NO Production In Macrophages Research Articles

Ethnopharmacological analysis from Thai traditional medicine called prasachandaeng remedy as a potential antipyretic drug

Ethnopharmacological relevancePrasachandaeng (PSD) remedy is a famous antipyretic drug for adults and children in Thai traditional medicine used and is described in Thailand's National List of Essential Medicine. Relationship between the taste of this herbal medicine, ethnopharmacological used and its pharmacological properties was reviewed. Aims of studySince there has been no scientific report on the antipyretic activity of PSD, aim of this study was to investigate the efficacy related antipyretic drug of the remedy and its 12 herbal ingredients. It involved quality evaluation of raw materials, extraction of PSD and its ingredients, in vitro evaluation of their inhibitory activities on fever mediators, i.e. NO and PGE2 production in murine macrophage (RAW 264.7) cell line stimulated by lipopolysaccharide, and its stability study of the 95% ethanolic extract of PSD remedy. Materials and methodsPSD remedy was extracted by maceration with 50% and 95% ethanol (PSD50 and PSD95), by decoction with distilled water (PSDW), and hydrolysis of PSDW with 0.1 N HCl (PSDH). The 12 plant ingredients were extracted with 95% ethanol. Quality evaluation of PSD ingredients was performed according to the standard procedures for the quality control of herbal materials. The inhibitory activity on nitric oxide production was determined by the Griess reaction and the inhibition of prostaglandin E2 production was determined using the ELISA test kit. ResultsPSD ingredients passed the quality standard stipulated for herbal materials. PSD95 exhibited the highest inhibitory activities on the production of NO and PGE2 with the IC50 values of 42.40 ± 0.72 and 4.65 ± 0.76 μg/mL, respectively. A standard drug acetaminophen (ACP) exhibited inhibition of NO and PGE2 production with the IC50 values of 99.50 ± 0.43 and 6.110 ± 0.661 μg/mL, respectively. The stability study was suggested two years shelf-life of PSD95. This is the first report on the activity related antipyretic activity of PSD remedy and its ingredients against two fever mediators, NO and PGE2. ConclusionThe results suggested that the 95% ethanolic extracts of PSD remedy and some of its ingredients, were better than ACP in reducing fever. PSD should be further studied using in vivo models and clinical trials to support its use as an antipyretic drug in Thai traditional medicine.

Neutral Lipids, Glycolipids, and Phospholipids, Isolated from Sandfish (Arctoscopus japonicus) Eggs, Exhibit Anti-Inflammatory Activity in LPS-Stimulated RAW264.7 Cells through NF-κB and MAPKs Pathways.

Total lipids were extracted from sandfish (Arctoscopus japonicus), and then they were separated into the following three lipid fractions: neutral lipids, glycolipids, and phospholipids. In this study, we analyzed the lipid fractions of A. japonicus eggs and we determined their anti-inflammatory activity in RAW264.7 macrophage cells. In these three lipid-fractions, the main fatty acids were as follows: palmitic acid (16:0), oleic acid (18:1n-9), docosahexaenoic acid (DHA, 22:6n-3), and eicosapentaenoic acid (EPA, 20:5n-3). Among the lipid fractions, phospholipids showed the highest concentration of DHA and EPA (21.70 ± 1.92 and 18.96 ± 1.27, respectively). The three lipid fractions of A. japonicus significantly suppressed the production of NO in macrophages. Moreover, they also significantly inhibited the expression of iNOS, COX-2, IL-6, IL-1β, and TNF-α, in a dose-dependent manner. Furthermore, the lipid fractions of A. japonicus suppressed the nuclear translocation of NF-κB p65 subunits in a dose-dependent manner. In addition, they attenuated the activation of MAPKs (p38, ERK1/2, and JNK) phosphorylation in LPS-stimulated RAW264.7 cells. These results indicate that all the lipid fractions of A. japonicus exert anti-inflammatory activity by suppressing the activation of NF-κB and MAPK pathways. Therefore, the lipid fractions of A. japonicus might be potentially used as anti-inflammatory agents.

Thiosemicarbazone and thiazole: in vitro evaluation of leishmanicidal and ultrastructural activity on Leishmania infantum

Leishmaniasis is a much-neglected tropical disease, especially in developing countries. Visceral Leishmaniasis (VL) is the most severe form of this infection caused by Leishmania infantum specie. It affects the splenic and hepatic systems and, when left untreated, it is fatal in 95% of cases. There is no human vaccine available and the recommended treatment not only presents adverse effects but it enables the development of resistant strains if interrupted. Hence, the urgent search for new leishmanicidal compounds. In the present work, we evaluated the in vitro leishmanicidal action of new thiosemicarbazone and thiazolidine compounds against the evolutionary forms of L. infantum, as well as their hemolytic activity, ultrastructural alterations, cytotoxicity, and nitric oxide levels on peritoneal macrophages. The inhibitory concentration 50% (IC50) against promastigote forms varied from 8.62 to 34.36 µM. In the evaluation of cytotoxicity, values of CC50 in peritoneal macrophages varied from 31.80 to 196.38 µM. Data showed that compounds JW-16.2 and GT-14 were the most promising in the series as they displayed the highest CC50 (184.58 and 196.38 μM), SI (19 and 15), low hemolytic activity, induced NO production in uninfected and infected macrophages and reduced survival of amastigotes (24.39 and 16.51 μM). Ultrastructural alterations, such as shrinkage of the cell body, shortening of the flagellum, and vacuolization of the cytoplasm, were identified. Promastigote forms treated with compounds GT-14 showed depolarization of mitochondria. Therefore, both compounds are promising due to low cytotoxicity to mammalian cells and leishmanicidal action.

Effects of carboxymethyl chitosan oligosaccharide on regulating immunologic function and inhibiting tumor growth

Herein, the effects of carboxymethyl chitosan oligosaccharide (CM-COS) on regulating immunologic function and inhibiting hepatocellular tumor growth were evaluated. Results showed that CM-COS caused dramatic viability loss of hepatocellular carcinoma BEL-7402 with non-toxicity towards normal liver L-02 cells. CM-COS repressed tumor growth of hepatoma-22, and elevated the spleen index and thymus index of tumor-bearing mice. Contents of VEGF and MMP-9 were significantly down-regulated by CM-COS. Histological analyses revealed that CM-COS promoted tumor cell necrosis and produced no significant toxicity to spleen tissues. Moreover, expressions of Caspase-3 in tumor tissues and IL-2 in spleen tissues were significantly activated by CM-COS. Additionally, in vitro cell viability, phagocytic capability and NO production of mouse peritoneal macrophages exposed to CM-COS were significantly higher. CM-COS remarkably increased the in vivo phagocytosing capacity of peritoneal macrophages of Kunming mice. Taken together, our findings suggested that CM-COS might be potentially effective and non-toxic candidate as anti-hepatoma agents.

Four new indole alkaloids from the roots of Isatis tinctoria

Chemical fractionation of the ethanolic extract of the roots of Isatis tinctoria L. (I. tinctoria) yielded fourteen indole alkaloids including four new ones, isatisindigoticanine L–N and isatindigoside M. Their structures were established on the basis of extensive spectroscopic techniques including NMR, HRESIMS and IR as well as chemical methods. The absolute configuration of 1 was confirmed by ECD experiments. All the compounds were tested for their inhibitory effects on NO production in mouse mononuclear macrophages (RAW264.7) induced by lipopolysaccharide (LPS). The results showed that only compound 5 exhibited inhibitory effects with IC50 value of 18.5 μM.

Synthesis and inhibitory effect of cis-guggulsterone on lipopolysaccharide-induced production of nitric oxide in macrophages

Guggulsterone is a bioactive plant sterol naturally found in migratory plants. It exists in various forms, and its active compounds include the isomers cis-guggulsterone (E-GS) and trans-guggulsterone (Z-GS). In this study, the anti-inflammatory effects of these two isomeric pregnadienedione steroids were investigated against lipopolysaccharide-induced inflammatory reaction in RAW264.7 mouse macrophages. Our results showed that both guggulsterones inhibited the production of NO in macrophages treated with lipopolysaccharide, with IC50 values ranging from 3.0 to 6.7 μM. E-GS exerted higher efficacy than Z-GS, and its anti-inflammatory effects was mediated through inhibition of iNOS and COX-2 expression.

Ethanol extract of Elephantopus scaber Linn. Attenuates inflammatory response via the inhibition of NF-κB signaling by dampening p65-DNA binding activity in lipopolysaccharide-activated macrophages.

Elephantopus scaber Linn. (E.scaber) is a widely-used traditional herb whose use has been documented for various inflammatory diseases such as fever, sore throat, dysentery, carbuncle and so on. However, the effect and mechanism of E.scaber in LPS-activated macrophages remain unclear. This study aims to investigate the anti-inflammatory mechanism of the ethanol extract of E.scaber (ESE) in lipopolysaccharide (LPS)-induced inflammatory models. Griess reagent was used to determine NO production, and the levels of TNF-α, IL-6, MCP-1 and IL-1β were determined by ELISA kits. The molecular mechanism research was performed by RT-PCR, Western blot, and electrophoretic mobility shift assay (EMSA). LPS-induced endotoxemia mouse model was used for evaluating the in vivo anti-inflammatory action of ESE. ESE suppressed LPS-induced iNOS, TNF-α, IL-6, MCP-1 and IL-1β transcription as well as supernatant NO, TNF-α, IL-6, MCP-1 and IL-1β production in macrophages. Although ESE inhibited NF-κB activation, it did not affect the IκBα phosphorylation and degradation and the NF-κB p65 nuclear translocation. The result of EMSA revealed that ESE inhibited the NF-κB p65-DNA binding activity. Additionally, ESE also decreased the proinflammatory cytokines in serum and peritoneal lavage fluid of LPS-induced endotoxemic mice. ESE has a potently anti-inflammatory effect through inhibiting the NF-κB p65-DNA binding activity in LPS-activated macrophages.

A Comparison of Characterization and Its Actions on Immunocompetent Cells of Polysaccharides from Sijunzi Decoction

Sijunzi decoction (SJZD) is a classic recipe in Traditional Chinese Medicine (TCM) with strong immune-enhancement activity. To further understand the characterization and immunomodulatory effect of polysaccharides from SJZD, the monosaccharide compositions of crude polysaccharide (SJZDP), polysaccharide fraction (S-3), and homogeneous polysaccharide (S-3-AG) from SJZD were compared by GC analysis, as well as their immunomodulatory effects on Peyer's patch cells, splenocytes, and macrophages which are related to intestinal immunity, specific immunity, and nonspecific immunity. The results showed that S-3-AG mainly contained Ara with a proportion of 38.9%, while Glc accounted for the largest proportion in S-3 (55.6%) and SJZDP (87.6%). The SJZDP, S-3, and S-3-AG all showed strong capability to stimulate Peyer's patch cells to proliferate and produce IgA and promoted the proliferation and IFN-γ production of splenocytes and increased the NO production and TNF-α production of macrophages. However, S-3 and S-3-AG were able to stimulate splenocytes to secret IL-4, SJZDP had no effect on IL-4 production of splenocytes in the tested concentrations. In addition, S-3 could stimulate the phagocytic activity of macrophages, and S-3-AG restrained the proliferation of macrophages at the concentration of 50–200 µg/mL. These results suggested that SJZDP, S-3, and S-3-AG might have different immunomodulatory effects on intestinal immunity, specific immunity, and nonspecific immunity due to their different monosaccharide compositions. It will provide references for the material basis and mechanism of SJZD immunomodulation activity.

Supercritical Fluid Extract of Angelica sinensis and Zingiber officinale Roscoe Ameliorates TNBS-Induced Colitis in Rats.

Inflammatory bowel disease (IBD) is a worldwide healthcare problem calling for the development of new therapeutic drugs. Angelica sinensis and Zingiber officinale Roscoe are two common dietetic Chinese herbs, which are traditionally used for complementary treatment of gastrointestinal disorders. As bioactive constituents, volatile and pungent substances of these two herbs could be effectively extracted together by supercritical fluid extraction. In this study, the supercritical fluid extract of Angelica sinensis and Zingiber officinale Roscoe (AZ-SFE) was obtained by an optimized extraction process and it was chemically characterized. The anti-inflammatory effect and underlying mechanism of AZ-SFE were evaluated in a lipopolysaccharide (LPS)-induced RAW264.7 cell model and a 2, 4, 6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rat model. AZ-SFE notably inhibited the production of NO in LPS-stimulated macrophages, and it inhibited the proliferation of Concanavalin A (Con A)-induced splenocytes with suppression of the Th1 immune response. In vivo, the study demonstrated that AZ-SFE significantly alleviated disease activity, colonic shortening, macroscopic damage and histological injury of TNBS-treated rats with reduction of oxidative stress, suppression of inflammatory cytokines, and modulation of hepcidin and serum iron. These findings suggested that AZ-SFE may be a promising supplement for current IBD therapy.

Dihydroagarofuran sesquiterpenoids esterified with organic acids from the leaves of Tripterygium wilfordii

Six new and one known dihydroagarofuran sesquiterpenoids esterified with organic acids were obtained from the leaves of Tripterygium wilfordii. Spectroscopic techniques (UV, IR, MS, NMR, ORD and CD) were used for the structure elucidation of the compounds. The structures of compounds 1 and 2 were confirmed by X-ray single crystallographic analyses. The inhibitory effects on NO production in LPS-induced macrophages of 1–7 were conducted. At 10 μmol/L, compounds 1, 2 and 7 showed moderate inhibitory effects on NO production in LPS-induced macrophages with inhibitory rate at 31.2 ± 3.6, 40.9 ± 4.3, and 66.79 ± 3.1%, respectively.

Xylose rich heteroglycan from flaxseed gum mediates the immunostimulatory effects on macrophages via TLR2 activation

Immunostimulatory activity of the flaxseed gum neutral fraction (NFG) was investigated. NFG was characterized as a xylose rich heteroglycan through monosaccharide composition analysis, FT-IR, methylation/GC–MS, and 1D/2D-NMR. NFG stimulated NO production and phagocytic activity of macrophages. Secretion of interleukin-6, interleukin-1β, and tumor necrosis factor-α (254.7 pg/mL, 2.5 ng/mL, and 42.9 pg/mL, respectively) was significantly induced by NFG. Mitogen-activated protein kinases of JNK and P38 were activated by NFG with increased phosphorylation of JNK and P38, while NO production was reduced to 6.05 and 4.42 μM by JNK and P38 inhibitor, respectively. Nuclear factor-κB signaling pathway was also activated by NFG with the suppression of IκBα and up-regulation of phosphorylation of IκBα and nuclear factor-κB P65. Toll like receptor-2 was the molecular target of NFG and responsible for the activation of down-stream signaling pathways. Thus, NFG from flaxseed gum may potentially be used as a natural immunomodulator in functional foods.

Immune-enhancing effects of anionic macromolecules extracted from Codium fragile on cyclophosphamide-treated mice.

Immune-regulation and homeostasis are critical in cancer therapy and immunomodulatory biomaterials have been used to decrease side effects of immunosuppressant drugs. Anionic macromolecules (CFAMs) were isolated from the seaweed Codium fragile, and its immune-enhancing biological activities were examined in CY-induced immunosuppressed mice. CFAMs improved the splenic lymphocyte proliferation, NK cell activity, and spleen index. The expression of immune-associated genes was highly upregulated in splenic lymphocytes, and gene expression was differently regulated according to mitogens such as T-cell (Con A) and B-cell (LPS) mitogens. Additionally, CFAMs boosted the proliferation, NO production, and phagocytosis of peritoneal macrophages. CFAMs also considerably stimulated immune-associated gene expression in peritoneal macrophages. Moreover, our results showed CFAMs mediated its immune-enhancing effects via the MAPK pathway. These suggested CFAMs can be used as a potent immunomodulatory material under immune-suppressive condition. Furthermore, CFAMs may also be used as a bio-functional and pharmaceutical material for improving human health and immunity.

CD200 Modulates S. aureus-Induced Innate Immune Responses Through Suppressing p38 Signaling.

Rapid activation of macrophages plays a central role in eliminating invading bacteria as well as in triggering the inflammatory responses, but how the anti-bacterial and the inflammatory responses are coordinated, in terms of macrophages, is not completely understood. In this study, we demonstrated that Staphylococcus aureus (S. aureus) induced the expression of CD200 in murine macrophages in a dose-dependent manner. We found that CD200 significantly suppressed the S. aureus-induced production of nitric oxide and proinflammatory cytokines in mouse macrophages. Concurrently, the bactericidal capability of macrophages was boosted upon the deletion of CD200. Furthermore, our data demonstrated that p38 mitogen-activated protein kinase (MAPK) was selectively down-regulated by CD200 administration, while enhanced upon CD200 silence in response to staphylococcal infection. The negative effect of CD200 siRNA on NO production in macrophages was largely abrogated upon the inhibition of p38 signaling, implying its critical involvement in this regulation. Together, our data demonstrate that CD200 plays a central role in regulating the inflammatory responses and the anti-bacterial activity of macrophages, at least partially, through suppressing p38 activity.

Localized Therapy of Vaginal Infections and Inflammation: Liposomes-In-Hydrogel Delivery System for Polyphenols

Natural polyphenols, such as resveratrol (RES) or epicatechin (EPI), are attractive for treatments of various diseases, including vaginal infections and inflammation, because of their strong anti-oxidative and anti-inflammatory properties. However, their low solubility and consequent poor bioavailability limit their therapeutic uses. To overcome these limitations, a vaginal delivery system comprising either RES or EPI liposomes-in-hydrogel was developed. This system permits therapeutic action of both liposomal polyphenol (RES or EPI) and chitosan-based hydrogel. Liposomes of around 200 nm and entrapment efficiency of 81% and 77% for RES and EPI, respectively, were incorporated into chitosan hydrogel, respectively. Medium molecular weight chitosan (2.5%, w/w) was found to have optimal texture properties and mucoadhesiveness in ex vivo conditions. The in vitro release studies confirmed the sustained release of polyphenols from the system. Both liposomal polyphenols and polyphenols-in-liposomes-in-hydrogel exhibited only minor effects on cell toxicity. EPI showed superior radical scavenging activity at lower concentrations compared to antioxidants vitamin C and E. Anti-inflammatory activity expressed as the inhibitory activity of formulations on the NO production in the LPS-induced macrophages (RAW 264.7) confirmed the superiority of EPI liposomes-in-hydrogel. The plain liposomes-in-hydrogel also exhibited potent anti-inflammatory activity, suggesting that chitosan hydrogel acts in synergy regarding anti-inflammatory effect of formulation.

In vitro approach for evaluation of carob by-products as source bioactive ingredients with potential to attenuate metabolic syndrome (MetS)

The potential bioactivities for alleviating Metabolic Syndrome associated risk factors were evaluated in carob (Ceratonia siliqua L.) fruit by-products, i.e. seed peel, germ and pod. Carob germ and seed peel showed higher phenolic content than pod (99.72, 80.24 and 47.06 μmol GAE g−1, respectively). Pod mostly contained gallic acid and gallotannins; seed peel and germ's showed as most abundant polyphenols quercetin and apigenin derivatives. Carob pod and seed peel revealed stronger antioxidant capacities compared to germ. The strongest antihypertensive activity was found in seed peel, followed by pod and germ. Anti-inflammatory activity showed inhibition of NO production in LPS-induced macrophages, although only pod was able of reducing pro-inflammatory mediators (TNF-α andPGD2). Finally, fat accumulation on mature adipocytes was reduced by carob seed peel and pod extracts. This work shows the potential use of pod carob by-products as food ingredients with special relevance of carob pod for attenuating metabolic syndrome.

Chemical constituents from the bark of bauhinia purpurea and their NO inhibitory activities

Phytochemical studies led to the isolation of a new phenylpropanoid glucoside, named purpuroside (1), along with eight known compounds (2–9) from the bark of Bauhinia purpurea. The structure of the new compound was elucidated on the basis of its spectroscopic data. The absolute configuration of compound 2 was verified by X-ray diffraction analysis. Compounds 1, 2, 7, 8, and 9 inhibited NO production in mouse peritoneal macrophages with IC50 values from 35.5 to 63.0 μM.

Anti-inflammatory bibenzyls from the stems of Dendrobium huoshanense via bioassay guided isolation

The stems of Dendrobium huoshanense have long been used to prevent various diseases, including inflammatory diseases. This study was aimed to explain the anti-inflammatory effect of D. huoshanense stems in LPS-induced RAW 264.7 macrophages and to discover potential anti-inflammatory compounds. Results exhibited that D. huoshanense stems ethanol extract could significantly inhibit LPS-induced production of NO, TNF-α and IL-1β. Based on bioassay guided strategy, four bibenzyls (1–4) were isolated from D. huoshanense stems for the first time. Anti-inflammatory assay showed 1–4 could remarkably inhibit the production of NO in LPS-induced macrophages. Moreover, quantitative RT-PCR analysis displayed that the mRNA levels of iNOs, TNF-α and IL-1β could also be significantly reduced by 1–4. These results suggested that D. huoshanense stems ethanol extract and bibenzyls 1–4 might be well developed as therapeutic agent to prevent inflammatory diseases.

Potential immunomodulatory activity of Phyllanthus niruri aqueous extract on macrophage infected with Streptococcus sanguinis

Background: Streptococcus sanguinis is an oral commensal bacterium commonly found in periodontal lesions and deep abscesses that are usually dominated by anaerobic bacteria. As an important causative agent of systemic diseases, and with the increasingly numerous cases of antimicrobial resistance, some means of modulating the immune response to bacterial infection is thus necessary. Phyllanthus niruri Linn is widely used as a medicinal herb to both prevent and treat disease and demonstrates immunomodulatory properties. Purpose: This study aimed to observe the potential for aqueous extract of Phylanthus niruri to induce macrophage proliferation and NO production following S. sanguinis infection. Methods: Macrophages were isolated from the peripheral blood of healthy subjects, stimulated with P. niruri aqueous extract in graded doses and infected with S. sanguinis ATCC 10556 bacterial suspension. Cell proliferation and nitric oxide release was observed at 24 and 48 hours to determine macrophage activities. Results: NO production and cell proliferation started to increase upon 50 and 100µg/ml P niruri respective stimulation. Statistical analysis using One-way Anova demonstrated a significant difference of cell proliferation after stimulation with P. niruri aqueous extract at various doses (p<0.05). Conclusion: P. niruri aqueous extract induced macrophage proliferation and NO secretion upon S sanguinis infection, showing potential antibacterial and immunomodulatory activities. At the same concentrations, NO production and macrophage were higher at 48 hours than at 24 hours.

Pogonatherumol, a Novel Highly Oxygenated Norsesquiterpene with Flavone C-Glycosides from Pogonatherum crinitum

A novel highly oxygenated norsesquiterpene, pogonatherumol (1), with two known flavone C-glycosides (2-3), was isolated from Pogonatherum crinitum. The structure of the new compound was illuminated based on its spectroscopic data and X-ray analysis. Compounds 1 and 3 inhibited NO production in the mouse peritoneal macrophage (64.5 ± 7.2% and 61.6 ± 5.8%, respectively, at a concentration of 50 μM). The three compounds were inactive when tested against two human cancer cell lines (IC50 values > 50 μM).

Conjugation of chitosan oligosaccharides enhances immune response to porcine circovirus vaccine by activating macrophages

Porcine circovirus type 2 (PCV2)-associated diseases have led to great economic losses to the pig industry. Our lab previously found that conjugation of chitosan oligosaccharides (COS) or via a carrier protein enhanced the immunogenicity of PCV2 vaccine against infectious pathogens. However, precise mechanisms and signal transduction pathways underlying the efficacy of COS conjugation remains poorly defined. In this study, to better understand the effects and mechanism of COS conjugates maintain the adjuvant potential in vivo, we investigated its augmentation of macrophage function, including cell activation, NO production, cytokine production and phagocytosis. Additionally, the role of Toll-like receptors (TLR) proteins in this process was also assessed. The results indicate that, as compared to the PCV and PCV/COS, conjugation of COS effectively enhanced the NO production, cytokines generation and phagocytosis activity of macrophages. Noticeably, the generation of NO and proinflammatory cytokines was closely related to the TLR2/4 signaling pathways, strongly suggesting that conjugation of COS regulates innate and adaptive immunity by activation of macrophages, resulting in immune enhancement. In summary, the present study provides a potential mechanism of COS conjugation as a novel adjuvant to improve immune responses against various diseases.