Effects In LPS-stimulated RAW Research Articles

Anti-inflammatory activities of melatonin derivatives in lipopolysaccharide-stimulated RAW 264.7 cells and antinociceptive effects in mice.

Preclinical Research This study describes the anti-inflammatory activities of two semisynthesized melatonin (MT) derivatives, benzoyl-melatonin (BMT) and acetyl-melatonin (AMT), on the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated macrophage cells (RAW 264.7) and their antinociceptive effects in mice. The MT derivatives inhibited production of nitric oxide NO and prostaglandin E2 in LPS-stimulated RAW264.7 cells in a dose-dependent manner with IC50 values lower than those of MT. BMT produced increased tail flick latency time, decreased number of writhes, and reduced nociceptive response in mice when compared with AMT and MT. BMT and AMT had enhanced anti-inflammatory effects in LPS-stimulated RAW264.7 compared with MT. However, in mouse studies BMT exhibited the highest potency as an anti-inflammatory agent and was longer-acting as an antinociceptive compound compared with AMT or MT, suggesting that BMT has potential as an anti-inflammatory and analgesic compound.

A new sulfonic acid derivative, (Z)-4-methylundeca-1,9-diene-6-sulfonic acid, isolated from the cold water sea urchin inhibits inflammatory responses through JNK/p38 MAPK and NF-κB inactivation in RAW 264.7

In this study, we isolated a new sulfonic acid derivative, (Z)-4-methylundeca-1,9-diene-6-sulfonic acid (1), from the sea urchin collected from the Sea of Okhotsk. We established the structure of this new compound by analysis of NMR and HRMS data, along with comparison of the data with those of the related compounds reported in the literature. In addition, we investigated its anti-inflammatory effects in LPS-stimulated RAW264.7 macrophages. Compound 1 inhibited the production of NO, iNOS, PGE2, and COX-2, and it also suppressed the production of pro-inflammatory cytokines, such as TNF-α and IL-1β. It inhibited the translocation of the NF-κB subunit p65 into the nucleus by interrupting the phosphorylation and degradation of IκB-α. In addition, compound 1 significantly decreased the phosphorylation of JNK and p38 in LPS-stimulated RAW264.7 macrophages, suggesting that suppression of the inflammation process by compound 1 was mediated through the MAPK pathway. Taken together, this study showed that the anti-inflammatory effects of a new sulfonic acid derivative, (Z)-4-methylundeca-1,9-diene-6-sulfonic acid were mediated through the inhibition of NF-κB and JNK/p38 MAPK signaling pathways.

Chemical composition and antioxidative and anti-inflammatory properties of ten commercial mung bean samples

Ten commercial mung bean samples (MB1–10) were investigated and compared for their chemical composition, and anti-oxidant and anti-inflammatory properties. Mung beans had a low oil content of 0.32–0.75 g/100 g, and differed in their total phenolic, total flavonoid, and vitexin and isovitexin contents. The acetone–water (1:1, v/v) extract of MB6 exhibited the highest total phenolic content of 5.07 mg gallic acid equivalents (GAE)/g, and the greatest total flavonoid concentration of 5.97 mg rutin equivalents (RE)/g. The acetone–water extracts differed in their RDSC, FRAP, HOSC and ORAC values, and showed significant anti-inflammatory effects in LPS-stimulated RAW 264.7 mouse macrophage cells at 100 μg/mL concentration. Furthermore, the results indicated that compounds other than vitexin and isovitexin might also significantly contribute to the overall anti-inflammatory effect of mung beans. This study indicated the potential utilization of mung beans in health promoting functional food or supplemental products.

Identification and Quantification of Phytochemical Composition and Anti-inflammatory and Radical Scavenging Properties of Methanolic Extracts of Chinese Propolis

Fifteen propolis samples collected from different regions of China were investigated and compared for their phytochemical composition and anti-inflammatory and radical scavenging properties. Eleven compounds including caffeic, p-coumaric, ferulic, isoferulic, and 3,4-dimethylcaffeic acids, pinobanksin, chrysin, pinocembrin, galangin, pinobanksin 3-acetate, and caffeic acid phenylethyl ester were quantified for the 15 propolis samples using a UHPLC method, whereas 38 compounds were identified by UPLC/Q-TOF-MS. The 15 propolis samples significantly differed in their total phenolic and total flavonoid contents, as well as their phytochemical profiles. The methanol extracts of propolis also showed significant anti-inflammatory effects in LPS-stimulated RAW 264.7 mouse macrophage cells at 10 μg propolis extract/mL concentration. Additionally, the propolis samples differed in their DPPH, ABTS cation, hydroxyl, and peroxide radical scavenging capacities and ferric reducing abilities. The results from this study may be used to improve the commercial production and consumption of Chinese propolis products.

Alantolactone suppresses inducible nitric oxide synthase and cyclooxygenase-2 expression by down-regulating NF-κB, MAPK and AP-1 via the MyD88 signaling pathway in LPS-activated RAW 264.7 cells

Several sesquiterpene lactones are the active components of several medicinal plants and have been demonstrated to perform various pharmacological functions. In this study, we investigated the anti-inflammatory effects of alantolactone, a sesquiterpene lactone isolated from the root of Aucklandia lappa, in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and peritoneal macrophages. Alantolactone inhibited inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) protein and mRNA transcription, as well as the downstream products, nitric oxide (NO), prostaglandin E2 (PGE2) and tumor necrosis factor-α (TNF-α). Investigation of the effects on nuclear factor κB (NF-κB) signaling showed that alantolactone inhibits the phosphorylation of inhibitory κB (IκB)-α and IκB kinase (IKK) and the subsequent translocation of the p65 and p50 NF-κB subunits to the nucleus. Moreover, inhibition of mitogen-activated protein kinases (MAPKs), including c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 MAPK, and activator protein-1 (AP-1) was also observed. A further study indicated that alantolactone attenuated the phosphorylation of Akt and inhibited the expression of MyD88 and Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP), an upstream signaling molecule required for IKK and MAPKs activation. Taken together, these results suggest that alantolactone exerts its anti-inflammatory effect in LPS-stimulated RAW 264.7 cells by suppressing NF-κB activation and MAPKs phophorylation via downregulation of the MyD88 signaling pathway. Thus, alantolactone may provide a useful therapeutic approach for inflammation-associated diseases.

Abutilon avicennae seed coat extract inhibits the lipopolysaccharide-induced inflammatory response in macrophages by multiple mechanisms

Abutilon avicennae is used in Korean folk medicine to treat middle ear infection, diarrhea, and inflammation of the lymphatic glands. The anti-inflammatory abilities of abutilon avicennae have not been investigated in detail. Therefore, we undertook the present study to examine whether an extract of the seed coat of abutilon avicennae (SCAA) could suppress LPS-stimulated inflammatory responses in RAW 264.7 cells and to determine the mechanisms by which it did so. We found that SCAA completely suppressed NO production in LPS-stimulated RAW 264.7 cells in a dose-dependent manner. We also confirmed that SCAA was not cytotoxic to RAW 264.7 cells at any of the concentrations we used in this study. Molecular analysis demonstrated that SCAA inhibited mRNA and protein expression of both iNOS and COX-2 in a dose-dependent manner. In addition, SCAA reduced the mRNA levels of the pro-inflammatory cytokines interleukin-1β (IL-1β) and interleukin-6 (IL-6). Moreover, SCAA suppressed the phosphorylation of mitogen-activated protein kinases (MAPKs; JNK, ERK1/2 and p38), whereas it had no effect on the phosphorylation status of Akt. SCAA suppressed the LPS-stimulated degradation of IkBα in a concentration-dependent manner, thereby preventing translocation of nuclear factor-kB (NF-kB) into the nucleus. Taken together, these results suggest that SCAA exerted its anti-inflammatory effects in LPS-stimulated RAW 264.7 cells by inhibiting IkBk degradation, and inhibiting the transcription of proinflammatory mediators and cytokines. Key words: Inflammation, abutilon avicennae, nuclear factor-kB, RAW 264.7.

Abutilon avicennae seed coat extract inhibits the lipopolysaccharide-induced inflammatory response in macrophages by multiple mechanisms

Abutilon avicennae is used in Korean folk medicine to treat middle ear infection, diarrhea, and inflammation of the lymphatic glands. The anti-inflammatory abilities of abutilon avicennae have not been investigated in detail. Therefore, we undertook the present study to examine whether an extract of the seed coat of abutilon avicennae (SCAA) could suppress LPS-stimulated inflammatory responses in RAW 264.7 cells and to determine the mechanisms by which it did so. We found that SCAA completely suppressed NO production in LPS-stimulated RAW 264.7 cells in a dose-dependent manner. We also confirmed that SCAA was not cytotoxic to RAW 264.7 cells at any of the concentrations we used in this study. Molecular analysis demonstrated that SCAA inhibited mRNA and protein expression of both iNOS and COX-2 in a dose-dependent manner. In addition, SCAA reduced the mRNA levels of the pro-inflammatory cytokines interleukin-1β (IL-1β) and interleukin-6 (IL-6). Moreover, SCAA suppressed the phosphorylation of mitogen-activated protein kinases (MAPKs; JNK, ERK1/2 and p38), whereas it had no effect on the phosphorylation status of Akt. SCAA suppressed the LPS-stimulated degradation of IkBα in a concentration-dependent manner, thereby preventing translocation of nuclear factor-kB (NF-kB) into the nucleus. Taken together, these results suggest that SCAA exerted its anti-inflammatory effects in LPS-stimulated RAW 264.7 cells by inhibiting IkBk degradation, and inhibiting the transcription of proinflammatory mediators and cytokines. Key words: Inflammation, abutilon avicennae, nuclear factor-kB, RAW 264.7.

Inducible Nitric Oxide Synthase and Cyclooxygenase-2 Participate in Anti-inflammatory Activity of Imperatorin from Glehnia littoralis

In this study, we have investigated the anti-inflammatory effects of imperatorin, a compound isolated from the roots of Glehnia littoralis, using a lipopolysaccharide (LPS)-stimulated mouse macrophage (RAW264.7) in vitro and a carrageenan (Carr)-induced mouse paw edema model in vivo. When RAW264.7 macrophages were treated with imperatorin together with LPS, a significant concentration-dependent inhibition of NO production was detected. Western blotting revealed that imperatorin blocked the protein expression of iNOS and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW264.7 macrophages significantly. In the anti-inflammatory test, imperatorin decreased the paw edema at 4 and 5 h after Carr administration and increased the activities of catalase, superoxide dismutase, and glutathione peroxidase in paw edema. We also demonstrated that imperatorin significantly attenuated the malondialdehyde level in the edema paw at the fifth hour after Carr injection. Imperatorin decreased the NO and tumor necrosis factor and prostaglandin E2 levels on serum at 5 h after Carr injection. Western blotting revealed that imperatorin decreased Carr-induced iNOS and COX-2 expressions at 5 h in edema paw. An intraperitoneal injection treatment with imperatorin also diminished neutrophil infiltration into sites of inflammation as did indomethacin. The results suggested that imperatorin had anti-inflammatory effects in LPS-stimulated RAW 264.7 cells and Carr-injected mice, respectively. In addition, inhibition of elevated iNOS and COX-2 protein expression as well as neutrophil infiltration of Carr-injected paws may be involved in the beneficial effects of imperatorin.

<i>Anethum graveloens</i> Flower Extracts Inhibited a Lipopolysaccharide-Induced Inflammatory Response by Blocking iNOS Expression and NF-κB Activity in Macrophages

Inflammation is a system used by a host to defend against the presence of bacteria, viruses, or yeasts. Toll-like receptors (TLRs) in the plasma membranes of macrophages are activated when they recognize the molecular structure of a virus or bacterium. Lipopolysaccharide (LPS), an outer cell-wall component of Gram-negative bacteria, initiates an inflammatory process via TLR4. We investigated the effect of the extract of Anethum graveloens flowers (AGFs) on LPS-mediated inflammation in RAW 264.7 cells. The extract markedly suppressed nitric oxide generation in a concentration-dependent manner in LPS-stimulated RAW 264.7 cells. It inhibited inducible nitric oxide synthase (iNOS) and the mRNA expression of cytokines such as interleukin-1 beta and interleukin-6 in LPS-stimulated RAW 264.7 cells. It also inhibited iNOS protein levels in LPS-stimulated RAW 264.7 cells. In addition, AGF decreased the LPS-induced phosphorylation of mitogen-activated protein kinases in LPS-stimulated RAW 264.7 cells. AGF inhibited the phosphorylation of Akt, an upstream molecule of the nuclear factor kappa B (NF-κB) pathway, and thus inhibited NF-κB activity in LPS-stimulated RAW 264.7 cells. These results suggest that AGF exerts an anti-inflammatory effect in LPS-stimulated RAW 264.7 cells by inhibiting iNOS expression and blocking the NF-κB pathway.

Pyranocoumarins Isolated from Peucedanum praeruptorum Dunn Suppress Lipopolysaccharide-Induced Inflammatory Response in Murine Macrophages Through Inhibition of NF-κB and STAT3 Activation

Praeruptorin C, D, and E (PC, PD, and PE) are three pyranocoumarins isolated from the dried root of Peucedanum praeruptorum Dunn of Umbelliferae. In the present study, we investigated the anti-inflammatory effect of these compounds in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Pyranocoumarins significantly inhibited LPS-induced production of nitric oxide, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). The mRNA and protein expressions of inducible nitric oxide synthase, IL-6, and TNF-α were also suppressed by these compounds. Both PD and PE exhibited greater anti-inflammatory activities than PC. Further study showed that pyranocoumarins suppressed the cytoplasmic loss of inhibitor κB-α protein and inhibited the translocation of NF-κB from cytoplasm to nucleus. In addition, pyranocoumarins suppressed LPS-induced STAT3 tyrosine phosphorylation. Taken together, the results suggest that pyranocoumarins may exert anti-inflammatory effects in LPS-stimulated RAW 264.7 macrophages through the inhibition of NF-κB and STAT3 activation.

Chain structures of glucans from Lentinus edodes and their effects on NO production from RAW 264.7 macrophages

Abstract Four glucans (LNT-H, LNT-S, LNT-E, and LNT-B) were successively isolated from the fruiting bodies of Lentinus edodes by hot-water, sonication, enzyme, and NaOH. The chemical structures were characterized by GC, FTIR, and 13 C NMR, which demonstrated that the four samples are (1 → 3)-β- d -glucans with (1 → 6)-glucopyranoside side groups having different molecular weight. The results of viscometry and atomic force microscopy (AFM) proved that LNT-H, LNT-S, LNT-E, and LNT-B exist as stiff triple helices in water and random coil in dimethyl sulfoxide (DMSO). The morphology of the original triple helix was almost dominated by linear shape, but the renatured LNT-B contains some branched structures in AFM image. Among the four glucans, LNT-H, LNT-E, and LNT-B are clearly contaminated by endotoxin, while LNT-S significantly exhibits nitric oxide (NO) inhibition effect in LPS-stimulated RAW 264.7 macrophages in a dose-dependent manner. Therefore, LNT-S is probably used as a promising molecule which can be helpful to the diseases associated with NO overproduction. This is the first primary report dealing with the suppression of LPS-induced NO from macrophage RAW 264.7 cells by Lentinan.

Neolitsea Sericea Essential Oil Attenuates LPS-induced Inflammation in RAW 264.7 Macrophages by Suppressing NF-κB and MAPK Activation

The chemical composition and anti-inflammatory activities of hydrodistilled essential oil from Neolitsea sericea leaves (NSE) have been investigated for the first time. The chemical constituents of NSE were analysed by GC-MS and found to include sericenine (32.3%), sabinene (21.0%), trans-beta-ocimene (13.3%), beta-caryophyllene (4.8%), and 4-terpineol (4.2%). The effects of NSE on nitric oxide (NO), prostaglandin E2 (PGE2), tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6 production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages were also examined. Pro-inflammatory cytokine and mediator tests indicated that NSE has excellent dose-dependent inhibitory activities. To further examine the mechanism responsible for the inhibition of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression by NSE, we examined the effect of NSE on nuclear factor-kappaB (NF-kappaB) activation and the phosphorylation of mitogen-activated protein kinases (MAPK). NSE inhibited NF-kappaB activation by LPS, and this was associated with the abrogation of IkappaB-alpha phosphorylation and subsequent decreases in nuclear p50 and p65 protein levels. Further, the phosphorylation of p38, ERK and JNK was suppressed by NSE in a concentration-dependent manner. These results suggest that NSE exerts anti-inflammatory effects in LPS-stimulated RAW 264.7 macrophages by inhibition of NF-kappaB activation and MAPK phosphorylation, and, therefore, may be useful for treatment of inflammatory diseases.

Chebulagic acid (CA) attenuates LPS-induced inflammation by suppressing NF-κB and MAPK activation in RAW 264.7 macrophages

Chebulagic acid (CA), a natural anti-oxidant, showed potent anti-inflammatory effects in LPS-stimulated RAW 264.7, a mouse macrophage cell line. These effects were exerted via inhibition of NO and PGE 2 production and down-regulation of iNOS, COX-2, 5-LOX, TNF-α and IL-6. CA inhibited NF-κB activation by LPS, and this was associated with the abrogation of IκB-α phosphorylation and subsequent decreases in nuclear p50 and p65 protein levels. Further, the phosphorylation of p38, ERK 1/2 and JNK in LPS-stimulated RAW 264.7 cells was suppressed by CA in a concentration-dependent manner. LPS-induced generation of reactive oxygen species (ROS) was also effectively inhibited by CA. These results suggest that CA exerts anti-inflammatory effects in LPS-stimulated RAW 264.7 macrophages by inhibition of NF-κB activation and MAP kinase phosphorylation.

Inducible nitric oxide synthase and cyclooxygenase-2 participate in anti-inflammatory and analgesic effects of the natural marine compound lemnalol from Formosan soft coral Lemnalia cervicorni

Lemnalol (8-isopropyl-5-methyl-4-methylene-decahydro-1,5-cyclo-naphthalen-3-ol) is a natural compound isolated from the marine soft coral Lemnalia cervicorni. In the present study, the anti-inflammatory and anti-nociceptive properties of lemnalol were investigated in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and carrageenan-injected rats, respectively. Our results demonstrate that lemnalol significantly inhibited the expression of the pro-inflammatory proteins, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in LPS-stimulated RAW 264.7 cells. An in vivo inflammation model was induced by intraplantar injection of carrageenan into rat hind paws. An intramuscular injection of lemnalol (15 mg/kg) 10 min before carrageenan injection resulted in significant inhibition of carrageenan-induced rat paw edema and thermal hyperalgesia behavior. Western blot experiments revealed that the carrageenan-induced expression of iNOS and COX-2 in paw tissue was significantly down-regulated by lemnalol. Moreover, post-intrathecal injection of lemnalol produced a dose-dependent anti-nociceptive effect in carrageenan-injected rats (1 and 5 μg). The present results indicate that the marine-derived compound lemnalol had anti-inflammatory and analgesic effects in LPS-stimulated RAW 264.7 cells and carrageenan-injected rats, respectively. In addition, inhibition of elevated iNOS and COX-2 protein expression as well as neurophil infiltration of carrageenan-injected paws may be involved in the beneficial effects of lemnalol.

Wen-Pi-Tang-Hab-Wu-Ling-San extract inhibits the release of inflammatory mediators from LPS-stimulated mouse macrophages

Aim of the study Wen-Pi-Tang is a traditional herbal prescription that has been used traditionally for the treatment of various inflammatory diseases, including chronic renal failure, renal injury, renal tubular cell damage and diabetic nephropathy. In this study, we investigated the pharmacological activity of modified Wen-Pi-Tang, Wen-Pi-Tang-Hab-Wu-Ling-San (WHW) extract. Materials and methods Production of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), IL-6 and NO in supernatant, mRNA expression of TNF-α, IL-1β, IL-6 and iNOS, protein expression of iNOS, phosphorylation of mitogen-activated protein kinases (MAPKs) and activation of nuclear factor-kappa B in the extract were assayed. Results We found that WHW extract had potent anti-inflammatory effects in LPS-stimulated RAW264.7 cells and primary peritoneal macrophages. WHW extract strongly inhibited the excessive production of inflammatory mediators, nitric oxide (NO), TNF-alpha (TNF-α), interleukin 1-beta (IL-1β) and IL-6 in LPS-stimulated macrophages. The inhibition of inducible nitric oxide synthase (iNOS) and these cytokines resulted from the reduced expressions of mRNAs of iNOS and these cytokines, respectively. WHW extract attenuated the phosphorylation of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinase (JNK), as well as the activation of nuclear factor-kappa B (NF-κB) in LPS-stimulated RAW264.7 cells. Conclusions These data suggest that WHW extract may exhibit anti-inflammatory effects through the modulation of MAPK and the NF-κB-dependent pathway involved in inflammation.

Anti-inflammatory Effects of Indomethacin's Methyl Ester Derivative and Induction of Apoptosis in HL-60 Cells

Indomethacin is used as an anti-inflammatory drug and a nonselective cyclooxygenase inhibitor. When indomethacin in methanol was photo-irradiated with an Hg lamp, methyl ester, ethyl ester, and gamma-lactone derivatives of indomethacin were produced. In the present study, we found that the methyl ester derivative of indomethacin (M-IN) could more potently inhibit prostaglandin E(2) (PGE(2)) and nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX 2) protein expression from lipopolysaccharide (LPS)-stimulated RAW 264.7 cells than indomethacin, similar to the effect of a non-steroidal anti-inflammatory drugs (NSAID). On the other hand, the results showed that M-IN with an IC(50) value maintained at 36.9 microg/ml for 12 h exhibited stronger cytotoxicity than ethyl ester, gamma-lactone derivatives of indomethacin, and indomethacin in promyelocytic leukemia HL-60 cells. Moreover, a series of biochemical analyses determined that M-IN caused apoptotic bodies, DNA fragmentation, and enhanced PARP and pro-caspase 3 degradation in HL-60 cells. These above results indicate that the photosynthesized product, M-IN, had stronger anti-inflammatory effects in LPS-stimulated RAW 264.7 cells and cytotoxicity effects in HL-60 cells than the parent drug, indomethacin.