Emodin inhibits LPS-induced inflammatory response by activating PPAR-γ in mouse mammary epithelial cells

Emodin, an anthraquinone derivative isolated from the rhizomes of Rheum palmatum, has been reported to have a protective effect against lipopolysaccharide (LPS)-induced mastitis. However, the underlying molecular mechanisms are not well understood. The aim of this study was to investigate the molecular mechanisms of emodin in modifying lipopolysaccharide (LPS)-induced signaling pathways in mouse mammary epithelial cells (MEC). The pro-inflammatory cytokines were determined by ELISA. Nuclear factor-κB (NF-κB), inhibitory kappa B (IκBα) protein, p38, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and PPAR-γ were determined by Western blotting. The results showed that emodin suppressed tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), iNOS and COX-2 expression. We also found that emodin inhibited LPS-induced NF-κB activation, IκBα degradation, phosphorylation of ERK, JNK and P38. Furthermore, emodin could activate PPAR-γ and the anti-inflammatory effects of emodin can be reversed by GW9662, a specific antagonist for PPAR-γ. In conclusion, our results demonstrate that emodin activates PPAR-γ, thereby attenuating LPS-induced inflammatory response.     

Pyridoxine inhibits endothelial NOS uncoupling induced by oxidized low-density lipoprotein via the PKCα signalling pathway in human umbilical vein endothelial cells

BACKGROUND AND PURPOSE

One key mechanism for endothelial dysfunction is endothelial NOS (eNOS) uncoupling, whereby eNOS generates superoxide (O₂^•−) rather than NO. We explored the effect of pyridoxine on eNOS uncoupling induced by oxidized low-density lipoprotein (ox-LDL) in human umbilical vein endothelial cells (HUVECs) and the potential molecular mechanism.

EXPERIMENTAL APPROACH

HUVECs were incubated with ox-LDL with/without pyridoxine, N^G-nitro-L-arginine methylester (L-NAME), chelerythrine chloride (CHCI) or apocynin. Endothelial O₂^•− was measured using lucigenin chemiluminescence, and O₂^•−-sensitive fluorescent dye dihydroethidium (DHE). NO levels were measured by chemiluminescence, PepTag Assay for non-radioactive detection of PKC activity, depletion of PKCα and p47phox by siRNA silencing and the states of phospho-eNOS Thr495, total-eNOS, phospho-PKCα/βII, total PKC, phospho-PKCα, total PKCα and p47phox were measured by Western blot.

KEY RESULTS

Ox-LDL significantly increased O₂^•− production and reduced NO levels released from HUVECs; an effect reversed by eNOS inhibitor, L-NAME. Pyridoxine pretreatment significantly inhibited ox-LDL-induced O₂^•− generation and preserved NO levels. Pyridoxine also prevented the ox-LDL-induced reduction in phospho-eNOS Thr495 and PKC activity. These protective effects of pyridoxine were abolished by the PKC inhibitor, CHCI, or siRNA silencing of PKCα. However, depletion of p47phox or treatment with the NADPH oxidase inhibitor, apocynin, had no influence on these effects. Also, cytosol p47phox expression was unchanged by the different treatments.

CONCLUSIONS AND IMPLICATIONS

Pyridoxine mitigated eNOS uncoupling induced by ox-LDL. This protectant effect was related to phosphorylation of eNOS Thr495 stimulated by PKCα, not via NADPH oxidase. These results provide support for the use of pyridoxine in ox-LDL-related vascular endothelial dysfunction.     

Tetrandrine inhibits Ca^2＋-activated chloride channel in cultured human umbilical vein endothelial cells

INTRODUCTION Tetrandrine (Tet, 6,6',7,12-tetramethoxy-2,2'- dim-ethyl-berbaman) is a purified bis-benzylisoquinoline al-kaloid derivedfrom the root ofa Chinese herb (Stephaniatetrandra S Moore)[1,2]. It was first shown as an anti-hypertensive agent in both normal and hypertensivesubjects in 1950s[3,4]. The primary anti-hypertensiveaction of Tet is presumably due to its vasodilatoryproperty, which was confirmed both in vivo (15 mg/kg in conscious rats) and in vitro (1-100 μmol/L,effecti…     

Oridonin inhibits LPS-induced inflammation in human gingival fibroblasts by activating PPARγ

Oridonin, the major terpene isolated from Rabdosia rubescens, has been used as dietary supplement. Recently, it has been known to exhibit anti-inflammatory effect. This study we employed an in vitro model of LPS-stimulated human gingival fibroblasts to investigate the anti-inflammatory effects and mechanism of oridonin. Oridonin (10–30 μg/mL) was administrated 1 h before LPS treatment. The results showed that oridonin significantly inhibited inflammatory mediators PGE₂, NO, IL-6, and IL-8 production. Immunoblotting experiments revealed that oridonin reduced the expression of phosphorylation levels of NF-κB p65 and IκBα. Furthermore, the expression of PPARγ was up-regulated by the treatment of oridonin. Further studies showed that PPARγ inhibitor GW9662 could reverse the inhibition of oridonin on PGE₂, NO, IL-6, and IL-8 production. In conclusion, oridonin inhibited LPS-induced microglia activation through activating PPARγ.     

Modulation of Ecto-5′-Nucleotidase by phospholipids in human umbilical vein endothelial cells (HUVEC)

Ecto-5′-nucleotidase, the major enzyme controlling extracellular adenosine production, can be activated by phospholipids, e.g. lysophosphatidylcholine (LPC). This study examined the structural requirements of phospholipids to evoke this enzyme activation and figured out two new activators of ecto-5′-nucleotidase: platelet activating factor (PAF) and sphingosylphosphorylcholine (SPC). Potential signal transduction pathways including an involvement of protein kinase C and PAF-receptor were evaluated on the model of human umbilical vein endothelial cells (HUVEC). Cells were pre-incubated with 10 μM of various phospholipids including lysophosphatidylcholine, β-arachidonyl-γ-palmityl-α-phosphatidylcholine, β, γ-dipalmityl-α-phosphatidyl-choline, β,γ-dipalmityl-α-phosphatidylethanolamine, β,γ-dipalmityl-α-phosphatidylserine, γ-acyl-β-lyso-α-phosphatidylethanolamine, β-acetyl-γ-O-hexadecyl-α-phosphatidylcholine (platelet activating factor), lysophosphatidylic acid, sphingosine-1-phosphate and sphingosylphosphorylcholine. In the cell supernatant the extracellular dephosphorylation rate of the fluorescent AMP-analogue 1, N⁶-etheno-5′AMP to 1, N⁶-etheno-adenosine was measured by HPLC. Out of these ten structurally related phospholipids only lysophosphatidylcholine, sphingosylphosphatidylcholine and platelet activating factor dose-dependently increased the activity of ecto-5′-nucleotidase. Pharmacological blocking experiments revealed that neither the activation of PAF-receptor nor of protein kinase C were important for mediating the activation of ecto-5′-nucleotidase. Thus, using information on the known molecular structures of tested phospholipids, a phosphatidylcholine residue in α-position and a short chain length fatty acid esterified in β-position seem essential for activation of ecto-5′-nucleotidase by glycerophospholipids. Since all tested phospholipids have similar fatty acid chain lengths and residues in α-position, they should act similarly on membrane fluidity. It is concluded that the observed effects are not based on changes in membrane fluidity by the added phospholipids, but rather involve a yet to be determined phospholipid-receptor.     

OSU-A9 inhibits angiogenesis in human umbilical vein endothelial cells via disrupting Akt–NF-κB and MAPK signaling pathways

Since the introduction of angiogenesis as a useful target for cancer therapy, few agents have been approved for clinical use due to the rapid development of resistance. This problem can be minimized by simultaneous targeting of multiple angiogenesis signaling pathways, a potential strategy in cancer management known as polypharmacology. The current study aimed at exploring the anti-angiogenic activity of OSU-A9, an indole-3-carbinol-derived pleotropic agent that targets mainly Akt–nuclear factor-kappa B (NF-κB) signaling which regulates many key players of angiogenesis such as vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). Human umbilical vein endothelial cells (HUVECs) were used to study the in vitro anti-angiogenic effect of OSU-A9 on several key steps of angiogenesis. Results showed that OSU-A9 effectively inhibited cell proliferation and induced apoptosis and cell cycle arrest in HUVECs. Besides, OSU-A9 inhibited angiogenesis as evidenced by abrogation of migration/invasion and Matrigel tube formation in HUVECs and attenuation of the in vivo neovascularization in the chicken chorioallantoic membrane assay. Mechanistically, Western blot, RT-PCR and ELISA analyses showed the ability of OSU-A9 to inhibit MMP-2 production and VEGF expression induced by hypoxia or phorbol-12-myristyl-13-acetate. Furthermore, dual inhibition of Akt–NF-κB and mitogen-activated protein kinase (MAPK) signaling, the key regulators of angiogenesis, was observed. Together, the current study highlights evidences for the promising anti-angiogenic activity of OSU-A9, at least in part through the inhibition of Akt–NF-κB and MAPK signaling and their consequent inhibition of VEGF and MMP-2. These findings support OSU-A9's clinical promise as a component of anticancer therapy.     

Protective effect of α-lipoic acid on oxidized low density lipoprotein-induced human umbilical vein endothelial cell injury

The present study investigated the effect and possible mechanisms of α-lipoic acid (LA) in preventing endothelial cell injury induced by oxidized low-density lipoprotein (oxLDL). A model of human umbilical vein endothelial cell (HUVEC) injury was established by incubating the HUVECs with 200 μg/ml oxLDL. HUVECs were pre-treated with 0.1, 0.2 or 0.5 mmol/l of LA in the presence of oxLDL for 24 h. Apoptosis and cellular surface ceramide content were investigated separately by flow cytometry and by LC-MS/MS. LOX-1, Bcl-2 and CRP protein expression levels were evaluated by western blotting. LOX-1 mRNA expression was evaluated by RT-PCR assay. The results showed that oxLDL induced cytotoxicity in both concentration-dependent and time-dependent manners. LA boosted the cell survival rate and significantly reduced the content of MDA and lactate dehydrogenase (LDH) leakage. Apoptotic rates were significantly reduced by the addition of LA compared to oxLDL group. LA might also have inhibited ceramide generation induced by oxLDL in a dose-dependent manner. Furthermore, LA down-regulated LOX-1 protein and mRNA expression and up-regulated Bcl-2 protein expression levels in a dose-dependent manner. Expression of CRP protein was weak and undetectable. These results suggested that LA exhibited cytoprotective effects against oxLDL by decreasing apoptotic rates and decreasing cellular surface ceramide content, two effects that are related to decreased LOX-1 expression, and also by stimulating the expression of Bcl-2 protein. The cytoprotective effects are not thought to be due to inhibited C-reactive protein (CRP) protein expression in HUVECs.     

E-selectin is a viable route of infection for polymer-coated adenovirus retargeting in TNF-α-activated human umbilical vein endothelial cells

Background: E-selectin is an attractive endothelial cell surface marker in inflammation and cancer. Purpose: We sought to investigate retargeting of adenovirus via E-selectin as a viable pathway of infection in tumor necrosis factor-α (TNF-α)-activated human umbilical vein endothelial cells (HUVECs).

Methods: E1, E3-deleted Ad5 expressing cytomegalovirus immediate-early (CMV IE) promoter-driven luciferase (Adluc) was coated with an amino-reactive multivalent hydrophilic polymer based on poly [N-(2-hydroxypropyl) methacrylamide] to generate pHPMA-adenovirus (pcAdluc). This was then retargeted by covalent attachment of a mouse antihuman E-selectin monoclonal antibody (MHES mAb), purified from the H18/7 hybridoma cell line (MHESpcAdluc).

Results: MHESpcAdluc was efficiently taken up into HUVECs, generating a high level of transduction in TNF-α-treated E-selectin positive cells but not in untreated receptor-negative cells. Specific retargeting of MHESpcAdluc was demonstrated through reduced transduction of stimulated HUVEC when incubated in the presence of free E-selectin antibodies.

Discussion and conclusion: Our results suggest that E-selectin could be a valuable target for gene transfer strategies internalizing polymer-coated modified adenovirus particles through a viable receptor-mediated endocytosis pathway, generating adequate levels of transgene expression per virus genome copy without compromising the specific activity of the parental virus.     

Glycine inhibits the LPS-induced increase in cytosolic Ca2+ concentration and TNFα production in cardiomyocytes by activating a glycine receptor

Aim:

Previous studies have demonstrated that glycine (GLY) markedly reduces lipopolysaccharide (LPS)-induced myocardial injury. However, the mechanism of this effect is still unclear. The present study investigated the effect of GLY on cytosolic calcium concentration ([Ca²⁺]_c) and tumor necrosis factor-α (TNFα) production in cardiomyocytes exposed to LPS, as well as whether the glycine-gated chloride channel is involved in this process.

Methods:

Neonatal rat cardiomyocytes were isolated, and the [Ca²⁺]_c and TNFα levels were determined by using Fura-2 and a Quantikine enzyme-linked immunosorbent assay, respectively. The distribution of the GLY receptor and GLY-induced currents in cardiomyocytes were also investigated using immunocytochemistry and the whole-cell patch-clamp technique, respectively.

Results:

LPS at concentrations ranging from 10 ng/mL to 100 μg/mL significantly stimulated TNFα production. GLY did not inhibit TNFα production induced by LPS at concentrations below 10 ng/mL but did significantly decrease TNFα release stimulated by 100 μg/mL LPS and prevented an LPS-induced increase in [Ca²⁺]_c, which was reversed by strychnine, a glycine receptor antagonist. GLY did not block the isoproterenol-induced increase in [Ca²⁺]_c, but did prevent the potassium chloride-induced increase in [Ca²⁺]_c in cardiomyocytes. Strychnine reversed the inhibition of the KCl–stimulated elevation in [Ca²⁺]_c by GLY. In chloride-free buffer, GLY had no effect on the dipotassium hydrogen phosphate-induced increase in [Ca²⁺]_c. Furthermore, GLY receptor α₁ and β subunit-immunoreactive spots were observed in cardiomyocytes, and GLY-evoked currents were blocked by strychnine.

Conclusion:

Cardiomyocytes possess the glycine-gated chloride channel, through which GLY prevents the increase in [Ca²⁺]_c and inhibits the TNFα production induced by LPS at high doses in neonatal rat cardiomyocytes.     

Glycine inhibits the LPS-induced increase in cytosolic Ca^2＋ concentration and TNFα production in cardiomyocytes by activating a glycine receptor

Aim： Previous studies have demonstrated that glycine （GLY） markedly reduces lipopolysaccharide （LPS）-induced myocardial injury. However, the mechanism of this effect is still unclear. The present study investigated the effect of GLY on cytosolic calcium concentration （[Ca^2＋]c） and tumor necrosis factor-α（TNFα） production in cardiomyocytes exposed to LPS, as well as whether the glycine-gated chloride channel is involved in this process. Methods： Neonatal rat cardiomyocytes were isolated, and the [Ca^2＋]c and TNFα levels were determined by using Fura-2 and a Quan tikine enzyme-linked immunosorbent assay, respectively. The distribution of the GLY receptor and GLY-induced currents in cardiomyocytes were also investigated using immunocytochemistry and the whole-cell patch-clamp technique, respectively. Results： LPS at concentrations ranging from 10 ng/mL to 100 pg/mL significantly stimulated TNFα production. GLY did not inhibit TNFα production induced by LPS at concentrations below 10 ng/mL but did significantly decrease TNFα release stimulated by 100 pg/mL LPS and prevented an LPS-induced increase in [Ca^2＋]c, which was reversed by strychnine, a glycine receptor antagonist. GLY did not block the isoproterenol-induced increase in [Ca^2＋]c, but did prevent the potassium chloride-induced increase in [Ca2＋]cin cardiomyocytes. Strychnine reversed the inhibition of the KCl-stimulated elevation in [Ca^2＋]cby GLY. In chloride-free buffer, GLY had no effect on the dipotassium hydrogen phosphate-induced increase in [Ca^2＋]c. Furthermore, GLY receptor α1 and β subunitimmunoreactive spots were observed in cardiomyocytes, and GLY-evoked currents were blocked by strychnine. Conclusion： Cardiomyocytes possess the glycine-gated chloride channel, through which GLY prevents the increase in [Ca^2＋]c and inhibits the TNFα production induced by LPS at high doses in neonatal rat cardiomyocytes.     

Proteomics analysis of human umbilical vein endothelial cells （HUVEC） after treatment with low molecular weight heparin

AIM: The endothelium is involved in the generation and the regulation of multiple physiological and pathological processes of blood vessels. Previously we confirmed low molecular weight heparin (LMWH) could inhibit tumor metastasis by protecting human umbilical vein endothelial cells (HUVEC). To understand the effects of LMWH on the protein expression of HUVEC, we performed a comprehensive proteomics to survey global changes in proteins after LMWH treatment in HUVEC cells. METHODS:     

PGC-1α ameliorates AngiotensinII-induced eNOS dysfunction in human aortic endothelial cells

Increasing evidences support that PGC-1α participates in regulating endothelial homeostasis, in part by mediating endothelial nitric oxide (NO) synthase (eNOS) activity and NO production. However, the molecular mechanisms by which PGC-1α regulates eNOS activity are not completely understood. In the present study, we investigated the effects of PGC-1α on eNOS dysfunction and further explore the underlying mechanisms. The results showed that PGC-1α expression was downregulated after AngiotensinII (AngII) treatment and paralleled with the decreased NO generation in human aortic endothelial cells. Overexpression of PGC-1α with adenovirus or pharmacological agonist ameliorated AngII-induced the decrease of NO generation, evidenced by the restoration of cGMP and nitrite concentration. Rather than affecting eNOS expression and uncoupling, PGC-1α inhibited AngII-induced decrease of eNOS serine 1177 phosphorylation through activation of PI3K/Akt signaling. In addition, PGC-1α overexpression suppressed AngII-induced the increase of PP2A-A/eNOS interaction and PP2A phosphatase activity, with a concomitant decrease in PP2A phosphorylation, leading to eNOS serine 1177 phosphorylation. However, pharmacological inhibition of PI3K/Akt signaling blunted the observed effect of PGC-1α on PP2A activity. Taken together, our findings suggest that PGC-1α overexpression improves AngII-induced eNOS dysfunction and that improved eNOS dysfunction is associated with activated PI3K/Akt pathway, impaired PP2A activity and reduced PP2A-A/eNOS association. These date indicate that forced PGC-1α expression may be a novel therapeutic approach for endothelial dysfunction.     

Salvianolic acid B suppresses maturation of human monocyte-derived dendritic cells by activating PPARγ

BACKGROUND AND PURPOSE

Salvianolic acid B (Sal B), a water-soluble antioxidant derived from a Chinese medicinal herb, is known to be effective in the prevention of atherosclerosis. Here, we tested the hypothesis that the anti-atherosclerotic effect of Sal B might be mediated by suppressing maturation of human monocyte-derived dendritic cells (h-monDC).

EXPERIMENTAL APPROACH

h-monDC were derived by incubating purified human monocytes with GM-CSF and IL-4. h-monDC were pre-incubated with or without Sal B and stimulated by oxidized low-density lipoprotein (ox-LDL) in the presence or absence of PPARγ siRNA. Expression of h-monDC membrane molecules (CD40, CD86, CD1a, HLA-DR) were analysed by FACS, cytokines were measured by elisa and the TLR4-associated signalling pathway was determined by Western blotting.

KEY RESULTS

Ox-LDL promoted h-monDC maturation, stimulated CD40, CD86, CD1a, HLA-DR expression and IL-12, IL-10, TNF-α production; and up-regulated TLR4 signalling. These effects were inhibited by Sal B. Sal B also triggered PPARγ activation and promoted PPARγ nuclear translocation, attenuated ox-LDL-induced up-regulation of TLR4 and myeloid differentiation primary-response protein 88 and inhibited the downstream p38-MAPK signalling cascade. Knocking down PPARγ with the corresponding siRNA blocked these effects of Sal B.

CONCLUSIONS AND IMPLICATIONS

Our data suggested that Sal B effectively suppressed maturation of h-monDC induced by ox-LDL through PPARγ activation.     

Activation of LXRα induces lipogenesis in HaCaT cells

The oxysterol nuclear receptors, LXRα (liver X receptor α; NR1H3) and LXRβ (NR1H2), coordinately regulate the expression of genes involved in lipid metabolism, anti-inflammation, and cholesterol transport. Previous studies have demonstrated that ligands of LXRα are important in the maintenance of the normal epidermal barrier function and keratinocyte differentiation. In this study, we examined whether LXRα and its ligands regulate lipid synthesis in HaCaT cells, a spontaneously transformed human keratinocyte cell line. When HaCaT cells were treated with the LXRα ligand TO901317, lipid droplets accumulated in the majority of cells, which were stained by Oil Red O. A luciferase reporter construct containing the LXR response element was activated about fourfold in HaCaT cells by TO901317 treatment, suggesting that LXR has a role in lipid synthesis in these cells. The expression of LXRα target genes, such as those encoding sterol regulatory binding protein and fatty acid synthase, were induced time dependently by TO901317, as measured by RT-PCR and western blotting. The expression of PPAR-α, -β, and -γ which regulate lipid metabolism, was also increased by TO901317 treatment. In contrast, TO901317 reduced the lipopolysaccharide-induced expression of cyclooxygenase 2 and inducible nitric oxide synthase in HaCaT cells. These results indicate that LXRα activation leads to lipogenesis in keratinocytes, which may enhance the epidermal barrier function of the skin.     

Fisetin inhibits IL-1β-induced inflammatory response in human osteoarthritis chondrocytes through activating SIRT1 and attenuates the progression of osteoarthritis in mice

Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage degradation and inflammation. Fisetin, a polyphenol extracted from fruits and vegetables, has been reported to have anti-inflammatory effects. Our study aimed to investigate the effect of fisetin on OA both in vitro and in vivo. In vitro, chondrocytes were pretreated with fisetin alone or fisetin combined with sirtinol (an inhibitor of SIRT1) for 2 h before IL-1β stimulation. Production of NO, PGE2, TNF-α and IL-6 were evaluated by the Griess reaction and ELISAs. The mRNA (COX-2, iNOS, MMP-3, MMP-13, ADAMTS-5, Sox-9, aggrecan and collagen-II) and protein expression (COX-2, iNOS, MMP-3, MMP-13, ADAMTS-5 and SIRT1) were measured by qRT-PCR and Western blot respectively. Immunofluorescence was used to assess the expression of collagen-II and SIRT1. SIRT1 activity was quantified with SIRT1 fluorometric assay kit. The in vivo effect of fisetin was evaluated by gavage in mice OA models induced by destabilization of the medial meniscus (DMM). We found that fisetin inhibited IL-1β-induced expression of NO, PGE2, TNF-α, IL-6, COX-2, iNOS, MMP-3, MMP-13, ADAMTS-5. Besides, fisetin remarkably decreased IL-1β-induced degradation of Sox-9, aggrecan and collagen-II. Furthermore, fisetin significantly inhibited IL-1β-induced SIRT1 decrease and inactivation. However, the inhibitory effect of fisetin was obvious abolished by sirtinol, suggesting that fisetin exerts anti-inflammatory effects through activating SIRT1. In vivo, fisetin-treated mice exhibited less cartilage destruction and lower OARSI scores. Moreover, fisetin reduced subchondral bone plate thickness and alleviated synovitis. Taken together, these findings indicate that fisetin may be a potential agent in the treatment of OA.