LPS-induced Nitric Oxide Production Research Articles

Requirement of scavenger receptors for activation of the IRF-3/IFN-β/STAT-1 pathway in TLR4-mediated production of NO by LPS-activated macrophages

Production of nitric oxide (NO) by LPS-activated macrophages is due to a complex cellular signaling initiated by TLR4 that leads to the transcription of IFN-β, which activates IRF-1 and STAT-1, as well as to the activation of NF-κB, required for iNOS transcription. High concentrations of LPS can also be uptaken by scavenger receptors (SRs), which, in concert with TLR4, leads to inflammatory responses. The mechanisms by which TLR4 and SRs interact, and the pathways activated by this interaction in macrophages are not elucidated. Therefore, our main goal was to evaluate the role of SRs, particularly SR-A, in LPS-stimulated macrophages for NO production. We first showed that, surprisingly, LPS can induce the expression of iNOS and the production of NO in TLR4−/− mice, provided exogenous IFN-β is supplied. These results indicate that LPS stimulate receptors other than TLR4. The inhibition of SR-A using DSS or neutralizing antibody to SR-AI showed that SR-A is essential for the expression of iNOS and NO production in stimulation of TLR4 by LPS. The restoration of the ability to express iNOS and produce NO by addition of rIFN-β to inhibited SR-A cells indicated that the role of SR-AI in LPS-induced NO production is to provide IFN-β, probably by mediating the internalization of LPS/TLR4, and the differential inhibition by DSS and neutralizing antibody to SR-AI suggested that other SRs are also involved. Our results reinforce that TLR4 and SR-A act in concert in LPS activation and demonstrated that, for the production of NO, it does mainly by synthesizing IRF-3 and also by activating the TRIF/IRF-3 pathway for IFN-β production, essential for LPS-mediated transcription of iNOS. Consequently STAT-1 is activated, and IRF-1 is expressed, which together with NF-κB from TLR4/MyD88/TIRAP, induce iNOS synthesis and NO production. Summary sentenceTLR4 and SRs act in concert activating IRF-3 to transcribe IFN-β and activate STAT-1 to produce NO by LPS-activated macrophages.

Botryococcus terribilis Ethanol Extract Exerts Anti-inflammatory Effects on Murine RAW264 Cells

The present study aimed to evaluate the effects of Botryococcus terribilis ethanol extract (BTEE) on lipopolysaccharide (LPS)-induced inflammation in RAW264 cells. BTEE significantly attenuated LPS-induced nitric oxide production and inflammatory cytokines release, including Ccl2, Cox2, and Il6. On the other hand, several anti-inflammatory mediators, such as Pgc1β and Socs1, were increased in BTEE-treated cells. Further, we performed an untargeted whole-genome microarray analysis to explore the anti-inflammatory molecular mechanism of BTEE. Enrichment analysis showed BTEE significantly downregulated 'response to stimulus', 'locomotion', and 'immune system response' and upregulated 'cell cycle' gene ontologies in both 6- and 17-h post-LPS stimulation conditions. Pathway analysis revealed BTEE could downregulate the expressions of chemokines of the CC and CXC subfamily, and cytokines of the TNF family, TGFβ family, IL1-like, and class I helical. PPI analysis showed AXL receptor tyrosine kinase (Axl), a receptor tyrosine kinase from the TAM family, and its upstream transcription factors were downregulated in both conditions. Node neighborhood analysis showed several Axl coexpressed genes were also downregulated. Further, kinase enrichment and chemical perturbation analyses supported Axl inhibition in BTEE-treated conditions. Altogether, these findings suggest anti-inflammatory effects of BTEE that are mediated via the suppression of pro-inflammatory cytokines and predict its potential as an Axl inhibitor.

Meliasendanins E-J, Nor-neolignan Constituents from Melia toosendan and their Anti-inflammatory Activity

A phytochemical investigation of the fruits extract of Melia toosendan afforded the isolation of two new nor-neolignans, meliasendanins E (1) and F (2), as well as twelve known compounds (3 - 14) using various separation technique such as Diaion HP20, silica, RP-18 gel column chromatography and semi-preparative HPLC. Their chemical structures were elucidated by extensive NMR spectroscopic data including 2D-NMR, and HR-ESI-MS as well as ECD data. Among the twelve known compounds, the absolute structures of 3 - 6 were determined first, and given the trivial names as meliasendanins G-J (3 - 6). Based on the evaluation of anti-inflammatory activity, compounds 7 - 8 exhibited inhibitory effects on LPS-induced nitric oxide production in RAW 264.7 macrophages with IC50 values of 34.6 and 39.5 μM, respectively.

Lindenane-Type Sesquiterpene Dimers Mitigate Lipopolysaccharide-Induced Inflammation by Inhibiting Toll-Like Receptor Signaling.

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and trigger an inflammatory response via the myeloid differential factor 88 (MyD88)-dependent and toll-interleukin-1 receptor domain-containing adapter-inducing interferon-β (TRIF)-dependent pathways. Lindenane type sesquiterpene dimers (LSDs) are characteristic metabolites of plants belonging to the genus Sarcandra (Chloranthaceae). The aim of this study was to evaluate the potential anti-inflammatory effects of the LSDs shizukaol D (1) and sarcandrolide E (2) on lipopolysaccharides (LPS)-stimulated RAW264.7 macrophages in vitro, and explore the underlying mechanisms. Both LSDs neutralized the LPS-induced morphological changes and production of nitric oxide (NO), as determined by CCK-8 assay and Griess assay, respectively. Furthermore, shizukaol D (1) and sarcandrolide E (2) downregulated interferon β (IFNβ), tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) mRNA levels as measured by reverse transcription polymerase chain reaction (RT-PCR), and inhibited the phosphorylation of nuclear factor kappa B p65 (p65), nuclear factor kappa-Bα (IκBα), Jun N-terminal kinase (JNK), extracellular regulated kinase (ERK), mitogen-activated protein kinase p38 (p38), MyD88, IL-1RI-associated protein kinase 1 (IRAK1), and transforming growth factor-β-activated kinase 1 (TAK1) proteins in the Western blotting assay. In conclusion, LSDs can alleviate the inflammatory response by inhibiting the TLR/MyD88 signalling pathway.

New Triterpenoids and Anti-Inflammatory Constituents from Glinus oppositifolius.

Three new triterpenoids-spergulagenin B (1), spergulagenin C (2), and spergulagenin D (3)-were isolated from the aerial part of Glinus oppositifolius, along with 17 known compounds (4-20). The structures of these new compounds were identified by spectroscopic and MS analyses. Compounds 3, 5, 19, and 20 were evaluated for inhibition of nitric oxide production in LPS-stimulated RAW 264.7 cells with IC50 values of 17.03, 18.21, 16.30, and 12.64 μM, respectively. Compounds 3, 5, and 20 exhibited inhibitory effects on LPS-induced nitric oxide production in RAW 264.7 cells with IC50 values of 18.35 ± 1.34, 17.56 ± 1.41, and 14.27 ± 1.29 μM, respectively.

Compounds isolated from the pericarp of Zanthoxylum bungeanum and inhibitory activity against LPS-induced NO production in RAW264.7

Seventeen compounds were isolated and identified from the ethyl acetate part of Zanthoxylum bungeanum Maxim., including one new compound 18-acetyloxyneocryptotanshinone (1) and 16 known compounds (2–17). Their structures were elucidated by extensive spectroscopy. The absolute configuration of 1 was confirmed by electronic circular dichroism (ECD). All compounds were evaluated for the inhibition of LPS-induced nitric oxide (NO) production in RAW264.7 macrophages, of which 1 and 10 exhibited the most significant inhibitory effect, with IC50 of 17.29 and 10.27 μM, respectively.

Essential Oil from Glossogyne tenuifolia Inhibits Lipopolysaccharide-Induced Inflammation-Associated Genes in Macro-Phage Cells via Suppression of NF-κB Signaling Pathway.

Glossogyne tenuifolia Cassini (Hsiang-Ju in Chinese) is a perennial herb native to Taiwan. It was used in traditional Chinese medicine (TCM) as an antipyretic, anti-inflammatory, and hepatoprotective agent. Recent studies have shown that extracts of G. tenuifolia possess various bioactivities, including anti-oxidant, anti-inflammatory, immunomodulation, and anti-cancer properties. However, the pharmacological activities of G. tenuifolia essential oils have not been studied. In this study, we extracted essential oil from air-dried G. tenuifolia plants, then investigated the anti-inflammatory potential of G. tenuifolia essential oil (GTEO) on lipopolysaccharide (LPS)-induced inflammation in murine macrophage cells (RAW 264.7) in vitro. Treatment with GTEO (25, 50, and 100 μg/mL) significantly as well as dose-dependently inhibited LPS-induced pro-inflammatory molecules, such as nitric oxide (NO) and prostaglandin E2 (PGE2) production, without causing cytotoxicity. Q-PCR and immunoblotting analysis revealed that the inhibition of NO and PGE2 was caused by downregulation of their corresponding mediator genes, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), respectively. Immunofluorescence and luciferase reporter assays revealed that the inhibition of iNOS and COX-2 genes by GTEO was associated with the suppression of nuclear export and transcriptional activation of the redox-sensitive transcription factor, nuclear factor -κB (NF-κB). In addition, GTEO treatment significantly inhibited phosphorylation and proteosomal degradation of the inhibitor of NF-κB (I-κBα), an endogenous repressor of NF-κB. Moreover, treatment with GTEO significantly blocked the LPS-mediated activation of inhibitory κB kinase α (IKKα), an upstream kinase of the I-κBα. Furthermore, p-cymene, β-myrcene, β-cedrene, cis-β-ocimene, α-pinene, and D-limonene were represented as major components of GTEO. We found that treatment with p-cymene, α-pinene, and D-limonene were significantly inhibiting LPS-induced NO production in RAW 264.7 cells. Taken together, these results strongly suggest that GTEO inhibits inflammation through the downregulation of NF-κB-mediated inflammatory genes and pro-inflammatory molecules in macrophage cells.

Screening of Antioxidant, Antibacterial, Anti-Adipogenic, and Anti-Inflammatory Activities of Five Selected Medicinal Plants of Nepal.

Herbal products have been widely used for the treatment of diseases throughout the ages. In this research, we investigated antioxidant, antibacterial, anti-adipogenic, and anti-inflammatory activities of methanolic extracts of five ethnomedicinally important plants; namely, Alnus nepalensis, Dryopteris sparsa, Artocarpus lacucha, Litsea monopetala, and Lyonia ovalifolia. We investigated the DPPH free radical scavenging potential, sensitivity of selected bacterial strains towards the extracts using a disc diffusion assay, anti-inflammatory activity in RAW-264.7 cells, and anti-adipogenic activity by the ORO assay in 3T3-L1 preadipocytes. The extract of A. nepalensis showed significant antioxidant activity (IC50=4.838 µg/mL), followed by A. lacucha, L. monopetala, and L. ovalifolia, exhibiting comparable IC50 values to that of ascorbic acid (IC50=5.063 µg/mL). Alnus nepalensis also showed good antibacterial activity in disc diffusion methods, with remarkable zones of inhibition in A. baumannii (14.66 mm) and P. mirabilis (15.50 mm) bacterial species. In addition, A. nepalensis was found to increase adipogenesis in 3T3-L1 cells, evidenced by increased lipid deposition in differentiated 3T3-L1 cells. A similar pattern of increased adipogenesis was observed on treatment with L. ovalifolia extracts. On the other hand, A. lacucha effectively reduced lipid deposition in 3T3-L1 cells at 100 µg/mL (75.18±6.42%) by inhibiting adipogenesis, showing its potential use in the management of obesity. Furthermore, A. lacucha 100 µg/mL (15.91±0.277 µM) and L. monopetala 75 µg/mL (12.52±0.05 µM) and 100 µg/mL (11.77±0.33 µM) significantly inhibited LPS-induced nitric oxide production in RAW 264.7 cells. Also, A. nepalensis and L. ovalifolia inhibited NO production significantly, endorsing their anti-inflammatory potential. The findings from these in-vitro studies suggest that the selected five plants possess remarkable antioxidant, antibacterial, anti-adipogenic, and anti-inflammatory activities. This study opens the door to conduct further advanced in-vivo experiments to find possible lead compounds for the development of valuable therapeutic agents for common health problems.

Cinnamomum japonicum Siebold Branch Extracts Attenuate NO and ROS Production via the Inhibition of p38 and JNK Phosphorylation.

Cinnamomum japonicum (CJ) is widely distributed in Asian countries like Korea, China, and Japan. Modern pharmacological studies have demonstrated that it exhibits various biological activities, including antioxidant and anti-inflammatory effects. However, most studies have confirmed the efficacy of its water extract but not that of its other extracts. Therefore, in this study, Cinnamomum japonicum Siebold branches (CJB: 70% EtOH extract) were separated using hexane, chloroform, ethyl acetate (CJB3), butanol, and water. Then, their antioxidative activities and phenolic contents were measured. Results revealed that the antioxidant activities and phenolic contents of CJB3 were higher than those of the other extracts. Further, the inhibitory and anti-inflammatory effect of CJB3 on lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) production and LPS-activated macrophages, respectively, was determined. CJB3 suppressed oxidative stress in LPS-activated cells and dose-dependently decreased LPS-stimulated ROS production. CJB3 reduced oxidative stress and reversed the glutathione decrease in LPS-activated RAW264.7 cells. The inhibitory and reducing effect of CJB3 on LPS-induced nitric oxide (NO) production and inducible NO synthase protein and messenger RNA levels, respectively, was investigated. CJB3 inhibited LPS-induced cytokine production and p38 and c-Jun N-terminal kinase (JNK) phosphorylation but not extracellular signal-regulated kinase phosphorylation. Overall, the study results suggest that CJB3 may exert its anti-inflammatory effects via the suppression of p38, JNK, and c-Jun activation.

New monoterpenoid indole alkaloids from the stems of Tabernaemontana bovina Lour (Apocynaceae)

Two new monoterpenoid indole alkaloids, named taberibogines E and F (1 and 2), together with three known ones (3–5) were isolated from the stems of Tabernaemontana bovina Lour (Apocynaceae). Their structures including absolute configurations were elucidated from a combination of NMR and HRESIMS data and NMR calculations as well as DP4+ probability analyses. Compounds 1 and 2 exhibited inhibitory effects on LPS-induced nitric oxide (NO) production in RAW 264.7 macrophages.

Isolation of the Anti-Inflammatory Agent Myceliostatin from a Methionine-Enriched Culture of Myceliophthora thermophila ATCC 42464.

Safe and effective nonsteroidal anti-inflammatory drugs are needed. Meanwhile, addition of amino acids to cultures of microorganisms is likely to increase the possibility of novel secondary metabolite isolation. In the course of screening for anti-inflammatory agents using cellular lipopolysaccharide (LPS)-induced nitric oxide (NO) production, two new related compounds with the myceliothermophin structure from a methionine-enriched culture of Myceliophthora thermophila ATCC 42464 were isolated. The new compounds have an additional methylthio group on the myceliothermophin structure and were named myceliostatins A and B. Both compounds inhibited LPS-induced NO production at nontoxic concentrations in macrophage-like mouse monocytic leukemia RAW264.7 cells. Myceliostatin B inhibited the expression of LPS-induced iNOS, IL-6, and IL-1β and the upstream NF-κB activity in situ and in vitro. Finally, it was found to inhibit NF-κB binding to DNA in the reconstruction system with purified p65. Myceliostatin B also inhibited LPS-induced reactive oxygen species (ROS) production. Thus, myceliostatin B, a novel compound derived from M. thermophila, was found to be a new anti-inflammatory and antioxidant compound directly inhibiting NF-κB.

Anti-neuroinflammatory effects of alkaloid-enriched extract from Huperzia serrata on lipopolysaccharide-stimulated BV-2 microglial cells

Context Alkaloid-enriched extract of Huperzia serrata (Thunb.) Trevis (Lycopodiaceae) (HsAE) can potentially be used to manage neuronal disorders. Objective This study determines the anti-neuroinflammatory effects of HsAE on lipopolysaccharide (LPS)-stimulated BV-2 microglial cells and the underlying mechanisms. Materials and methods BV-2 cells were pre- or post-treated with different concentrations of HsAE (25-150 µg/mL) for 30 min before or after LPS induction. Cell viability was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and no cytotoxicity was found. Nitric oxide (NO) concentration was determined using Griess reagent. The levels of prostaglandin E2 (PGE2), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 were determined using enzyme-linked immunosorbent assay. The levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 and the phosphorylation of mitogen-activated protein kinase (MAPK) were analyzed using western blotting. Results HsAE reduced LPS-induced NO production with half-maximal inhibitory concentration values of 99.79 and 92.40 µg/mL at pre- and post-treatment, respectively. Pre-treatment with HsAE at concentrations of 50, 100, and 150 µg/mL completely inhibited the secretion of PGE2, TNF-α, IL-6, and IL-1β compared to post-treatment with HsAE. This suggests that prophylactic treatment is better than post-inflammation treatment. HsAE decreased the expression levels of iNOS and COX-2 and attenuated the secretion of pro-inflammatory factors by downregulating the phosphorylation of p38 and extracellular signal-regulated protein kinase in the MAPK signaling pathway. Discussion and Conclusions HsAE exerts anti-neuroinflammatory effects on LPS-stimulated BV-2 cells, suggesting that it may be a potential candidate for the treatment of neuroinflammation in neurodegenerative diseases.

Turmeronols (A and B) from <i>Curcuma longa</i> have anti-inflammatory effects in lipopolysaccharide-stimulated BV-2 microglial cells by reducing NF-κB signaling

Turmeronols (A and B), bisabolane-type sesquiterpenoids found in turmeric, reduce inflammation outside the brain in animals; however, their effects on neuroinflammation, a common pathology of various neurodegenerative diseases, are not understood. Inflammatory mediators produced by microglial cells play a key role in neuroinflammation, so this study evaluated the anti-inflammatory effects of turmeronols in BV-2 microglial cells stimulated with lipopolysaccharide (LPS). Pretreatment with turmeronol A or B significantly inhibited LPS-induced nitric oxide (NO) production; mRNA expression of inducible NO synthase; production of interleukin (IL)-1β, IL-6, and tumor necrosis factor α and upregulation of their mRNA expression; phosphorylation of nuclear factor-κB (NF-κB) p65 proteins and inhibitor of NF-κB kinase (IKK); and nuclear translocation of NF-κB. These results suggest that these turmeronols may prevent the production of inflammatory mediators by inhibiting the IKK/NF-κB signaling pathway in activated microglial cells and can potentially treat neuroinflammation associated with microglial activation.

Guettarda crispiflora Vahl Methanol Extract Ameliorates Acute Lung Injury and Gastritis by Suppressing Src Phosphorylation.

Many species in the genus Guettarda are known to exert anti-inflammatory effects and are used as traditional medicinal plants to treat various inflammatory symptoms. However, no studies on the inflammatory activities of Guettarda crispiflora Vahl have been reported. The aim of the study was to investigate in vitro and in vivo the anti-inflammatory effects of a methanol extract of Guettarda crispiflora Vahl (Gc-ME). To determine the anti-inflammatory activity of Gc-ME, lipopolysaccharide (LPS)-, poly(I:C)-, or Pam3CSK4-treated RAW264.7 cells, HCl/EtOH- and LPS-treated mice were employed for in vitro and in vivo tests. LPS-induced nitric oxide production in RAW264.7 cells was determined by Griess assays and cytokine gene expression in LPS-activated RAW264.7 cells, confirmed by RT- and real-time PCR. Transcriptional activation was evaluated by luciferase reporter gene assay. Target protein validation was assessed by Western blot analysis and cellular thermal shift assays (CETSA) with LPS-treated RAW264.7 and gene-transfected HEK293 cells. Using both a HCl/EtOH-induced gastritis model and an LPS-induced lung injury model, inflammatory states were checked by scoring or evaluating gastric lesions, lung edema, and lung histology. Phytochemical fingerprinting of Gc-ME was observed by using liquid chromatography-mass spectrometry. Nitric oxide production induced by LPS and Pam3CSK4 in RAW264.7 cells was revealed to be reduced by Gc-ME. The LPS-induced upregulation of iNOS, COX-2, IL-6, and IL-1β was also suppressed by Gc-ME treatment. Gc-ME downregulated the promotor activities of AP-1 and NF-κB triggered by MyD88- and TRIF induction. Upstream signaling proteins for NF-κB activation, namely, p-p50, p-p65, p-IκBα, and p-Src were all downregulated by Ch-EE. Moreover, Src was revealed to be directly targeted by Gc-ME. This extract, orally treated strongly, attenuated the inflammatory symptoms in HCl/EtOH-treated stomachs and LPS-treated lungs. Therefore, these results strongly imply that Guettarda crispiflora can be developed as a promising anti-inflammatory remedy with Src-suppressive properties.

Targeted isolation of sesquiterpene lactone dimers from Aucklandia lappa guided by LC-HRMS/MS-based molecular networking

An LC-HRMS/MS-based molecular networking strategy was applied to investigate the potential sesquiterpene dimers of Aucklandia lappa, leading to the isolation of three undescribed guaiane-guaiane dimers and one guaiane-eudesmane dimer together with six known sesquiterpenes. The structures were determined by analyzing their 1D, 2D NMR, and HRESIMS data as well as ECD calculations. The biogenetic pathway of the sesquiterpene dimers was postulated to involve the Diels-Alder cycloaddition as the key step. All compounds exhibited their inhibitory effects on LPS-induced nitric oxide production in RAW 264.7 macrophages with IC50 values ranging from 0.3 to 25.1 μM.

Human β-Defensin 3 Inhibits Porphyromonas Gingivalis Lipopolysaccharide-Induced Oxidative and Inflammatory Responses of Microglia by Suppression of Cathepsins B and L.

Recently, the effects of antibacterial peptides are suggested to have therapeutic potential in Alzheimer's disease. Furthermore, systemic treatment of Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) induced Alzheimer's disease-like neuropathological changes in middle-aged mice. Then, we examined whether human β-defensins (hBDs), antimicrobial peptides produced by the oral mucosa and salivary glands, can suppress Pg LPS-induced oxidative and inflammatory responses by microglia. hBD3 (1 μM) significantly suppressed Pg LPS-induced production of nitric oxide and interleukin-6 (IL-6) by MG6 cells, a mouse microglial cell line. hBD3 (1 μM) also significantly inhibited Pg LPS-induced expression of IL-6 by HMC3 cells, a human microglial cell line. In contrast, neither hBD1, hBD2 nor hBD4 failed to inhibit their productions. Furthermore, hBD3 suppressed Pg LPS-induced p65 nuclear translocation through the IκBα degradation. Pg LPS-induced expression of IL-6 was significantly suppressed by E64d, a cysteine protease inhibitor, and CA-074Me, a known specific inhibitor for cathepsin B, but not by pepstatin A, an aspartic protease inhibitor. Interestingly, hBD3 significantly inhibited enzymatic activities of recombinant human cathepsins B and L, lysosomal cysteine proteases, and their intracellular activities in MG6 cells. Therefore, hBD3 suppressed oxidative and inflammatory responses of microglia through the inhibition of cathepsins B and L, which enzymatic activities are necessary for the NF-κB activation.

In Vitro Structure–Activity Relationships Between Dammarane-Type Saponins Isolated From Panax notoginseng and Their Anti-inflammatory Properties

Objectives: Total saponin extracts from the roots of Panax notoginseng have been proven to have anti-inflammatory properties. Dammarane-type saponins isolated from the roots of P notoginseng have been shown to be active inhibitors of nitric oxide (NO) and tumor necrosis factor-α (TNF-α). To clarify the structural requirements for inhibition, some structure–activity relationships (SARs) were determined. Methods: RAW264.7 macrophages were maintained in vitro under standard cell culture conditions. The release of NO and TNF-α from these cells was induced by lipopolysaccharides (LPS) and was measured. The primary SAR inhibitory activity of LPS-induced NO and TNF-α and production in RAW264.7 macrophage cells were evaluated by cytotoxicity assays. Results: SAR studies of dammarane-type saponins revealed that the glycosides substituent at C-3, C-6, and C-20 were important features for the NO and TNF-α production in RAW264.7 cells induced LPS. Moreover, the 5(6)-double bond and the OH group at C-7 improved the TNF-α inhibitory activity. In the case of ginsenoside Rh2, the C-20 configuration was needed for potent TNF-α inhibition. Conclusions: These results provide an approach for understanding the structural requirements of dammarane-type saponins isolated from P notoginseng for optimum anti-inflammatory properties of these compounds.

Characterization and therapeutic effect of Sargassum coreanum fucoidan that inhibits lipopolysaccharide-induced inflammation in RAW 264.7 macrophages by blocking NF-κB signaling

Inflammation is a complex host-protective response against harmful stimuli involving macrophage activation that results in secretion of inflammatory mediators, like nitric oxide (NO), pro-inflammatory cytokines, and prostaglandin E2 (PGE2). In this study, we evaluated fucoidan isolated using Viscozyme-assisted enzymatic extraction of Sargassum coreanum extract against lipopolysaccharide (LPS)-stimulated inflammation in RAW 264.7 macrophages and zebrafish model. Among the fucoidan fractions isolated using ion exchange chromatography, SCVF5 showed the highest sulfate and fucose contents based on chemical composition and monosaccharide analysis. Fourier-transform infrared (FT-IR) spectroscopy confirmed the presence of sulfate esters by the stretching vibrations of the SO peak at 1240 cm−1. SCVF5 showed anti-inflammatory effects by inhibiting NO and PGE2 generation in LPS-stimulated RAW 264.7 macrophages by downregulating inducible NO synthase and cyclooxygenase-2 expression. Treatment with SCVF5 suppressed pro-inflammatory cytokine production, such as TNF-α, (IL)-1β, and IL-6 by modulating the nuclear factor-kappa B signaling cascade in LPS-induced RAW 264.7 cells. Furthermore, in vivo results showed that SCVF5 can potentially downregulate LPS-induced toxicity, cell death, and NO production in LPS-induced zebrafish model. Collectively, these results suggest that S. coreanum fucoidan has remarkable anti-inflammatory activity in vitro and in vivo and may have potential applications in the functional food, cosmetic, and pharmaceutical industries.

Pinostrobin ameliorates lipopolysaccharide (LPS)-induced inflammation and endotoxemia by inhibiting LPS binding to the TLR4/MD2 complex

Pinostrobin is a natural flavonoid with valuable pharmacological properties, including anti-cancer, anti-viral, and anti-oxidant activities. However, the anti-inflammatory effects of pinostrobin have not been well studied. In this study, we investigated whether pinostrobin attenuates lipopolysaccharide (LPS)-induced inflammation and endotoxemia. Additionally, the target molecule of pinostrobin was identified through molecular docking simulation. Pinostrobin decreased LPS-induced nitric oxide (NO) and prostaglandin E2 production, and reduced the expression of inducible NO synthase and cyclooxygenase-2. Furthermore, pinostrobin inhibited the production of proinflammatory cytokines, including interleukin-12 and tumor necrosis factor-α in LPS-stimulated RAW 264.7 macrophages accompanied by inhibiting nuclear translocation of nuclear factor-κB. The anti-inflammatory effect of pinostrobin was further confirmed in LPS-microinjected zebrafish larvae by diminishing the recruitment of macrophages and neutrophils, and proinflammatory gene expression. Moreover, LPS-microinjected zebrafish larvae showed a decrease in heart rate and an increase in mortality and abnormalities. However, pinostrobin significantly attenuated these adverse effects. Molecular docking showed that pinostrobin fits into myeloid differentiation factor (MD2) and Toll-like receptor 4 (TLR4) with no traditional hydrogen bonds (pose 1). The 2D ligand interaction diagram showed that pinostrobin forms a carbon hydrogen bond with LYS89 in MD2 and many non-covalent interactions, including π-alkyl or alkyl and van der Waals interactions, indicating that pinostrobin hinders LPS binding between MD2 and TLR4 and consequently inhibits TLR4/MD2-mediated inflammatory responses. These data suggest that pinostrobin attenuates LPS-induced inflammation and endotoxemia by binding to the TLR4/MD2 complex.

Anti-Inflammatory Activity of Panduratin A against LPS-Induced Microglial Activation.

Uncontrolled and excessive microglial activation is known to contribute to inflammation-mediated neurodegeneration. Therefore, reducing neurotoxic microglial activation may serve as a new approach to preventing neurodegeneration. Here, we investigated the anti-inflammatory effects of panduratin A against microglial activation induced by lipopolysaccharides (LPS) in the SIMA9 microglial cell line. We initially examined the anti-inflammatory properties of panduratin A by measuring LPS-induced nitric oxide (NO) production and the levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6). Panduratin A significantly reduced NO levels and pro-inflammatory cytokines’ production and secretion. In addition, panduratin A enhanced the production of anti-inflammatory cytokines IL-4 and IL-10. The anti-inflammatory effects of panduratin A are related to the suppression of the NF-κB signaling pathway. Together, these results demonstrate the anti-inflammatory properties of panduratin A against LPS-induced microglial activation, suggesting panduratin A has the potential to be further developed as a new agent for the prevention of neuroinflammation-associated neurodegenerative diseases.