Anti-inflammatory effects of sophocarpine in LPS-induced RAW 264.7 cells via NF-κB and MAPKs signaling pathways

Sophocarpine, a tetracyclic quinolizidine alkaloid, is one of the most abundant active ingredients in Sophora alopecuroides L. Our previous studies have showed that sophocarpine exerts anti-inflammatory activity in animal models. In the present study, anti-inflammatory mechanisms of sophocarpine were investigated in lipopolysaccharide (LPS)-induced responses in RAW 264.7 cells. Furthermore, the cytotoxicity of sophocarpine was tested. The results indicated that sophocarpine could increase the LDH level and inhibit cell viability up to 800μg/ml, and which was far higher than that of the plasma concentration of sophocarpine in clinical effective dosage. The results also demonstrated that sophocarpine (50 and 100μg/ml) suppressed LPS-stimulated NO production and pro-inflammatory cytokines secretion, including tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). These were associated with the decrease of the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Furthermore, sophocarpine inhibited LPS-mediated nuclear factor-κB (NF-κB) activation via the prevention of inhibitor κB (IκB) phosphorylation. Sophocarpine had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2), whereas it attenuated the phosphorylation of p38 mitogen-activated protein (MAP) kinase and c-Jun NH(2)-terminal kinase (JNK). Our data suggested that sophocarpine exerted anti-inflammatory activity in vitro, and it might attribute to the inhibition of iNOS and COX-2 expressions via down-regulation of the JNK and p38 MAP kinase signal pathways and inhibition of NF-κB activation.     

Polysaccharide from Ganoderma atrum induces tumor necrosis factor-α secretion via phosphoinositide 3-kinase/Akt,mitogen-activated protein kinase and nuclear factor-κB signaling pathways in RAW264.7 cells

The aim of this study was to investigate the signaling pathways involved in the macrophage activation by Ganoderma atrum polysaccharide (PSG-1) and elucidate the molecular mechanism of PSG-1-induced signal transduction in the regulation of tumor necrosis factor (TNF)-α secretion. Our results illustrated that the mitogen-activated protein kinase (MAPK) pathways were simultaneously activated and involved in PSG-1-induced TNF-α secretion in RAW264.7 cells. Moreover, our results also demonstrated that the phosphoinositide 3-kinase (PI3K)/Akt pathway was stimulated and played an important role in the PSG-1 induced TNF-α secretion. Additionally, the present study showed that nuclear factor (NF)-κB activation by PSG-1 was triggered by PI3K/Akt/MAPK pathway and NF-κB participated in PSG-1 stimulated TNF-α production. In conclusion, we have elucidated the mechanism of PSG-1-mediated immunomodulatory activities, and provide a theoretical basis for the potential of PSG-1 as a novel immunomodulating agent.     

Phytoglycoprotein inhibits interleukin-1β and interleukin-6 via p38 mitogen-activated protein kinase in lipopolysaccharide-stimulated RAW 264.7 cells

This study was carried out to investigate the anti-inflammatory effects of 30-kDa glycoprotein isolated from Dioscorea batatas Decne (DBD glycoprotein), which consists of carbohydrate content (61%) and protein content (39%) on lipopolysaccharide (LPS, 2 μg/ml)-stimulated RAW 264.7 cells. We found that DBD glycoprotein (200 μg/ml) has an inhibitory effect on the production of intracellular hydrogen peroxide (H₂O₂), on the phosphorylation of p38 mitogen-activated protein (MAP) kinase, on the DNA binding activity of activator protein-1 (AP-1), and on c-Jun and c-Fos protein expression, respectively. In addition, DBD glycoprotein treatment markedly suppressed the interleukin (IL)-1β, IL-6, and inducible nitric oxide synthase (iNOS) expression and the production of nitric oxide (NO) in LPS-stimulated RAW 264.7 cells. Interestingly, IL-1β, IL-6, and iNOS expression was significantly attenuated by treatment with protein kinase C (PKC) inhibitor (staurosporine) as well as p38 MAP kinase inhibitor (SKF86002) in LPS-stimulated RAW 264.7 cells. On the basis of these results, we assume that DBD glycoprotein has anti-inflammatory potential, which can modulate proinflammatory signal transduction in LPS-stimulated RAW 264.7 cells.     

Anti-inflammatory effects of diaporisoindole B in LPS-stimulated RAW 264.7 macrophage cells via MyD88 activated NF-κB and MAPKs pathways

Diaporisoindole B (DPB), an isoprenylisoindole alkaloid isolated from the mangrove endophytic fungus Diaporthe sp. SYSU-HQ3, has been proved to inhibit the production of nitric oxide (NO) in lipopolysaccharide (LPS)-challenged RAW 264.7 mouse macrophages, showing potent anti-inflammatory effects. In this study, we further investigated the anti-inflammatory effects of DPB and explored the possible mechanisms in LPS-challenged RAW 264.7 mouse macrophages. The results showed that DPB (3.125, 6.2, 12.5 and 25 μM) could significantly reduce LPS-induced levels of PGE2, and inhibit the expressions of iNOS and COX-2 in a dose-dependent manner. In addition, DPB also inhibited LPS-induced production of inflammatory cytokines, including TNF-α, IL-1β, IL-6. Moreover, we further investigated signal transduction mechanisms by which DPB exerted anti-inflammatory effects. DPB could affect LPS-mediated nuclear factor kappa B (NF-κB) signaling pathway activation via down-regulating the upstream myeloid differentiation protein 88 (MyD88) at the protein level. Additionally, DPB also strongly inhibited the phosphorylation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK) and p38. Therefore, DPB might exert anti-inflammatory effects by suppressing NF-κB activation and MAPKs pathways via down-regulating MyD88 in RAW 264.7 cells.     

Neurotropin inhibits neuroinflammation via suppressing NF-κB and MAPKs signaling pathways in lipopolysaccharide-stimulated BV2 cells

Neurotropin (NTP) is a widely used drug in China and Japan mainly for the treatment of chronic pain and peripheral inflammation. Nevertheless, the effects of NTP on neuroinflammation have not been explored. In this study, we investigated the anti-inflammatory effects of NTP in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells and its underlying mechanisms. BV-2 cells were pretreated with NTP for 12 h before exposure to LPS. The expression of pro-inflammatory cytokines (TNF-α and IL-6) were detected by RT-PCR and EILSA at mRNA and protein levels, respectively. Western blotting was conducted to measure the protein levels of major genes in MAPKs and NF-κB signaling pathways. Results demonstrated that NTP could attenuate the production of pro-inflammatory cytokines. Furthermore, NTP inhibited the activation of NF-κB signaling by decreasing the translocation of NF-κB p65 to the nucleus and suppressed the MAPKs signaling pathway via inhibition of the phosphorylation of p38, ERK and JNK. Taken together, these findings suggest that neurotropin exerts anti-inflammatory effects by suppressing the production of pro-inflammatory mediators via inhibition of NF-κB and MAPKs signaling pathways in LPS-stimulated BV-2 cells.     

Shikonofuran E plays an anti-inflammatory role by down-regulating MAPK and NF-κB signaling pathways in lipopolysaccharide-stimulated RAW264.7 macrophages

Journal of Natural Medicines - The anti-inflammatory effects of shikonofuran E from Onosma paniculatum on RAW 264.7 murine macrophage cells induced by lipopolysaccharide (LPS) were first time...     

Involvement of phosphatidylcholine-phospholipase C and protein kinase C in peptidoglycan-induced nuclear factor-κB activation and cyclooxygenase-2 expression in RAW 264.7 macrophages

In this study, we examined the role of phosphatidylcholine–phospholipase C (PC–PLC) and protein kinase C (PKC) in peptidoglycan (PGN)-induced nuclear factor-κB (NF-κB) activation and cyclooxygenase-2 (COX-2) expression in RAW 264.7 macrophages. PGN-induced COX-2 expression was attenuated by a PC–PLC inhibitor (D609) and by PKC inhibitors (Go 6976 and Ro 31-8220), but not by a phosphatidylinositol–PLC (PI–PLC) inhibitor (U-73122). PGN caused an increase in PKC activity, and this effect was inhibited by D609, Go 6976, and Ro 31-8220, but not by U-73122. Furthermore, the PGN-mediated increases in κB-luciferase activity were also inhibited by D609 and Ro 31-8220. Our data demonstrate that PGN activates PC–PLC which induces PKC activation; this in turn initiates NF-κB activation, and ultimately induces COX-2 expression in RAW 264.7 macrophages.     

Esculentoside B inhibits inflammatory response through JNK and downstream NF-κB signaling pathway in LPS-triggered murine macrophage RAW 264.7 cells

Natural compound esculentoside B (EsB), (2S,4aR,6aR,6aS,6bR,8aR,9R,10R,11S,12aR,14bS)-11-hydroxy-9-(hydroxymethyl)-2 methoxycarbonyl-2,6a,6b,9,12a-pentamethyl-10-[(2S,3R,4S,5R)-3,4,5-trihydroxyoxan-2-yl]oxy-1,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydropicene-4a-carboxylic acid with molecular weight of 664.833, isolated from roots of Phytolacca acinosa Roxb has been widely used as a constituent of traditional Chinese medicine (TCM). However, the anti-inflammatory capacity of EsB has not been reported yet. Therefore, the objective of this study was to investigate anti-inflammatory activities of EsB in LPS-treated macrophage RAW 264.7 cells. EsB could inhibit nitric oxide (NO) production. EsB also suppressed gene and protein expression levels of inducible isoform of NO synthase (NOS) and cyclooxygenase-2 in a dose-dependent manner. In addition, EsB decreased gene expression and protein secretion levels of pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6. EsB remarkably suppressed nuclear translocation of nuclear factor kappa-B (NF-κB) from cytosolic space. Phosphorylation of IκB was also inhibited by EsB. Moreover, EsB specifically down-regulated phospho-c-Jun N-terminal kinase (p-JNK), but not p-p38 or phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2). Taken together, these results suggest that EsB has inhibitory effect on inflammatory response by inactivating NF-κB and p-JNK. It could be used as a new modulatory drug for effective treatment of inflammation-related diseases.     

Anti-inflammatory effect of gamma-irradiated genistein through inhibition of NF-κB and MAPK signaling pathway in lipopolysaccharide-induced macrophages

Genistein was irradiated with γ-irradiation at doses of 0, 10, 30, 50, 100, and 150 kGy. We observed that the decrease in the genistein peak after gamma irradiation was concomitant with the appearance of several new peaks. 150 kGy gamma-irradiated genistein did not exert cytotoxicity in macrophages, and inhibited inducible nitric oxide synthase-mediated nitric oxide production and pro-inflammatory cytokines level, such as tumor necrosis factor-α, interleukin-6 and interleukin-1β, in lipopolysaccharide (LPS)-induced macrophages. The treatment of LPS-stimulated macrophages with 150 kGy gamma-irradiated genistein resulted in a significant decrease in cyclooxygenase-2 levels, as well as the expression of cell surface molecules, such as CD80 and CD86. Furthermore, we also found that the anti-inflammatory action of 150 kGy gamma-irradiated genistein occurred through an inhibition of mitogen-activated protein kinases (extracellular signal-regulated kinase 1/2, p38 and c-Jun N-terminal kinase) and nuclear factor-κB signaling pathways based on a toll-like receptor 4 in macrophages, which may be speculated that several radiolysis products of genistein transformed by gamma-irradiation induce the inhibition of pro-inflammatory mediators. From these findings, it seems likely that gamma-irradiated genistein could play a potent role in the treatment of inflammatory disease as a value-added product in the medical industry.     

Saucerneol G, a new lignan, from Saururus chinensis inhibits matrix metalloproteinase-9 induction via a nuclear factor κB and mitogen activated protein kinases in lipopolysaccharide-stimulated RAW264.7 cells

This study was conducted to demonstrate the inhibitory effect of saucerneol G (SG), a new lignan, isolated from the aerial part of Saururus chinensis (Saururaceae) on lipopolysaccharide (LPS)-stimulated matrix metalloproteinase-9 (MMP)-9 inductions in RAW 264.7 cells. Aimed at evaluating the mechanism of action by which SG inhibits the LPS-mediated induction of MMP-9, the effects of SG on nuclear factor-κB (NF-κB) DNA binding activity, NF-κB-dependent reporter gene activity, inhibitory factor-κB (IκB) phosphorylation, degradation and p65 nuclear translocation were assessed. SG profoundly suppressed the DNA binding activity and the reporter gene activity as well as translocation of NF-κB p65 subunit. Furthermore, SG also dose dependently inhibited LPS-stimulated activation of mitogen-activated protein kinases (MAPKs). These findings suggest that SG may inhibit LPS-stimulated MMP-9 induction by blocking NF-κB and MAPKs activation.     

Neocryptotanshinone inhibits lipopolysaccharide-induced inflammation in RAW264.7 macrophages by suppression of NF-κB and iNOS signaling pathways

Neocryptotanshinone (NCTS) is a natural product isolated from traditional Chinese herb Salvia miltiorrhiza Bunge. In this study, we investigated its anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated mouse macrophage (RAW264.7) cells. MTT results showed that NCTS partly reversed LPS-induced cytotoxicity. Real-time PCR results showed that NCTS suppressed LPS-induced mRNA expression of inflammatory cytokines, including tumor necrosis factor α (TNFα), interleukin-6 (IL-6) and interleukin-1β (IL-1β). Moreover, NCTS could decrease LPS-induced nitric oxide (NO) production. Western blotting results showed that NCTS could down-regulate LPS-induced expression of inducible nitric oxide synthase (iNOS), p-IκBα, p-IKKβ and p-NF-κB p65 without affecting cyclooxygenase-2 (COX-2). In addition, NCTS inhibited LPS-induced p-NF-κB p65 nuclear translocation. In conclusion, these data demonstrated that NCTS showed anti-inflammatory effect by suppression of NF-κB and iNOS signaling pathways.KEY WORDS: Neocryptotanshinone, Inflammation, NF-κB, Inducible nitric oxide synthase     

Anti-inflammatory effect of corymbocoumarin from Seseli gummiferum subsp. corymbosum through suppression of NF-κB signaling pathway and induction of HO-1 expression in LPS-stimulated RAW 264.7 cells

This study investigated the anti-inflammatory activity of corymbocoumarin, an angular-type pyranocoumarin isolated from Seseli gummiferum subsp. corymbosum in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Corymbocoumarin not only inhibited the production of nitric oxide (NO) and prostaglandin E₂ (PGE₂), but also inhibited the protein and mRNA expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Corymbocoumarin also attenuated pro-inflammatory cytokine tumor necrosis factor-α (TNF-α). Investigation of the effect on nuclear factor κB (NF-κB) signaling pathway showed that corymbocoumarin inhibited the phosphorylation of Akt and inhibitory κB (IκB)-α and decreased the subsequent translocation of the p65 and p50 NF-κB subunits to the nucleus. A further study revealed that corymbocoumarin exerted anti-inflammatory activity through induction of heme oxygenase (HO)-1 expression. The in vivo study showed that corymbocoumarin (20 mg/kg, i.p.) reduced paw swelling in carrageenan-induced acute inflammation model. Taken together, these results suggest that corymbocoumarin exerts its anti-inflammatory effect in LPS-stimulated RAW 264.7 cells by suppressing NF-κB activation and inducing HO-1 expression. Corymbocoumarin may provide a useful therapeutic approach for inflammation-associated diseases.     

Dibenzylbutyrolactone lignans from Forsythia koreana fruits attenuate lipopolysaccharide-induced inducible nitric oxide synthetase and cyclooxygenase-2 expressions through activation of nuclear factor-κb and mitogen-activated protein kinase in RAW264.7 cells

Previously, we reported that dibenzylbutyrolactone lignans (DBLLs) from the fruit of Forsythia koreana NAKAI (Oleaceae) has anti-inflammatory, antioxidant, and anti-asthmatic effects. In this study, to clarify the anti-inflammatory mechanisms of DBLL, we evaluated the effects of DBLLs on lipopolysaccharide-stimulated inducible nitric oxide synthetase (iNOS) and cyclooxygenase-2 (COX-2) expressions, nitric oxide (NO) and prostaglandin E(2) (PGE(2)) productions, nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) activations, inhibitor of κB (IκB) and inhibitor of κB kinase (IKK) phosphorylations in cytosolic proteins, and cytotoxicity in Raw264.7 cells. DBLLs potently suppressed both the enzyme expression and DNA-binding activity of NF-κB. Arctiin, arctigenin (1.0 μM) and matairesinol (10 μM) inhibited the expression of iNOS by 37.71±2.86%, 32.51±4.28%, and 27.44±2.65%, respectively, and arctiin, arctigenin (0.1 μM) and matairesinol (1.0 μM) inhibited COX-2 expression by 37.93±7.81%, 26.70±4.61% and 29.37±5.21%, respectively. The inhibitory effects of DBLLs on NO and PGE(2) productions were the same patterns as those seen for the reductions in iNOS and COX-2 expression, respectively. Arctiin, arctigenin (1.0 μM) and matairesinol (10 μM) significantly (p<0.05) inhibited NF-κB DNA binding by 44.85±6.67%, 44.16±6.61%, and 44.79±5.62%, respectively, and arctiin (0.1 μM) and arctigenin (1.0 μM) significantly (p<0.05) inhibited the phosphorylation of IκB by 20.58±3.86% and 25.99±6.18%, respectively. Furthermore, arctiin, matairesinol (1.0 μM) and arctigenin (10 μM) inhibited the phosphorylation of IKK by 38.80±6.64%, 38.33±6.65%, and 38.57±8.14%, respectively. In addition, DBLLs potently inhibited the lipopolysaccharide (LPS)-induced activation of MAPKs (SAPK/c-Jun NH(2)-terminal kinase (JNK), p38, and extracellular signal receptor-activated kinase (ERK)1/2). Overall, arctiin was the most effective; its effect was nearly the same as that of 10 μM helenalin. These findings suggest that treatment with non-toxic DBLLs inhibits not only NF-κB and NF-κB-regulated protein activation, but also potently inhibits the activations of specific MAPKs.