Anti-inflammatory activities of inotilone from Phellinus linteus through the inhibition of MMP-9, NF-κB, and MAPK activation in vitro and in vivo

Inotilone was isolated from Phellinus linteus. The anti-inflammatory effects of inotilone were studied by using lipopolysaccharide (LPS)-stimulated mouse macrophage RAW264.7 cells and λ-carrageenan (Carr)-induced hind mouse paw edema model. Inotilone was tested for its ability to reduce nitric oxide (NO) production, and the inducible nitric oxide synthase (iNOS) expression. Inotilone was tested in the inhibitor of mitogen-activated protein kinase (MAPK)?[extracellular signal-regulated protein kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), p38], and nuclear factor-κB (NF-κB), matrix-metalloproteinase (MMP)-9 protein expressions in LPS-stimulated RAW264.7 cells. When RAW264.7 macrophages were treated with inotilone together with LPS, a significant concentration-dependent inhibition of NO production was detected. Western blotting revealed that inotilone blocked the protein expression of iNOS, NF-κB, and MMP-9 in LPS-stimulated RAW264.7 macrophages, significantly. Inotilone also inhibited LPS-induced ERK, JNK, and p38 phosphorylation. In in vivo tests, inotilone decreased the paw edema at the 4(th) and the 5(th) h after Carr administration, and it increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx). We also demonstrated that inotilone significantly attenuated the malondialdehyde (MDA) level in the edema paw at the 5(th) h after Carr injection. Inotilone decreased the NO and tumor necrosis factor (TNF-α) levels on serum at the 5(th) h after Carr injection. Western blotting revealed that inotilone decreased Carr-induced iNOS, cyclooxygenase-2 (COX-2), NF-κB, and MMP-9 expressions at the 5(th) h in the edema paw. An intraperitoneal (i.p.) injection treatment with inotilone diminished neutrophil infiltration into sites of inflammation, as did indomethacin (Indo). The anti-inflammatory activities of inotilone might be related to decrease the levels of MDA, iNOS, COX-2, NF-κB, and MMP-9 and increase the activities of CAT, SOD, and GPx in the paw edema through the suppression of TNF-α and NO. This study presents the potential utilization of inotilone, as a lead for the development of anti-inflammatory drugs.     

Anethum graveloens Flower Extracts Inhibited a Lipopolysaccharide-Induced Inflammatory Response by Blocking iNOS Expression and NF-κB Activity in Macrophages

Inflammation is a system used by a host to defend against the presence of bacteria, viruses, or yeasts. Toll-like receptors (TLRs) in the plasma membranes of macrophages are activated when they recognize the molecular structure of a virus or bacterium. Lipopolysaccharide (LPS), an outer cell-wall component of Gram-negative bacteria, initiates an inflammatory process via TLR4. We investigated the effect of the extract of Anethum graveloens flowers (AGFs) on LPS-mediated inflammation in RAW 264.7 cells. The extract markedly suppressed nitric oxide generation in a concentration-dependent manner in LPS-stimulated RAW 264.7 cells. It inhibited inducible nitric oxide synthase (iNOS) and the mRNA expression of cytokines such as interleukin-1 beta and interleukin-6 in LPS-stimulated RAW 264.7 cells. It also inhibited iNOS protein levels in LPS-stimulated RAW 264.7 cells. In addition, AGF decreased the LPS-induced phosphorylation of mitogen-activated protein kinases in LPS-stimulated RAW 264.7 cells. AGF inhibited the phosphorylation of Akt, an upstream molecule of the nuclear factor kappa B (NF-κB) pathway, and thus inhibited NF-κB activity in LPS-stimulated RAW 264.7 cells. These results suggest that AGF exerts an anti-inflammatory effect in LPS-stimulated RAW 264.7 cells by inhibiting iNOS expression and blocking the NF-κB pathway.     

Anti-inflammatory activity of myricetin from Diospyros lotus through suppression of NF-κB and STAT1 activation and Nrf2-mediated HO-1 induction in lipopolysaccharide-stimulated RAW264.7 macrophages

Diospyros lotus is traditionally used for the treatment of diabetes, diarrhea, tumor, and hypertension. The purpose of this study was to investigate the anti-inflammatory effect and underlying molecular mechanisms of myricetin in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Myricetin dose-dependently suppressed the production of pro-inflammatory mediators (NO, iNOS, PGE₂, and COX-2) in LPS-stimulated RAW264.7 macrophages. Myricetin administration decreased the production of NO, iNOS, TNF-α, IL-6, and IL-12 in mice. Myricetin decreased NF-κB activation by suppressing the degradation of IκBα, nuclear translocation of p65 subunit of NF-κB, and NF-κB DNA binding activity in LPS-stimulated RAW264.7 macrophages. Moreover, myricetin attenuated the phosphorylation of STAT1 and the production of IFN-β in LPS-stimulated RAW264.7 macrophages. Furthermore, myricetin induced the expression of HO-1 through Nrf2 translocation. In conclusion, these results suggest that myricetin inhibits the production of pro-inflammatory mediators through the suppression of NF-κB and STAT1 activation and induction of Nrf2-mediated HO-1 expression in LPS-stimulated RAW264.7 macrophages.     

Identification of a novel compound that inhibits iNOS and COX-2 expression in LPS-stimulated macrophages from Schisandra chinensis

A novel α-iso-cubebenol, which has anti-inflammatory effects in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages, was isolated from the fruits of Schisandra chinensis. α-iso-cubebenol inhibited LPS-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) production. Consistent with these findings, α-iso-cubebenol also reduced the LPS-induced expression of inducible nitric oxide synthase and cyclooxygenase-2 at the protein and mRNA levels in a concentration-dependent manner. α-iso-cubebenol also inhibited LPS-induced nuclear translocation of the NF-κB p65 subunit. Furthermore, α-iso-cubebenol suppressed the phosphorylation of ERK, JNK, and p38 kinase induced by LPS. Since the novel α-iso-cubebenol blocked the production of several pro-inflammatory mediators induced by LPS in macrophages, the molecule can be useful material for the development of anti-inflammatory agents against bacterial infections or endotoxin.     

(S)-tetrahydroisoquinoline alkaloid inhibits LPS-induced arachidonic acid release through downregulation of cPLA2 expression

Sepsis, a systemic inflammatory response syndrome, remains a potentially lethal condition. (S)-1-α-Naphthylmethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (CKD712) is noted as a drug candidate for sepsis. Many studies have demonstrated its significant anti-inflammatory effects. Here we first examined whether CKD712 inhibits lipopolysaccharide (LPS)-induced arachidonic acid (AA) release in the RAW 264.7 mouse monocyte cell line, and subsequently, its inhibitory mechanisms. CKD712 reversed LPS-associated morphological changes in the RAW 264.7 cells, and inhibited LPS-induced release of AA in a concentrationdependent manner. The inhibition was apparently due to the diminished expression of a cytosolic form of phospholipase A₂ (cPLA₂) by CKD712, resulting from reduced NF-κB activation. Furthermore, CKD712 inhibited the activation of ERK1/2 and SAP/JNK, but not of p38 MAPK. CKD712 had no effect on the activity or phosphorylation of cPLA₂ and on calcium influx. Our results collectively suggest that CKD712 inhibits LPS-induced AA release through the inhibition of a MAPKs/NF-κB pathway leading to reduced cPLA₂ expression in RAW 264.7 cells.     

Magnesium isoglycyrrhizinate suppresses LPS-induced inflammation and oxidative stress through inhibiting NF-κB and MAPK pathways in RAW264.7 cells

Magnesium Isoglycyrrhizinate (MgIG), a novel molecular compound extracted from licorice root, has exhibited greater anti-inflammatory activity and hepatic protection than glycyrrhizin and β-glycyrrhizic acid. In this study, we investigated the anti-inflammatory effect and the potential mechanism of MgIG on Lipopolysaccharide (LPS)-treated RAW264.7 cells. MgIG down-regulated LPS-induced pro-inflammatory mediators and enzymes in LPS-treated RAW264.7 cells, including TNF-α, IL-6, IL-1β, IL-8, NO and iNOS. The generation of reactive oxygen species (ROS) in LPS-treated RAW264.7 cells was also reduced. MgIG attenuated NF-κB translocation by inhibiting IKK phosphorylation and IκB-α degradation. Simultaneously, MgIG also inhibited LPS-induced activation of MAPKs, including p38, JNK and ERK1/2. Taken together, these results suggest that MgIG suppresses inflammation by blocking NF-κB and MAPK signaling pathways, and down-regulates ROS generation and inflammatory mediators.     

Phytochemical Profile and Anti-Inflammatory Activity of the Fraction from Artemisia lavandulaefolia

Artemisia lavandulaefolia, a traditional herbal medicine, has been utilized as anti-inflammatory and analgesia agent in clinic. Bioassay-guided fractionation resulted in a fraction (ALDF) with anti-inflammatory effect obtained from A. lavandulaefolia. Its main constituents were analyzed and identified by UPLC-ESI-Q-TOF-MS technology. ALDF showed the strong inhibitory activity on the nitrogen oxide (NO) production in LPS-induced RAW 264.7 macrophages with an IC₅₀ value of 1.64±0.41 μg/mL. Further results displayed that ALDF also significantly suppressed the secretion of key pro-inflammatory mediators, including tumor necrosis factor-α (TNF-α), prostaglandin E₂ (PGE₂) and interleukin-1β (IL-1β), and the increase of the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression induced by LPS stimulation. Mechanism study indicated that ALDF was able to block NF-κB signaling pathway through inhibiting IκB and p65 phosphorylation, as well as NF-κB p65 nuclear translocation. Furthermore, in vivo results in mice revealed that treatments with ALDF evoked significant inhibition on ear edema induced by xylene and on the writhing responses induced by acetic acid. These results suggest that ALDF holds great potential in the prevention and treatment of inflammatory disorders.     

Anti-Inflammatory Activity of N-Butanol Extract from Ipomoea stolonifera In Vivo and In Vitro

Ipomoea stolonifera (I. stolonifera) has been used for the treatment of inflammatory diseases including rheumatism and rheumatoid arthritis in Chinese traditional medicine. However, the anti-inflammatory activity of I. stolonifera has not been elucidated. For this reason, the anti-inflammatory activity of n-butanol extract of I. stolonifera (BE-IS) was evaluated in vivo by using acute models (croton oil-induced mouse ear edema, carrageenan-induced rat paw edema, and carrageenan-induced rat pleurisy) and chronic models (cotton pellet-induced rat granuloma, and complete Freund’s adjuvant (CFA)-induced rat arthritis). Results indicated that oral administration of BE-IS significantly attenuated croton oil-induced ear edema, decreased carrageenan-induced paw edema, reduced carrageenan-induced exudates and cellular migration, inhibited cotton pellet-induced granuloma formation and improved CFA-induced arthritis. Preliminary mechanism studies demonstrated that BE-IS decreased the levels of myeloperoxidase (MPO) and malondialdehyde (MDA), increased the activity of anti-oxidant enzyme superoxide dismutase (SOD) in vivo, and reduced the production of nitric oxide (NO), prostaglandin E₂ (PGE₂), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 in lipopolysaccharide-activated RAW264.7 macrophages in vitro. Results obtained in vivo and in vitro demonstrate that BE-IS has considerable anti-inflammatory potential, which provided experimental evidences for the traditional application of Ipomoea stolonifera in inflammatory diseases.     

Escin exerts synergistic anti-inflammatory effects with low doses of glucocorticoids in vivo and in vitro

Escin, a natural mixture of triterpenoid saponins isolated from the seed of the horse chestnut (Aesculus hippocastanum), had been demonstrated to possess anti-edematous and anti-inflammatory effects. The present study was designed to investigate whether escin exhibits synergistic anti-inflammatory effects when combined with glucocorticoids. The carrageenan-induced paw edema and pleuritis in bilaterally adrenalectomized rats were used to investigate the anti-inflammatory effects of escin and glucocorticoid alone or combined. The carrageenan-induced paw edema was inhibited only when escin and corticosterone (Cort) were administered together. Co-administration of escin with Cort significantly reduced the volume of exudates and the number of white blood cells of exudates in bilaterally adrenalectomized rats with pleuritis, but treatment with escin or Cort alone at a suboptimal concentration did not show any effect on the pleuritis rats. After the murine macrophagic RAW264.7 cells stimulated by lipopolysaccharide (LPS), they were treated with escin, Cort or escin and Cort. Then nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β) of cell culture supernatants were analyzed. Escin or Cort markedly reduced the content of NO, TNF-α and IL-1β secreted by LPS-stimulated RAW264.7 macrophage cells. The combination of suboptimal concentrations of escin with Cort, which alone could not markedly inhibit the release of inflammatory factors, inhibited the secretion of NO, TNF-α and IL-1β in LPS-stimulated RAW264.7 macrophage cells. The findings suggest escin can synergize with glucocorticoids to enhance their anti-inflammatory effect.     

Anethum graveloens flower extracts inhibited a lipopolysaccharide-induced inflammatory response by blocking iNOS expression and NF-κB activity in macrophages

Inflammation is a system used by a host to defend against the presence of bacteria, viruses, or yeasts. Toll-like receptors (TLRs) in the plasma membranes of macrophages are activated when they recognize the molecular structure of a virus or bacterium. Lipopolysaccharide (LPS), an outer cell-wall component of Gram-negative bacteria, initiates an inflammatory process via TLR4. We investigated the effect of the extract of Anethum graveloens flowers (AGFs) on LPS-mediated inflammation in RAW 264.7 cells. The extract markedly suppressed nitric oxide generation in a concentration-dependent manner in LPS-stimulated RAW 264.7 cells. It inhibited inducible nitric oxide synthase (iNOS) and the mRNA expression of cytokines such as interleukin-1 beta and interleukin-6 in LPS-stimulated RAW 264.7 cells. It also inhibited iNOS protein levels in LPS-stimulated RAW 264.7 cells. In addition, AGF decreased the LPS-induced phosphorylation of mitogen-activated protein kinases in LPS-stimulated RAW 264.7 cells. AGF inhibited the phosphorylation of Akt, an upstream molecule of the nuclear factor kappa B (NF-κB) pathway, and thus inhibited NF-κB activity in LPS-stimulated RAW 264.7 cells. These results suggest that AGF exerts an anti-inflammatory effect in LPS-stimulated RAW 264.7 cells by inhibiting iNOS expression and blocking the NF-κB pathway.     

In Vitro Investigation of the Immunomodulatory Potential of Probiotic Lactobacillus casei

The current study investigated the immunomodulatory potential of ethyl acetate soluble supernatant of Lactobacillus casei (LC-EAS) in vitro. The effect of LC-EAS on nitric oxide release was analyzed in RAW 264.7 cells, wherein, an inhibition in nitric oxide production through suppression of inducible nitric oxide synthase mRNA expression was observed. Evaluation of LC-EAS on LPS-induced peripheral blood mononuclear cells showed a down-regulation in TNF-α and IL-6 genes and an upregulation of IL-10. An inhibition in the protein expression of NF-κB, ERK1/2 and STAT3 phosphorylation confirms the immunomodulatory potential of LC-EAS. The effect of LC-EAS on in vitro intestinal epithelial cells was investigated using HT-29 human colon adenocarcinoma cancer cells. LC-EAS exhibited an inhibition of NF-κB and ERK1/2 phosphorylation, whereas STAT3 phosphorylation was unregulated. To evaluate the downstream target of STAT3 upregulation, expression of the intestinal trefoil factor TFF3 which is a NF-κB regulator and STAT3 downstream target was studied. LC-EAS was observed to elevate TFF3 mRNA expression. Overall the study shows that the anti-inflammatory potential of LC-EAS is through inhibition of NF-κB in different cell types.     

Design and synthesis of 1,3-benzothiazinone derivatives as potential anti-inflammatory agents

A series of 1,3-benzothiazinone derivatives were designed and synthesized for pharmacological assessments. Among the synthesized 19 compounds, some compounds showed high activities on inhibiting LPS-induced nitrite oxide and TNF-α production, down-regulating COX-2 and increasing IL-10 production in RAW264.7 cells. All the compounds had no obvious cytotoxicity in in vitro assay. LD₅₀ value of compound 25 was greater than 2000 mg/kg, which was safer than meloxicam. Compound 25 significantly inhibited phosphorylation of NF-κB and STAT3 in LPS-induced RAW264.7 cells. Inhibition of synthesized compounds on COX activity was weaker than meloxicam. Compound 25 displayed lower gastrointestinal toxicity than meloxicam. Besides, compound 25 decreased the swelling in carrageenan-induced paw edema models of inflammation and reduced PGE₂ level significantly. In summary, 1,3-benzothiazinone derivatives are unique scaffolds with anti-inflammatory activity and low toxicity.