LPS In RAW264 Research Articles

Diterpenoid alkaloids from Delphinium forrestii var. viride and their anti-inflammation activity

Six undescribed diterpenoid alkaloids including five C19-diterpenoid alkaloids forrestlines A-E, and one C20-diterpenoid alkaloid forrestline F, together with nine known alkaloids have been isolated from the whole herbs of Delphinium forrestii var. vride. Their structures were elucidated by spectroscopic data, and their inhibitory activities on NO production stimulated by LPS in RAW264.7 macrophage cells were determined. Then, forrestline F, with the strongest inhibitory activity (IC50 of 9.57 ± 1.43 μM), was selected to study its possible anti-inflammatory mechanism. ELISA results showed that forrestline F suppressed inflammatory cytokines, including interleukin-1β (IL-1β), tumor necrosisfactor-α (TNF-α), and interleukin-6 (IL-6). Moreover, forrestline F could down-regulate LPS-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) by western blotting assay. It also inhibited expression of phosphorylation of MAPKs (including p-p38, p-ERK and p-JNK), and NF-κB p65, and decreased ROS accumulation by upregulating the expression of HO-1 expression via nuclear translocation of Nrf2. In conclusion, forrestline F showed anti-inflammatory effect by inhibiting NF-κB/MAPK and Nrf2/HO-1 signaling pathway. Therefore, forrestline F could be a promising molecule for the development of anti-inflammatory agents in the future.

Tulipiferamide A, an Alkamide from Liriodendron tulipifera, Exhibits an Anti-Inflammatory Effect via Targeting IKKβ Phosphorylation.

Three new alkamides, tulipiferamides A-C (1-3, respectively), and 30 known compounds (4-33) were obtained from the roots of Liriodendron tulipifera (Magnoliaceae). Dehydrotemisin (4), an elemane sesquiterpene lactone, was isolated for the first time from nature. The structures were deduced by the interpretation of NMR spectroscopic and MS spectral data. The geometries of the double bonds in tulipiferamides A-C (1-3, respectively) were determined on the basis of 1H-1H coupling constants and 13C chemical shifts. The presence of the alkamide type in this plant is reported for the first time. An analysis of the inflammatory response revealed that seven compounds (1, 4, 7, 9, 14, 23, and 27) suppressed the nitric oxide production induced by LPS in RAW264.7 macrophages. Furthermore, tulipiferamide A (1) inhibits NF-κB activation by selectively targeting IKKβ, an upstream kinase of NF-κB, resulting in the suppression of inflammatory mediators, including iNOS, COX-2, IL-1β, TNFα, and IL-6. Our results provide a rationale for the further development of tulipiferamide A as a selective IKKβ inhibitor to modulate inflammatory diseases.

In vitro and in vivo relaxation and anti-inflammation of natural flavonoids from Elaeagnus pungens leaf via L-type calcium channel and targeting MAPK signal pathway

Ethnopharmacological relevanceIn traditional Chinese medicine (TCM), the leaf of Elaeagnus pungens Thunb. (Family Elaeagnaceae) is a herb documented as an antiasthmatic remedy to treat the severe asthma, bronchitis and other respiratory diseases in the early material medica “Bencao Gangmu” (Ming dynasty, about 442 years ago). Aim of the studyThis work is purposed to investigate the pharmacological effects and mechanism of total flavonoids from Elaeagnus pungens leaves (FLA) on asthma in vivo and vitro.Materials and methods: Female BALB/c mice were sensitized by intraperitoneal injection of OVA with aluminum hydroxide and intranasal challenged with OVA. After treatment with FLA (10, 20 mg/kg p.o.), the behaviors of mice were observed by score evaluation. Enumeration of total cells and OVA-specific IgE assay in the blood were measured as well as enumeration of total cells and cytokines assay in the BALF. Furthermore, histopathological analysis was performed by HE staining. The in vitro relaxing action on muscle force of FLA (0.0316–10.0 mg/ml) was evaluated using isometric tension in tracheal rings, and VDLCC currents were recorded to explore the relaxation mechanism in the isolated tracheal rings and mouse ASM cells, respectively. In vitro anti-inflammatory actions were assessed with LPS-stimulated RAW 264.7 macrophages. The production of inflammatory mediators and MAPK signaling pathway was estimated using ELISA and Western blotting analysis, respectively. ResultsThe high dose of flavones from E. pungens leaf (20 mg/kg) can significantly improve the symptom of asthma breakout and relieve the lung swelling. FLA treatment decreased eosinophils and leukocytes numbers in blood and BLAF with a dosedependent manner. Furthermore, the inhibiting effect of FLA on the level of Ig E and inflammatory-related cytokines including TNF-α, IL-5 showed dose-independent. FLA relaxed high K + -induced contraction in a dose-dependent manner. The maximal relaxation produced by FLA was 99.7% (IC 50 = 0.46 mg/ml). The whole-cell VDLCC currents were abolished by FLA (3.16 mg/ml) and FLA significantly decreased the maximal amplitude of VDLCCs. No cytotoxic effect of FLA was observed in RAW264.7 cells under the tested concentrations (1–300 μg/mL). The increased IL-6 and NO by the stimulation of LPS in RAW264.7 cells were significantly inhibited by FLA in the dosedependent manner. Treatment with LPS in the presence of FLA, LPS-induced phosphorylation of ERK1/2 and JNK was inhibited in the macrophages. Conclusion: FLA from Elaeagnus pungens leaf can alleviate the inflammation symptom via reducing the eosinophils and leukocytes numbers as well as the production of pro-inflammatory cytokines. This anti-inflammatory effect is related to the modulation of the MAPK signaling pathway. FLA can relax the precontracted TRs by blocking the VDLCCs, which interrupts extracellular Ca 2+ influx and inhibit the rise of [Ca 2+ ]i. It strongly suggests that these flavonoids components are the substances basis of Elaeagnus pungens leaves for allergic action, bronchospasm and inflammation in asthma.

Sec-O-glucosylhamaudol suppressed inflammatory reaction induced by LPS in RAW264.7 cells through inhibition of NF-κB and MAPKs signaling.

As a major bioactive compound from the Saposhnikovia divaricata (Turcz.) Schischk, sec-O-glucosylhamaudol (SOG), has been reported to have anti-nociceptive activity and high 5-lipoxygenase (5-LOX) activity. Nevertheless, the mechanism of the potential anti-inflammatory effects of SOG is unclear. The anti-inflammatory impacts of SOG in RAW 264.7 cell lines stimulated by LPS were explored in the present study. It was found that SOG dose-dependently reduced the emergence of inflammation cytokines, such as IL-6 and TNF-α in Raw264.7 murine macrophages stimulated by LPS. Real-time PCR assay demonstrated the SOG dose-dependently inhibited transcription of these cytokines as well. In addition, it was also found that NF-κB activation and MAPKs phosphorylation including p38, JNK and ERK1/2 induced by LPS were suppressed by SOG. Due to its anti-inflammatory activity, our results suggest that SOG might have therapeutic effects on inflammatory disease, such as acute lung injury or rheumatoid arthritis.

Tetrastigma hemsleyanum tubers polysaccharide ameliorates LPS-induced inflammation in macrophages and Caenorhabditis elegans

Tetrastigma hemsleyanum Diels et Gilg, a rare herbal plant native to China, possesses high medicinal value. More researches have focused on the flavones in the tubers of Tetrastigma hemsleyanum whereas polysaccharides attract few attentions. In this study, a purified polysaccharide (TTP-1) with the average molecular weights of 478.33 kDa was extracted from Tetrastigma hemsleyanum and conducted multiple analysis to identify its structural information. In vitro, TTP-1 could suppress the inflammation, cytotoxicity, genotoxicity, mitochondrial dysfunction and oxidative damage induced by LPS in RAW264.7 cells. Meanwhile, TTP-1 could attenuate inflammation via COX-2, iNOS, MAPKs pathways and ameliorate oxidative damage through Nrf2-Keap1, Sirt1-FoxO1 pathways in RAW264.7 cells. In vivo, the inflammation induced by LPS triggered the suppression of survival ratio and growing development, as well as the restrain of athletic ability in Caenorhabditis elegans. The treatment of TTP-1 reversed the situation induced by LPS. In a word, TTP-1 could ameliorate LPS-induced inflammation both in vitro and in vivo.

Siglec-1 negatively regulates TLR4-mediated inflammatory response by uniquely controlling Src phosphorylation at Ser17

Abstract Introduction Sepsis are mainly caused by Gram-negative bacteria, and Toll-like receptor 4 (TLR4) sensing of LPS plays pivotal roles in the pathogenesis of sepsis. Identifying key regulators that govern LPS-induced TLR4 activation of inflammatory pathway will facilitate the understanding of the regulatory mechanisms of inflammation and provide potential targets for sepsis therapy. Results LPS induced robust Ser17-phosphorylation (dispensable for kinase activity) of Src kinase in RAW 264.7 and THP1 cells. Silencing of Siglec-1, a membrane-bound lectin, dramatically abrogated Src phosphorylation at Ser17 induced by LPS in RAW 264.7 cells, whereas did not affect Src phosphorylation at Tyr416 and Tyr527, two critical sites that determine Src kinase activity. Furthermore, the phosphorylated levels of P38, NF-kB, JNK, ERK, Syk and PKC triggered by LPS remained comparable between scramble shRNA-transduced and Siglec-1 shRNA-transduced Raw 264.7 cells. Strikingly, LPS failed to induce Ser17-phosphorylation of Src kinase in Siglec-1−/− BMDMs compared with WT BMDMs; no defects on Tyr416 and Tyr527 sites were observed. RAW 264.7 cells with Src depletion showed enhanced cytokine production by LPS treatment. Reconstitution of WT Src and Src-S17D mutant, but not Src-S17A mutant, rescued the elevated inflammatory cytokines in Src knockdown cells with LPS challenge. Co-IP identified a novel interaction between Siglec-1 and Src, and over-expression of DAP12 enhanced the interaction in 293T cells. Conclusion Siglec-1 negatively regulates TLR4-mediated inflammatory response by uniquely controlling Src phosphorylation at Ser17. Pharmacological targeting Ser17 in Src may provide promising strategies to fend off sepsis.

Lanceoleins A–G, hydroxychalcones, from the flowers of Coreopsis lanceolata and their chemopreventive effects against human colon cancer cells

Seven new chalcones, lanceolein A–G (compounds 5 and 7–12), as well as five known chalcones (1–4 and 6), were isolated from the methanolic extract of Coreopsis lanceolata flowers. The chemical structures of 5 and 7–12 were determined on the basis of spectroscopic data interpretation. All compounds inhibited the production of nitrite oxide (NO) induced by LPS in RAW264.7 macrophage cells. Also, compounds 1–6 showed moderated cytotoxicity against human colon cancer cell lines, while compounds 7–12 hardly showed the cytotoxicity. Especially, compounds 2, 5, and 6 exhibited a little higher cytotoxicity on HCT15 cells, with IC50 values of 43.7 ± 2.17 μM, 35.6 ± 0.24 μM, and 47.9 ± 1.18 μM, respectively. In the Tali assay, compounds 2 and 5 increased the numeral of apoptotic cells. These compounds also significantly promoted the expression of apoptotic proteins including PARP and caspase-3.

Anti-Inflammatory Effects of Canavalia gladiata in Macrophage Cells and DSS-Induced Colitis Mouse Model.

Canavalia gladiata, known as sword bean, has been used as a Chinese traditional medicine for anti-inflammatory effects. However, the action mechanisms of sword bean have not yet been clearly defined. In the present study, the whole parts of a ripened sword bean (RSB) and the green sword bean (GSB) containing bean pod were extracted with ethanol by reflux extraction. The two crude extracts (RSBE and GSBE) from RSB and GSB were validated by a liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis of gallic acid as a reference chemical. The anti-inflammatory effects of two sword bean extracts were extensively investigated using LPS-stimulated macrophage cells. First, RSBE and GSBE significantly inhibited the production of pro-inflammatory mediators, such as tumor necrosis factor- (TNF-), interleukin-6 (IL-6), prostaglandinE2 (PGE2), and nitric oxide (NO) in LPS-induced RAW264.7 cells. RSBE and GSBE showed no cytotoxicity to RAW264.7 cells and mouse peritoneal macrophage cells. In addition, the overexpression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) induced by LPS in RAW264.7 cells was significantly decreased by RSBE and GSBE. Western blotting and immunostaining analysis showed that RSBE and GSBE inhibited the nuclear translocation of NF-B subunits, which correlated with the inhibitory effects on inhibitor kappa B (IB) degradation. In dextran sulfated sodium (DSS)-induced colitis mice model, RSBE restored body weight, colon length, and the levels of pro-inflammatory cytokines, such as TNF-, IL-6, interleukin-1 (IL-1), and interferon- (IFN-). In addition, RSBE significantly suppressed the expression of COX-2, iNOS, and NF-B.

β-carotene attenuates lipopolysaccharide-induced inflammation via inhibition of the NF-κB, JAK2/STAT3 and JNK/p38 MAPK signaling pathways in macrophages.

β-carotene is one of the most abundant carotenoids, has potential anti-inflammatory effect, it has been reported that β-carotene could suppress LPS-induced inflammatory responses by inhibiting nuclear factor kappa B (NF-κB) translocation, but the more detailed molecular mechanisms underlying the anti-inflammatory action of β-carotene remain to be fully understood. In this study, we investigated the influence of β-carotene on the activation of JAK2/STAT3, MAPK, and NF-κB signaling pathway induced by LPS in RAW264.7 cells and peritoneal macrophages. Cells were treated with different concentrations of β-carotene for 3hr after LPS treatment for 24hr. The mRNA expression and the release of IL-1β, IL-6, and TNF-α were evaluated by RT-PCR and ELISA, and the level of signaling proteins of JAK2/STAT3, MAPK, and NF-κB signaling pathway were detected by Western blot. The results showed that β-carotene significantly suppressed (p<0.05) LPS-induced release of IL-1β, IL-6, and TNF-α and their mRNA expression. LPS-induced JAK2/STAT3, IκB/NF-κB p65, JNK/p38 MAPK signal activation were significantly attenuated (p<0.05) by β-carotene in a dose-dependent manner. In conclusion, β-carotene could attenuate LPS-induced inflammation via inhibition of the NF-κB, JAK2/STAT3, and JNK/p38 MAPK signaling pathways in macrophages.

Tuberatolide B isolated from Sargassum macrocarpum inhibited LPS-stimulated inflammatory response via MAPKs and NF-κB signaling pathway in RAW264.7 cells and zebrafish model

Brown algae are known to contain rich anti-inflammatory compounds. The objective of this study was to identify anti-inflammatory compound in the Sargassum macrocarpum and anti-inflammatory effect of tuberatolide B (TTB) was investigated. TTB significantly suppressed the production of NO and pro-inflammatory cytokines such as interleukin (IL)-6 and IL-1β. In addition, the mechanism involved in the inhibition of the inflammatory effect of LPS by TTB was investigated. The results showed that TTB aforementioned this effect through NF-κB and MAPKs phosphorylation pathways. We also assessed the anti-inflammatory activity of TTB using in vivo zebrafish model. In LPS-stimulated zebrafish, TTB enhanced survival and significantly inhibited the NO production and mRNA expression of inducible NO synthase. Therefore, anti-inflammatory activity of TTB against LPS in RAW264.7 cells and the zebrafish model was determined. These finding suggest that TTB may be used as functional anti-inflammatory foods and nutraceuticals.

Sirt1 S-nitrosylation induces acetylation of HMGB1 in LPS-activated RAW264.7 cells and endotoxemic mice

Excessive inflammation plays a detrimental role in endotoxemia. A recent study indicated that alarmins such as high mobility group box 1 (HMGB1) have drawn attention as therapeutic targets of sepsis. Post-translational modification (i.e., acetylation of lysine residues) of HMGB1 leads to the release of HMGB1 into the cellular space, operating as a warning signal that induces inflammation. Sirtuin 1 (SIRT1) has been shown to negatively regulate HMGB1 hyperacetylation and its extracellular release in sepsis. Therefore, we hypothesized that the S-nitrosylation (SNO) of SIRT1 may disrupt the ability of SIRT1 to negatively regulate the hyperacetylation of HMGB1. As long as the S-nitrosylation of SIRT1 occurs during septic conditions, it may worsen the situation. We found that the activity of SIRT1 decreased as the SNO-SIRT1 levels increased, resulting in HMGB1 release by LPS in RAW264.7 cells. Both the iNOS inhibitor (1400 W) and silencing iNOS significantly inhibited SNO-SIRT1, allowing increases in SIRT1 activity that decreased the HMGB1 release by LPS. SNAP, a NO donor, significantly increased both SNO-SIRT1 levels and the HMGB1 release that was accompanied by decreased sirt1 activity. However, sirtinol, a Sirt1 inhibitor, by itself decreased Sirt1 activity compared to that of the control, so that it did not affect already increased SNO-SIRT levels by SNAP. Most importantly, in lung tissues of LPS-endotoxic mice, significantly increased levels of SNO-SIRT were found, which was inhibited by 1400 W treatment. Plasma nitrite and HMGB1 levels were significantly higher than those in the sham controls, and the elevated levels were significantly lowered in the presence of 1400 W. We concluded that the S-nitrosylation of Sirt1 under endotoxic conditions may uninhibit the acetylation of HMGB1 and its extracellular release.

Heterophyllin B Ameliorates Lipopolysaccharide-Induced Inflammation and Oxidative Stress in RAW 264.7 Macrophages by Suppressing the PI3K/Akt Pathways.

Heterophyllin B (HB), an active cyclic peptide, is a compound existing in the ethyl acetate extract of Pseudostellaria heterophylla (Miq.) Pax and exhibited the activity of inhibiting the production of NO and cytokines, such as IL-1β and IL-6, in LPS-stimulated RAW 264.7 macrophages. In addition, HB suppressed the production of ROS and the apoptosis induced by LPS in RAW 264.7 macrophages. The underlying mechanism was investigated in the LPS-induced RAW 264.7 cells. The results showed that HB decreased the level of IL-1β and IL-6 expression by qRT-PCR analysis. HB up-regulated the relative ratio of p-AKT/AKT and p-PI3K/PI3K as indicated by western blot analysis. In summary, HB inhibited the LPS-induced inflammation and apoptosis through the PI3K/Akt signaling pathways and represented a potential therapeutic target for treatment of inflammatory diseases.

Design, synthesis and ability of non-gold complexed substituted purine derivatives to inhibit LPS-induced inflammatory response

In order to study the anti-inflammatory activity of novel 6-substituted and 6,9-disubstituted purine derivatives, 20 compounds, L1–10 and W1–10, derived from purine and lacking a gold complex were designed, synthesized and their anti-inflammatory activity was screened. LPS-induced TNF-α, IL-1β, IL-6, PGE2, NO, COX-2 and iNOS mRNA were evaluated, and western blot and NF-κB p65 translocation assay were performed in RAW 264.7 macrophages. Furthermore, carrageenan-induced hind paw edema experiments were performed in mice. Compound L1, L4, W2, and W4 markedly exerted a dose-dependent inhibition of TNF-α, IL-1β, IL-6 and PGE2 release induced by LPS in RAW 264.7 macrophages. Moreover, these compounds strongly inhibited LPS-induced NO, COX-2 and iNOS mRNA in the same cells. Anti-inflammatory activity tests in vivo showed that L1 and L4 were more effective than Au(L3)(PPh3), a known anti-inflammatory agent, at 2–5 h, and W4 was the most effective at 3–5 h after dosing. Thus, W2, W4, and L1, L4, could effectively inhibit LPS-induced inflammatory response in vitro and in vivo suggesting a promising role as anti-inflammatory agents.

Isoliquiritigenin Activates Nuclear Factor Erythroid-2 Related Factor 2 to Suppress the NOD-Like Receptor Protein 3 Inflammasome and Inhibits the NF-κB Pathway in Macrophages and in Acute Lung Injury

Among the cellular response mechanisms, the nuclear factor erythroid-2 related factor 2 (Nrf2) pathway is considered a survival pathway that alleviates oxidative injury, while both the NOD-like receptor protein 3 (NLRP3) and NF-κB pathways are pro-inflammatory pathways that cause damage to cells. These pathways are implicated in the development and resolution of acute lung injury (ALI). Isoliquiritigenin (ISL), a flavonoid from the liquorice compound, is suggested to be a regulator of the above pathways, but the mechanisms of how the NLRP3/NF-κB pathway interacts with Nrf2 and its protective effects in ALI remain unknown. In the present study, ISL inhibited reactive oxygen species (ROS) generation and cytotoxicity induced by t-BHP and pro-inflammatory enzymes production induced by LPS in RAW 264.7 cells. Such cytoprotective effects coincided with the induction of AMP-activated protein kinase (AMPK)/Nrf2/antioxidant response element (ARE) signaling and the suppression of the NLRP3 and NF-κB pathways. Consistent with these findings, ISL treatment significantly alleviated lung injury in LPS-induced ALI mice, which was reflected by reductions in histopathological changes, pulmonary edema, and protein leakage. At the same time, the increased levels of inflammatory cell exudation and pro-inflammatory mediators, the enhanced production of ROS, myeloperoxidase, and malondialdehyde, and the depleted expression of GSH and superoxide dismutase induced by LPS were ameliorated by ISL. Furthermore, ISL notably activated AMPK/Nrf2/ARE signaling and inhibited LPS-induced NLRP3 and NF-κB activation in the lung. Moreover, although inhibition of the LPS-induced histopathological changes and ROS production were attenuated in Nrf2-deficient mice, the repression of the NLRP3 and NF-κB pathways by ISL was Nrf2-dependent and Nrf2-independent, respectively. In conclusion, our results are the first to highlight the beneficial role and relevant mechanisms of ISL in LPS-induced ALI and provide novel insight into its application.

Atorvastatin ameliorates LPS-induced inflammatory response by autophagy via AKT/mTOR signaling pathway.

Macrophages play crucial roles in immune response and atherosclerosis-related cardiovascular disease. Recent evidence of macrophage autophagy has demonstrated a novel pathway through which contributes to vascular inflammation. The aim of this study was to elucidate the role of autophagy in the inhibition of inflammatory response in macrophages by atorvastatin. We found that atorvastatin promoted autophagy flow determined by up-regulating the expression of autophagy-related protein microtubule-associated protein light chain (LC3B), inducing the formation of autophagosomes and down-regulating the expression of SQSTM1/P62, which is consumed during autophagy. Atorvastatin also inhibited the expression of inflammatory factors IL-1β and TNFα induced by LPS in RAW264.7 cells. Furthermore, pretreatment with an autophagy inhibitor 3MA or LY294002 attenuated the suppressive effect of atorvastatin on LPS-induced IL-1β and TNFα expression. Additionally, knockdown autophagy-related gene 5(Atg5) with a special siRNA also prevented the role of atorvastatin in decreasing IL-1β and TNFα release induced by LPS. Finally, we detected that AKT/mTOR/P70S6K signaling pathway was involved in atorvastatin-induced autophagy in macrophages. These data suggest that atorvastatin attenuates LPS-induced inflammatory factors secretion, at least in part, through enhancing autophagy by AKT/mTOR signaling pathway. Our findings provide a novel evidence that statins exert anti-inflammatory effect in atherosclerosis by autophagy activation.

Osthole pretreatment alleviates TNBS-induced colitis in mice via both cAMP/PKA-dependent and independent pathways.

Osthole, a natural coumarin found in traditional Chinese medicinal plants, has shown multiple biological activities. In the present study, we investigated the preventive effects of osthole on inflammatory bowel disease (IBD). Colitis was induced in mice by infusing TNBS into the colonic lumen. Before TNBS treatment, the mice received osthole (100 mg·kg-1·d-1, ip) for 3 d. Pretreatment with osthole significantly ameliorated the clinical scores, colon length shortening, colonic histopathological changes and the expression of inflammatory mediators in TNBS-induced colitis. Pretreatment with osthole elevated serum cAMP levels; but treatment with the PKA inhibitor H89 (10 mg·kg-1·d-1, ip) did not abolish the beneficial effects of osthole on TNBS-induced colitis. In mouse peritoneal macrophages, pretreatment with osthole (50 μmol/L) significantly attenuated the LPS-induced elevation of cytokines at the mRNA level; inhibition of PKA completely reversed the inhibitory effects of osthole on IL-1β, IL-6, COX2, and MCP-1 but not on TNFα. In Raw264.7 cells, the p38 inhibitor SB203580 markedly suppressed LPS-induced upregulation of the cytokines, whereas the PKA inhibitors H89 or KT5720 did not abolish the inhibitory effects of SB203580. Moreover, in LPS-stimulated mouse peritoneal macrophages, SB203580 strongly inhibited the restored expression of IL-1β, IL-6, COX2, and MCP-1, which was achieved by abolishing the suppressive effects of osthole with the PKA inhibitors. Western blot analysis showed that osthole significantly suppressed the phosphorylation of p38, which was induced by TNBS in mice or by LPS in Raw264.7 cells. Inhibition of PKA partially reversed the suppressive effects of osthole on p38 phosphorylation in LPS-stimulated cells. Collectively, our results suggest that osthole is effective in the prevention of TNBS-induced colitis by reducing the expression of inflammatory mediators and attenuating p38 phosphorylation via both cAMP/PKA-dependent and independent pathways, among which the cAMP/PKA-independent pathway plays a major role.

Two new secolignans with in vitro anti-inflammatory activities from Urtica fissa rhizomes.

Two new secolignans, urticin A (1) and urticin B (2), were isolated from the ethanol extract of Urtica fissa rhizomes. Their structures were elucidated on the basis of extensive spectroscopic evidence (UV, IR, HR-ESI-MS, and NMR). Urticin A and urticin B possessed in vitro anti-inflammatory activities, which significantly inhibited the TNF-α and NO release induced by LPS in RAW 264.7 cells.

Inhibition of LPS-stimulated ROS production by fluorinated and hydroxylated chalcones in RAW 264.7 macrophages with structure-activity relationship study

Based on the importance of the previous fluorinated and/or hydroxylated chalcones studies, thirty-six compounds were designed as phenyl or hydroxyphenyl bearing fluoro, trifluoromethyl or trifluoromethoxy phenyl propenones and synthesized by applying modified Claisen-Schmidt condensation reaction as a single step. Inhibitory effects of the synthesized compounds on ROS production stimulated by LPS in RAW 264.7 macrophage were evaluated. Structure-activity relationship (SAR) study revealed that the compounds possessing para-hydroxyphenyl group combined with meta-fluoro or meta-trifluoromethyl phenyl group, and meta/para-hydroxyphenyl group combined with ortho-trifluoromethoxyphenyl group have an essential role in inhibiting the LPS-stimulated ROS production in RAW 264.7 macrophages. The most significant inhibitory effect on LPS-stimulated ROS production in RAW 264.7 macrophages was observed in compound 30 that possessed para-hydroxyphenyl group along with ortho-trifluoromethoxyphenyl group.

Morita-Baylis-Hillman Adducts Display Anti-Inflammatory Effects by Modulating Inflammatory Mediator Expression in RAW264.7 Cells.

Inflammatory response plays an important role not only in the normal physiology but also in pathologies such as cancers. The Morita-Baylis-Hillman adducts (MBHA) are a novel group of synthetic molecules that have demonstrated many biological activities against some parasitic cells such as Plasmodium falciparum, Leishmania amazonensis, and Leishmania chagasi, and antimitotic activity against sea urchin embryonic cells was also related. However, little is known about the mechanisms induced by MBHA in inflammatory process and its relation with anticancer activity. The present work investigated the cytotoxicity of three MBHA derivatives (A2CN, A3CN, and A4CN), on human colorectal adenocarcinoma, HT-29 cells, and their anti-inflammatory activities were examined in lipopolysaccharide- (LPS-) stimulated RAW264.7 macrophage cells, being these derivatives potentially cytotoxic to HT-29 cells. Coincubation with A2CN, A3CN, or A4CN and LPS in RAW264.7 cells inhibited NO production, as well as the production of reactive oxygen species (ROS) was also repressed. The mRNA expressions of IL-1β and IL-6 were significantly downregulated by such MBHA compounds in RAW264.7 cells, but only A2CN was able to inhibit the COX-2 gene expression. We also showed that MBHA compounds decreased almost to zero the production of IL-1β and IL-6. These findings display that such MBHA compounds exhibit anticancer and anti-inflammatory activities.

Salidroside attenuates inflammatory response via suppressing JAK2-STAT3 pathway activation and preventing STAT3 transfer into nucleus

Salidroside (SAL) is an active ingredient isolated from the Rhodiola rosea, has potent anti-inflammatory effect, but the mechanism is still elusive. The purpose of this study is to verify the effects of SAL on LPS-induced inflammatory response and investigate the possible underlying molecular mechanism. RAW264.7 cells were pre-incubated with SAL for 2h, then stimulated with or without LPS for another 16h. The levels of TNF-α, MCP-1, IL-6, and PGE2 were detected by ELISA, and the production of NO was determined by nitrite analysis. The expression levels of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected by Western blotting. In RAW264.7 cells and murine peritoneal macrophages, the activation of signal molecules was also measured by Western blot. The nuclear translocation of STAT3 was determined by Laser confocal and nucleocytoplasmic separation experiments. Our results showed that SAL attenuated the productions of TNF-α, IL-6, MCP-1, PGE2 and NO dose dependently. SAL also suppressed LPS-induced expressions of iNOS and COX-2 significantly. Further studies revealed that SAL down-regulated the phosphorylation of JAK2-STAT3 signaling pathway and reduced the nuclear translocation of STAT3 induced by LPS in RAW264.7 cells and primary peritoneal macrophages. In addition, consistent with the results in vitro, in the model of mice acute lung injury (ALI) induced by LPS, SAL reduced the infiltration of inflammatory cells and decreased the levels of serum TNF-α and IL-6 obviously. Taken together, these data indicated that SAL exerted anti-inflammatory action via down-regulating LPS-induced activation of JAK2-STAT3 pathway and suppressing STAT3 transfer into the nucleus at least in part.