Aloe-emodin from rhubarb (Rheum rhabarbarum) inhibits lipopolysaccharide-induced inflammatory responses in RAW264.7 macrophages.

Rheum rhabarbarum (rhubarb) has long been used for the treatment of inflammation in China and other Asian countries. However, the mechanism underlying the anti-inflammatory activity of this medicinal plant is not fully understood. The present study was designed to investigate the anti-inflammatory effects of anthraquinones, the major constituents in rhubarb, and the molecular mechanism involved in their anti-inflammatory effects. RAW264.7 cells were stimulated by lipopolysaccharide (LPS) in the presence or absence of the compounds examined. The proliferation of RAW264.7 cells was assayed by the Alamar-Blue method. The quantity of nitric oxide (NO) was determined by Griess assay. The expression of pro-inflammatory cytokines was determined by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR. Inducible nitric oxide synthase (iNOS), inhibitor of nuclear factor κBα (IκBα), extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK), and Akt/phosphoinositide 3-kinase (PI3K) protein expression levels were determined by Western blotting. Aloe-emodin markedly suppressed the production of NO, interleukin-6 (IL-6), and interleukin-1β (IL-1β) in LPS-stimulated RAW264.7 cells with no apparent cytotoxicity. The mRNA expression levels of iNOS, IL-6, and IL-1β genes were also significantly inhibited by aloe-emodin. Western blot analysis showed that aloe-emodin suppressed LPS-induced iNOS protein expression, IκBα degradation, and the phosphorylation of ERK, p38, JNK, and Akt. These results demonstrate that aloe-emodin is the bioactive component of rhubarb that confers an anti-inflammatory effect through a likely mechanism involving a decrease in pro-inflammatory cytokine production in LPS-induced RAW264.7 macrophages via inhibition of NF-κB, MAPK, and PI3K pathways.

Hu B ，Zhang H ，Meng X ，Wang F ，Wang P ... -  《-》

Anti-inflammatory effect of the six compounds isolated from Nauclea officinalis Pierrc ex Pitard, and molecular mechanism of strictosamide via suppressing the NF-κB and MAPK signaling pathway in LPS-induced RAW 264.7 macrophages.

Nauclea officinalis Pierrc ex Pitard. is a Chinese medicinal herb that contains high level of alkaloids which is the most abundant and active constituent. Strictosamide isolated from Nauclea officinalis Pierrc ex Pitard. showed significant effects on inflammatory response, compared with pumiloside, 3-epi-pumiloside, vincosamide, 3α,5α-tetrahydrodeoxycordifoline lactam and naucleamide A-10-O-β-D-glucopyranoside of this plant. we investigated the biological activities of the six compounds mentioned-above, and the underlying molecular mechanism exerted by the most potent one, strictosamide. The effects of strictosamide and other five compounds on the inhibitory activity of nitric oxide (NO) were screened by Griess test. The contents of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in media were detected by using Enzyme-linked immunosorbent (ELISA) kits. The effects on the mRNA expression of nitric oxide synthase (iNOS), TNF-α and IL-1β of strictosamide were further investigated by RT-qPCR. Western blot assay was conducted to illustrate the effects of strictosamide on iNOS and phosphorylation of p65, inhibitor of NF-κB (IκB)-α, IκB-kinase (IKK)-α as well as p-extracellular signal-regulated kinase (ERK), p-c-jun N-terminal kinase (JNK) and p-p38 in the protein levels. Strictosamide potently suppressed the productions of NO, TNF-α and IL-1β in LPS-induced RAW 264.7 macrophages, and it dose-dependently alleviated the LPS-simulated protein level of iNOS as well as the mRNA expressions of iNOS, TNF-α and IL-1β. In addition, molecular data revealed that strictosamide markedly decreased the expressions of p-p65, p-IκBα and p-IKKα. Furthermore, strictosamide significantly attenuated LPS-induced the phosphorylation of p38, ERK and JNK. At present study, the results indicated that the anti-inflammatory activity of strictosamide was associated with the restraint of NO, TNF-α and IL-1β via negative regulation of both NF-κB and mitogen-activated protein kinases (MAPKs) in LPS-induced RAW 264.7 cells.

Li D ，Chen J ，Ye J ，Zhai X ，Song J ，Jiang C ，Wang J ，Zhang H ，Jia X ，Zhu F ... -  《-》

The p38 MAPK inhibitor JLU1124 inhibits the inflammatory response induced by lipopolysaccharide through the MAPK-NF-κB pathway in RAW264.7 macrophages.

Our previous results showed that JLU1124 is a potent p38 mitogen-activated protein kinase (MAPK) inhibitor. Compared to the classic p38 MAPK inhibitor SB203580, JLU1124 inhibits p38 phosphorylation at low concentrations without cytotoxicity on cells. p38 MAPK is a known target for inflammation treatment. Thus, we became interested in whether JLU1124 has anti-inflammatory effects. We used LPS stimulated RAW264.7 macrophages as a model of inflammation to evaluate the anti-inflammatory effects of JLU1124. Our results showed that p38 phosphorylation, the production of nitric oxide (NO), interleukin (IL)-6 and tumor necrosis factor (TNF)-α, the mRNA and protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 were enhanced by lipopolysaccharide (LPS). At concentrations of less than 10 μmol/L, JLU1124 inhibits p38 phosphorylation in a dose-dependent manner and significantly suppresses LPS-induced production of NO, IL-6 and TNF-α, and decreases the expressions of iNOS and COX-2 in RAW264.7 macrophages which indicate that JLU1124 has anti-inflammatory effects. However, JLU1124 has no significant effect on the phosphorylation of extracellular signal-regulated kinase1/2 and c-Jun NH2-terminal kinase which was involved in inflammation. Furthermore, our results showed that JLU1124 inhibits NF-κB inhibitor (IκB)α phosphorylation, nuclear translocation and transcriptional activity of NF-κB induced by LPS which may be through suppression of Akt phosphorylation. In conclusion, our study indicates that JLU1124 efficiently inhibits p38 phosphorylation and has anti-inflammatory effects in LPS-treated RAW264.7 macrophages. The anti-inflammatory mechanism of JLU1124 is mainly through decreasing Akt phosphorylation and inhibiting IκBα phosphorylation, thus suppressing NF-κB activation and nuclear translocation.

Li XN ，Su J ，Zhao L ，Xiang JB ，Wang W ，Liu F ，Li HY ，Zhong JT ，Bai X ，Sun LK ... -  《-》

Coptisine from Coptis chinensis inhibits production of inflammatory mediators in lipopolysaccharide-stimulated RAW 264.7 murine macrophage cells.

Coptis chinensis has been used for the treatment of inflammatory diseases in China and other Asian countries for centuries. However, the chemical constituents and mechanism underlying the anti-inflammatory activity of this medicinal plant are poorly understood. Here, coptisine, the main constituent of C. chinensis, was shown to potently inhibit the production of nitric oxide (NO) by suppressing the protein and mRNA expressions of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Coptisine also inhibited the production of the pro-inflammatory cytokines interleukin-1β (IL-1β) and interleukin-6 (IL-6) by suppressing expression of cytokine mRNA. Coptisine suppressed the degradation of inhibitor of nuclear factor κBα (IκBα) and phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and phosphoinositide 3-kinase/Akt (PI3K/Akt). Coptisine had no effect on the expression of toll-like receptor 4 (TLR-4) and myeloid differentiation factor 88 (MyD88) as well as LPS binding to TLR-4. Coptisine also inhibited carrageenan-elicited rat paw edema and reduced the release of TNF-α and NO in rat inflamed tissue. These results suggest that coptisine inhibits LPS-stimulated inflammation by blocking nuclear factor-kappa B, MAPK, and PI3K/Akt activation in macrophages, and can be used as an agent for the prevention and treatment of inflammatory diseases.

Wu J ，Zhang H ，Hu B ，Yang L ，Wang P ，Wang F ，Meng X ... -  《-》

Assessment of the anti-inflammatory effects of three rhubarb anthraquinones in LPS-Stimulated RAW264.7 macrophages using a pharmacodynamic model and evaluation of the structure-activity relationships.

Rhubarb (Rhei Radix et Rhizoma) is a traditional Chinese medicine, has been used as a strong astringent in China to treat inflammation-related diseases, such as acute pancreatitis, acute cholecystitis, appendicitis and so on. Rhein, emodin and aloe-emodin are the important active anthraquinone in rhubarb, and are considered to be the main ingredients contributing to anti-inflammatory. Rhein, emodin and aloe-emodin, anthraquinones with the same parent structure that are found in rhubarb, have beneficial anti-inflammatory effects in vitro and in vivo. Anthraquinone derivatives also have important clinical roles. However, their pharmacodynamic differences and the structure-activity relationships associated with their anti-inflammatory properties have not been systematically explored. The present study was designed to quantify the effects of three rhubarb anthraquinones on inflammation and to explore the structure-activity relationships of these compounds. In this study, we detected NF-κB phosphorylation, iNOS protein expression, and IL-6 and NO production in LPS-stimulated RAW264.7 cells and then calculated median effect equations and built a dynamic pharmacodynamic model to quantitatively evaluate the efficacy of these three anthraquinones. Additionally, to determine the structure-activity relationships, we investigated the physicochemical properties and molecular electrostatic potentials of the drug molecules. We found that rhein, emodin, and aloe-emodin exerted at least dual-target (NF-κB, iNOS) inhibition of LPS-induced inflammatory responses. Compared with rhein and emodin, aloe-emodin had a stronger anti-inflammatory effect, and its inhibition of iNOS protein expression was approximately twice that of NF-κB phosphorylation. In addition, aloe-emodin had the strongest hydrophobic effect among the three anthraquinones. Overall, we concluded that the receptor binding the rhubarb anthraquinones had a hydrophobic pocket. Anthraquinone molecules with stronger hydrophobic effects had higher affinity for the receptor, resulting in greater anti-inflammatory activity. These results suggest that the addition of a hydrophobic group is a potential method for structural modification to design anti-inflammatory anthraquinone derivatives with enhanced potency.

Hu Y ，Huang W ，Luo Y ，Xiang L ，Wu J ，Zhang Y ，Zeng Y ，Xu C ，Meng X ，Wang P ... -  《-》