Resveratrol Inhibits LPS-Induced MAPKs Activation via Activation of the Phosphatidylinositol 3-Kinase Pathway in Murine RAW 264.7 Macrophage Cells

Background

Resveratrol is a natural polyphenolic compound that has cardioprotective, anticancer and anti-inflammatory properties. We investigated the capacity of resveratrol to protect RAW 264.7 cells from inflammatory insults and explored mechanisms underlying inhibitory effects of resveratrol on RAW 264.7 cells.

Methodology/Principal Findings

Murine RAW 264.7 cells were treated with resveratrol (1, 5, and 10 µM) and/or LPS (5 µg/ml). Nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by Griess reagent and ELISA. The mRNA and protein levels of proinflammatory proteins and cytokines were analysed by ELISA, RT-PCR and double immunofluorescence labeling, respectively. Phosphorylation levels of Akt, cyclic AMP-responsive element-binding protein (CREB), mitogen-activated protein kinases (MAPKs) cascades, AMP-activated protein kinase (AMPK) and expression of SIRT1(Silent information regulator T1) were measured by western blot. Wortmannin (1 µM), a specific phosphatidylinositol 3-kinase (PI3-K) inhibitor, was used to determine if PI3-K/Akt signaling pathway might be involved in resveratrol’s action on RAW 264.7 cells. Resveratrol significantly attenuated the LPS-induced expression of nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in RAW 264.7 cells. Resveratrol increased Akt phosphorylation in a time-dependent manner. Wortmannin, a specific phosphatidylinositol 3-kinase (PI3-K) inhibitor, blocked the effects of resveratrol on LPS-induced RAW 264.7 cells activation. In addition, PI3-K inhibition partially abolished the inhibitory effect of resveratrol on the phosphorylation of cyclic AMP-responsive element-binding protein (CREB) and mitogen-activated protein kinases (MAPKs) cascades. Meanwhile, PI3-K is essential for resveratrol-mediated phosphorylation of AMPK and expression of SIRT1.

Conclusion and Implications

This investigation demonstrates that PI3-K/Akt activation is an important signaling in resveratrol-mediated activation of AMPK phosphorylation and SIRT1 expression, and inhibition of phosphorylation of CREB and MAPKs activation, proinflammatory mediators and cytokines production in response to LPS in RAW 264.7 cells.     

Resveratrol Possesses Protective Effects in a Pristane-Induced Lupus Mouse Model

Background

Systemic lupus erythematosus (SLE) is a multisystemic autoimmune disease characterized by the production of autoantibodies. To date, no therapy has been found to satisfactorily treat SLE. SIRT1 deficiency results in the development of an autoimmune syndrome in mice, including a high titer of anti-nuclear antibody in serum, immunoglobulin deposition in the kidney, and immune complex glomerulonephritis. Resveratrol is an activator of SIRT1 and possesses anti-inflammation and immune-regulatory properties.

Objective

To evaluate the preventative effects of resveratrol on a pristane-induced lupus animal model and assess its putative immune modulation effects.

Methods

BALB/c mice received a single intraperitoneal injection of 0.5 ml of pristane on day 1 and then various doses of resveratrol were given to the mice daily starting on day 2 and continuing for seven months. The autoantibodies in serum and supernatants were measured. Single cells isolated from spleen, isolated CD4+ T cells, and CD19+ B cells were cultured with or without resveratrol in vitro and assessed by flow cytometry.

Results

Resveratrol attenuated proteinuria, immunoglobuin depositon in kidney, and glomerulonephritis as well as IgG1 and IgG2a in serum in pristane-induced lupus mice. Resveratrol also suppressed CD69 and CD71 expression on CD4+ T cells as well as CD4+ T cell proliferation, induced CD4+ T cell apoptosis, and decreased CD4 IFNγ⁺ Th1 cells and the ratio of Th1/Th2 cells in vitro. In vitro antibody production and proliferation of B cells were also inhibited.

Conclusion

Resveratrol possesses protective effects in pristane-induced lupus mice and may represent a novel approach for the management of SLE.     

Lipoxin A4-Induced Heme Oxygenase-1 Protects Cardiomyocytes against Hypoxia/Reoxygenation Injury via p38 MAPK Activation and Nrf2/ARE Complex

Objective

To investigate whether lipoxin A₄ (LXA₄) increases expression of heme oxygenase-1(HO-1) in cardiomyocytes, whether LXA₄-induced HO-1 protects cardiomyocytes against hypoxia/reoxygenation (H/R) injury, and what are the mechanisms involved in the LXA₄-induced HO-1 induction.

Methods

Rat cardiomyocytes were exposed to H/R injury with or without preincubation with LXA₄ or HO-1 inhibitor ZnPP-IX or various signal molecule inhibitors. Expressions of HO-1 protein and mRNA were analyzed by using Western blot and RT-PCR respectively. Activity of nuclear factor E2-related factor 2 (Nrf2) binding to the HO-1 E1 enhancer was assessed by chromatin immunoprecipitation. Nrf2 binding to the HO-1 antioxidant responsive element (ARE) were measured by using electrophoretic mobility shift assay.

Results

Pretreatment of the cells undergoing H/R lesion with LXA₄ significantly reduced the lactate dehydrogenase and creatine kinase productions, increased the cell viability, and increased the expressions of HO-1 protein and mRNA and HO-1 promoter activity. HO-1 inhibition abolished the protective role of LXA₄ on the cells undergoing H/R lesion. LXA₄ increased p38 mitogen-activated protein kinase (p38 MAPK) activation, nuclear translocation of Nrf2, Nrf2 binding to the HO-1 ARE and E1 enhancer in cardiomyocytes with or without H/R exposure.

Conclusion

The protection role of LXA₄ against H/R injury of cardiomyocytes is related to upregulation of HO-1, via activation of p38 MAPK pathway and nuclear translocation of Nrf2 and Nrf2 binding to the HO-1 ARE and E1 enhancer, but not via activation of phosphatidyinositol-3-kinase or extracellular signal-regulated kinase pathway.     

Comparision of Piceid and Resveratrol in Antioxidation and Antiproliferation Activities In Vitro

Background

The clinic therapeutic effect of resveratrol is limited due to its low oral bioavailability. Piceid, a precursor of resveratrol, is the most abundant form of resveratrol in nature. A number of studies have hypothesized that piceid may have the same bioactivities like those of resveratrol. The aim of this work is to compare piceid with resveratrol in antioxidation and antiproliferation activities in vitro.

Methods

The antioxidative effects of resveratrol and piceid were evaluated by phenanthroline-Fe²⁺ method and H₂O₂-induced oxidative injury cell model. The antiproliferation effects were determined by MTT method in human liver tumor HepG2 cells, human breast cancer MDA-MB-231 cells and MCF-7 cells. The effects of resveratrol and piceid on the cell cycle and the apoptosis were evaluated by flow cytometry. Additionally, the uptake profiles of resveratrol and piceid in cancer cells were observed using fluorescence microscopy and clarified by LC-MS/MS.

Conclusion

Piceid exhibited higher scavenging activity against hydroxyl radicals than resveratrol in vitro. Resveratrol showed a significant protective effect against H₂O₂-induced cell damage. What is more, resveratrol had biphasic effects on tumor cells. Resveratrol and piceid only showed significant cytotoxicity on tumor cells at high concentration (≥50 µmol/L), while low concentration of resveratrol (<30 µmol/L) increased the cell viability. The principal effect of resveratrol and piceid on the viability of tumor cells was caused by the cell cycle arrest, while the effect on apoptosis was relatively minor. The reason that piceid showed lower biological activity than resveratrol at the same concentration was probably because piceid was more difficult in being uptaken by cells.     

Resveratrol inhibits inflammatory responses via the mammalian target of rapamycin signaling pathway in cultured LPS-stimulated microglial cells

Background

Resveratrol have been known to possess many pharmacological properties including antioxidant, cardioprotective and anticancer effects. Although current studies indicate that resveratrol produces neuroprotection against neurological disorders, the precise mechanisms for its beneficial effects are still not fully understood. We investigate the effect of anti-inflammatory and mechamisms of resveratrol by using lipopolysaccharide (LPS)-stimulated murine microglial BV-2 cells.

Methodology/Principal Findings

BV-2 cells were treated with resveratrol (25, 50, and 100 µM) and/or LPS (1 µg/ml). Nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by Griess reagent and ELISA. The mRNA and protein levels of proinflammatory proteins and cytokines were analysed by RT-PCR and double immunofluorescence labeling, respectively. Phosphorylation levels of PTEN (phosphatase and tensin homolog deleted on chromosome 10), Akt, mammalian target of rapamycin (mTOR), mitogen-activated protein kinases (MAPKs) cascades, inhibitor κB-α (IκB-α) and cyclic AMP-responsive element-binding protein (CREB) were measured by western blot. Resveratrol significantly attenuated the LPS-induced expression of NO, PGE2, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and nuclear factor-κB (NF-κB) in BV-2 cells. Resveratrol increased PTEN, Akt and mTOR phosphorylation in a dose-dependent manner or a time-dependent manner. Rapamycin (10 nM), a specific mTOR inhibitor, blocked the effects of resveratrol on LPS-induced microglial activation. In addition, mTOR inhibition partially abolished the inhibitory effect of resveratrol on the phosphorylation of IκB-α, CREB, extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal protein kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK).

Conclusion and Implications

This study indicates that resveratrol inhibited LPS-induced proinflammatory enzymes and proinflammatory cytokines via down-regulation phosphorylation of NF-κB, CREB and MAPKs family in a mTOR-dependent manner. These findings reveal, in part, the molecular basis underlying the anti-inflammatory properties of resveratrol.     

Role of dopamine D2 receptors in ischemia/reperfusion induced apoptosis of cultured neonatal rat cardiomyocytes

Background

Myocardial ischemia/reperfusion injury is the major cause of morbidity and mortality for cardiovascular diseases. Dopamine D₂ receptors are expressed in cardiac tissues. However, the roles of dopamine D₂ receptors in myocardial ischemia/reperfusion injury and cardiomyocyte apoptosis are unclear. Here we investigated the effects of both dopamine D₂ receptors agonist (bromocriptine) and antagonist (haloperidol) on apoptosis of cultured neonatal rat ventricular myocytes induced by ischemia/reperfusion injury.

Methods

Myocardial ischemia/reperfusion injury was simulated by incubating primarily cultured neonatal rat cardiomyocytes in ischemic (hypoxic) buffer solution for 2 h. Thereafter, these cells were incubated for 24 h in normal culture medium.

Results

Treatment of the cardiomyocytes with 10 μM bromocriptine significantly decreased lactate dehydrogenase activity, increased superoxide dismutase activity, and decreased malondialdehyde content in the culture medium. Bromocriptine significantly inhibited the release of cytochrome c, accumulation of [Ca²⁺]_i, and apoptosis induced by ischemia/reperfusion injury. Bromocriptine also down-regulated the expression of caspase-3 and -9, Fas and Fas ligand, and up-regulated Bcl-2 expression. In contrast, haloperidol (10 μM) had no significant effects on the apoptosis of cultured cardiomyocytes under the aforementioned conditions.

Conclusions

These data suggest that activation of dopamine D₂ receptors can inhibit apoptosis of cardiomyocytes encountered during ischemia/reperfusion damage through various pathways.     

Mechanical ventilation with high tidal volumes attenuates myocardial dysfunction by decreasing cardiac edema in a rat model of LPS-induced peritonitis

Background

Injurious mechanical ventilation (MV) may augment organ injury remote from the lungs. During sepsis, myocardial dysfunction is common and increased endothelial activation and permeability can cause myocardial edema, which may, among other factors, hamper myocardial function. We investigated the effects of MV with injuriously high tidal volumes on the myocardium in an animal model of sepsis.

Methods

Normal rats and intraperitoneal (i.p.) lipopolysaccharide (LPS)-treated rats were ventilated with low (6 ml/kg) and high (19 ml/kg) tidal volumes (Vt) under general anesthesia. Non-ventilated animals served as controls. Mean arterial pressure (MAP), central venous pressure (CVP), cardiac output (CO) and pulmonary plateau pressure (P_plat) were measured. Ex vivo myocardial function was measured in isolated Langendorff-perfused hearts. Cardiac expression of endothelial vascular cell adhesion molecule (VCAM)-1 and edema were measured to evaluate endothelial inflammation and leakage.

Results

MAP decreased after LPS-treatment and Vt-dependently, both independent of each other and with interaction. MV Vt-dependently increased CVP and Pplat and decreased CO. LPS-induced peritonitis decreased myocardial function ex vivo but MV attenuated systolic dysfunction Vt-dependently. Cardiac endothelial VCAM-1 expression was increased by LPS treatment independent of MV. Cardiac edema was lowered Vt-dependently by MV, particularly after LPS, and correlated inversely with systolic myocardial function parameters ex vivo.

Conclusion

MV attenuated LPS-induced systolic myocardial dysfunction in a Vt-dependent manner. This was associated with a reduction in cardiac edema following a lower transmural coronary venous outflow pressure during LPS-induced coronary inflammation.     

α-Lipoic Acid Reduces Infarct Size and Preserves Cardiac Function in Rat Myocardial Ischemia/Reperfusion Injury through Activation of PI3K/Akt/Nrf2 Pathway

Background

The present study investigates the effects and mechanisms of α-Lipoic acid (LA) on myocardial infarct size, cardiac function and cardiomyocyte apoptosis in rat hearts subjected to in vivo myocardial ischemia/reperfusion (MI/R) injury.

Methodology/Principal Findings

Male adult rats underwent 30 minutes of ischemia followed by 3, 24, or 72 h of reperfusion. Animals were pretreated with LA or vehicle before coronary artery ligation. The level of MI/R- induced LDH and CK release, infarct size, cardiomyocyte apoptosis and cardiac functional impairment were examined and compared. Western blot analysis was performed to elucidate the mechanism of LA pretreatment. The level of inflammatory cytokine TNF-α released to serum and accumulated in injured myocardium as well as neutrophil accumulation in injured myocardium were also examined after MI/R injury. Our results reveal that LA administration significantly reduced LDH and CK release, attenuated myocardial infarct size, decreased cardiomyocytes apoptosis, and partially preserved heart function. Western blot analysis showed that LA pretreatment up-regulated Akt phosphorylation and Nrf2 nuclear translocation while producing no impact on p38MAPK activation or nitric oxide (NO) production. LA pretreatment also increased expression of HO-1, a major target of Nrf2. LA treatment inhibited neutrophil accumulation and release of TNF-α. Moreover, PI3K inhibition abolished the beneficial effects of LA.

Conclusions/Significance

This study indicates that LA attenuates cardiac dysfunction by reducing cardiomyoctyes necrosis, apoptosis and inflammation after MI/R. LA exerts its action by activating the PI3K/Akt pathway as well as subsequent Nrf2 nuclear translocation and induction of cytoprotective genes such as HO-1.     

Human alveolar epithelial cell injury induced by cigarette smoke

Background

Cigarette smoke (CS) is a highly complex mixture and many of its components are known carcinogens, mutagens, and other toxic substances. CS induces oxidative stress and cell death, and this cell toxicity plays a key role in the pathogenesis of several pulmonary diseases.

Methodology/Principal Findings

We studied the effect of cigarette smoke extract (CSE) in human alveolar epithelial type I-like (ATI-like) cells. These are isolated type II cells that are differentiating toward the type I cell phenotype in vitro and have lost many type II cell markers and express type I cell markers. ATI-like cells were more sensitive to CSE than alveolar type II cells, which maintained their differentiated phenotype in vitro. We observed disruption of mitochondrial membrane potential, apoptosis and necrosis that were detected by double staining with acridine orange and ethidium bromide or Hoechst 33342 and propidium iodide and TUNEL assay after treatment with CSE. We also detected caspase 3 and caspase 7 activities and lipid peroxidation. CSE induced nuclear translocation of Nrf2 and increased expression of Nrf2, HO-1, Hsp70 and Fra1. Moreover, we found that Nrf2 knockdown sensitized ATI-like cells to CSE and Nrf2 overexpression provided protection against CSE-induced cell death. We also observed that two antioxidant compounds N-acetylcysteine and trolox protected ATI-like cells against injury by CSE.

Conclusions

Our study indicates that Nrf2 activation is a major factor in cellular defense of the human alveolar epithelium against CSE-induced toxicity and oxidative stress. Therefore, antioxidant agents that modulate Nrf2 would be expected to restore antioxidant and detoxifying enzymes and to prevent CS-related lung injury and perhaps lessen the development of emphysema.     

Resveratrol enhances antitumor activity of TRAIL in prostate cancer xenografts through activation of FOXO transcription factor

Background

Resveratrol (3, 4′, 5 tri-hydroxystilbene), a naturally occurring polyphenol, exhibits anti-inflammatory, antioxidant, cardioprotective and antitumor activities. We have recently shown that resveratrol can enhance the apoptosis-inducing potential of TRAIL in prostate cancer cells through multiple mechanisms in vitro. Therefore, the present study was designed to validate whether resveratrol can enhance the apoptosis-inducing potential of TRAIL in a xenograft model of prostate cancer.

Methodology/Principal Findings

Resveratrol and TRAIL alone inhibited growth of PC-3 xenografts in nude mice by inhibiting tumor cell proliferation (PCNA and Ki67 staining) and inducing apoptosis (TUNEL staining). The combination of resveratrol and TRAIL was more effective in inhibiting tumor growth than single agent alone. In xenografted tumors, resveratrol upregulated the expressions of TRAIL-R1/DR4, TRAIL-R2/DR5, Bax and p27^{/K IP1}, and inhibited the expression of Bcl-2 and cyclin D1. Treatment of mice with resveratrol and TRAIL alone inhibited angiogenesis (as demonstrated by reduced number of blood vessels, and VEGF and VEGFR2 positive cells) and markers of metastasis (MMP-2 and MMP-9). The combination of resveratrol with TRAIL further inhibited number of blood vessels in tumors, and circulating endothelial growth factor receptor 2-positive endothelial cells than single agent alone. Furthermore, resveratrol inhibited the cytoplasmic phosphorylation of FKHRL1 resulting in its enhanced activation as demonstrated by increased DNA binding activity.

Conclusions/Significance

These data suggest that resveratrol can enhance the apoptosis-inducing potential of TRAIL by activating FKHRL1 and its target genes. The ability of resveratrol to inhibit tumor growth, metastasis and angiogenesis, and enhance the therapeutic potential of TRAIL suggests that resveratrol alone or in combination with TRAIL can be used for the management of prostate cancer.     

Alveolar macrophages regulate neutrophil recruitment in endotoxin-induced lung injury

Background

Alveolar macrophages play an important role during the development of acute inflammatory lung injury. In the present study, in vivo alveolar macrophage depletion was performed by intratracheal application of dichloromethylene diphosphonate-liposomes in order to study the role of these effector cells in the early endotoxin-induced lung injury.

Methods

Lipopolysaccharide was applied intratracheally and the inflammatory reaction was assessed 4 hours later. Neutrophil accumulation and expression of inflammatory mediators were determined. To further analyze in vivo observations, in vitro experiments with alveolar epithelial cells and alveolar macrophages were performed.

Results

A 320% increase of polymorphonuclear leukocytes in bronchoalveolar lavage fluid was observed in macrophage-depleted compared to macrophage-competent lipopolysaccharide-animals. This neutrophil recruitment was also confirmed in the interstitial space. Monocyte chemoattractant protein-1 concentration in bronchoalveolar lavage fluid was significantly increased in the absence of alveolar macrophages. This phenomenon was underlined by in vitro experiments with alveolar epithelial cells and alveolar macrophages. Neutralizing monocyte chemoattractant protein-1 in the airways diminished neutrophil accumulation.

Conclusion

These data suggest that alveolar macorphages play an important role in early endotoxin-induced lung injury. They prevent neutrophil influx by controlling monocyte chemoattractant protein-1 production through alveolar epithelial cells. Alveolar macrophages might therefore possess robust anti-inflammatory effects.     

Resveratrols in Grape Berry Skins and Leaves in Vitis Germplasm

Background

Resveratrol is an important stilbene that benefits human health. However, it is only distributed in a few species including grape and is very expensive. At present, grape has been an important source resveratrol. However, the details are scarce on resveratrol distribution in different Vitis species or cultivars.

Methodology/Principal Finding

The composition and content of resveratrols were investigated by HPLC for assessing genotypic variation in berry skins and leaves of 75 grape cultivars, belonging to 3 species and 7 interspecific hybrids. Trans-resveratrol, cis-piceid and trans-piceid were detected in berry skins and leaves, but cis-resveratrol was not. Resveratrol content largely varied with genetic background as well as usage. In most cultivars, total resveratrol including the above three compounds was higher in berry skins than leaves. In berry skins of most cultivars and leaves of almost all cultivars, cis-piceid was the most abundant resveratrol; trans-resveratrol and trans-piceid were minor components. Some specific cultivars were found with extremely high levels of trans-resveratrol, cis- piceid, trans-piceid or total resveratrols in berry skins or leaves. In skins and leaves, rootstock cultivars had a higher content of total resveratrols, and the cultivated European type cultivars and their hybrids with V. labrusca had relatively low totals. There were no significant correlations of the amounts of total resveratrols or any individual resveratrol between berry skins and leaves. All 75 cultivars can be divided into four groups based on the composition of resveratrols and their concentration by principal component analysis.

Conclusion

Resveratrol content of grape berries and leaves varied largely with their genetic background and usage. Rootstock cultivars had a higher content of total resveratrols than the other germplasm. Total resveratrols were lower in leaves than berry skins in most cultivars. Cis-piceid was the most abundant resveratrol in most cultivars, and trans-res and trans-pd were minor components.