Nitric Oxide Production In RAW Research Articles

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.

Cinnamein Inhibits the Induction of Nitric Oxide and Proinflammatory Cytokines in Macrophages, Microglia and Astrocytes.

Chronic inflammation driven by proinflammatory cytokines (TNFα, IL-1β, IL-6, etc.), and nitric oxide (NO) plays an important role in the pathogenesis of several autoimmune, inflammatory as well as neurodegenerative disorders like rheumatoid arthritis, multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, etc. Therefore, identification of nontoxic anti-inflammatory drugs may be beneficial for these autoimmune, inflammatory and neurodegenerative disorders. Cinnamein, an ester derivative of cinnamic acid and benzyl alcohol, is used as a flavoring agent and for its antifungal and antibacterial properties. This study underlines the importance of cinnamein in inhibiting the induction of proinflammatory molecules in RAW 264.7 macrophages and primary mouse microglia and astrocytes. Stimulation of RAW 264.7 macrophages with lipopolysaccharide (LPS) and interferon γ (IFNγ) led to marked production of NO. However, cinnamein pretreatment significantly inhibited LPS- and IFNγ-induced production of NO in RAW 264.7 macrophages. Cinnamein also reduced the mRNA expression of inducible nitric oxide synthase (iNOS) and TNFα in RAW cells. Accordingly, LPS and viral double-stranded RNA mimic polyinosinic: polycytidylic acid (polyIC) stimulated the production of TNFα, IL-1β and IL-6 in primary mouse microglia, which was inhibited by cinnamein pretreatment. Similarly, cinnamein also inhibited polyIC-induced production of TNFα and IL-6 in primary mouse astrocytes. These results suggest that cinnamein may be used to control inflammation in different autoimmune, inflammatory and neurodegenerative disorders.

Pharmacological bioactivity of enzymatically bio-transformed ginsenosides

Many ginsenosides have shown positive effects, including anti-cancer potential and anti-inflammatory effects. Of note, protopanaxadiol (PPD) and protopanaxatriol (PPT) are not easily absorbed by the body through the digestive tract due to their hydrophilicity. From this point of view, the cytotoxic potencies of the hydrolysates of PPD and PPT on CRC are much stronger than their source compounds. Moreover, several minor ginsenosides that are absent naturally but have high disease ameliorative efficacy can be obtained from major ginsenoside by enzymatic hydrolysis. Therefore, the first aim of this study was to determine the effectiveness of the biotransformation of ginsenosides via enzymatic hydrolysis to improve their bioactivity. Second, the anti-inflammatory and anti-cancer effects of the raw and bio-transformed ginseng metabolites were determined in vitro. The results suggest that enzymes can effectively biotransform major ginsenosides (i.e., PPD and PPT) into minor ginsenosides (i.e., compound K) by hydrolyzing the β-glucosidic linkage. Moreover, the bio-transformed ginsenosides were effective in inhibiting the proliferation of HCT-116 cells and suppressing lipopolysaccharide-induced nitric oxide production in RAW 264.7 murine macrophages. Therefore, the enzymatic hydrolysis of ginsenosides can be employed to functionally produce hydrolysates with increased bioactivity.

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.

Sinensiols H-J, three new lignan derivatives from Selaginella sinensis (Desv.) Spring.

One new lignan sinensiol H (1) and two new bisnorlignans, sinensiols I and J (2 and 3), along with three known compounds were isolated from the whole plants of Selaginella sinensis. Their structures were elucidated on the basis of 1D and 2D NMR spectroscopy as well as high-resolution mass spectrometry. The absolute configuration of 1 was established by ECD calculation. Compounds 2 and 3 represent rare examples of naturally occurring 9,9'-bisnorlignans. All the isolated compounds were assayed for their inhibitory effects on LPS-induced nitric oxide production in RAW 264.7 macrophages.

Triterpenoids from Chios mastic gum of Pistacia lentiscus and their inhibition of LPS-induced NO production in RAW 264.7 cells

A phytochemical investigation of mastic, the resinous secretion obtained from the wounds of the trunk and branches of Pistacia lentiscus, led to the isolation of six undescribed triterpenoids (1–6), including two unusual nor-dammarane-type triterpenoids (1 and 2), four tirucallane-type triterpenoids (3–6), together with seven known compounds (7–13). The structures of those undescribed compounds were elucidated by integrating spectroscopic analyses, including NMR, UV, IR, as well as HR-MS data. The absolute configurations of compounds 1 and 2 were confirmed by electronic circular dichroism. Anti-inflammatory evaluation revealed that compounds 5, 8, 9, and 12 exhibited moderate inhibition of the nitric oxide production in RAW264.7 cells with IC50 values ranging from 20.24 to 36.63 μM, compared to that of positive control, dexamethasone (IC50 = 19.85 μM).

Hypoxylonone, a new oxa-bridged seven-membered ring analog from fungus Hypoxylon cf. subgilvum SWUF15-004

A new oxa-bridged seven-membered ring analog, hypoxylonone (1), and thirteen known compounds (2–14) were isolated from fungus Hypoxylon cf. subgilvum SWUF15-004. The structures were elucidated by the analysis of spectroscopic (IR, 1 D and 2 D NMR), HRESIMS and X-ray diffraction (MoKα) data. Several isolated compounds were evaluated for cytotoxicity against four human cancer cell lines (HeLa, HT29, MCF-7, A549). Compound 1 exhibited weak inhibitory effects of the nitric oxide production in RAW264.7 cells. Compounds 8 and 9 exhibited slight cytotoxicity.

Anti-infammatory scalemic chromanoids and chromenoids from Rhododendron dauricum

Four pairs of undescribed chromane and chromene meroterpenoid scalemic mixtures (1a/1b-4a/4b), together with three pairs of known chromane meroterpenoid ones (5a/5b-7a/7b) were isolated from the twigs and leaves of Rhododendron dauricum L. Among them, 1a/1b-3a/3b and 5a/5b-7a/7b were the chromane ones derived from an intramolecular [2 + 2] cyclic addition of their respective chromene precursors, forming a 6/6/6/4 and 6/6/5/4 ring fused scaffold. The absolute configurations of the chiral center at C-15 of 2a/2b were determined by Snatzke's method, and comparing the experimental and calculated electronic circular dichroism (ECD) data. The inhibitory effects of the isolated compounds were tested against lipopolysaccharide (LPS)-induced nitric oxide production in RAW264.7 macrophage cells to evaluate their anti-inflammatory activity. Compounds 4a, 4b and 6a displayed inhibitory effects on nitric oxide (NO) production, and compound 4b exhibited the obvious anti-inflammatory activity, with an IC50 value of 6.91 ± 0.97 μM, by downregulating nuclear factor kappa B (NF-κB) and reducing the expression of inducible nitric oxide synthase (iNOS) in LPS-induced RAW264.7 cells. These results intimated that 4b could be used as a leading compound to develop anti-inflammatory drugs and is worthy of further investigated.

NO Release Inhibitory Activity of Flavonoids from Aesculus Wilsonii Seeds through MAPK (P38), NF-κB, and STAT3 Cross-Talk Signaling Pathways.

The flavonoid constituents of Aesculus wilsonii, a source of the Chinese medicinal drug Suo Luo Zi, and their in vitro anti-inflammatory effects were investigated. Fifteen flavonoids, including aeswilflavonosides IA-IC (1: - 3: ) and aeswilflavonosides IIA-IIE (4: - 8: ), along with seven known derivatives were isolated from a seed extract. Their structures were elucidated by extensive spectroscopic methods, acid and alkaline hydrolysis, and calculated electronic circular dichroism spectra. Among them, compounds 3: and 7: possess a 5-[2-(carboxymethyl)-5-oxocyclopent-yl]pent-3-enylate or oleuropeoylate substituent, respectively, which are rarely reported in flavonoids. Compounds 2, 3, 7: , and 12: - 15: were found to inhibit lipopolysaccharide-induced nitric oxide production in RAW 264.7 cell lines. In a mechanistic assay, the flavonoid glycosides 2, 3: , and 7: reduced the expressions of interleukin-6 and tumor necrosis factor-alpha induced by lipopolysaccharide. Further investigations suggest that 2: and 3: downregulated the protein expression of tumor necrosis factor-alpha and interleukin-6 by inhibiting the phosphorylation of p38. Compound 7: was found to reduce the production of inducible nitric oxide synthase, and the secretion of tumor necrosis factor-alpha and interleukin-6 through inhibiting nuclear factor kappa-light-chain-enhancer of activated B signaling pathway. Compounds 2, 3: , and 7: possessed moderate inhibitory activity on the expression of signal transducer and activator of transcription-3. Taken together, the data indicate that the flavonoid glycosides of A.wilsonii seeds exhibit nitric oxide release inhibitory activity through mitogen-activated protein kinase (p38), nuclear factor kappa-light-chain-enhancer of activated B, and signal transducer and activator of transcription-3 cross-talk signaling pathways.

Anti-Inflammatory Constituents of Antrodia camphorata on RAW 264.7 Cells Induced by Polyinosinic-Polycytidylic Acid.

Antrodia camphorata is an endemic mushroom in Taiwan. This study was designed to screen anti-inflammatory compounds from the methanolic extract of the mycelium of A. camphorata on nitric oxide (NO) production in RAW 264.7 cells induced by polyinosinic-polycytidylic acid (poly I:C), a synthetic analog of double-stranded RNA (dsRNA) known to be present in viral infection. A combination of bioactivity-guided isolation with an NMR-based identification led to the isolation of 4-acetylantroquinonol B (1), along with seven compounds. The structure of new compounds (4 and 5) was elucidated by spectroscopic experiments, including MS, IR, and NMR analysis. The anti-inflammatory activity of all isolated compounds was assessed at non-cytotoxic concentrations. 4-Acetylantroquinonol B (1) was the most potent compound against poly I:C-induced NO production in RAW 264.7 cells with an IC50 value of 0.57 ± 0.06 μM.

Tetra-, penta-, and hexa-nor-lanostane triterpenes from the medicinal fungus Ganoderma australe

Chemical investigation on the medicinal fungus Ganoderma australe led to the identification of ten new nor-lanostane triterpenes, namely two hexa-nor ones, ganoaustratetraenones A (1) and B (2), five penta-nor ones, ganoaustraldehydes A–E (3–7), and three tetra-nor ones ganoaustrenoic acids A–C (8–10). The chemical structures along with the absolute configurations were determined by extensive spectroscopic analysis of 1D & 2D NMR and HRESIMS data. The postulated biosynthesis pathways of these compounds were proposed. Ganoaustraldehydes A (3) and B (4) showed moderate inhibition against nitric oxide production in RAW264.7 macrophage cells with the respective IC50 values of 32.5, 34.2 µM (the IC50 of positive control pyrrolidine dithiocarbamate was 20.0 µM).Graphical

Effect of Fractions from Lycopus lucidus Turcz. Leaves on Genomic DNA Oxidation and Matrix Metalloproteinase Activity.

We investigated the inhibitory effects of fractions from Lycopus lucidus Turcz. leaves on genomic DNA oxidation, Nitric Oxide (NO) production, and Matrix Metalloproteinase (MMP) activity. Oxidative damage of genomic DNA was detected after Fenton reaction with H2O2 using DNA electrophoresis. Western blotting was performed to compare the expression levels of MMP-2 in phorbol 12-myristate 13-acetate (PMA)-induced HT-1080 cells. Lipopolysacchride (LPS)-induced NO production in RAW 264.7 cells was measured using Griess reagent. All fractions (n-Hexane, 85% aq. MeOH, n-BuOH, and water fractions) from the leaves of L. lucidus Turcz. significantly inhibited intracellular production of reactive oxygen species (ROS) (p<0.05). Particularly, 85% aq. MeOH and n-BuOH fractions showed higher ROS inhibitory activity than the other fractions. n-Hexane, 85% aq. MeOH, n-BuOH and water (0.05 mg/mL) fractions significantly inhibited oxidative DNA damage by 57.97%, 68.48%, 58.97%, and 68.39%, respectively (p <0.05). Treatment of RAW 264.7 cells with each fraction reduced LPS-induced NO production in a dose-dependent manner (p<0.05). n-Hexane and 85% aq. MeOH fractions notably reduced MMP-2 secretion levels in the culture supernatants from HT-1080 cells. Overall, these results indicated that L. lucidus Turcz. leaves can be exploited as plant based sources of antioxidants in the pharmaceutical, cosmetic, nutraceutical, and food industries.

Anti-inflammatory Polyketides from the Marine-Derived Fungus Eutypella scoparia.

Three new polyketides, eutyketides A and B (1 and 2) and cytosporin X (3), along with four known compounds (4–7), were obtained from the marine-derived fungus Eutypella scoparia. The planar structures of 1 and 2 were elucidated by extensive HRMS and 1D and 2D NMR analyses. Their relative configurations of C-13 and C-14 were determined with chemical conversions by introducing an acetonylidene group. The absolute configurations of 1–3 were determined by comparing their experimental electronic circular dichroism (ECD) data with their computed ECD results. All of the isolated compounds were tested for their anti-inflammatory activities on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophages. Compounds 5 and 6 showed stronger anti-inflammatory activities than the other compounds, with the inhibition of 49.0% and 54.9% at a concentration of 50.0 µg/mL, respectively.

The Anti-Inflammatory Activities of Fermented Curcuma That Contains Butyrate Mitigate DSS-Induced Colitis in Mice.

Inflammatory bowel disease is characterized by a radical imbalance of inflammatory signaling pathways in the gastrointestinal tract, and it is categorized into two diseases, such as Crohn’s disease and ulcerative colitis. In this study, we investigated anti-inflammatory activities using fermented Curcuma that contains butyrate (FB). Nitric oxide production in RAW 264.7 cells and the expression of inducible nitric oxide synthase in the intestinal mucosa appears to be enhanced in active ulcerative colitis. Here, the cytotoxicity, physiological activity, and anti-inflammatory efficacy of FB in colitis animals were investigated. To verify the anti-inflammatory effect, this study was conducted using the dextran sulfate sodium (DSS)-induced colitis mice model. As a result, non-toxicity was confirmed, and anti-inflammatory effects were revealed by inducing a reduction of LPS-induced NO production. In the DSS-induced colitis, reduced weight was recovered and a decrease in inflammatory factors Ig-E and TNF-α in the mesenteric lymph node (MLN) and spleen was induced, and it was confirmed to help with the morphological remodeling of the intestine. In conclusion, this paper suggests that FB can help to alleviate intestinal inflammation and to improve the intestinal environment, with the help of morphological remodeling.

Molecular Networking-Guided Isolation of Cycloartane-type Triterpenoids from Curculigo orchioides and Their Inhibitory Effect on Nitric Oxide Production.

The MolNetEnhancer workflow was applied to molecular networking analysis of the CH2Cl2-soluble fraction of the rhizomes of Curculigo orchioides, which showed a potent inhibitory effect on the lipopolysaccharide (LPS)-induced nitric oxide production. Among the molecular network, clusters of cycloartane-type triterpenoids were classified using the ClassyFire module of MolNetEnhancer, and their structures were predicted by the in silico fragment analysis tool, Network Annotation Propagation (NAP). Using mass spectrometry (MS)-guided isolation methods, six cycloartane-type triterpenoids (1–6) were isolated, and their structures were elucidated based on the interpretation of NMR, HRESIMS, and single-crystal X-ray diffraction. Among the isolates, compounds 1 and 4, which have an α,β-unsaturated carbonyl moiety on the A-ring, exhibited significant inhibitory effects on LPS-induced nitric oxide production in RAW264.7 cells with IC50 values of 12.4 and 11.8 μM, respectively.

강황 추출물의 항산화 및 면역 활성 증진 효과

This study sought to investigate the antioxidant and immune-stimulatory effects of different Curcuma longa (C. longa) extracts based on their color (random, light, medium, dark orange), the soil of cultivation (clay, granite), drying temperature (50∼80℃), and extraction methods (water, ethanol). The antioxidant factors of C. longa were determined by the total phenolic content and DPPH and ABTS radical scavenging activities. The nitric oxide (NO) production of the macrophage cell line (RAW264.7) and the proliferation of splenocytes were investigated to assess the immune-enhancing effects of C. longa. The antioxidant activity was higher in the dark color and the ethanol extracts. All the types of extracts were nontoxic and enhanced the production of NO in RAW264.7 at less than 250 ㎍/mL of C. longa. When mouse splenocytes were exposed to various concentrations of hot water or ethanol extracts of C. longa with media, concanavalin A (Con A), lipopolysaccharide (LPS), and cyclophosphamide (CPA), their proliferation increased. Specifically, the 70% ethanol extract of C. longa showed the highest levels of splenocyte proliferation among all samples. These results suggest that the antioxidant and immune-stimulating activities of C. longa were most affected by the extracting solvent rather than color, soil in which it was cultured, and drying temperature. Appropriate extraction methods for C. longa should be researched further to develop good healthy foods with high antioxidant and immune-enhancing activities.

Design, synthesis and anti-inflammatory evaluation of aloe-emodin derivatives as potential modulators of Akt, NF-κB and JNK signaling pathways

To discover novel anti-inflammatory agents, a series of nitrogen-containing derivatives of aloe-emodin were designed and synthesized. The anti-inflammatory activities of all synthesized derivatives were screened by evaluating their inhibitory effects on LPS-induced nitric oxide production in RAW264.7 macrophages. The preliminary structure-activity relationship was determined. Among them, 2i exhibited the best nitric oxide inhibitory activity (IC50 = 3.15 μM), with no obvious toxicity. Further evaluation of the inhibitory effect of 2i on inflammatory cytokines showed that 2i significantly reduced the levels of TNF-α, IL-1β, IL-6 and PGE2. In addition, 2i markedly downregulated the expression levels of iNOS and COX-2. Preliminary mechanistic studies indicated that the anti-inflammatory effect of 2i might be related to the inhibition of the Akt, NF-κB and JNK signaling pathways. Overall, our findings suggested that 2i might be a promising anti-inflammatory agent, or might serve as a new lead compound for the further development of anti-inflammatory agents.

Neuropeptide FF-related gene in fish (Larimichthys polyactis): identification, characterization, and potential anti-inflammatory function.

Neuropeptide FF (NPFF), an octapeptide of the RFamide-related peptides (FaRPs), is involved in regulatory function in various biological processes. The regulatory role of NPFF in the immune and inflammatory response was currently being revealed. Neuropeptide FF-related gene (termed LpNPFF) and its two receptors, NPFF receptor 1 (LpNPFFR1) and NPFF receptor 2 (LpNPFFR2) were identified by PCR and Semi-quantitative RT-PCR assay. Effect of LpNPFF on the production of nitric oxide (NO) in macrophage RAW264.7 cell was divided into PBS group, lipopolysaccharide (LPS) group, LPS treated with LpNPFF group, and LPS treated with LpNPFF and receptor antagonist RF9 group. Then specimens were measured by color reaction at 570nm absorbance value. Sequence analysis showed that LpNPFF cDNA consists of 835 nucleotides with a 5'- untranslated region (UTR) of 150 base pair (bp), an open reading frame (ORF) of 384bp and a 3'-UTR of 300bp (Accession No. MT012894). The ORF encodes 127 amino acid (aa) residues with a hydrophobic signal peptide at N-terminus and two presumptive peptides with -PQRFa structure, LpNPFF (1) and LpNPFF (2). LpNPFFR1 and LpNPFFR2 encode 427 and 444 aa residues respectively, which both have seven hydrophobic TMDs and identified as G protein coupled receptors (GPCRs). Results of tissue distribution showed that LpNPFF and receptors were highly expressed in the brain and gonad. Furtherly, in vitro assay found LpNPFF could inhibit NO production in RAW 264.7 macrophages under inflammatory stress with LPS, while its receptor antagonist RF9 caused the evoke of NO generation. These results contribute to the further study of neuropeptide evolution in marine organisms, and also provide a new research idea for exploring the related functions of NPFF gene.