B (NF-κB) p65 subunit was observed by immunofluorescent staining. Amber extract negatively regulated the LPS-induced differentiation of RAW 264.7 cells to dendritic-like cells and reduced the LPS-induced increase in ROS and NO levels. It also reduced the level of mRNA and protein expressions of TNF-α, IL-6, COX-2, and iNOS in LPS-induced RAW 264.7 macrophages, in a dose-dependent manner. Furthermore, amber extract suppressed the nuclear translocation of the NF-κB p65 subunit. These findings suggest that the potent anti-inflammatory effect of the amber extract is mediated by the inhibition of the NF-κB p65 signaling pathway. Collectively, this study renders amber extract as a potential pharmacological alternative to treat inflammation-related diseases. • Amber extract suppressed the generation of NO and ROS in LPS-treated RAW 267.4. • Amber extract inhibited the expression of TNF-α and IL-6 in LPS-treated RAW 267.4. • Amber extract interrupted the NF-κB p65 translocation in LPS-treated RAW 267.4. [ABSTRACT FROM AUTHOR]

Species of Podocarpus are used traditionally in their native areas for the treatment of fevers, asthma, coughs, cholera, chest pain, arthritis, rheumatism, and sexually transmitted diseases. To identify natural products having efficacy against inflammatory bowel disease (IBD), we identified a new, 16-hydroxy-4β-carboxy-O-β-D-glucopyranosyl-19-nor-totarol (4) together with three known diterpenoids from P. macrophyllus. Furthermore, all the extracts, fractions, and isolates 1–4 were investigated for their anti-inflammatory effects by assessing the expression on nitric oxide (NO) production and proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated RAW 264.7 and HT-29 cells. Among them, nagilactone B (2) exhibited a potent anti-inflammatory effect against NO production on RAW 264.7 cells; therefore, nagilactone B was further assessed for anti-inflammatory activity. Western blot analysis revealed that nagilactone B significantly decreased the expression of LPS-stimulated protein, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and phosphorylated extracellular regulated kinase (pERK)1/2. In addition, nagilactone B downregulated tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-8 levels in LPS-induced macrophages and colonic epithelial cells. To our best knowledge, this is the first report on the inhibitory effect of nagilactone B (pure state) and rakanmakilactone G against NO production in LPS-stimulated RAW 264.7 cells. Thus, diterpenoids isolated from P. macrophyllus could be employed as potential therapeutic phytochemicals for IBD. [ABSTRACT FROM AUTHOR]

[Display omitted] • This study provided a novel, efficient and simple synthesis of a new family of steroidal chalcone derivatives of 20-keto 3β-3-phenyl acrylate -pregn-5-enes. • The structures of all targeted synthesized compounds had been characterized. • The anti-inflammatory biological activity of targeted synthesized compounds was assayed. • In vitro activity assay screened compound 11e with excellent anti-inflammatory biological activity and low cytotoxicity against RAW 264.7 cells. To identify new potential anti-inflammatory agents, we herein report the synthesis of novel steroidal chalcones with 3β-pregnenolone esters of cinnamic acid derivatives using pregnenolone as the starting material. The structures of the newly synthesised compounds were confirmed by 1H NMR, 13C NMR, HRMS and infrared imaging. All the derivatives were examined to determine their in vitro anti-inflammatory profiles against LPS-induced inflammation in RAW 264.7 cells; the derivates were evaluated by the quantification of the pro-inflammatory mediator nitric oxide (NO) in the cell culture supernatant based on the Griess reaction, which measures nitrite levels, followed by an in vitro cytotoxicity study. Among these novel derivatives, compound 11e [3β-3-phenyl acrylate-pregn-5-en-17β-yl-3′ -(p-fluoro)-phenylprop-2′-en-1′-one] was identified as the most potent anti-inflammatory agent, which showed significant anti-inflammatory activity by inhibiting the LPS-induced pro-inflammatory mediator NO in a dose-dependent manner without any cytotoxicity. Moreover, compound 11e markedly inhibited the expression of pro-inflammatory cytokines, including inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2), in LPS-induced RAW 264.7 cells. Further studies confirmed that compound 11e significantly suppressed the transcriptional activity of NF-κB in activated RAW 264.7 cells. Molecular docking study revealed the strong binding affinity of compound 11e to the active site of the pro-inflammatory proteins, which confirmed that compound 11e acted as an anti-inflammatory mediator. These results indicated that steroidal chalcones with 3β-pregnenolone esters of cinnamic acid derivatives might be considered for further research in the design of anti-inflammatory drugs, and compound 11e might be a promising therapeutic anti-inflammatory drug candidate. [ABSTRACT FROM AUTHOR]

Scurrula ferruginea (Jack) Danser (locally known as 'Dedalu' or 'dian nan ji sheng' in Malaysia and China) is a hemi-parasitic shrub that is widely used as herbal medicine to treat inflammation, rheumatism, and stroke. However, the scientific basis of its anti-inflammatory function and mechanism remain to be proven. Aim of the study : To evaluate the anti-inflammatory activity as well as the preliminary mechanism of S. ferruginea parasitizing on Tecoma stans. The anti-inflammatory capability of freeze-dried stem aqueous extract was assessed via inhibition of inflammatory cytokines interleukin- (IL-) 1 β , IL-6, IL-10, and tumor necrosis factor-alpha (TNF- α) production in lipopolysaccharide (LPS) and interferon- γ (IFN- γ) stimulated RAW 264.7 macrophages. The underlying anti-inflammatory mechanism was deciphered through reverse transcriptase and real time quantitative polymerase chain reactions (RT-PCR and qPCR) for inducible NO synthase (i NOS), cyclooxygenase-2 (COX-2), IL-1 β , and TNF- α mRNA expression. The results exhibited that aqueous extract of freeze-dried S. ferruginea stem sample concentration-dependently inhibited IL-1 β protein production along with the down regulation of i NOS and IL-1 β mRNA expression. Moreover, it significantly suppressed the protein release of IL-6 and IL-10 in a concentration-dependent manner. However, it slightly reduced TNF- α at higher sample concentration (250 μg/mL) without affecting the mRNA expression levels of COX-2 and TNF- α. This study suggests that S. ferruginea parasitizing on Tecoma stans exerted anti-inflammatory capability attributed to inhibition of i NOS and IL-1 β mRNA expression, NO creation, IL-1 β , IL-6, IL-10, and TNF- α protein production, indicating this plant might be a useful plant-derived candidate against inflammation. Image 1 • The anti-inflammatory effect in vitro of Scurrula ferruginea had been proven. • S. ferruginea inhibited NO, IL-1 β , IL-6, IL-10, and TNF- α productions. • Decrease of iNOS and IL-1β in LPS/IFN-γ induced RAW 264.7. [ABSTRACT FROM AUTHOR]

Hippophae rhamnoides L. (Elaeagnaceae; commonly known as "sea buckthorn" and "vitamin tree"), is a spiny deciduous shrub whose fruit is used in foods and traditional medicines. The H. rhamnoides fruit (berry) is rich in vitamin C, with a level exceeding that found in lemons and oranges. H. rhamnoides berries are usually washed and pressed to create pomace and juice. Today, the powder of the aqueous extract of H. rhamnoides berries are sold as a functional food in many countries. As part of our ongoing effort to identify bioactive constituents from natural resources, we aimed to isolate and identify those from the fruits of H. rhamnoides. Phytochemical analysis of the extract of H. rhamnoides fruits led to the isolation and identification of six compounds, namely, a citric acid derivative (1), a phenolic (2), flavonoids (3 and 4), and megastigmane compounds (5 and 6). Treatment with compounds 1–6 did not have any impact on the cell viability of RAW 264.7 mouse macrophages. However, pretreatment with these compounds suppressed lipopolysaccharide (LPS)-induced NO production in RAW 264.7 mouse macrophages in a concentration-dependent manner. Among the isolated compounds, compound 1 was identified as the most active, with an IC50 of 39.76 ± 0.16 μM. This value was comparable to that of the NG-methyl-L-arginine acetate salt, a nitric oxide synthase inhibitor with an IC50 of 28.48 ± 0.05 μM. Western blot analysis demonstrated that compound 1 inhibited the LPS-induced expression of IKKα/β (IκB kinase alpha/beta), I-κBα (inhibitor of kappa B alpha), nuclear factor kappa-B (NF-κB) p65, iNOS (inducible nitric oxide synthase), and COX-2 (cyclooxygenase-2) in RAW 264.7 cells. Furthermore, LPS-stimulated cytokine production was detected using a sandwich enzyme-linked immunosorbent assay. Compound 1 decreased interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) production in LPS-stimulated RAW 264.7 cells. In summary, the mechanism of action of 1 included the suppression of LPS-induced NO production in RAW 264.7 cells by inhibiting IKKα/β, I-κBα, NF-κB p65, iNOS, and COX-2, and the activities of IL-6 and TNF-α. [ABSTRACT FROM AUTHOR]