Establishment of indirect enzyme-linked immunosorbent assay for artificial musk.

Anti-inflammatory component (AIC) known as an important constituent of artificial musk exhibits bioactive effects on many pharmacological models. This study describes an immunochemical assay for the quality control of artificial musk in traditional Chinese medicine using an enzyme-linked immunosorbent assay. A polyclonal antibody against AIC was produced by rabbit. The method, at an effective measuring range of 3.13-200 ng/ml of AIC, 3 ng/ml limit of detection, and no cross-reaction with natural musk, successfully detected artificial musk in many traditional Chinese prescriptions containing artificial musk. The results demonstrated that a novel and reliable assay system for detection of artificial musk was generated.

Resolution and chiral recognition of muscone as well as actions on neural system.

In this letter, (R)-muscone and (S)-muscone were prepared by optical resolution of dl-muscone using N,N'-dibenzyl-l-tartaramide or N,N'-dibenzyl-d-tartaramide, according to the method reported by Kunihiko Takabe. But we separated the diastereomers using EtOH instead of MeOH in the recrystallization step to get the goal product with higher optical purity and yield. The regulatory effect of muscone and its enantiomer on the neural system showed that they could prolong mouse hypoxia tolerance and dose-dependently enhance mouse spinal cord stimulation induced by strychnine nitrate. (R)-muscone and (S)-muscone had a synergistic action on shortening sleep time induced by sodium pentobarbital in mice.

Effect of artificial musk aqueous extract on the expressions of inflammatory mediators released from lipopolysaccharide-stimulated RAW264.7 cells.

OBJECTIVE: To identify the anti-inflammatory effects of artificial musk aqueous extract(AME)on lipopolysaccharide-stimulated cytokines secreted or released by RAW264.7 cells. METHODS: Cytokines including interleukin(IL)-6,IL-10,and tumor necrosis factor Α were determined using cytokine enzyme-linked immunosorbent assay kits. RESULT: Compared with model group,the levels of major cytokines such as tumor necrosis factor Α,IL-6,and IL-10 significantly decreased in different AME groups in a dose-dependent manner. CONCLUSION: In lipopolysaccharide-stimulated RAW264.7 macrophages,AME can remarkably inhibit the release of inflammatory cytokines and thus exerts its anti-inflammatory effects.

An aptamer-based assay for thrombin via structure switch based on gold nanoparticles and magnetic nanoparticles.

An aptamer-based assay for thrombin with high specificity and sensitivity was presented. In the protocol, the aptamer for thrombin was immobilized on magnetic nanoparticle, and its complementary oligonucleotide was labeled with gold nanoparticles, then the aptamer was hybridized with the complementary oligonucleotide to form the duplex structure as a probe, this probe could be used for the specific recognition for thrombin. In the presence of thrombin, the aptamer prefer to form the G-quarter structure with thrombin, resulting in the dissociation of the duplex of the probe and the release of the gold labeled oligonucleotide. Upon this, we were able to detect thrombin through the detection of the electrochemical signal of gold nanoparticles. The strategy combines with the high specificity of aptamer and the excellent characteristics of nanoparticles. This assay is simple, rapid, sensitive and highly specific, it does not require labeling of thrombin, and it could be applied to detect thrombin in complex real sample. The method shows great potential in other protein analysis and in disease diagnosis.

[Effects of dihydroxy-stilbene compound Vam3 on airway inflammation, expression of ICAM-1, activities of NF-kappaB and MMP-9 in asthmatic mice].

The aim of the present study is to investigate the effects of Vam3 which is one of the dihydroxystilbene compounds on expressions of ICAM-1 in the lungs of OVA-induced asthmatic mice and the mechanisms of anti-airway inflammation. Balb/c mice were challenged with OVA inhalation. Lung tissues were stained with Mayer's hematoxylin and eosin for histopathologic examination. The expression of ICAM-1 in the lungs of mice was analyzed by Western blotting and immunohistochemistry method. The NF-kappaB activities were detected by NF-kappaB-luc reporter genetic transient transfection method. The activities of MMP-9 induced by LPS, TNF-alpha and PMA in THP-1 cells were determined by gelatin zymography method. The results showed that Vam3 could inhibit the expression of ICAM-1 in the OVA-induced mouse model. In addition, Vam3 could significantly suppress the activities of NF-kappaB in A549 cells and MMP-9 in THP-1 cells induced by LPS, TNF-alpha and PMA. These results suggested that Vam3 could alleviate the asthmatic inflammation by decreasing ICAM-1 expression in asthmatic mice, down regulating NF-kappaB and MMP-9 activities. Compound Vam3 showed inhibitory effects on inflammatory signal pathways involved in asthma.

Synthesis of calophyllolide analogue and its preliminary anti-inflammatory activity.

A new pyranocoumarin as an analogue of calophyllolide, compound 6, is firstly prepared and reported to have potent anti-inflammatory activity on carrageenin-induced edema in rats.

[Synthesis and anti-inflammatory activity of hydroxylated E,E-1-(3'-indolyl)-5-(substituted phenyl)-1,4-pentadien-3-one derivatives].

AIM: A series of new 1,4-pentadien-3-one derivatives were synthesized to search for new Eight novel hydroxylated non-steroidal anti-inflammatory drugs (NSAIDs) with potent activity. METHODS: E,E-1-(3'-indolyl)-5-( substituted phenyl)-1,4-pentadien-3-one derivatives were synthesized by means of aldol condensation and characterized by 1H NMR, ESI-MS and element analysis. Their anti-inflammatory activity in vitro were evaluated. RESULTS: Preliminary in vitro pharmacological tests showed that all compounds exhibited anti-inflammatory activity. CONCLUSION: Compounds 4d and 4e exhibited potent anti-inflammatory activity and their anti-inflammatory activity was comparable to resveratrol, and were worthy of further study.

Anti-inflammatory effect of amurensin H on asthma-like reaction induced by allergen in sensitized mice.

AIM: To explore the anti-inflammatory effects of amurensin H on asthma-like reaction induced by allergen in sensitized mice. METHODS: BALB/c mice were sensitized by ovalbumin (OVA, ip) on d 0 and d 14 and challenged with 1% OVA on d 18 to 22. Mice developed airway eosinophilia, mucus hypersecretion, and elevation in cytokine levels. Mice were administered amurensin H orally at the doses of 49, 70, or 100 mg/kg once every day from d 15 to the last day. Bronchoalveolar lavage fluid (BALF) were collected at 24 h and 48 h after the last OVA challenge. Levels of tumor necrosis factor-alpha (TNF-alpha), interleukin 4 (IL-4), interleukin 5 (IL-5), and interleukin 13 (IL-13) in BALF were measured using ELISA method. Differential cell counts of macrophages, lymphocytes, neutrophils and eosinophils were performed in 200 cells per slide (one slide per animal). Lung tissue sections of 6-mum thickness were stained with Mayer's hematoxylin and eosin for assessment of cell infiltration, mucus production, and tissue damage. RESULTS: Oral administration of amurensin H significantly inhibited OVA-induced increases in total cell counts, eosinophil counts, and TNF- alpha, IL-4, IL-5 and IL-13 levels in BALF. In addition, amuresin H dramatically decreased OVA-induced lung tissue damage and mucus production. CONCLUSION: Amurensin H may have therapeutic potential for the treatment of allergic airway inflammation.

[Effect of anti-inflammatory drugs on the NF-kappaB activation of HEK293 cells].

AIM: To investigate the regulatory effects of various anti-inflammatory drugs on both endogenous and TNFalpha-induced NF-kappaB activation as well as the relative biological activity. METHODS: HEK293 cells were cultured in 96-well plate and 6-well plate, treated with meloxicam, indomethacin, dexamethasone and hydrocortisone, without or with 10 ng.mL(-1) TNFalpha for 24 hours. Then cell proliferation was measured by MTT and cell apoptosis was analyzed by pI stain-flow cytometry. HEK293/ kappaB-luc cells transfected stably with pElam-kappaB-luc vector, were cultured in 96-well plate and treated as above. Equal amounts of cell lysates were tested for luciferase activity which represents NF-kappaB activation. RESULTS: Endogenous NF-kappaB activation was present in HEK293 cells and its level can be increased about 2 times by 10 ng.mL(-1) TNFalpha-induction. Dexamethasone (1 x 10(-8) mol.L(-1)) and meloxicam (1 x 10(-7) - 1 x 10(-6) mol.L(-1)) can decrease both endogenous and TNFalpha-induced NF-kappaB activation. Hydrocortisone (1 x 10(-9) mol.L(-1)) increases endogenous NF-kappaB activation but decreases TNFalpha-induced one significantly. No influence of indomethacin on endogenous NF-kappaB activation was observed. However, its influence on TNFalpha-induced NF-kappaB activation is needed for further study. Cell apoptosis was observed after treatment with TNFalpha and 1 x 10(-8), 1 x 10(-6) mol.L(-1) dexamethasone and 1 x 10(-7) mol.L(-1) indomethacin, or only with dexamethasone. No significant effect of these anti-inflammatory drugs on cell proliferation was observed. CONCLUSION: Various anti-inflammatory drugs differ in their ability to regulate NF-kappaB activation in HEK293 cells, which indicates that NF-kappaB activation might be a potential useful target to study mechanism and for drug screening.

A novel immunostimulator, N-[alpha-O-benzyl-N-(acetylmuramyl)-L-alanyl-D-isoglutaminyl]-N6-trans-(m-nitrocinnamoyl)-L-lysine, and its adjuvancy on the hepatitis B surface antigen.

N(2)-[alpha-O-benzyl-N-(acetylmuramyl)-L-alanyl-D-isoglutaminyl]-N(6)-trans-(m-nitrocinnamoyl)-L-lysine (muramyl dipeptide C, or MDP-C) has been synthesized as a novel, nonspecific immunomodulator. The present study shows that MDP-C induces strong cytolytic activity by macrophages on P388 leukemia cells and cytotoxic activity by cytotoxic T lymphocytes (CTLs) on P815 mastocytoma cells. Our results also indicate that MDP-C is an effective stimulator for production of interleukin-2 and interleukin-12 by murine bone marrow derived dendritic cells (BMDCs) and production of interferon-gamma by CTLs. Additionally, MDP-C increases the expression levels of several surface molecules, including CD11c, MHC class I, and intercellular adhesion molecule-1 in BMDCs. Moreover, MDP-C remarkably enhances the immune system's responsiveness to hepatitis B surface antigen (HBsAg) in hepatitis B virus transgenic mice for both antibody production and specific HBsAg T-cell responses ex vivo. Our results indicate that MDP-C is an apyrogenic, nonallergenic, and low-toxicity immunostimulator with great potential for diagnostic, immunotherapeutic, and prophylactic applications in diseases such as hepatitis B and cancers.

Ginsenoside-Ro enhances cell proliferation and modulates Th1/Th2 cytokines production in murine splenocytes.

AIM: To study the effects of ginsenoside-Ro on cell proliferation and cytokine production in murine splenocytes. METHODS: The effect of ginsenoside-Ro on murine splenocytes proliferation was studied using [3H] thymidine incorporation assay. Effects of ginsenoside-Ro on the production of cytokines interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) from murine splenocytes were detected by ELISA method. Effects of ginsenoside-Ro on mRNA level of Th1 cytokine IFN-gamma and Th2 cytokine IL-4 were evaluated by reverse transcription polymerase chain reaction (RT-PCR) analysis. RESULTS: Ginsenoside-Ro showed no mitogenic effect on unstimulated murine splenocytes. It enhanced the proliferation of Con A-induced murine splenocytes and the production of IL-2 at concentrations of 1-10 micromol x L(-1). Moreover, ginsenoside-Ro increased the production and expression of Th2 cytokine IL-4 and decreased the production and expression of Th1 cytokine IFN-gamma in Con A-induced murine splenocytes at concentrations of 2-10 micromol x L(-1). CONCLUSION: Ginsenoside-Ro showed immunomodulatory effects by regulating the production and expression of Th1/Th2 cytokines in murine splenocytes.

Protopanaxatriol-type ginsenosides differentially modulate type 1 and type 2 cytokines production from murine splenocytes.

Seven protopanaxatriol-type ginsenosides and their aglycones including PPT, PT, -Re, -Rg (1), -F (1), -Rh (1), 20(R)-Rh (1) which are closely related in structure were studied for their effects on type 1 and type 2 cytokines production from murine splenocytes and their related mechanisms were examined. The results indicate that PPT, PT and ginsenoside-Re show hardly any or weak effects on concanavalin A (Con A)-induced production of IFN-gamma and IL-4. Ginsenoside-Rh (1) and 20(R)-Rh (1) induce a Con A-induced type 1 cytokine pattern by increasing the production of interleukin-12 (IL-12), the expression of IFN-gamma, T-bet and enhancing NF-kappaB DNA binding activity. In contrast ginsenosides-Rg (1) and -F (1) cause a Con A-induced type 2 cytokines response by increasing the expression of IL-4, GATA-3 and enhancing NF-kappaB DNA binding activity. Thus, these protopanaxatriol-type ginsenosides have different immunomodulatory effects, which might explain the complex immunomodulatory effect of Panax ginseng.

[Effect of zinc on immune function in mice spleen lymphocytes].

OBJECTIVE: In order to study the effects of different levels of zinc nutrition status on the immune function of mice spleen lymphocytes. METHODS: The experiment included two periods: zinc depleted period and repletion period. In the depleted period, mice were divided into 3 dose groups: zinc deficient group (ZD, 5.2mg/kg), zinc pair-fed group (PZ) and normal zinc group (NZ, 25.6 mg/kg). In the repletion period, mice were divided into 3 dose groups: ZD group, DZ-NZ group and PZ-NZ group. It was measured some biomarkers of immunity to assess the zinc nutritional status, including lymphocytes amount, lymphocytes proliferation index and IL-2 activity. RESULTS: The lymphocytes counts of ZD group spleen showed significant decline at seventy days fed zinc deficiency diets. The counts of ZD-NZ group were increased after 22 days of taking normal zinc diet. Zinc deficiency could suppressed the proliferation of the spleen lymphocytes. The spleen lymphocytes proliferation of the ZD-NZ group showed a moderate but non-significant increase compared with the ZD group. Zinc deficiency could decrease IL-2 production. The production of IL-2 of ZD group was lower than those of ZP (P < 0.05), IL-2 production increased following zinc supplement. CONCLUSION: It is found that zinc status can affect the immune function. Production of IL-2 in spleen lymphocyte is affected evidently by zinc.

Immunoenhancing activity of protopanaxatriol-type ginsenoside-F3 in murine spleen cells.

AIM: To investigate the immunoenhancing activity of ginsenoside-F3 in murine spleen cells and explore its mechanism. METHODS: The enhancing effect of ginsenoside-F3 on murine spleen cell proliferation was studied using [3H]thymidine incorporation assay. Effects of ginsenoside-F3 on the production of type 1 cytokines IL-2, IFN-gamma, and type 2 cytokines IL-4 and IL-10 from murine spleen cells were detected by ELISA method. Effects of ginsenoside-F3 on mRNA level of cytokines IL-4, IFN-gamma, and transcription factors T-bet and GATA-3 were evaluated by RT-PCR analysis. Effect of ginsenoside-F3 on NF-kappaB DNA binding activity in murine spleen cells was investigated by electrophoretic mobility shift assays (EMSA). RESULTS: Ginsenoside-F3 at 0.1-100 micromol/L not only promoted the murine spleen cell proliferation, but also increased the production of IL-2 and IFN-gamma, while decreased the production of IL-4 and IL-10 from murine spleen cells with the maximal effect at 10 micromol/L. RT-PCR analysis displayed that ginsenoside-F3 enhanced the IFN-gamma and T-bet gene expression and decreased IL-4 and GATA-3 gene expression. EMSA experiment showed that ginsenoside-F3 10 micromol/L enhanced the NF-kappaB DNA binding activity induced by ConA in murine spleen cells. CONCLUSION: Ginsenoside-F3 has immunoenhancing activity by regulating production and gene expression of type 1 cytokines and type 2 cytokines in murine spleen cells.

Imrecoxib: a novel and selective cyclooxygenase 2 inhibitor with anti-inflammatory effect.

AIM: To investigate the inhibitory effect of imrecoxib, a synthetic compound of completely new structure, on cyclooxygenase 1 (COX-1) and 2 (COX-2) and its anti-inflammatory effect in vivo. METHODS: The inhibitory effects of imrecoxib on cyclooxygenase 1 and 2 were studied using whole cell assay with murine peritoneal macrophages induced by calcimycin and LPS. The inhibitory effects of imrecoxib on mRNA level of COX-1 and COX-2 in human macrophage cell line U937 were detected by reverse transcription polymerase chain reaction (RT-PCR) analysis. Effects of imrecoxib on acute and chronic inflammation were evaluated in rat carrageenan induced edema model and rat adjuvant-induced arthritis model, respectively. RESULTS: Imrecoxib was found to inhibit COX-1 and COX-2 with IC50 value of 115+/-28 nmol/L and 18+/-4 nmol/L, respectively. Imrecoxib was shown to selectively and dose-dependently inhibit COX-2 mRNA level. Imrecoxib effectively inhibited carrageenan-induced acute inflammation at the doses of 5, 10, and 20 mg/kg i.g. and adjuvant-induced chronic inflammation at the doses of 10 and 20 mg/kg/d i.g. CONCLUSION: Imrecoxib is a novel and moderately selective COX-2 inhibitor that possesses anti-inflammatory effect by inhibition of COX-2 mRNA expression.

Parallel approach for solution-phase synthesis of 2-quinoxalinol analogues and their inhibition of LPS-induced TNF-alpha release on mouse macrophages in vitro.

A parallel solution-phase synthesis of 2-quinoxalinol analogues is described. The key step-simultaneous reductions of m-Ar(NO2)2 to m-Ar(NH2)2 was investigated extensively. We obtained preliminary pharmacological activity of those analogues for the inhibition of LPS-induced TNF-alpha release on mouse macrophage in vitro. Two compounds revealed inhibitory activity, with IC50 values of 0.40 microM (7-amino-6-[(3-methoxypropyl)amino]-3-methyl-2-quinoxalinol) and 2.2 microM (7-amino-6-[(3-butoxypropyl)amino]-3-methyl-2-quinoxalinol), respectively.

Resveratrol inhibits matrix metalloproteinase-9 transcription in U937 cells.

AIM: To examine the inhibitory effect of resveratrol on matrix metalloroteinase-9 (MMP-9) and explore its mechanism. METHODS: MMP-9 activity was analyzed by gelatin zymography; MMP-9 protein was detected by Western blot; MMP-9 mRNA expression was investigated by RT-PCR. Activation of activator protein -1 (AP-1) was measured by electrophoretic mobility shift assay (EMSA). RESULTS: MMP-9 activity in U937 cells increased significantly after exposed to PMA at 10 nmol/L for 24 h without FCS (P<0.01). Resveratrol at 1 and 10 micromol/L showed significant inhibition on MMP-9 activity (P<0.05 and P<0.01, respectively). Western blot and RT-PCR experiments displayed that MMP-9 protein (P<0.01) and mRNA expression (P<0.01) increased significantly in PMA-treated U937 cells. Resveratrol at 1 and 10 micromol/L showed inhibitory effects on MMP-9 protein production and MMP-9 mRNA expression (P<0.05). The activation of AP-1 induced by PMA was also extensively inhibited by resveratrol at 0.1, 1, and 10 micromol/L. CONCLUSION: The inhibitory effect of resveratrol on MMP-9 activity may be partly through suppression of activation of nuclear transcription factor AP-1, and inhibition of MMP-9 mRNA expression and MMP-9 protein production.