14,15-Secopregnane-type C21-steriosides from the roots of Cynanchum stauntonii.

Nine 14,15-secopregnane-type C21-steriosides, stauntosides U, V, V1-V3, W and C1-C3, as well as two known C21-steriosides, were isolated from the roots of Cynanchum stauntonii. Stauntosides U, V and V1-V3 share the same basic structural features of 8α:14α,14:16,15:20,18:20-tetraepoxy-14,15-secopregn-6-ene-3ß,5α,9α-triol, with the numbering system following that of C21-pregnanes. The aglycones of stauntosides U, V and V1-V3 are classified into two subcategories, the 5,9-dihydroxy groups and 5α:9α-peroxy bridge, according to the oxidative states of the two hydroxy groups at the C-5 and C-9 positions. The anti-inflammatory activity of the major compounds was assessed in an in vitro inflammatory model of mouse peritoneal macrophages using IC50 values of the inhibition of nitric oxide (NO) production as an indicator. Stauntosides V1 and V3 exhibited target activity with IC50 values of 9.3 µM and 12.4 µM, respectively, compared with dexamethasone, which was used as a positive control.

Anti-inflammatory effect of a resveratrol derivative 3,4,5-trimethoxy-4',5'-dihydroxy-trans-stilbene (WL-09-5) via ROS-mediated NF-κB pathway.

Inflammation derived from macrophages activation leads to various diseases. Synthetic modifications of resveratrol have been shown to have better anti-inflammatory activities. In this study, croton oil-induced mouse ear edema and lipopolysaccharides (LPS)-stimulated RAW264.7 macrophages were used to evaluate the anti-inflammatory effects of WL-09-5, a derivative of resveratrol. Furthermore, the activation of NF-κB was determined. Results showed that WL-09-5 significantly reduced the croton oil-induced ear edema, scavenged NO and ROS production, and reduced the levels of TNF-α, IL-6, and IL-1ß. Furthermore, WL-09-5 may significantly inhibit the translocation of NF-κB in macrophage cells stimulated by LPS in a dose-dependent manner, which is a potent mechanism of its anti-inflammatory effects. In conclusion, WL-09-5 is an underlying candidate for inflammatory diseases that need further investigations.

[Effect of WS070117M1 on chronic obstructive pulmonary disease in mice and the underling mechanisms of anti-inflammation].

The aim of this study is to investigate the anti-inflammatory effect of the adenosine derivative N6-(3-hydroxylaniline) adenosine (WS070117M1) on cigarette smoke plus LPS (lipopolysaccharide)-induced chronic obstructive pulmonary disease (COPD) in mice and its mechanism. COPD model was established by exposing male BALB/c mice to cigarette smoke and challenged with LPS inhalation. Supernatants of bronchoalveolar lavage fluid (BALF) were harvested and IL-1ß, IL-6, IL-8 and TGF-ß1 levels were measured by ELISA (enzyme-linked immunesorbent assay). The number of total white blood cells and neutrophils in bronchoalveolar lavage fluid was counted separately. Lung tissue was stained with Mayer 's hematoxylin and eosin for histopathologic examination. pAMPKa protein expression and distribution of lung tissue were analyzed by immunohistochemistry method. In vitro, levels of AMPKα phosphorylation in phorbol-12- myristate-13-acetate (PMA) differentiated THP-1 cells was detected by immunohistochemistry, IL-8 level in supernatants of cigarette smoke condensate stimulating PMA differentiated THP-1 cells was measured by ELISA. The results showed that WS070117M1 treatment significantly activated AMPKa in the lung tissue. It also resulted in down regulation of IL-1ß, IL-6, IL-8 and TGF-ß1 levels in bronchoalveolar lavage fluid and IL-8 level in cigarette smoke condensate stimulating PMA differentiated THP-1 cells. In addition, WS070117M1 could inhibit the recruitment of total white blood cells and neutrophils. These results suggest that WS070117M1 may alleviate the airway inflammation by activating AMPK in the lung tissue.

Anti-inflammatory Hydrolyzable Tannins from Myricaria bracteata.

Twelve hydrolyzable tannins were obtained from the twigs of Myricaria bracteata, including two new hellinoyl-type dimers, bracteatinins D1 (1) and D2 (2); a new hellinoyl-type trimer, bracteatinin T1 (3); two known monomers, nilotinin M4 (4) and 1,3-di-O-galloyl-4,6-O-(aS)-hexahydroxydiphenoyl-ß-d-glucose (5); six known dimers, tamarixinin A (6), nilotinin D8 (7), hirtellins A (10), B (9), and E (8), and isohirtellin C (11); and a known trimer, hirtellin T3 (12). The structures of the tannins were elucidated by spectroscopic data analysis and comparisons to known tannins. All compounds were evaluated as free radical scavengers using 1,1-diphenyl-2-picrylhydrazyl and hydroxy radicals and compared to the activity of BHT and Trolox. Compound 6 showed a significant anti-inflammatory effect on croton oil-induced ear edema in mice (200 mg/kg, inhibition rate 69.8%) and on collagen-induced arthritis in DBA/1 mice (20 mg/kg, inhibition rate 46.0% at day 57).

[Effects of Vam3 on sodium nitroprusside-induced apoptosis and SIRT1 and p53 expression in rat articular chondrocytes].

This study is to investigate the effect of Vam3, a dimeric derivative of resveratrol, on SNP-induced apoptosis and its potential mechanism in rat articular chondrocytes. Isolated rat articular chondrocytes were treated with sodium nitroprusside (SNP), a NO donor, to induce apoptosis. Apoptosis percentage was evaluated by Annexin V-PI and nucleus fracture was examined by DAPI staining. Level of intracellular reactive oxygen species (ROS) was detected using 2, 7'-dichlorofluorescin diacetate (DCFH-DA) as a fluorescence probe by fluorescence microplate reader. The change in mitochondrial membrane potential was detected by TMRE staining. Expressions of SIRT1, acetylated p53 (ac-p53), cleaved caspase 9 and cleaved caspase 3 were determined by Western blotting. It showed that Vam3 up to 10 micromol x L(-1) could significantly reduce SNP-induced rat articular chondrocytes apoptosis (P < 0.01) and nucleus fracture, inhibit the increase of intracellular ROS level (P < 0.01) and reverse the decrease in mitochondrial membrane potential (P < 0.01). Simultaneously, Vam3 could upregulate the expression of SIRT1, deacetylate p53, and inhibit the cleavage of caspase 9 and caspase 3 (P < 0.01) of rat articular chondrocytes exposed to SNP. This study indicates Vam3 could protect rat articular chondrocytes against SNP-induced apoptosis, perhaps through the upregulation of SIRT1 and deacetylation of p53.

Vam3, a resveratrol dimer, inhibits cigarette smoke-induced cell apoptosis in lungs by improving mitochondrial function.

AIM: To investigate the effects of Vam3 (a resveratrol dimer extracted from Vitis amurensis Rupr) on cigarette smoke (CS)-induced cell apoptosis in lungs in vitro and in vivo and the underlying mechanisms of action. METHODS: Human bronchial epithelial cell line BEAS-2B was exposed to cigarette smoke condensate (CSC, 300 mg/L), and cell apoptosis was determined using flow cytometry and Hoechst staining. Mitochondrial membrane potential was examined with TMRE staining. ROS and ceramide levels were detected with DCFH-DA fluorescence and HPLC-MS/MS, respectively. Cytochrome c release was detected using immunofluorescence. Caspase-9 and neutral sphingomyelinase 2 expression was measured with Western blotting. The breast carcinoma cell line MCF7 stably expressing GFP-tagged Bax was used to elucidate the role of mitochondria in CS-induced apoptosis. For in vivo study, male mice were exposed to CS for 5 min twice a day for 4 weeks. The mice were orally administered Vam3 (50 mg·kg(-1)·d(-1)) or resveratrol (30 mg·kg(-1)·d(-1)) each day 1 h before the first CS exposure. RESULTS: Pretreatment of BEAS-2B cells with Vam3 (5 µmol/L) or resveratrol (5 µmol/L) significantly suppressed CSC-induced apoptosis, and prevented CSC-induced Bax level increase in the mitochondria, mitochondrial membrane potential loss, cytochrome c release and caspase-9 activation. Furthermore, pretreatment of BEAS-2B cells with Vam3 or resveratrol significantly suppressed CSC-stimulated intracellular ceramide production, and CSC-induced upregulation of neutral sphingomyelinase 2, the enzyme responsible for ceramide production in bronchial epithelial cells. Similar results were obtained in C6-pyridinium ceramide-induced apoptosis of GFP-Bax-stable MCF7 cells in vitro, and in the lungs of CS-exposed mice that were treated with oral administration of Vam3 or resveratrol. CONCLUSION: Vam3 protects bronchial epithelial cells from CS-induced apoptosis in vitro and in vivo by preventing mitochondrial dysfunction.

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.

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.

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.

[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.

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.

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.

[Inhibition of dexamethasone, indomethacin and resveratrol on matrix metalloproteinase-9 and the mechanism of inhibition].

AIM: To investigate the expression of matrix metalloproteinase-9 (MMP-9) in mouse ears induced with croton oil and the inhibitory effect of dexamethasone, indomethacin and resveratrol on MMP-9 expression, and further explore the relationship between anti-inflammation and MMP-9 inhibition of these three medicines. METHODS: Immuno-histochemistry was used to detect the expression of MMP-9 in mouse ears. Expression of MMP-9 in U937 cells was analyzed by gelatin zymography. RESULTS: Mouse ear edema induced with croton oil was inhibited significantly by dexamethasone and indomethacin at the dose of 10 mg.kg-1 and resveratrol at 50 mg.kg-1 administered subcutaneously. The inhibitory rate was 76.2% (P < 0.001), 56.7% (P < 0.001) and 36.9% (P < 0.001) respectively. The MMP-9 expression increased in mouse ears induced with croton oil and inhibited by dexamethasone, indomethacin and resveratrol at above doses. Gelatin zymography results showed that MMP-9 expression in U937 cells increased significantly after exposed to PMA at 1 x 10(-8) mol.L-1 (P < 0.001); MMP-9 expression induced with phorbol myristate acetate(PMA) was inhibited by dexamethasone at 1 x 10(-9), 1 x 10(-7) and 1 x 10(-5) mol.L-1, indomethacin at 1 x 10(-6) and 1 x 10(-5) mol.L-1 and resveratrol at 1 x 10(-6) and 1 x 10(-5) mol.L-1. CONCLUSION: The inhibition of MMP-9 expression may be one of the anti-inflammatory mechanisms of dexamethasone, indomethacin and resveratrol.

[Effect of indomethacin on expression of interleukin-6 caused by lipopolysaccharide in rheumatoid arthritic patients' synoviocyte].

AIM: To study the effects of indomethacin on interleukin-6 (IL-6) expression stimulated with lipopolysaccharide (LPS) in rheumatoid arthritic patients' synoviocyte. METHODS: Fibroblast-like cells (FLS) from rheumatoid arthritic patients' joint tissue were cultured for 24 h and incubated 24 h with LPS (1 mg.L-1) or the supernatant of U937 cells stimulated by LPS (1 mg.L-1). After indomethacin or dexamethasone added into the supernatant of U937 cells, FLS was incubated with the super natant for 24 h. The expression of IL-6 protein was detected by radioimmunoassay. The mRNA expression of IL-6 was accessed by RT-PCR. RESULTS: LPS did not obviously affect the growth of FLS, and the protein secretion and mRNA expression of IL-6 were not changed in FLS treated with LPS. The IL-6 secretion and IL-6 mRNA expression were significantly increased in FLS cultured with the supernatant from U937 cell treated with LPS. Indomethacin at concentrations of 1 x 10(-7)-1 x 10(-5) mol.L-1 obviously inhibited the protein secretion and mRNA expression of IL-6 in FLS cultured with the supernatant from U937 cell stimulated with LPS, and the inhibitory effects increased as the concentrations of indomethacin increased. CONCLUSION: Indomethacin can inhibit the increase of IL-6 expression caused by supernatant of U937 cells stimulated with LPS in FLS.