Antimicrobial peptide HI-3 from Hermetia illucens alleviates inflammation in lipopolysaccharide-stimulated RAW264.7 cells via suppression of the nuclear factor kappa-B signaling pathway

Hermetia illucens-3 (HI-3), an active insect antimicrobial peptide extracted from H. illucens larvae, exerts antibacterial and anticancer activity. However, the inflammatory effects and their relative molecular mechanisms remain unclear. To explore the inflammatory effects of HI-3, an inflammatory model was induced using 1 ng/mL LPS in RAW264.7 cells. The cell viability and phagocytosis of LPS-stimulated RAW264.7 cells were then detected after HI-3 treatment. Furthermore, the antioxidant activity, the levels of proinflammatory cytokines, and the expression levels of both p65 and inhibitor of nuclear factor kappa B (IκB) were measured. Results showed that HI-3 could inhibit the differentiation, proliferation, phagocytosis, and antioxidant ability, as well as the secretion and messenger RNA expression levels of IL-6, TNF-α, and IL-1β of LPS-induced RAW264.7 cells in a dose-dependent manner. At the same time, the level of the anti-inflammatory cytokine IL-10 was increased after HI-3 treatment. Western blotting results showed that HI-3 suppressed LPS-induced p65 and IκB activation in a dose-dependent manner. Therefore, HI-3 exerts its anti-inflammatory effect by inhibiting the expression of proinflammatory cytokines and the activation of p65 and IκB, which indicated that HI-3 could be a promising therapeutic medicine for inflammation.     

Synthesis and evaluation of 1,2,4-oxadiazole derivatives as potential anti-inflammatory agents by inhibiting NF-κB signaling pathway in LPS-stimulated RAW 264.7 cells

In this study, a series of compounds with 1,2,4-oxadiazole core was designed and synthesized for the optimization of JC01, an anti-inflammatory hit identified from our in-house compound library using NF-κB pathway luciferase assay and NO production assay. All the synthetic compounds 1–29 have been screened for their anti-inflammatory effects by evaluating their inhibition against LPS-induced NO release, and compound 17 exhibited the highest activity. Western blotting and immunofluorescence analysis revealed that 17 prominently inhibited LPS-induced activation of NF-κB in RAW264.7 cells and blocked the phosphorylation of p65. Consistent with these results, it was found that 17 prevented the nuclear translocation of NF-κB induced by LPS. These data highlighted 17 as a promising anti-inflammatory agent by inhibiting NF-κB activity.     

New pentadienone oxime ester derivatives: synthesis and anti-inflammatory activity

To develop novel anti-inflammatory agents, a series of new pentadienone oxime ester compounds were designed and synthesized. The structures were determined by IR, ¹H NMR, ^13?C NMR, and HRMS. All compounds have been screened for their anti-inflammatory activity by evaluating their inhibition against LPS-induced nitric oxide (NO) release in RAW 264.7 cell. Among them, compound 5j was found to be one of the most potent compounds in inhibiting NO and IL-6 (IC₅₀ values were 6.66?µM and 5.07?µM, respectively). Preliminary mechanism studies show that title compound 5j could significantly suppress expressions of nitric oxide synthase, COX-2, and NO, IL-6 through Toll-like receptor 4/mitogen-activated protein kinases/NF-κB signalling pathway. These data support further studies to assess rational design of more efficient pentadienone oxime ester derivatives with anti-inflammatory activity in the future.     

The spin trap 5,5-dimethyl-1-pyrroline N-oxide inhibits lipopolysaccharide-induced inflammatory response in RAW 264.7 cells

AimExposure of macrophages to lipopolysaccharide (LPS) induces oxidative and inflammatory stresses, which cause cell damage. Antioxidant and anti-inflammatory properties have been attributed to the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), commonly used in free radical analysis, but these aspects of DMPO have been little explored. In this study, we sought to establish the anti-inflammatory activity of DMPO, presumably by removing free radicals which otherwise help activate inflammatory response and damage cells.Main methodsRAW 264.7 macrophages were treated with LPS and/or DMPO for different time points, cell damage, production of inflammatory mediators, inducible nitric oxide synthase (iNOS) expression, NF-κB p65 activation, phosphorylation of MAPKs and Akt, and intracellular reactive oxygen species (ROS) were determined.Key findingsAfter cells were treated with LPS and/or DMPO for 24 h, DMPO reduced the LPS-induced inflammatory response as indicated by downregulated iNOS expression and production of inflammatory mediators. Accordingly, DMPO protected cells from LPS-induced cytotoxicity. In order to understand the mechanistic basis of these DMPO effects, the NF-κB p65 activation and the phosphorylation of MAPKs and Akt were examined. We found, by assaying cells treated with LPS and/or DMPO for 15–60 min, that DMPO inhibited the phosphorylation of MAPKs, Akt, and IκBα, and reduced the NF-κB p65 translocation. Furthermore, we demonstrated that DMPO inhibited LPS-induced ROS production.SignificanceDMPO showed the anti-inflammatory activity and attenuated LPS-induced cell damage, most likely by reducing ROS production and thus preventing the subsequent inflammatory activation and damage.     

Synergistic anti-inflammatory effects of silibinin and thymol combination on LPS-induced RAW264.7 cells by inhibition of NF-κB and MAPK activation

BackgroundCombination drug therapy has become an effective strategy for inflammation control. The anti‑inflammatory capacities of silibinin and thymol have each been investigated on its own, but little is known about the synergistic anti-inflammatory effects of these two compounds.PurposeThis study aims to investigate the synergistic anti-inflammatory effects of silibinin and thymol when administered in combination to lipopolysaccharide (LPS)-induced RAW264.7 cells.MethodsRAW264.7 cells were pre-treated with silibinin and thymol individually or in combination for 2 h before LPS stimulation. Cell viability was detected by the MTT assay. Nitric oxide (NO) production was measured by Griess reagent. Reactive oxygen species (ROS) was evaluated by 2’,7’-dichlorofluorescein-diacetate. ELISA was used to detect tumour necrosis factor-α (TNF-α), and interleukin-6 (IL-6). Western blot was performed to analyse the protein expression of LPS-induced RAW264.7 cells.ResultsWe observed a synergistic anti-inflammatory effect of silibinin and thymol when administered in combination to LPS-induced RAW264.7 cells. Silibinin combined with thymol (40 μM and 120 μM respectively, with the molar ratio 1:3) had more potent effects on the inhibition of NO, TNF-α, and IL-6 than those exerted by individual administration of these compounds in LPS-induced RAW264.7 cells. The combination of silibinin and thymol (40 μM and 120 μM respectively, with the molar ratio 1:3) strongly inhibited ROS and cyclooxygenase-2 (COX-2). More importantly, the combination of silibinin and thymol (40 μM and 120 μM respectively, with the molar ratio 1:3) was also successful in inhibiting nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) activities. Our results suggest that the synergistic anti-inflammatory effects of silibinin with thymol were associated with the inhibition of NF-κB and MAPK signalling pathways.ConclusionThe combination of silibinin and thymol (40 μM and 120 μM, respectively, with the molar ratio 1:3) could inhibit inflammation by suppressing NF-κB and MAPK signalling pathways in LPS-induced RAW264.7 cells.     

Carabrol suppresses LPS-induced nitric oxide synthase expression by inactivation of p38 and JNK via inhibition of I-κBα degradation in RAW 264.7 cells

Carabrol, isolated from Carpesium macrocephalum, showed anti-inflammatory potential in LPS-induced RAW 264.7 murine macrophages. In present study, carabrol demonstrated the inhibitory activity on pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α. In addition, mRNA and protein levels of iNOS and COX-2 were reduced by carabrol. Molecular analysis revealed that these suppressive effects were correlated with the inactivation of p38 and JNK via inhibition of NF-κB activation. Immunoblotting showed that carabrol suppressed LPS-induced degradation of I-κBα and decreased nuclear translocation of p65. Taken together, these results suggest that carabrol can be a modulator of pro-inflammatory signal transduction pathway in RAW 264.7 cells.     

Anti-Inflammatory Activities of Methanol Extract of Mastixia arborea C.B. Clarke as to Mouse Macrophage and Paw Edema

The biological activity of Mastixia arborea (MA) relates to inflammation, but the underlying mechanisms are largely unknown. We confirmed the anti-inflammatory effects of a methanol extract of MA extract on lipopolysaccharide (LPS)-stimulated RAW264.7 mouse macrophage cells and carrageenan-induced mice paw edema. The MA extract significantly inhibited nitric oxide (NO), prostaglandin E₂ (PGE₂), interleukin-1β (IL-1β), and IL-6 production in LPS-stimulated RAW264.7 cells. In vitro expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was suppressed by the extract. The extract attenuated acute inflammatory responses in carrageenan-induced mice paw edema. A mechanism study indicated that translocation of the NF-κB (p65) subunit into the nucleus and phosphorylation of ERK and JNK were inhibited by the extract. These results indicate that the extract is an effective suppressor of the inflammatory response, blocking the phosphorylation of ERK and JNK and the translocation of NF-κB in macrophages, thereby producing an anti-inflammatory effect in vivo.     

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.     

Design,synthesis, anti-inflammatory activity,and molecular docking studies of perimidine derivatives containing triazole

We report here the design, synthesis, and anti-inflammatory activities of a series of perimidine derivatives containing triazole (5a–s). The chemical structures of the synthesized compounds have been assigned on the basis of IR, ¹H NMR, ¹³C NMR, and HRMS spectral analyses. The anti-inflammatory properties of the synthesized perimidine derivatives were evaluated in a lipopolysaccharide (LPS)-stimulated inflammation model. Among the tested compounds, compound 7-(3-methylbenzyl)-7H-[1,2,4]triazolo[4,3-a]perimidine (hereafter referred to as 5h) and compound 7-(2-fluorobenzyl)-7H-[1,2,4]triazolo[4,3-a]perimidine (hereafter referred to as 5n) caused a reduction in the levels of the pro-inflammatory cytokines—tumor necrosis factor (TNF)-α and interleukin (IL)-6—in RAW264.7 cells. The anti-inflammatory potential of compounds 5h and 5n was also evaluated in vivo in a xylene-induced ear inflammation model. Compound 5n showed the most potent anti-inflammatory activity with an inhibition of 49.26% at a dose of 50 mg/kg. This activity is more potent than that of the reference drug ibuprofen (28.13%), and slightly less than that of indometacin (49.36%). To further elucidate the mechanisms underlying these inhibitory effects, LPS-induced nuclear factor-κB (NF-κB) activation and mitogen-activated protein kinase (MAPK) phosphorylation were studied. The results of western blotting showed that the extract obtained from compound 5n inhibited NF-κB (p65) activation and MAPK (extracellular signal-regulated kinase (ERK) and p38) phosphorylation in a dose-dependent manner. Moreover, the results of a docking study of compound 5n into the COX-2 binding site revealed that its mechanism was possibly similar to that of naproxen, a COX-2 inhibitor. The effect of compound 5n on COX-2 antibody was showed it could significantly inhibit COX-2 activity.     

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.     

Synthesis and bio-evaluation of human macrophage migration inhibitory factor inhibitor to develop anti-inflammatory agent

Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is involved in the development of an array of inflammatory disorders including rheumatoid arthritis, inflammatory bowel disease, psoriasis, multiple sclerosis and sepsis. The synthesis of MIF-inhibitor is a rationale approach to develop novel anti-inflammatory agent to treat multitude of inflammatory diseases. In this work, we have synthesized and evaluated MIF-inhibitory activity of a series of small molecules containing isoxazoline skeleton. Mode of binding of this inhibitor to human MIF (huMIF) was determined by docking studies. The synthesized molecules inhibit tautomerase activity of huMIF. The anti-inflammatory activity of the most active inhibitor, 4-((3-(4-hydroxy-3-methoxyphenyl)-4, 5-dihydroisoxazol-5-yl) methoxy) benzaldehyde (4b) was evaluated against huMIF-induced inflammation in a cellular model (RAW 264.7 cell). Compound 4b significantly inhibits huMIF-mediated NF-κB translocation to the nucleus, up-regulation of inducible nitric oxide synthase and nitric oxide production in RAW 264.7 cell which are the markers for inflammation. The compound 4b is not cytotoxic as evident from cell viability assay. Hence, the compound 4b has potential to be a novel anti-inflammatory agent.     

Discovery of talmapimod analogues as polypharmacological anti-inflammatory agents

Abstract

Twenty novel talmapimod analogues were designed, synthesised and evaluated for the in vivo anti-inflammatory activities. Among them, compound 6n, the most potent one, was selected for exploring the mechanisms underlying its anti-inflammatory efficacy. In RAW264.7 cells, it effectively suppressed lipopolysaccharides-induced (LPS-induced) expressions of iNOS and COX-2. As illustrated by the western blot analysis, 6n downregulated both the NF-κB signalling and p38 MAPK phosphorylation. Further enzymatic assay identified 6n as a potent inhibitor against both p38α MAPK (IC₅₀=1.95?µM) and COX-2 (IC₅₀=0.036?µM). By virtue of the concomitant inhibition of p38α MAPK, its upstream effector, and COX-2, along with its capability to downregulate NF-κB and MAPK-signalling pathways, 6n, a polypharmacological anti-inflammatory agent, deserves further development as a novel anti-inflammatory drug.     

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.     

Paeonol attenuates inflammation by confining HMGB1 to the nucleus

Inflammation is a biological process that exists in a large number of diseases. If the magnitude or duration of inflammation becomes uncontrolled, inflammation may cause pathological damage to the host. HMGB1 and NF-κB have been shown to play pivotal roles in inflammation-related diseases. New drugs aimed at inhibiting HMGB1 expression have become a key research focus. In the present study, we showed that paeonol (Pae), the main active component of Paeonia suffruticosa, decreases the expression of inflammatory cytokines and inhibits the translocation of HMGB1 induced by lipopolysaccharide (LPS). By constructing HMGB1-overexpressing (HMGB1⁺) and HMGB1-mutant (HMGB1^m) RAW264.7 cells, we found that the nuclear HMGB1 could induce an LPS-tolerant state in RAW264.7 cells and that paeonol had no influence on the expression of inflammatory cytokines in HMGB1^m RAW264.7 cells. In addition, the anti-inflammatory property of paeonol was lost in HMGB1 conditional knockout mice, indicating that HMGB1 is a target of paeonol and a mediator through which paeonol exerts its anti-inflammatory function. Additionally, we also found that HMGB1 and P50 competitively bound with P65, thus inactivating the NF-κB pathway. Our research confirmed the anti-inflammation property of paeonol and suggests that inhibiting the translocation of HMGB1 could be a new strategy for treating inflammation.     

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.