Chondroprotective and anti-inflammatory effects of amurensin H by regulating TLR4/Syk/NF-κB signals.

The low-grade, chronic inflammation initiated by TLR4-triggered innate immune responses has a central role on early osteoarthritis. Amurensin H is a resveratrol dimer with anti-inflammatory and anti-apoptotic effects, while its effects on TLR-4 signals to inhibit osteoarthritis are still unclear. In the present study, treatment with amurensin H for 2 weeks in monosodium iodoacetate-induced mice significantly slows down cartilage degeneration and inflammation using macroscopic evaluation, haematoxylin and eosin (HE) staining and micro-magnetic resonance imaging. In IL-1ß-stimulated rat chondrocytes, amurensin H suppresses the production of inflammatory mediators including nitric oxide, IL-6, IL-17, PGE2 and TNF-α using Greiss and ELISA assay. Amurensin H inhibits matrix degradation via decreasing levels of MMP-9 and MMP-13 using Western blot assay, promotes synthesis of type II collagen and glycosaminoglycan using immunostaining and safranin O staining, respectively. Amurensin H inhibits intracellular and mitochondrial reactive oxygen species (ROS) generation, and mitochondrial membrane depolarization using DCFH-DA, MitoSOX Red and JC-1 assay as well. IL-1ß stimulates TLR4 activation and Syk phosphorylation in chondrocytes, while amurensin H inhibits TLR4/Syk signals and downstream p65 phosphorylation and translocation in a time and dose-dependent manner. Together, these results suggest that amurensin H exerts chondroprotective effects by attenuating oxidative stress, inflammation and matrix degradation via the TLR4/Syk/NF-κB pathway.

Amurensin H, a Derivative From Resveratrol, Ameliorates Lipopolysaccharide/Cigarette Smoke-Induced Airway Inflammation by Blocking the Syk/NF-κB Pathway.

Amurensin H, a resveratrol dimer derived from Vitis amurensis Rupr, has several biological effects, including anti-inflammatory and antioxidant activities. Studies have found that amurensin H attenuated asthma-like allergic airway inflammation. However, its protective activity on chronic obstructive pulmonary disease (COPD) airway inflammation is not fully explored. The present study used a lipopolysaccharide (LPS)/cigarette smoke-induced mice model and an LPS-stimulated THP-1-derived macrophages model to measure the lung tissue's morphology changes. The results showed that amurensin H ameliorated the histological inflammatory alterations in the lung tissues, leading to a decrease in the expression of interleukin 6 (IL-6), IL-17A, tumor necrosis factor α (TNF-α), and interferon γ in bronchoalveolar lavage fluid. Amurensin H also significantly inhibited the release of IL-1ß, IL-6, IL-8, and TNF-α in LPS-stimulated THP-1-derived macrophages. Furthermore, amurensin H markedly inhibited the expressions of p-Syk, nuclear factor κB (NF-κB), and p-NF-κB both in vivo and in vitro. Results from cotreatment with Syk inhibitor BAY61-3606 and NF-κB inhibitor BAY11-7082 in vitro revealed that amurensin H's protective effect against airway inflammation could be due partly to the inhibition of the Syk/NF-κB pathway. These findings suggest that amurensin H shows therapeutic effects on COPD airway inflammation, and inhibiting the Syk/NF-κB pathway might be part of its underlying mechanisms.

Vam3 ameliorates total body irradiation-induced hematopoietic system injury partly by regulating the expression of Nrf2-targeted genes.

Vam3, a resveratrol dimer, has been implicated in the regulation of chronic obstructive pulmonary disease. However, the effect of Vam3 on total body irradiation (TBI)-induced hematopoietic progenitor cells (HPCs), and hematopoietic stem cells (HSCs) injury is unknown. In this study, we examined whether Vam3could ameliorate hematopoietic system injury induced by TBI. Our results indicated that Vam3 alleviated TBI-induced injury by improving the self-renewal and differentiation of HPCs, and HSCs. Vam3 decreased the intracellular ROS levels in irradiated mice HPCs/HSCs or c-kit positive cells and inhibited apoptosis and DNA damage in LSKs and HPCs after TBI. Vam3 up-regulated the expression of Nrf2 and related genes and proteins in irradiated c-kit positive cells in vitro. However, Vam3 did not increase the cell viability or the number of CFU-GM c-kit positive cells in irradiated Nrf2-/- mice but decreased the cellular ROS level. The above data showed that Vam3 ameliorates total body irradiation-induced hematopoietic system injury and that Nrf2 is essential in mediating Vam3's protective effect on the proliferation of c-kit positive cells after irradiation but not its ability to scavenge for free radicals.

Vam3, a Compound Derived from Vitis amurensis Rupr., Attenuated Colitis-Related Tumorigenesis by Inhibiting NF-κB Signaling Pathway.

BACKGROUND: Chronic inflammation is one of the important mediators of colitis-related colon cancer (CRC). Abundant mast cells (MCs) were observed in the tumor microenvironment and mediators released upon MC activation play an important role in the process of chronic inflammation. Previously, we found that activation of intestine mucosal MCs recruited and modulated the inflammatory CD11b(+)Gr1(+) cells to promote the CRC development. In the current study we investigated the effects of Vam3, a resveratrol dimer with potent anti-inflammatory effects, on CRC development. METHODS: RBL-2H3 cells, a basophilic leukemia cell line, were pretreated with 2.5 or 5 µM Vam3 and then stimulated with dinitrophenol-conjugated bovine serum albumin (DNP-BSA) plus lipopolysaccharide (LPS). The MC degranulation was determined by measuring ß-hexosaminidase release. Generation of TNF-α and IL-6 in RBL-2H3 cells or in peritoneal macrophages was determined by ELISA and real-time qPCR. NF-κB p65 and phospho-NF-κB p65 expression was determined by Western blotting. NF-κB activity in RAW264.7 cells was determined by luciferase reporter assay. CRC was induced in C57BL/6 mice by intraperitoneal injection of azoxymethane (AOM), followed by oral exposure to dextran sodium sulfate (DSS). Vam3 at 50 mg/kg, or disodium cromoglycate (DSCG, MC stabilizer) at 100 mg/kg, or vehicle were administrated to the mice 4 weeks after DSS withdrawal. Levels of TNF-α, IL-6, and mouse MC protease-1 were determined by ELISA. Infiltration of CD11b(+)Gr1(+) cells was determined by flow cytometry analysis. One-way ANOVA was used to compare difference between groups. RESULTS: Pretreatment with Vam3 significantly inhibited RBL-2H3 cell degranulation and inflammatory cytokine production from RBL-2H3 cells and from peritoneal macrophages. After Vam3 treatment, NF-κB activity in RAW264.7 cells, and expressions of phospho-NF-κB p65 in RBL-2H3 cells and in peritoneal macrophages were significantly down-regulated. In the AOM plus DSS-induced CRC murine model, the Vam3 and DSCG-treated mice had less tumor numbers than those treated with vehicle. Expression of phospho-NF-κB p65, production of inflammatory cytokines, and infiltration of MCs and CD11b(+)Gr1(+) cells were attenuated in the Vam3-treated mice. CONCLUSION: Vam3 treatment could attenuate the CRC development. This effect may be due to its inhibition on NF-κB signaling pathway in MCs and macrophages of the inflamed intestines.

[Analysis of porphyrin photosensitizers using HPLC method].

Photodynamic therapy (PDT), because of its good targeting, minimal invasion, and safety, is becoming a very active area in cancer prevention and treatment, in which the photosensitizers have proved to be the core element for PDT. We developed a new HPLC method for analyzing porphyrin photosensitizers using Shiseido Capcell PAK C18 (150 mm x 4.6 mm, 5 µm) as the column at 30 °C, methanol-1% aqueous solution of acetic acid as the mobile phase in a flow rate of 1.0 mL · min(-1) in a gradient elution mode, and the detection wavelength at 380 nm. This method, showing good specificity, precision, accuracy and robusty via methodology validations, can be applied to the purity test and assay of porphyrin photosensitizers, and has played a key guide role in the R&D of the new porphyrin photosensitizer--sinoporphyrin sodium.

[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.

Cyclin d1 downregulation contributes to anticancer effect of isorhapontigenin on human bladder cancer cells.

Isorhapontigenin (ISO) is a new derivative of stilbene compound that was isolated from the Chinese herb Gnetum Cleistostachyum and has been used for treatment of bladder cancers for centuries. In our current studies, we have explored the potential inhibitory effect and molecular mechanisms underlying isorhapontigenin anticancer effects on anchorage-independent growth of human bladder cancer cell lines. We found that isorhapontigenin showed a significant inhibitory effect on human bladder cancer cell growth and was accompanied with related cell cycle G(0)-G(1) arrest as well as downregulation of cyclin D1 expression at the transcriptional level in UMUC3 and RT112 cells. Further studies identified that isorhapontigenin downregulated cyclin D1 gene transcription via inhibition of specific protein 1 (SP1) transactivation. Moreover, ectopic expression of GFP-cyclin D1 rendered UMUC3 cells resistant to induction of cell-cycle G(0)-G(1) arrest and inhibition of cancer cell anchorage-independent growth by isorhapontigenin treatment. Together, our studies show that isorhapontigenin is an active compound that mediates Gnetum Cleistostachyum's induction of cell-cycle G(0)-G(1) arrest and inhibition of cancer cell anchorage-independent growth through downregulating SP1/cyclin D1 axis in bladder cancer cells. Our studies provide a novel insight into understanding the anticancer activity of the Chinese herb Gnetum Cleistostachyum and its isolate isorhapontigenin.

Vam3, a derivative of resveratrol, attenuates cigarette smoke-induced autophagy.

AIM: To appraise the efficacy of Vam3 (Amurensis H), a dimeric derivative of resveratrol, at inhibiting cigarette smoke-induced autophagy. METHODS: Human bronchial epithelial cells were treated with cigarette smoke condensates, and a chronic obstructive pulmonary disease (COPD) model was established by exposing male BALB/c mice to cigarette smoke. The protein levels of the autophagic marker microtubule-associated protein 1A/1B-light chain 3 (LC3), Sirtuin 1 (Sirt1), and foxhead box O 3a (FoxO3a) were examined using Western blotting and Immunohistochemistry. LC3 punctae were detected by immunofluorescence. The levels of FoxO3a acetylation were examined by immunoprecipitation. The level of intracellular oxidation was assessed by detecting ROS and GSH-Px. RESULTS: Vam3 attenuated cigarette smoke condensate-induced autophagy in human bronchial epithelial cells, and restored the expression levels of Sirt1 and FoxO3a that had been reduced by cigarette smoke condensates. Similar protective effects of Vam3, reducing autophagy and restoring the levels of Sirt1 and FoxO3a, were observed in the COPD animal model. Additionally, Vam3 also diminished the oxidative stress that was induced by the cigarette smoke condensates. CONCLUSION: Vam3 decreases cigarette smoke-induced autophagy via up-regulating/restoring the levels of Sirt1 and FoxO3a and inhibiting the induced oxidative stress.

Bioactive neolignans and lignans from the bark of Machilus robusta.

Sixteen new neolignans and lignans (1-16), together with 12 known analogues, have been isolated from an ethanol extract of the bark of Machilus robusta. Compounds 1 and 2 showed activity against HIV-1 replication in vitro, with IC(50) values of 2.52 and 2.01 µM, respectively. At 10 µM, 6, 8, and 9 reduced dl-galactosamine-induced hepatocyte (WB-F344 cells) damage, and 9 could additionally attenuate rotenone-induced PC12 cell damage. The known compounds (-)-pinoresinol (17) and (+)-lyoniresinol (18) were active against serum deprivation induced PC12 cell damage.

[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.

BACE1 (beta-secretase) inhibitory chromone glycosides from Aloe vera and Aloe nobilis.

Four new chromone glycosides allo-aloeresin D (2) , C-2'-decoumaroyl-aloeresin G (8), 2'-O-coumaroyl-(S)-aloesinol (9), 2'-O-[ P-methoxy-(E)-cinnamoyl]-(S)-aloesinol (10) and nine known chromone glycosides ( 1, 3 - 7, 11 - 13) were isolated from two Aloe spp. plants, A. vera and A. nobilis. Among them, 1 and 8 showed significant inhibitory activity against BACE1 (beta-secretase) with IC (50) values of 39.0 and 20.5 x 10 (-6) M, as well as inhibition of Abeta (1-42) production by 7.4 and 12.3 %, respectively, in B103 neuroblastoma cells at 30 ppm. The preliminary structure-activity relationships of ALOE chromone glucosides were also discussed.

Isolation and biomimetic synthesis of anti-inflammatory stilbenolignans from Gnetum cleistostachyum.

One new stilbenolignan, gnetucleistol F (1), and four known stilbenolignans, gnetofuran A (2), lehmbachol D (3), gnetifolin F (4) and gnetumontanin C (5) were isolated from the lianas of Gnetum cleistostachyum C. Y. CHENG (Gnetaceae). Their structures and relative configurations were determined by means of spectroscopic evidence. Compounds 1, 2, 3 and 4 were synthesized for the first time on the basis of their biogenetic pathway, and their possible biomimetical synthetic mechanisms were discussed. The pharmacological activities of all stilbenolignans have been tested. Among them, 1, 2, 3, 4 and 5 showed moderate inhibitory activities on TNF-alpha and 1 also showed potent inhibitory activity on malondialdehyde.

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.

Bioactive stilbene dimers from Gnetum cleistostachyum.

Two new stilbene dimers, named bisisorhapontigenin A (1) and cis shegansu B (2), together with gnetuhainin P (3) and gnetulin (4), were isolated from the lianas of Gnetum cleistostachyum C. Y. Cheng. Their structures were elucidated by means of spectroscopic evidences, including UV, IR, MS, 1H NMR, 13C NMR, NOE and 2D NMR, respectively. The pharmacological activities of compounds 1, 2 and 3 also have been tested.