The anti-inflammatory potential of Cortex Phellodendron in vivo and in vitro: Down-regulation of NO and iNOS through suppression of NF-κB and MAPK activation

Cortex Phellodendri amurensis (CPA), derived from the dried bark of Phellodendron amurense Rupr., is a traditional medicine widely used to treat various inflammation-related diseases. The aim of this study was to investigate the anti-inflammatory activity and molecular mechanism of CPA in vivo and in vitro.Mice were pretreated with CPA (200 mg/kg, p.o.) for three consecutive days; 2 h after the last CPA treatment, mice were intraperitoneally injected with lipopolysaccharide (LPS) to induce endotoxemia (35 mg/kg). After treatment, we assessed survival rate, protein levels and cytokine expression. In addition, we confirmed the molecular mechanism of anti-inflammatory effects of CPA in LPS-stimulated macrophage RAW 264.7 cells.The results showed that CPA significantly increased mice survival rates and down-regulated LPS-induced interleukin (IL)-6, IL-1β and macrophage chemo-attractant protein (MCP)-1 in serum. In addition, CPA inhibited inducible nitric oxide synthase (iNOS), activation of nuclear factor (NF)-κB by degradation and phosphorylation of IκBα, and attenuated phosphorylation of mitogen-activated protein kinases (MAPKs; ERK 1/2, p38 and JNK) from mice challenged with LPS. Moreover, in RAW 264.7 cells, CPA dose-dependently down-regulated LPS-stimulated NO, iNOS expression, as well as inflammatory cytokines and protein expression, consistent with the results in vivo.The anti-inflammatory properties of CPA in vitro and in vivo suggest its utility for attenuating inflammation-related diseases.     

Anti-inflammatory Activity of Methylene Chloride Fraction From Glehnia littoralis Extract via Suppression of NF-κB and Mitogen-Activated Protein Kinase Activity

Glehnia littoralis (Umbelliferae) has been used traditionally in Korean, Japanese, and Chinese medicine for the treatment of immune-related diseases; however, its anti-inflammatory activity and underlying mechanism remain to be defined. We investigated the anti-inflammatory effect and inhibitory mechanism on inflammation by the methylene chloride fraction from Glehnia littoralis extract (MCF-GLE), which was more effective than Glehnia littoralis extract (GLE). MCF-GLE inhibited 12-O-Tetradecanoyl-phorbol-13-acetate (TPA)–induced inflammation in an inflammatory edema mouse model. Also, MCF-GLE strongly inhibited the releases of nitric oxide (NO), prostaglandin E₂ (PGE₂), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) and significantly suppressed the mRNA and protein expression of inducible nitric oxide synthase and cyclooxygenase-2 in lipopolysaccharide-stimulated RAW 264.7 macrophage cells in a dose-dependent manner. Furthermore, MCF-GLE suppressed NF-κB activation and IκB-α degradation. MCF-GLE also attenuated the activation of ERK and JNK in a dose-dependent manner. These results indicate that MCF-GLE has an inhibitory effect on the in vivo and in vitro inflammatory reaction and is a possible therapeutic agent. Our results suggest that the anti-inflammatory properties of MCF-GLE may result from the inhibition of pro-inflammatory mediators, such as NO, PGE₂, TNF-α, and IL-1β via suppression of NF-κB– and mitogen-activated protein kinases-dependent pathways.     

Britanin suppresses LPS-induced nitric oxide,PGE2 and cytokine production via NF-κB and MAPK inactivation in RAW 264.7 cells

Little is known about the biological properties of britanin, which is isolated from the flowers of Inula japonica (Inulae Flos). Based on our previous studies that Inulae Flos had anti-inflammation and anti-asthmatic activities, we tried to find the bioactive compounds from it. In this study, the anti-inflammatory effects of britanin on the inflammatory mediators as well as on nuclear factor (NF)-кB and mitogen-activated protein (MAP) kinase activation were evaluated in RAW 264.7 cells. Britanin inhibited the production of nitric oxide (NO) and prostaglandin E2 (PGE₂) along with the expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. In addition, britanin reduced the release of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6. Furthermore, the phosphorylations of MAP kinases (p38 and JNK) in LPS-stimulated RAW 264.7 cells were suppressed by britanin. Moreover, britanin inhibited the NF-κB activation induced by LPS, which was associated with the abrogation of IκBα degradation and subsequent decreases in nuclear p65 levels. This study suggests that the anti-inflammatory activities of britanin might be attributed to the inhibition of iNOS and COX-2 and cytokine expression at least in part, through the attenuation of the phosphorylations of MAP kinases and NF-κB activation via IκBα degradation in macrophages. We conclude that britanin may have potential for the treatment of inflammatory diseases through the down-regulation of MAP kinases and NF-κB mediated activation of macrophages.     

Okra polysaccharide-2 plays a vital role on the activation of RAW264.7 cells by TLR2/4-mediated signal transduction pathways

Polysaccharide is the main active component of okra (Abelmoschus esculentus L.) and it can effectively stimulate the activation of macrophages. However, the immune regulatory mechanism is still not clear. Therefore, the present study aimed to reveal the possible mechanism by investigating the effect of okra polysaccharide-2 (RPS-2) on Toll-like receptor (TLR) 2/4-mediated signal transduction pathways in RAW264.7 murine macrophage cells. In order to confirm whether RPS-2 stimulated macrophages activation via TLR2 or TLR4, RAW264.7 murine macrophage cells were pretreated with TLR2/4 inhibitors for 1 h before RPS-2 treatment, and then the NO, IL-10, TNF-α levels were tested. The results indicated that both TLR2 and TLR4 were the keys of immune regulatory effect of RPS-2. Afterwards, the effect of RPS-2 on NF-κB and MAPKs signaling pathways were studied by western blot analysis. It showed RPS-2 induced the phosphorylation of p65, IκBα, p38, ERK1/2 and JNK. At the same time, the specific inhibitors reduced these phosphorylation levels as well as NO, IL-10 and TNF-α amounts. In a word, RPS-2 activated macrophages by NF-κB and MAPKs signal transduction pathways.     

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.     

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.     

狐臭柴茎挥发油体外抗炎活性及基于NF-κB和MAPKs信号通路作用机制的研究

目的:探究狐臭柴茎挥发油的体外抗炎活性及其作用机制。方法:通过水蒸气蒸馏法制备狐臭柴茎挥发油,采用GC-MS对其化学成分进行分析。通过Griess法和ELISA法测定挥发油对LPS诱导RAW264.7细胞上清液中一氧化氮(NO)、肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)水平的影响;qRT-PCR检测iNOS、COX-2、TNF-α、IL-6、IL-1β mRNA的表达;Western blot测定挥发油对iNOS、COX-2、NF-κB和MAPKs信号通路蛋白的影响。结果:从挥发油中共鉴定出74种化学成分,其中主要的化学成分是茅术醇(13.50%)。挥发油显著抑制NO、TNF-α和IL-6的分泌(P＜0.01),同时降低了促炎因子(TNF-α、IL-6和IL-1β)和促炎酶(iNOS和COX-2)的mRNA表达水平(P＜0.01)。Western blot研究表明挥发油能下调NF-κB信号通路细胞核p65蛋白表达、p65和IκBα磷酸化(P＜0.05或P＜0.01)。此外,还降低了MAPKs信号通路p38、JNK和ERK蛋白的磷酸化(P＜0.01)。结论:狐臭柴茎挥发油对LPS诱导RAW264.7细胞炎症模型具有较好的抗炎效果,其内在的分子机制与下调NF-κB和MAPKs信号通路有关。     

Anti-inflammatory effect of sargachromanol G isolated from Sargassum siliquastrum in RAW 264.7 cells

A study on the anti-inflammatory activity of brown alga Sargassum siliquastrum led to the isolation of sargachromanol G (SG). In this study, the anti-inflammatory effect and the action mechanism of SG have been investigated in murine macrophage cell line RAW 264.7. SG dosedependently inhibited the production of inflammatory markers [nitric oxide (NO), inducible nitric oxide synthase (iNOS), prostaglandin E(2) (PGE(2)), and cyclooxygenase-2 (COX-2)] and pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6] induced by LPS treatment. To further elucidate the mechanism of this inhibitory effect of SG, we studied LPS-induced nuclear factor-κB (NF-κB) activation and mitogen-activated protein kinases (MAPKs) phosphorylation. SG inhibited the phosphorylation IκB-α and NF-κB (p65 and p50) and MAPK (ERK1/2, JNK, and p38) in a dose dependent manner. These results suggest that the anti-inflammatory activity of SG results from its modulation of pro-inflammatory cytokines and mediators via the suppression of NF-κB activation and MAPK phosphorylation.     

Artemisia fukudo essential oil attenuates LPS-induced inflammation by suppressing NF-κB and MAPK activation in RAW 264.7 macrophages

In the present study, the chemical constituents of Artemisia fukudo essential oil (AFE) were investigated using GC–MS. The major constituents were α-thujone (48.28%), β-thujone (12.69%), camphor (6.95%) and caryophyllene (6.01%). We also examined the effects of AFE on the production of nitric oxide (NO), prostaglandin E₂ (PGE₂), tumour necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Western blotting and RT-PCR tests indicated that AFE has potent dose-dependent inhibitory effects on pro-inflammatory cytokines and mediators. We investigated the mechanism by which AFE inhibits NO and PGE₂ by examining the level of nuclear factor-κB (NF-κB) activation within the mitogen-activated protein kinase (MAPK) pathway, which is an inflammation-induced signal pathway in RAW 264.7 cells. AFE inhibited LPS-induced ERK, JNK, and p38 phosphorylation. Furthermore, AFE inhibited the LPS-induced phosphorylation and degradation of Iκ-B-α, which is required for the nuclear translocations of the p50 and p65 NF-κB subunits in RAW 264.7 cells. Our results suggest that AFE might exert an anti-inflammatory effect by inhibiting the expression of pro-inflammatory cytokines. Such an effect is mediated by a blocking of NF-κB activation which consequently inhibits the generation of inflammatory mediators in RAW264.7 cells. AFE may be useful for treating inflammatory diseases.     

Vitisin A suppresses LPS-induced NO production by inhibiting ERK,p38, and NF-κB activation in RAW 264.7 cells

Vitisin A, a resveratrol tetramer isolated from Vitis vinifera roots, exhibits antioxidative, anticancer, antiapoptotic, and anti-inflammatory effects. It also inhibits nitric oxide (NO) production. Here, we examined the mechanism by which vitisin A inhibits NO production in lipopolysaccharide (LPS)-induced RAW 264.7 macrophage cells. Vitisin A dose dependently inhibited LPS-induced NO production and inducible NO synthase (iNOS) expression. In contrast, the production of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) was not altered by vitisin A. To investigate the signaling pathway for NO inhibition by vitisin A, we examined nuclear factor-κB (NF-κB) activation in the mitogen-activated protein kinase (MAPK) pathway, an inflammation-induced signal pathway in RAW 264.7 cells. Vitisin A inhibited LPS-induced extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 phosphorylation and suppressed LPS-induced NF-κB activation in RAW 264.7 cells. This suggests that vitisin A decreased NO production via downregulation of ERK1/2 and p38 and the NF-κB signal pathway in RAW 264.7 cells.     

Anti-inflammatory effect of essential oil and its constituents from fingered citron (Citrus medica L. var. sarcodactylis) through blocking JNK,ERK and NF-κB signaling pathways in LPS-activated RAW 264.7 cells

We investigated the composition of essential oil from fingered citron (Citrus medica L. var. sarcodactylis) (FCEO) peels by GC–MS and its anti-inflammatory effects on lipopolysaccharide (LPS) – stimulated mouse macrophage (RAW 264.7) cells. Fifteen compounds, representing 98.97% of the essential oil, were tentatively identified; the main constituents were limonene (52.44%) and γ-terpinene (28.41%). FCEO significantly inhibited nitric oxide (NO) and prostaglandin E2 (PGE2) by suppressing the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, respectively. Additionally, FCEO suppressed the production of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6. FCEO attenuated LPS-induced nuclear factor-κB (NF-κB) activation via inhibition of inhibitor κB-α phosphorylation. Furthermore, FCEO blocked activation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) but not that of p38 mitogen-activated protein kinase. These results indicate that FCEO inhibits LPS-stimulated inflammation by blocking the NF-κB, JNK, and ERK pathways in macrophages, and demonstrate that FCEO possesses anti-inflammatory properties.     

Saucerneol F, a new lignan, inhibits iNOS expression via MAPKs, NF-κB and AP-1 inactivation in LPS-induced RAW264.7 cells

Saucerneol F (SF), a new tetrahydrofuran-type sesquilignan isolated from Saururus chinensis, dose-dependently inhibited nitric oxide (NO) production, with concomitant reduction of inducible nitric oxide synthase (iNOS) protein and mRNA expression in lipopolysaccharide (LPS)-stimulated murine macrophage RAW264.7 cells. To elucidate the molecular mechanism underlying the inhibition of iNOS expression by SF, we assessed the effects of SF on nuclear factor-κB (NF-κB) DNA-binding activity, NF-κB-dependent reporter gene activity, inhibitory factor-κB (IκB) phosphorylation and degradation, and p65 nuclear translocation. Treatment with SF decreased the luciferase activities of NF-κB reporter promoters in a dose-dependent manner and translocation of NF-κB p65. In addition, pretreatment of SF reduced LPS-stimulated activation of mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK, and c-Jun NH(2)-terminal kinase (JNK). Furthermore, SF attenuated the luciferase activities of AP-1 reporter promoters and the DNA-binding capacity of AP-1. Taken together, the present results indicate that SF attenuates NO production and iNOS expression by blocking LPS-induced activation of NF-κB, MAPKs, and AP-1, suggesting that SF is potentially applicable as an anti-inflammatory drug.     

α-Dihydroxychalcone-glycoside (α-DHC) isolated from the heartwood of Pterocarpus marsupium inhibits LPS induced MAPK activation and up regulates HO-1 expression in murine RAW 264.7 macrophage

Three phenolic glycosides isolated from the heartwood of Pterocarpus marsupium showed significant free radical and superoxide ion scavenging activity and antioxidant potential that were comparable to, or several folds higher than those of standard antioxidants, trolox and ascorbic acid. The effective concentrations of these compounds were far below their cytotoxic levels. Compound 3, which was characterized to be α-dihydroxychalcone-glycoside (α-DHC), was the most potent one. Subsequent studies demonstrated that α-DHC effectively reduced nitric oxide and cytokine production by the LPS stimulated RAW 264.7 mouse macrophage cell line. The compound effectively attenuated the expression of inflammation-mediating enzymes COX-2 and iNOS at the mRNA as well as protein levels in a concentration dependent manner. It prevented phosphorylation of all the three MAPKs (JNK, ERK, p38) and eventually blocked the activation of downstream elements contributing to inflammation. Phosphorylation of IκB-α and subsequent translocation of NF-κB into the nucleus were restricted, while the expression of stress responsive gene HO-1 was up-regulated. α-DHC targeted Keap-1 by modifying its cysteine thiols, dissociating it from Nrf-2 and facilitating nuclear entry of the latter; and this in turn induced HO-1 expression. Thus α-DHC exerts its anti-inflammatory activity in a dual manner: by down regulating MAPKs and restricting nuclear stabilization of NF-κB at one end, and by disrupting Nrf-2–Keap-1 complex on the other. In conclusion, the anti-inflammatory potential together with its high therapeutic index envisages α-DHC as a prospective candidate molecule for the development of therapeutic strategy against inflammatory disorders.