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。     

Grape-seed proanthocyanidins inhibit the lipopolysaccharide-induced inflammatory mediator expression in RAW264.7 macrophages by suppressing MAPK and NF-κb signal pathways

Grape-seed proanthocyanidins (GSPs) have been shown to function as an anti-oxidant and anti-inflammatory agent with little toxicity in vivo and in vitro. However, little is known about their anti-inflammatory properties and mechanisms of action. The specific focus being its effects on the MAP kinases and nuclear factor-kappaB (NF-κB) signal transduction pathways in lipopolysaccharide (LPS) -stimulated RAW264.7 cells. GSPs extract has been found to suppress the mRNA expression of pro-inflammatory cytokines like tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and inflammatory molecule of cyclooxygenase-2 (COX-2) while mRNA level of IL-10 was greatly promoted. Furthermore, GSPs extract inhibited the expression of phosphorylated ERK, JNK and P38, as well as phosphorylated IKKα/β and NF-κB p65 subunit. In conclusion, our results show that GSPs extract showed its anti-inflammatory and immunomodulatory properties by suppressing the activation of MAP kinases and NF-κB signal transduction pathways.     

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.     

Monotropein isolated from the roots of Morinda officinalis ameliorates proinflammatory mediators in RAW 264.7 macrophages and dextran sulfate sodium (DSS)-induced colitis via NF-κB inactivation

We previously demonstrated that monotropein isolated from the roots of Morinda officinalis (Rubiaceae) has anti-inflammatory effects in vivo. In the present study, we investigated the molecular mechanisms underlying the anti-inflammatory effects of monotropein in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and dextran sulfate sodium (DSS)-induced colitis mouse model. Monotropein was found to inhibit the expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) mRNA in LPS-induced RAW 264.7 macrophages. Treatment with monotropein decreased the DNA binding activity of nuclear factor-κB (NF-κB). Consistent with these findings, monotropein also suppressed phosphorylation and degradation of inhibitory κB-α (IκB-α), and consequently the translocations of NF-κB. In the DSS-induced colitis model, monotropein reduced disease activity index (DAI), myeloperoxidase (MPO) activity, and inflammation-related protein expressions by suppressing NF-κB activation in colon mucosa. Taken together, these findings suggest that the anti-inflammatory effects of monotropein are mainly related to the inhibition of the expressions of inflammatory mediators via NF-κB inactivation, and support its possible therapeutic role in colitis.     

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.     

红景天苷对脂多糖诱导的支气管上皮细胞损伤的保护作用

目的：探讨红景天苷对脂多糖（LPS）诱导的正常人支气管上皮细胞（NHBE）损伤的保护作用及机制。方法：培养NHBE细胞,调整其细胞的密度为每毫升含有1＆#215;104个,给予不同浓度的红景天苷（3.3、10、30μg·mL-1）作用24 h,MTT检测细胞的活力,观察红景天苷在正常的情况下对人支气管上皮细胞的影响。在此基础上,以终浓度为2μg·mL-1的LPS作用NHBE细胞6 h,诱导其损伤,然后给予红景天苷24 h,MTT检测细胞活力,收集上清液,ELISA试剂盒检测炎症因子IL-6（白介素-6）、IL-1β（白介素-1β）、TNF-α（肿瘤坏死因子-α）的水平,收集细胞,用Western blot方法测定细胞中的蛋白NF-κB（核因子）、MAPK（丝裂原活化蛋白激酶）的表达。结果：在正常条件下,红景天苷对人支气管上皮细胞的活力无影响,没有损伤作用。红景天苷可明显降低培养液中IL-6、IL-1β、TNF-α的水平（P〈0.01）,降低细胞中NF-κB、MAPK的表达（P〈0.01）。结论：红景天苷可以通过降低IL-6、 IL-1β、TNF-α的水平来减轻LPS诱导的NHBE细胞的损伤,减轻炎症反应。     

Protective effect of taraxasterol on acute lung injury induced by lipopolysaccharide in mice

Taraxasterol, a pentacyclic-triterpene isolated from Taraxacum officinale, has been reported to have potent anti-inflammatory properties. However, the effect of taraxasterol on lipopolysaccharide (LPS)-induced mice acute lung injury has not been investigated. The aims of this study were to investigate whether taraxasterol could ameliorate the inflammation response in LPS-induced acute lung injury and to clarify the possible mechanism. Male BALB/c mice were pretreated with taraxasterol 1 h before intranasal instillation of LPS. 7 h after LPS administration, the myeloperoxidase (MPO) in lung tissues, lung wet/dry ratio and inflammatory cells in the bronchoalveolar lavage fluid (BALF) were detected. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) in the BALF were measured by ELISA. The extent of phosphorylation of IκB-α, p65 NF-κB, p46–p54 JNK, p42–p44 ERK, and p38 were determined by western blotting. The results showed that taraxasterol attenuated the infiltration of inflammatory cells, the activity of myeloperoxidase (MPO), lung wet/dry ratio, and the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) in a dose-dependent manner. Additionally, western blotting results showed that taraxasterol inhibited the phosphorylation of IκB-α, p65 NF-κB, p46–p54 JNK, p42–p44 ERK, and p38 caused by LPS. Our data suggest that anti-inflammatory effects of taraxasterol against the LPS-induced ALI may be due to its ability of inhibition of the NF-κB and MAPK signaling pathways.     

A novel naturally occurring salicylic acid analogue acts as an anti-inflammatory agent by inhibiting nuclear factor-kappaB activity in RAW264.7 macrophages

Methyl salicylate 2-O-β-D-lactoside (DL0309), is a molecule chemically related to salicylic acid that is isolated from Gaultheria yunnanensis (FRANCH.) REHDER (G. yunnanensis). G. yunnanensis, a traditional Chinese herbal medicine, is widely used for treating rheumatoid arthritis, swelling, pain, trauma, and chronic tracheitis. In the present study, we explored the mechanism whereby DL0309 exerts anti-inflammatory effects, using the model of lipopolysaccharide (LPS)-treated RAW264.7 cells. We examined the effects of DL0309 on LPS-induced nuclear factor-kappaB (NF-κB) activity by Western blot analysis, cell imaging analysis and an electrophoretic mobility shift assay (EMSA). Production of pro-inflammatory cytokines was also measured. Our observations indicate that DL0309 suppressed production of nitric oxide (NO), reactive oxygen species (ROS) and the pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), in a concentration-dependent manner. The phosphorylation of IKK-β and degradation of IκB-α by LPS were both inhibited by DL0309 in the cytoplasm. The increased protein level of NF-κB by LPS in the nucleus was also reduced by DL0309. Consistent with these results, we found that DL0309 prevents the nuclear translocation and DNA binding activity of NF-κB. Finally, our results demonstrate that DL0309 exerts anti-inflammatory effects, by inhibiting the production of pro-inflammatory cytokines and suppressing of the activation of the NF-κB signaling pathway in LPS-treated macrophage cells. Therefore, DL0309 may have therapeutic potential for treating inflammatory diseases by regulating the NF-κB pathway and pro-inflammatory cytokine production.     

D(−)-Salicin inhibits the LPS-induced inflammation in RAW264.7 cells and mouse models

D(−)-Salicin is a traditional medicine which has been known to exhibit anti-inflammation and other therapeutic activities. The present study aimed to investigate whether D(−)-Salicin inhibited the LPS-induced inflammation in vivo and in vitro. We evaluated the effect of D(−)-Salicin on cytokines (TNF-α, IL-1β, IL-6 and IL-10) in vivo and in vitro by enzyme-linked immunosorbent assay and signaling pathways (MAPKs and NF-κB) in vivo by Western blot. The results showed that D(−)-Salicin markedly decreased TNF-α, IL-1β and IL-6 concentrations and increased IL-10 concentration. In addition, western blot analysis indicated that D(−)-Salicin suppressed the activation of MAPKs and NF-κB signaling pathways stimulated by LPS. To examine whether D(−)-Salicin ameliorated LPS-induced lung inflammation, inhibitors of MAPKs and NF-κB signaling pathways were administrated intraperitoneally to mice. Interference with specific inhibitors revealed that D(−)-Salicin-mediated cytokine suppression was through MAPKs and NF-κB pathways. In the mouse model of acute lung injury, histopathologic examination indicted that D(−)-Salicin suppressed edema induced by LPS. So it is suggest that D(−)-Salicin might be a potential therapeutic agent against inflammatory diseases.     

Chromene suppresses the activation of inflammatory mediators in lipopolysaccharide-stimulated RAW 264.7 cells

Inflammation is complex process involving a variety of immune cells that defend the body from harmful stimuli. However, pro-inflammatory cytokines and inflammatory mediators can also exacerbate diseases such as cancer. The aim of this study was to identify a natural effective remedy for inflammation. We isolated a functional algal chromene compound from Sargassum siliquastrum, named sargachromanol D (SD). We evaluated the anti-inflammatory effect of SD on lipopolysaccharide (LPS)-exposed RAW 264.7 cells by measuring cell viability, cytotoxicity, and production of inflammatory mediators such as nitric oxide (NO), prostaglandin E₂ (PGE₂), inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6. SD inhibited production of NO and PGE₂ from LPS-induced cells by preventing the expression of inflammatory mediators such as iNOS and COX-2 in a dose-dependent manner. Concurrently, levels of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were reduced with increasing concentrations of SD. In addition, SD inhibited the activation of NF-κB and mitogen-activated protein kinases (MAPKs) pathways in a concentration-dependent manner. These results indicate that SD inhibits LPS-stimulated inflammation by inhibition of the NF-κB and MAPKs pathways in macrophages.