LPS-induced NO Production Research Articles

Discovery of 2-((2-methylbenzyl)thio)-6-oxo-4-(3,4,5-trimethoxyphenyl)-1,6-dihydropyrimidine-5-carbonitrile as a novel and effective bromodomain and extra-terminal (BET) inhibitor for the treatment of sepsis

Sepsis has long been a major health problem worldwide. It threatens the lives of hospitalized patients and has been one of the leading causes of death in hospitalized patients over the past decades. BRD4 has been regarded as a potential target for sepsis therapy, for its critical role in the transcriptional expression of NF-κB pathway-dependent inflammatory factors. In this study, compound 1 was obtained through virtual screening, and candidate compound 27 was obtained through several rounds of iterative SAR analysis. 27 decreased LPS-induced NO production and expression of the pro-inflammatory factors IL-6, IL-1β and TNF-α. In vivo, 27 effectively protected mice from LPS-induced sepsis, increased survival rate and decreased the level of pro-inflammatory factors in serum. Collectively, we reported here 27, a BRD4 inhibitor with a new scaffold, as a potential candidate for the treatment of sepsis.

Isolation, Structural Analysis and Anti-Inflammatory Activity of a Polysaccharide from Ilex cornuta Fruits.

In the present study, a polysaccharide from Ilex cornuta fruits (LCFP-3) was obtained by hot water extraction, Diethyaminoethyl cellulose-52 (DEAE-52) chromatography column and Sephadex G-100 gel column purification. Its structural characteristics were further explored using high performance anion exchange chromatography (HPAEC), gas chromatography and mass spectrometry (GC/MS), scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. Monosaccharide composition analysis revealed LCFP-3 contained mainly Galactose (31.92 %), Arabinose (25.87 %) and Galacturonic acid (23.35 %) while small percentage of Rhamnose, Glucose, Mannose and Xylose. Chemical composition analysis showed that the total sugar content of LCFP-3 was 90.31 % and the protein content was 0.246 %. Gel permeation chromatography (GPC) analysis showed that its average molecular weight was 41.199 kDa. Structural analysis showed that LCFP-3 may be composed of residues, T-α-Arap, T-α-Rhap, 1,3-α-Arap, 1,4-α-Arap, T-β-Galp, 1,4-α-GalpA(OMe), 1,4-β-Glcp, 1,3-β-Galp, 1,3,6-β-Manp, 1,6-β-Galp, 1,3,4-β-GalpA, 1,4,6-β-Manp, 1,3,6-β-Glcp, 1,2,3,4-α-Xylp. The anti-inflammatory activity of LCFP-3 was evaluated using lipopolysaccharide (LPS)-induced RAW246.7 macrophages. The results showed that 1-200 μg/mL LCFP-3 could dose-dependently protect against LPS-induced toxicity and 1 μg/mL LCFP-3 could significantly inhibit LPS-induced NO production. Therefore, LCFP-3 exerted an anti-inflammatory activity and has great potential as a functional ingredient.

Anti-Inflammatory and α-Glucosidase Inhibitory Activities of Chemical Constituents from Bruguiera parviflora Leaves

Bruguiera parviflora (Rhizophoraceae) is one of the Bruguiera genus-based mangrove plants which has not been investigated for the chemical compositions as well as biological activities so far. The present study was aimed at investigating the phytochemicals as well as anti-inflammatory and α-glucosidase inhibitory activities of B. parviflora leaves. The results showed that the crude extract and its fractions significantly increased the percentage inhibitory activity against α-glucosidase and decreased NO production in LPS-stimulated RAW 264.7 cells in a dose-dependent manner. The most effective fraction BP5 was further chromatographed and purified. As a result, eight compounds were isolated and elucidated, including five flavonoids (1–5) and three triterpenoids (6–8). All isolated compounds were evaluated for the anti-inflammatory and α-glucosidase inhibitory effects. The results indicated that flavonoids namely taxifolin (1), quercetin (2), myricetin (3), rutin (4), and kaempferol (5) exhibited potent anti-inflammatory as well as α-glucosidase inhibitory activities. Among them, compound 2 showed the most potent inhibitory effect against an α-glucosidase activity with an I C 50 value of 3.4 ± 0.5 μ g / mL and the LPS-induced NO production of 11.8 μM at the concentration of 100 μg/mL. These findings suggest that flavonoids (1–5) from B. parviflora leaves may be useful as the potential α-glucosidase inhibitor as well as anti-inflammatory agent.

Polysubstituted Cyclopentene Benzamides and Dianthramide Alkaloids from Delphinium anthriscifolium Hance.

Thirteen new benzamide alkaloids, delphiniumines A-M (1-13), together with one known analogue (14), were isolated from Delphinium anthriscifolium Hance. All of the structures were determined by spectroscopic and spectrometric analyses. Absolute configuration for 1 was established using experimental and calculated ECD data, as well as by X-ray crystallography analysis. Compound 1 possesses a previously undescribed polysubstituted cyclopentene carbon framework. Compound 2 was isolated as an artifact from 1 during the extraction process. Compound 7 is glycosylated with a β-D-glucose unit. Compound 13 bears a chlorine substituent. At a concentration of 10 μM, compounds 6, 8, and 10-12 suppressed LPS-induced NO production in RAW264.7 cells with inhibition rates ranging from 40.3% to 78.8%.

Polyhydroxylated eudesmane sesquiterpenoids and sesquiterpenoid glucoside from the flower buds of Tussilago farfara.

Chemical fractionation of the n-BuOH partition, which was generated from the EtOH extract of the flower buds of Tussilago farfara, afforded a series of polar constituents including four new sesquiterpenoids (1-4), one new sesquiterpenoid glucoside (5) and one known analogue (6) of the eudesmane type, as well as five known quinic acid derivatives (7-11). Structures of the new compounds were unambiguously characterized by detailed spectroscopic analyses, with their absolute configurations being established by X-ray crystallography, electronic circular dichroism (ECD) calculation and induced ECD experiments. The inhibitory effect of all the isolates against LPS-induced NO production in murine RAW264.7 macrophages was evaluated, with isochlorogenic acid A (7) showing significant inhibitory activity.

(±)-Heterocageflavone, anti-inflammatory isoprenylated flavonoids with a tricyclo[5.3.1.03,8]undecane moiety from Artocarpus heterophyllus

(±)-Heterocageflavone (1), a pair of enantiomeric isoprenylated flavonoids, were isolated from Artocarpus heterophyllus. Compound 1 incorporates an unusual tricyclo[5.3.1.03,8]undecane moiety, forming a unique caged polycyclic system. The structures of the pair were determined by spectroscopic analysis, ECD calculations, and single-crystal X-ray diffraction. Compound 1 showed significant inhibitory activity against LPS-induced NO production in RAW264.7 cells with an IC50 value of 49.2 μM.

Toxicological damages on copper exposure to IgM+ B cells of Nile tilapia (Oreochromis niloticus) and mitigation of its adverse effects by β-glucan administration

Present investigation was carried out to study toxicological damages of copper exposure and mitigation of its adverse effects with β-glucan administration in IgM+ B cells which processes multiple roles similar to macrophages in Nile tilapia (Oreochromis niloticus). IgM+ B cells were pretreated with β-glucan (25 μg/mL) for 24 h before exposed to cupric oxide nanoparticles (CuO NPs) or cupric chloride (Cu ions) at the doses of 0, 5, 10, and 20 μg/mL for 24 h, respectively. Our results demonstrated that β-glucan increased reduced glutathione (GSH) to against oxidative damage from CuO NPs and Cu ions exposure in IgM+ B cells. The apoptosis process through mitochondrial signaling pathway was depressed in IgM+ B cells since the mitochondrial membrane potential (ΔΨm) was protected from copper exposure by β-glucan treatment. Furthermore, the inhibition on phagocytic abilities of IgM+ B cells caused by copper exposure could be enhanced with β-glucan treatment via evaluation of microspheres and bioparticles uptake and LPS-induced NO production. Importantly, β-glucan might participate in immunomodulation in IgM+ B cells through B cell antigen receptor (BCR) to suppress toxicological effect derived from copper exposure. Taken together, this study provides more information on the toxicological damages in IgM+ B cells upon copper exposure and explains the molecular mechanism to reverse adverse effects caused by copper exposure with β-glucan administration.

A new polyketide of endophytic fungi Aspergillus sp. ZJ-58 from Coptis chinensis

A new polyketide, coptaspin A(1), along with two known compounds 4-acetyl-3,4-dihydro-6,8-dihydroxy-3-methoxy-5-methylisocoumarin(2), and cytochalasin Z_(12)(3), was isolated from the endophytic fungi Aspergillus sp. ZJ-58, which was isolated from the genuine medicinal plant Coptis chinensis in Chongqing after solid-state fermentation on rice and silica gel, MCI, and HPLC-based separation. Their structures were elucidated by MS, NMR, IR, UV, and ECD. The newly isolated compound 1 showed moderate inhibitory activities against LPS-induced NO production in RAW264.7 macrophages with the IC_(50) value of 58.7 μmol·L~(-1), suggesting its potential anti-inflammatory activity.

A Newly Synthesized Flavone from Luteolin Escapes from COMT-Catalyzed Methylation and Inhibits Lipopolysaccharide-Induced Inflammation in RAW264.7 Macrophages via JNK, p38 and NF-κB Signaling Pathways.

Luteolin is a common dietary flavone possessing potent anti-inflammatory activities. However, when administrated in vivo, luteolin becomes methylated by catechol-O-methyltransferases (COMT) owing to the catechol ring in the chemical structure, which largely diminishes its anti-inflammatory effect. In this study, we made a modification on luteolin, named LUA, which was generated by the chemical reaction between luteolin and 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). Without a catechol ring in the chemical structure, this new flavone could escape from the COMT-catalyzed methylation, thus affording the potential to exert its functions in the original form when administrated in the organism. Moreover, an LPS-stimulated RAW cell model was applied to detect the anti-inflammatory properties. LUA showed much more superior inhibitory effect on LPS-induced production of NO than diosmetin (a major methylated form of luteolin) and significantly suppressed upregulation of iNOS and COX-2 in macrophages. LUA treatment dramatically reduced LPS-stimulated reactive oxygen species (ROS) and mRNA levels of pro-inflammatory mediators such as IL-1β, IL-6, IL-8 and IFN-β. Furthermore, LUA significantly reduced the phosphorylation of JNK and p38 without affecting that of ERK. LUA also inhibited the activation of NF-κB through suppression of p65 phosphorylation and nuclear translocation.

New Trichothecenes Isolated from the Marine Algicolous Fungus Trichoderma brevicompactum.

Eight trichothecenes, including four new compounds 1–4 and four known entities 5–8, together with one known cyclonerane (9) were isolated from the solid-state fermentation of Trichoderma brevicompactum NTU439 isolated from the marine alga Mastophora rosea. The structures of 1–9 were determined by 1D/2D NMR (nuclear magnetic resonance), MS (mass spectrometry), and IR (infrared spectroscopy) spectroscopic data. All of the compounds were evaluated for cytotoxic activity against HCT-116, PC-3, and SK-Hep-1 cancer cells by the SRB assay, and compound 8 showed promising cytotoxic activity against all three cancer cell lines with the IC50 values of 3.3 ± 0.3, 5.3 ± 0.3, and 1.8 ± 0.8 μM, respectively. Compounds 1–2, 4–6, and 7–8 potently inhibited LPS-induced NO production, and compounds 5 and 8 showed markedly inhibited gelatinolysis of MMP-9 in S1 protein-stimulated THP-1 monocytes.

Synthesis of lathyrane diterpenoid nitrogen-containing heterocyclic derivatives and evaluation of their anti-inflammatory activities

As our ongoing work on lathyrane diterpenoid derivatization, three series of lathyrane diterpenoid derivatives were designed and synthesized based combination principles, including pyrazole, thiazole and furoxan moieties. Biological evaluation indicated that compound 23d exhibited excellently inhibitory activity on LPS-induced NO production in RAW264.7 cells (IC50 = 0.38 ± 0.18 μM). The preliminary structure–activity relationships (SARs) suggested that phenylsulfonyl substituted furoxan moiety had the strongest ability to improve anti-inflammatory activity of lathyrane diterpenoids. Furthermore, compound 23d significantly reduced the level of ROS. Its molecular mechanism was related to inhibiting the transcriptional activation of Nrf2/HO-1 pathway. Based on these considerations, 23d might be a promising anti-inflammatory agent, which is noteworthy for further exploration.

Erinacine A Prevents Lipopolysaccharide-Mediated Glial Cell Activation to Protect Dopaminergic Neurons against Inflammatory Factor-Induced Cell Death In Vitro and In Vivo.

Hericium erinaceus (HE) is a common edible mushroom consumed in several Asian countries and considered to be a medicinal mushroom with neuroprotective effects. Erinacine A (EA) is a bioactive compound in Hericium erinaceus mycelium (HEM) that has been shown to have a neuroprotective effect against neurodegenerative diseases, e.g., Parkinson’s disease (PD). Although the etiology of PD is still unclear, neuroinflammation may play an important role in causing dopaminergic neuron loss, which is a pathological hallmark of PD. However, glial cell activation has a close relationship with neuroinflammation. Thus, this study aimed to investigate the anti-neuroinflammatory and neuroprotective effects of EA on lipopolysaccharide (LPS)-induced glial cell activation and neural damage in vitro and in vivo. For the in vitro experiments, glial cells, BV-2 microglial cells and CTX TNA2 astrocytes were pretreated with EA and then stimulated with LPS and/or IFN-γ. The expression of proinflammatory factors in the cells and culture medium was analyzed. In addition, differentiated neuro-2a (N2a) cells were pretreated with EA or HEM and then stimulated with LPS-treated BV-2 conditioned medium (CM). The cell viability and the amount of tyrosine hydroxylase (TH) and mitogen-activated protein kinases (MAPKs) were analyzed. In vivo, rats were given EA or HEM by oral gavage prior to injection of LPS into the substantia nigra (SN). Motor coordination of the rats and the expression of proinflammatory mediators in the midbrain were analyzed. EA pretreatment prevented LPS-induced iNOS expression and NO production in BV-2 cells and TNF-α expression in CTX TNA2 cells. In addition, both EA and HEM pretreatment significantly increased cell viability and TH expression and suppressed the phosphorylation of JNK and NF- κB in differentiated N2a cells treated with CM. In vivo, both EA and HEM significantly improved motor dysfunction in the rotarod test and the amphetamine-induced rotation test and reduced the expression of TNF-α, IL-1β and iNOS in the midbrain of rats intranigrally injected with LPS. The results demonstrate that EA ameliorates LPS-induced neuroinflammation and has neuroprotective properties.

β-glucan mitigation on toxicological effects in monocytes/macrophages of Nile tilapia (Oreochromis niloticus) following copper exposure

The protective effect of β-glucan against toxicological effects caused by copper oxide nanoparticles (Cu NPs) and copper ions (Cu ions) were studied in monocytes/macrophages (MO/MФ) of Nile tilapia (Oreochromis niloticus). Our results demonstrated that CuO NPs and Cu ions exposure aroused strong oxidative lesion in MO/MФ by detection of cellular reactive oxygen species (ROS) and reduced glutathione (GSH), as well as identification of several antioxidant-related cytokines. Meanwhile, the serious pro-inflammatory responses were accompanied during the processes of oxidative lesion by TNFα, IL-1β, and IL-6 genes validation. Copper induced MO/MФ underwent apoptosis through mitochondrial signaling pathway by mitochondrial membrane potential (ΔΨm) detection and Bax, Bcl-2, Cyt-c, Apaf-1, Caspase 9, Caspase 3 genes validation. Furthermore, the phagocytic abilities were inhibition in MO/MФ by evaluation of microspheres (0.5 and 1.0 μm beads) and bioparticles (S. agalactiae and A. hydrophila) uptake and LPS-induced NO production. However, β-glucan might participate in immunomodulation through C-type lectin receptor (CLR) and complement receptor 3 (CR3) to suppress pro-inflammatory responses, thereby revered all the copper induced aforementioned adverse effects in MO/MΦ. Taken together, our results provide insights on the mechanisms through β-glucan administration to mitigate toxicological effects of CuO NPs and Cu ions exposure to the MO/MΦ, which will benefit aspects related to fish farming and aquaculture production.

Cembranoids from the Vietnamese soft coral Sarcophyton ehrenbergi

Using various chromatographic separations, two new cembranoids, ehrenbergols F and G (1 and 2), along with three known analogs ehrenbergol D (3), (+)-isosarcophine (4) and sinulariol Z2 (5) were isolated from the soft coral Sarcophyton ehrenbergi. The structural elucidation was done by extensive analysis of the 1 D and 2 D NMR, HR-ESI-QTOF-MS as well as CD experiments. In addition, compounds 1 (IC50 of 38.38 ± 2.89 µM), 3 (IC50 of 37.14 ± 3.22 µM) and 4 (IC50 of 45.01 ± 2.49 µM) revealed moderate inhibitory activity on LPS-induced NO production in RAW264.7 cells, whereas 2 (IC50 of 73.32 ± 1.95 µM) and 5 (IC50 of 64.48 ± 4.93 µM) exhibited weak effect.

Evaluation of Anti-Inflammatory Effects of Celery Leaf and Stem Extracts in LPS-Induced RAW 264.7 Cells Using Nitric Oxide Assay and LC-MS Based Metabolomics.

The present work demonstrated and compared the anti-inflammatory effects of celery leaf (CLE) and stem (CSE) extracts. LC-MS-based metabolomics were an effective approach to achieve the biomarker identification and pathway elucidation associated with the reduction in inflammatory responses. The celery extracts suppressed LPS-induced NO production in RAW 264.7 cells, and CLE was five times more effective than CSE. Distinct differences were revealed between the control and celery-treated samples among the 24 characteristic metabolites that were identified. In celery-treated LPS cells, reversals of intracellular (citrulline, proline, creatine) and extracellular (citrulline, lysine) metabolites revealed that the therapeutic outcomes were closely linked to arginine metabolism. Reversals of metabolites when treated with CLE (aspartate, proline) indicated targeted effects on the TCA and urea cycles, while, in the case of CSE (histidine, glucose), the glycolysis and the pentose phosphate pathways were implicated. Subsequently, apigenin and bergapten in CLE were identified as potential biomarkers mediating the anti-inflammatory response.

Sesquiterpene Lactones from Sigesbeckia glabrescens Possessing Potent Anti-inflammatory Activity by Directly Binding to IKKα/β.

Chromatographic fractionation of Sigesbeckia glabrescens led to the identification of 10 new sesquiterpene lactones, named siegesbeckialides I-O (1-7) and glabrescones A-C (8-10), along with 14 known analogues. An anti-inflammatory activity assay showed that siegesbeckialide I (1) most potently inhibited LPS-induced NO production in RAW264.7 murine macrophages. Furthermore, siegesbeckialide I suppressed the protein expression of iNOS and COX2, as well as the release of PGE2, IL-1β, IL-6, and TNF-α in LPS-stimulated RAW264.7 cells. Mechanistically, siegesbeckialide I directly binds to inhibitors of IKKα/β and suppresses their phosphorylation. This leads to the inhibition of IKKα/β-mediated phosphorylation and degradation of inhibitor α of NF-κB (IκBα), as well as the activation of NF-κB signaling.

Bioactivity-guided isolation of cyclooxygenase-2 inhibitors from Saussurea obvallata (DC.) Edgew. Using affinity solid phase extraction assay

Ethnopharmacological relevanceSaussurea obvallata (DC.) Edgew. is a traditional Tibetan medicine used for the treatment of inflammation-related diseases, but the scientific validation was very limited.Aim of the study: This study aimed to rapid screen and targeted isolate cyclooxygenase-2 (COX-2) inhibitors from S. obvallata extract. Materials and methodsAn efficient ligand-fishing method based on affinity solid phase extraction (A-SPE) combining with HPLC was developed. The identified COX-2 inhibitors were separated using preparative liquid chromatography. In vitro COX-2 inhibition assays were employed to confirm the inhibitory activities of the isolated compounds. In addition, the effect of the isolated compounds on the production of prostaglandin E2 (PGE2) and the expression of COX-2 in LPS-induced RAW 264.7 were evaluated. ResultsA total of four phenylpropanoids, isolariciresinol, syringaresinol, pinoresinol and balanophonin were targeted isolated as COX-2 inhibitors with IC50 values of 36.4 ± 2.6 μM, 23.1 ± 1.8 μM, 3.6 ± 0.3 μM and 12.1 ± 0.9 μM, respectively. The isolated compounds significantly inhibited LPS-induced NO production in a dose-dependent manner. And, the results of the inhibitory effect on the release of PGE2 and the expression of COX-2 in LPS-induced macrophages were consistent with A-SPE analysis. ConclusionThe present work demonstrated that the developed A-SPE-HPLC method could successfully targeted isolated COX-2 inhibitors from S. obvallata extract. And, the isolation results indicated that the therapeutic effect of S. obvallata on inflammation-related diseases was partly based on the COX-2 active ingredients.

Lanostane triterpenoids from the fruiting bodies of Fomitopsis pinicola and their anti-inflammatory activities

Thirteen previously undescribed lanostane triterpenoids, as well as nine known lanostane triterpenoids, were isolated from the fruiting bodies of Fomitopsis pinicola (Sw.) P. Karst. Their structures and absolute configurations were characterized by extensive analysis of spectroscopic data and single-crystal X-ray diffraction. Nor-pinicolic acids A−F possess unusual C-25-C-27 nor-lanostane skeletons, which were first reported from F. pinicola. Anti-inflammatory assays indicated that pinicopsic acid F and 16α-hydroxy-3-oxolanosta-7,9(11),24-trien-21-oic acid showed moderate inhibitory activities against LPS-induced NO production in RAW 264.7 cells, with IC50 values of 24.5 and 25.7 μM, respectively.

Chemical epigenetic manipulation triggers the production of sesquiterpenes from the deep-sea derived Eutypella fungus

Chemical epigenetic manipulation of a deep-sea-derived Eutypella sp. fungus by the co-treatment with a histonedeacetylase inhibitor (suberohydroxamic acid, SBHA) and a DNA methyltransferase inhibitor (5-azacytidine, 5-Aza), resulted in the activation of a sesquiterpene-related biosynthetic gene cluster. Chromatographic separation of the elicitor-treated cultures led the isolation of 21 sesquiterpenes, including 17 undescribed compounds, eutypeterpenes A-Q. Their structures were identified by the extensive analysis of the spectroscopic data, including the single-crystal X-ray diffraction, chemical conversion, and the calculated NMR and ECD data for configurational assignments. Eutypeterpene A is a first bergamotene-type sesquiterpene incorporated with a dioxolanone unit, and eutypeterpenes O-Q with a cyclopentane ring represent an undescribed subtype of sesquiterpenes. The bioassay results showed that most compounds exert inhibitory effects against the lipopolysaccharide (LPS)-induced NO production in RAW 264.7 macrophages, and eutypeterpene N is the most active. This study demonstrates that the epigenetic manipulation is an effective approach to trigger the production of cryptic metabolites from deep-sea derived fungus. The significant inhibition against LPS-induced NO production in vitro suggests eutypeterpenes to be potential for the development as anti-inflammatory agents.

Cladosporitides A–C, three polyketides from an endophytic fungi Cladosporium tenuissimum

Three new polyketides, named cladosporitides A–C (1–3), were isolated from an endophytic fungi Cladosporium tenuissimum derived from the stem of Berberis heteropoda. Their structures were determined by extensive spectroscopic analysis including HRMS, 1D and 2D NMR data. Compound 1 possesses an unusual octahydro-2H-2,5-epoxychromene tricyclic structure moiety. An anti-inflammatory assay indicated that compounds 1–3 showed moderate inhibitory activities against LPS-induced NO production in RAW 264.7 cells with IC50 values ranging from 22.32 to 33.97 μM.