Anti-Inflammatory Activity of Ferula assafoetida Oleo-Gum-Resin (Asafoetida) against TNF-α-Stimulated Human Umbilical Vein Endothelial Cells (HUVECs).

Inflammation is the body's biological reaction to endogenous and exogenous stimuli. Recent studies have demonstrated several anti-inflammatory properties of Ferula species. In this paper, we decided to study the anti-inflammatory effect of ethanolic extract of Ferula assafoetida oleo-gum-resin (asafoetida) against TNF-α-stimulated human umbilical vein endothelial cells (HUVECs). HUVECs were cultured in a flat-bottom plate and then treated with ethanolic extract of asafoetida (EEA, 0-500 µg/ml) and TNF-α (0-100 ng/ml) for 24 h. We used the MTT test to assess cell survival. In addition, the LC-MS analysis was performed to determine the active substances. HUVECs were pretreated with EEA and then induced by TNF-α. Intracellular reactive oxygen species (ROS) and adhesion of peripheral blood mononuclear cells (PBMCs) to HUVECs were evaluated with DCFH-DA and CFSE fluorescent probes, respectively. Gene expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin and surface expression of ICAM-1 protein were measured using real-time PCR and flow cytometry methods, respectively. While TNF-α significantly increased intracellular ROS formation and PBMC adhesion to TNF-α-induced HUVECs, the pretreatment of HUVECs with EEA (125 and 250 µg/ml) significantly reduced the parameters. In addition, EEA pretreatment decreased TNF-α-induced mRNA expression of VCAM-1 and surface protein expression of ICAM-1 in the target cells. Taken together, the results indicated that EEA prevented ROS generation, triggered by TNF-α, and inhibited the expression of VCAM-1 and ICAM-1, leading to reduced PBMC adhesion. These findings suggest that EEA can probably have anti-inflammatory properties.

The Unitary Micro-Immunotherapy Medicine Interferon-γ (4 CH) Displays Similar Immunostimulatory and Immunomodulatory Effects than Those of Biologically Active Human Interferon-γ on Various Cell Types.

As a cytokine, gamma-interferon (IFN-γ) is considered a key player in the fine-tuned orchestration of immune responses. The extreme cellular sensitivity to cytokines is attested by the fact that very few of these bioactive molecules per cell are enough to trigger cellular functions. These findings can, at least partially, explain how/why homeopathically-prepared cytokines, and especially micro-immunotherapy (MI) medicines, are able to drive cellular responses. We focused our fundamental research on a unitary MI preparation of IFN-γ, specifically employed at 4 CH, manufactured and impregnated onto sucrose-lactose pillules as all other MI medicines. We assessed the IFN-γ concentration in the medium after dilution of the IFN-γ (4 CH)-bearing pillules and we evaluated in vitro drug responses in a wide range of immune cells, and in endothelial cells. Our results showed that IFN-γ (4 CH) stimulated the proliferation, the activation and the phagocytic capabilities of primary immune cells, as well as modulated their cytokine-secretion and immunity-related markers' expression in a trend that is quite comparable with the well-recognized biological effects induced by IFN-γ. Altogether, these data provide novel and additional evidences on MI medicines, and specifically when active substances are prepared at 4 CH, thus suggesting the need for more investigations.

Triptolide attenuates lipopolysaccharide-induced inflammatory responses in human endothelial cells: involvement of NF-κB pathway.

BACKGROUND: Endothelial cell inflammation is a central event in the pathogenesis of numerous cardiovascular diseases, including sepsis and atherosclerosis. Triptolide, a principal bioactive ingredient of Traditional Chinese Medicine Tripterygium wilfordii Hook.F., displays anti-inflammatory actions in vivo. However, the mechanisms underlying these beneficial effects remain undetermined. The present study investigated the effects and possible mechanisms of triptolide on lipopolysaccharide (LPS)-induced inflammatory responses in human umbilical vein endothelial cells (HUVECs). METHODS: The effects of triptolide on the LPS-induced production and expression of inflammatory molecules, monocyte adhesion and activation of nuclear factor (NF)-κB pathway were examined in cultured HUVECs. RESULTS: In cultured HUVECs, pre-treatment with triptolide dose-dependently attenuated LPS-induced cytokine and chemokine production, adhesion molecule expression and monocyte adhesion. Mechanistically, triptolide was found to dose-dependently inhibit the LPS-induced increases in the DNA binding activity of NF-κB p65 associated with attenuating IκBα phosphorylation and its degradation. Additionally, the present study revealed that triptolide inhibited LPS-triggered NF-κB transcriptional activation in a dose-dependent manner. CONCLUSIONS: The results of the present study indicated that triptolide suppresses the inflammatory response of endothelial cells possibly via inhibition of NF-κB activation.

Aloin reduces inflammatory gene iNOS via inhibition activity and p-STAT-1 and NF-κB.

Aloin is the major anthraquinone glycoside obtained from the Aloe species and exhibits anti-inflammatory and anti-oxidative activities. Here, we aimed to determine the effects of aloin on heme oxygenase-1 (HO-1) induction and on the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX) 2 in lipopolysaccharide (LPS)-activated human umbilical vein endothelial cells (HUVECs). To the end, aloin was tested whether aloin reduces iNOS protein expression and inflammatory markers (interleukin (IL)-1ß and tumor necrosis factor (TNF)-α) in LPS-treated mice lung tissue. The results indicated that aloin affected HO-1 induction and reduced LPS-activated NF-κB-luciferase activity showed to preferential inhibition of iNOS/NO and COX-2/PGE2 that was partly related to inhibition of STAT-1 phosphorylation. In particular, aloin induced translocation of Nrf2 from cytosol into the nucleus by an increased Nrf2-ARE binding activity, and reduced IL-1ß production in LPS-activated HUVECs. The reduced expression of iNOS/NO by aloin was reversed by siHO-1RNA-transfection. In LPS-treated mice, aloin significantly reduced iNOS protein in lung tissues, and TNF-α levels in the BALF. We concluded that aloin may be beneficial for treatment of lung injury.

Involvement of heme oxygenase-1 induction in anti-vascular inflammation effects of Xanthoceras sorbifolia in human umbilical vein endothelial cells.

OBJECTIVE: To define the effects of Xanthoceras sorbifolia (EXS) on vascular inflammation and the mechanisms in endothelial cells. METHODS: Vascular protective effects of an ethanol extract of seeds from EXS (1-50 µg/mL) against tumor necrosis factor-α (TNF-α)-induced vascular inflammation were examined in human umbilical vein endothelial cells (HUVECs). RESULTS: EXS significantly decreased TNF-α-induced expression of cell adhesion molecules, such as intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and endothelial cell selectin, in a dose-dependent manner. Pre-treatment with EXS significantly inhibited translocation and transcriptional activity of nuclear factor-κB (NF-κB) increased by TNF-α. EXS also significantly inhibited formation of intracellular reactive oxygen species (ROS). Moreover, the vascular protective effects of EXS were linked to up-regulation of heme oxygenase-1 (HO-1) and nuclear factor E2-related factor-2 (Nrf-2) expression. EXS-induced HO-1 expression was significantly decreased in SnPP (HO-1 inhibitor)- and HO-1 siRNA-treated cells, whereas an increase was found in cobalt protoporphyrin IX (CoPP) (HO-1 inducer)-treated cells. In addition, pretreatment with EXS increased HO-1 and Nrf-2 expression under TNF-α stimulation with or without N-acetyl-L-cysteine. Furthermore, the inhibitory effects of EXS on TNF-α-induced vascular inflammation were partially reversed in SnPP- and of HO-1 siRNA-treated cells but increased by CoPP. CONCLUSION: These results suggest that EXS may have important implications for prevention of vascular complications associated with vascular inflammation by inhibition of the NF-¦ÊB/ROS pathway and activation of the Nrf-2/HO-1 pathway.

Ethanol Extract of Brassica rapa ssp. pekinensis Suppresses Tumor Necrosis Factor-α-Induced Inflammatory Response in Human Umbilical Vein Endothelial Cells.

Brassica rapa L. ssp. pekinensis, commonly known as Chinese cabbage, is a cruciferous vegetable traditionally consumed in east Asia. Although its habitual consumption could account for the low incidence of chronic vascular inflammation, the therapeutic and protective potential of phytochemicals derived from Chinese cabbage has been poorly studied. In this study, we identified the phenolic compounds, kaempferol and quercetin, from the ethanol extract of Chinese cabbage (EtCC). We show for the first time that EtCC contains effective phytochemicals that suppress tumor necrosis factor (TNF)-α-induced inflammatory response in human umbilical vein endothelial cells. The EtCC inhibited TNF-α-induced monocyte adhesion to endothelial cells in a dose-dependent manner. The antiadhesive activity of EtCC directly correlated with downregulation of expression and transcription of vascular cell adhesion molecule-1 (VCAM-1). It was caused by an Nrf-2-dependent mechanism, leading to activation of antioxidant responsive element-driven promoter. Taken together, these results suggest that EtCC inhibits the expression of TNF-α-induced adhesion molecules through the indirect transcriptional modulation of VCAM-1 in endothelial cells. In conclusion, regular consumption of vegetables containing dietary phytochemicals might be a potential therapeutic strategy to protect against various stresses, to prevent several pathological conditions, and to treat chronic vascular inflammation, such as atherosclerosis.

Oral butyrate reduces oxidative stress in atherosclerotic lesion sites by a mechanism involving NADPH oxidase down-regulation in endothelial cells.

Butyrate is a 4-carbon fatty acid that has antiinflammatory and antioxidative properties. It has been demonstrated that butyrate is able to reduce atherosclerotic development in animal models by reducing inflammatory factors. However, the contribution of its antioxidative effects of butyrate on atherogenesis has not yet been studied. We investigated the influence of butyrate on oxidative status, reactive oxygen species (ROS) release and oxidative enzymes (NADPH oxidase and iNOS) in atherosclerotic lesions of ApoE(-/-) mice and in oxLDL-stimulated peritoneal macrophages and endothelial cells (EA.hy926). The lesion area in aorta was reduced while in the aortic valve, although lesion area was unaltered, superoxide production and protein nitrosylation were reduced in butyrate-supplemented mice. Peritoneal macrophages from the butyrate group presented a lower free radical release after zymosan stimulus. When endothelial cells were pretreated with butyrate before oxLDL stimulus, the CCL-2 and superoxide ion productions and NADPH oxidase subunit p22phox were reduced. In macrophage cultures, in addition to a reduction in ROS release, nitric oxide and iNOS expression were down-regulated. The data suggest that one mechanism related to the effect of butyrate on atherosclerotic development is the reduction of oxidative stress in the lesion site. The reduction of oxidative stress related to NADPH oxidase and iNOS expression levels associated to butyrate supplementation attenuates endothelium dysfunction and macrophage migration and activation in the lesion site.

Tricin, flavonoid from Njavara reduces inflammatory responses in hPBMCs by modulating the p38MAPK and PI3K/Akt pathways and prevents inflammation associated endothelial dysfunction in HUVECs.

Previous studies revealed the potent anti-inflammatory activity of tricin, the active component of Njavara rice bran. Here, we report the involvement of specific signaling pathways in the protective effect of tricin against LPS induced inflammation in hPBMCs and the role of tricin in modulating endothelial dysfunction in LPS induced HUVECs. Pretreatment with tricin (15µM) significantly inhibited the release of TNF-α and was comparable to the specific pathway blockers like ERK inhibitor (PD98059), JNK inhibitor (SP600125) and p38 inhibitor (SB203580), whereas an increased release of TNF-α was observed in PI3K/Akt inhibitor (LY294002) treated cells. Tricin alone and combination treatment of tricin and SB203580 showed more significant inhibition of activation of COX-2 and TNF-α than that of SB203580 alone treated group. Combination treatment of tricin and LY294002 showed increased activation of COX-2 and TNF-α, proved that PI3K activation is essential for the anti-inflammatory effect of tricin. Studies conducted on HUVECs revealed the protective effect of tricin against endothelial dysfunction associated with LPS induced inflammation by inhibiting the activation of proinflammatory mediators like TNF-α, IFN-γ, MCP 1 by modulating NF-κB and MAPK signaling pathways. ELISA and flow cytometric analysis again confirmed the protection of tricin against endothelial damage, especially from the decreased activation of cell adhesion molecules like ICAM-1, VCAM-1 and E-Selectin upon tricin treatment. This work establishes the mechanism behind the potent anti-inflammatory activity of the flavonoid tricin.

Ameliorative Effect of Vicenin-2 and Scolymoside on TGFBIp-Induced Septic Responses.

Transforming growth factor ß-induced protein (TGFBIp) is an extracellular matrix protein whose expression in several cell types is greatly increased by TGF-ß. TGFBIp is released by the human umbilical vein endothelial cells (HUVECs) and functions as a mediator of experimental sepsis. Cyclopia subternata is a medicinal plant commonly used in traditional medicine to relieve pain in biological processes. In this study, we investigated the antiseptic effects and underlying mechanisms of vicenin-2 and scolymoside, two active compounds in C. subternata against TGFBIp-mediated septic responses in HUVECs and mice. The anti-inflammatory activities of vicenin-2 or scolymoside were determined by measuring permeability, human neutrophils adhesion and migration, and activation of pro-inflammatory proteins in TGFBIp-activated HUVECs and mice. According to the results, vicenin-2 or scolymoside effectively inhibited lipopolysaccharide-induced release of TGFBIp and suppressed TGFBIp-mediated septic responses, such as hyperpermeability, adhesion and migration of leukocytes, and expression of cell adhesion molecules. In addition, vicenin-2 or scolymoside suppressed the production of tumor necrosis factor-α and interleukin 6 and activation of nuclear factor-κB and extracellular regulated kinases 1/2 by TGFBIp. Vicenin-2 or scolymoside reduced cecal ligation and puncture (CLP)-induced septic mortality and pulmonary injury. Collectively, these results indicate that vicenin-2 and scolymoside could be a potential therapeutic agent for treatment of various severe vascular inflammatory diseases via inhibition of the TGFBIp signaling pathway.

Phytochemical profile, antioxidative and anti-inflammatory potentials of Gynura bicolor DC.

BACKGROUND: The phytochemical composition of aqueous and ethanol extracts from Gynura bicolor DC., a vegetable, was determined. Human umbilical vein endothelial (HUVE) cells were used to examine the antioxidative and anti-inflammatory potentials of these extracts at 1, 2 or 4% (v/v) against high-glucose-induced injury. RESULTS: Both aqueous and ethanol extracts contained phenolic acids, flavonoids, carotenoids and anthocyanins in the ranges 1428-1569, 1934-2175, 921-1007 and 2135-2407 mg per 100 g dry weight respectively. Both extracts were rich in quercetin, lutein, malvidin and pelargonidin. Addition of these extracts at test doses decreased reactive oxygen species formation, preserved glutathione content and retained glutathione peroxide and catalase activities in high-glucose-treated HUVE cells (P < 0.05). Treatments with these extracts at 2 and 4% lowered interleukin-6, tumor necrosis factor-alpha and prostaglandin E2 production and reduced cyclooxygenase-2 activity (P < 0.05). CONCLUSION: These findings suggest that this vegetable could be considered as a functional food and might provide antioxidative and anti-inflammatory protection.

Anti-inflammatory effect of the blueberry anthocyanins malvidin-3-glucoside and malvidin-3-galactoside in endothelial cells.

Blueberry fruits have a wide range of health benefits because of their abundant anthocyanins, which are natural antioxidants. The purpose of this study was to investigate the inhibitory effect of blueberry's two main anthocyanins (malvidin-3-glucoside and malvidin-3-galactoside) on inflammatory response in endothelial cells. These two malvidin glycosides could inhibit tumor necrosis factor-alpha (TNF-α) induced increases of monocyte chemotactic protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) production both in the protein and mRNA levels in a concentration-dependent manner. Mv-3-glc at the concentration of 1 µM could inhibit 35.9% increased MCP-1, 54.4% ICAM-1, and 44.7% VCAM-1 protein in supernatant, as well as 9.88% MCP-1 and 48.6% ICAM-1 mRNA expression (p<0.05). In addition, they could decrease IκBα degradation (Mv-3-glc, Mv-3-gal, and their mixture at the concentration of 50 µM had the inhibition rate of 84.8%, 75.3%, and 43.2%, respectively, p<0.01) and block the nuclear translocation of p65, which suggested their anti-inflammation mechanism was mediated by the nuclear factor-kappa B (NF-κB) pathway. In general malvidin-3-glucoside had better anti-inflammatory effect than malvidin-3-galactoside. These results indicated that blueberry is good resource of anti-inflammatory anthocyanins, which can be promising molecules for the development of nutraceuticals to prevent chronic inflammation in many diseases.

Identification of plumericin as a potent new inhibitor of the NF-κB pathway with anti-inflammatory activity in vitro and in vivo.

BACKGROUND AND PURPOSE: The transcription factor NF-κB orchestrates many pro-inflammatory signals and its inhibition is considered a promising strategy to combat inflammation. Here we report the characterization of the natural product plumericin as a highly potent inhibitor of the NF-κB pathway with a novel chemical scaffold, which was isolated via a bioactivity-guided approach, from extracts of Himatanthus sucuuba, an Amazonian plant traditionally used to treat inflammation-related disorders. EXPERIMENTAL APPROACH: A NF-κB luciferase reporter gene assay was used to identify NF-κB pathway inhibitors from H. sucuuba extracts. Monitoring of TNF-α-induced expression of the adhesion molecules VCAM-1, ICAM-1 and E-selectin by flow cytometry was used to confirm NF-κB inhibition in endothelial cells, and thioglycollate-induced peritonitis in mice to confirm effects in vivo. Western blotting and transfection experiments were used to investigate the mechanism of action of plumericin. KEY RESULTS: Plumericin inhibited NF-κB-mediated transactivation of a luciferase reporter gene (IC50 1 µM), abolished TNF-α-induced expression of the adhesion molecules VCAM-1, ICAM-1 and E-selectin in endothelial cells and suppressed thioglycollate-induced peritonitis in mice. Plumericin exerted its NF-κB pathway inhibitory effect by blocking IκB phosphorylation and degradation. Plumericin also inhibited NF-κB activation induced by transfection with the constitutively active catalytic subunit of the IκB kinase (IKK-ß), suggesting IKK involvement in the inhibitory action of this natural product. CONCLUSION AND IMPLICATIONS: Plumericin is a potent inhibitor of NF-κB pathways with a new chemical scaffold. It could be further explored as a novel anti-inflammatory lead compound.

Flos lonicerae extracts and chlorogenic acid protect human umbilical vein endothelial cells from the toxic damage of perfluorooctane sulphonate.

Chlorogenic acid (CGA), one of the most common phenolic acids, is found in many food and traditional Chinese herbs. Various bioactivities of CGA are studied. However, little is known about these properties of Flos Lonicerae extracts, and the difference in the effect between Flos Lonicerae extracts and CGA has not been reported. CGA was identified in Flos Lonicerae extracts by HPLC and determined qualitatively by quadrupole ion trap mass spectrometry. In this study, we evaluated the effect of Flos Lonicerae extracts and CGA on inflammatory-related gene expression, adhesion molecule expression and reactive oxygen species (ROS) production in perfluorooctane sulphonate (PFOS)-treated human umbilical vein endothelial cells (HUVECs). The suppression of transcription of IL-1ß, IL-6, COX-2, and P-Selectin genes with Flos Lonicerae extracts was greater than that of CGA in PFOS-treated HUVECs, while the degree of suppression on PFOS-induced expression of NOS3 and ICAM-1 was greater for CGA. Furthermore, the suppressive effect of Flos Lonicerae extracts on adhesion of monocytes onto PFOS-induced HUVECs was greater than that of CGA. In addition, Flos Lonicerae extracts and CGA were highly effective in reducing ROS although their effects were almost comparable. So, Flos Lonicerae extracts exhibited antioxidant activity and CGA was a major contributor to this activity. These results suggest that Flos Lonicerae extracts could be useful to prevent PFOS-mediated inflammatory diseases.

Purple corn anthocyanins inhibit diabetes-associated glomerular monocyte activation and macrophage infiltration.

Diabetic nephropathy (DN) is one of the major diabetic complications and the leading cause of end-stage renal disease. In early DN, renal injury and macrophage accumulation take place in the pathological environment of glomerular vessels adjacent to renal mesangial cells expressing proinflammatory mediators. Purple corn utilized as a daily food is rich in anthocyanins exerting disease-preventive activities as a functional food. This study elucidated whether anthocyanin-rich purple corn extract (PCA) could suppress monocyte activation and macrophage infiltration. In the in vitro study, human endothelial cells and THP-1 monocytes were cultured in conditioned media of human mesangial cells exposed to 33 mM glucose (HG-HRMC). PCA decreased the HG-HRMC-conditioned, media-induced expression of endothelial vascular cell adhesion molecule-1, E-selectin, and monocyte integrins-ß1 and -ß2 through blocking the mesangial Tyk2 pathway. In the in vivo animal study, db/db mice were treated with 10 mg/kg PCA daily for 8 wk. PCA attenuated CXCR2 induction and the activation of Tyk2 and STAT1/3 in db/db mice. Periodic acid-Schiff staining showed that PCA alleviated mesangial expansion-elicited renal injury in diabetic kidneys. In glomeruli, PCA attenuated the induction of intracellular cell adhesion molecule-1 and CD11b. PCA diminished monocyte chemoattractant protein-1 expression and macrophage inflammatory protein 2 transcription in the diabetic kidney, inhibiting the induction of the macrophage markers CD68 and F4/80. These results demonstrate that PCA antagonized the infiltration and accumulation of macrophages in diabetic kidneys through disturbing the mesangial IL-8-Tyk-STAT signaling pathway. Therefore, PCA may be a potential renoprotective agent treating diabetes-associated glomerulosclerosis.

2-methoxycinnamaldehyde from Cinnamomum cassia reduces rat myocardial ischemia and reperfusion injury in vivo due to HO-1 induction.

ETHNOPHARMACOLOGICAL RELEVANCE: Cinnamomum cassia Blume has been used as a traditional Chinese herbal medicine for alleviation of fever, inflammation, chronic bronchitis, and to improve blood circulation. AIM OF THE STUDY: We addressed whether 2-methoxycinnamaldehyde (2-MCA), one of active ingredients of Cinnamomum cassia, reduces vascular cell adhesion molecule-1 (VCAM-1) expression in tumor necrosis factor-alpha (TNF-α)-activated endothelial cells and protects ischemia/reperfusion (I/R)-injury due to heme oxygenase (HO)-1 induction. MATERIALS AND METHODS: Adult male rats were subjected to 30 min of ischemia by occlusion of the left anterior descending coronary artery followed by 24h of reperfusion. Rats were randomized to receive vehicle or 2-MCA (i.v.) 10 min before reperfusion. RESULTS: Administration of 2-MCA significantly improved I/R-induced myocardial dysfunction by increasing the values of the first derivative (±dp/dt) of left ventricular pressure and decreased infarct size. In addition, 2-MCA reduced the expression of high mobility group box 1 (HMGB1), an activator of the inflammatory cascade when released into the extracellular space, and VCAM-1 in I/R myocardium along with increase of HO-1 induction. The reduced injury was accompanied by significantly reduction of neutrophils infiltration and increased SOD activity in ischemic tissues and reduced serum level of cardiac troponin I (cTnI). Furthermore, 2-MCA significantly increased HO-1 induction by translocation of Nrf-2 from cytosol to nucleus in endothelial cells. Inhibition of VCAM-1 expression by 2-MCA was reversed both by SnPPIX, a HO-1 inhibitor and siHO-1 RNA trasfection in TNF-α-activated cells. In addition, 2-MCA significantly inhibited NF-κB luciferase activity in TNF-α-activated endothelial cells. As expected, 2-MCA significantly inhibited monocyte (U937) adhesion to endothelial cells. CONCLUSION: We concluded that 2-MCA protects of myocardial I/R-injury due to antioxidant and anti-inflammatory action possibly by HO-1 induction which can be explained why Cinnamomum cassia has been used in inflammatory disorders.

A predictive biomimetic model of cytokine release induced by TGN1412 and other therapeutic monoclonal antibodies.

Human peripheral blood mononuclear cells (PBMC) are routinely used in vitro to detect cytokine secretion as part of preclinical screens to delineate agonistic and antagonistic action of therapeutic monoclonal antibodies (mAbs). Preclinical value of standard human PBMC assays to detect cytokine release syndrome (CRS) has been questioned, as they did not predict the "cytokine storm" that occurred when healthy human volunteers were given a CD28-specific super-agonist mAb, TGN1412. In this article, we describe a three-dimensional biomimetic vascular test-bed that can be used as a more physiologically relevant assay for testing therapeutic Abs. For developing such a system, we used TGN1412 as a model mAb. We tested soluble TGN1412 on various combinations of human blood components in a module containing endothelial cells grown on a collagen scaffold and measured cytokine release using multiplex array. Our system, consisting of whole leukocytes, endothelial cells, and 100% autologous platelet-poor plasma (PPP) consistently produced proinflammatory cytokines in response to soluble TGN1412. In addition, other mAb therapeutics known to induce CRS or first infusion reactions, such as OKT3, Campath-1H, or Herceptin, generated cytokine profiles in our model system consistent with their in vivo responses. As a negative control we tested the non-CRS mAbs Avastin and Remicade and found little difference between these mAbs and the placebo control. Our data indicate that this novel assay may have preclinical value for predicting the potential of CRS for mAb therapeutics.

Dual functions of ginsenosides in protecting human endothelial cells against influenza H9N2-induced inflammation and apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax ginseng is a precious traditional Chinese herbal medicine which has been utilized as herbal tonic for improving immunity. The active component, ginsenosides have been shown to possess various pharmacological functions including immunomodulation and cardiovascular protection. AIM OF THE STUDY: To investigate the immunomodulatory effect and anti-apoptotic effect of ginsenosides on avian influenza-infected human endothelial cells, and to present evidence for the cardiovascular protection by ginseng during influenza infection. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs) were infected with avian influenza H9N2/G1 to induce IP-10 production and cell death, cells were then incubated with ginsenosides PPT and Re. The level of IP-10 and microRNA was determined by ELISA and real-time PCR respectively. Cell death was determined by MTT, TUNEL and flow cytometry. RESULTS: Ginsenoside metabolite protopanaxatriol showed significant suppression effect on IP-10 production upon H9N2/G1 infection through up-regulation of miR-15b expression. In addition, ginsenoside-induced cytoprotection was reflected in the increase of cell viability. Data from flow cytometry analysis and TUNEL assay also showed that ginsenoside Re could protect ECs from H9N2/G1-induced apoptosis and DNA damage. CONCLUSIONS: This report further supports the traditional belief for immunomodulatory effects of ginseng, also demonstrated the partial protective mechanism of ginsenosides on avian influenza infection and its related endothelial dysfunction.

Calycosin protects HUVECs from advanced glycation end products-induced macrophage infiltration.

ETHNOPHARMACOLOGICAL RELEVANCE: Astragali radix is a traditional Chinese medicine that has long been used for treatment of diabetes and diabetes-associated disease, but its active component and mechanism on the disease is not well defined. AIM OF THE STUDY: Infiltration of leukocytes within the glomeruli and vasculature is one of the early and characteristic features of diabetic nephropathy. Advanced glycation end products (AGEs) play pivotal role in the progression of diabetic-associated diseases. The present study was designed to explore the therapeutic effect of calycosin, an active component from A. radix, on AGEs-induced macrophages infiltration in HUVECs. MATERIALS AND METHODS: Transwell HUVEC-macrophage co-culture system was established to evaluate macrophage migration and adhesion. Immunocytochemistry was applied to examine TGF-beta1, ICAM-1 and RAGE protein expressions; real-time PCR was carried out to determine mRNA expression of TGF-beta1, ICAM-1 and RAGE. Immunofluorescence was carried out to observe estrogen receptor-alpha, ICAM-1, RAGE expression and the phosphorylation status of ERK1/2 and NF-κB. RESULTS: Calycosin significantly reduced AGEs-induced macrophage migration and adhesion to HUVEC. Pre-treatment with calycosin strikingly down-regulated HUVEC TGF-beta1, ICAM-1 and RAGE expressions in both protein and mRNA levels. Furthermore, calycosin incubation significantly increased estrogen receptor expression and reversed AGEs-induced ERK1/2 and NF-κB phosphorylation and nuclear translocation in HUVEC, and this effect of calycosin could be inhibited by estrogen receptor inhibitor, ICI182780. CONCLUSIONS: These findings suggest that calycosin can reduce AGEs-induced macrophage migration and adhesion to endothelial cells and relieve the local inflammation; furthermore, this effect was via estrogen receptor-ERK1/2-NF-κB pathway.

Grape seed extract ameliorates tumor necrosis factor-α-induced inflammatory status of human umbilical vein endothelial cells.

BACKGROUND: Inflammation has played a key role in the causation of atherosclerosis. However, the effects of grape seed extract (GSE) on the pro-inflammatory intracellular signaling, enzyme activity, and inflammatory mediators of endothelial cells have not been sufficiently studied, and less information exists on the comparison between GSE and vitamin C, a well-known antioxidant compound, on their anti-inflammatory properties. PURPOSE: We investigated the effects of GSE and vitamin C on the cell viability, oxidative stress, monocyte adhesion, the expression of nuclear factor-κB inhibitor (IκB), intercellular adhesion molecule-1 (ICAM-1) and cyclooxygenase-2 (COX-2), and the production of prostaglandin E(2) (PG E(2)) in TNF-α-treated human umbilical vein endothelial cells (HUVECs). METHODS: Cell viability was measured by MTT assay. The adhesion of THP-1 to HUVECs was evaluated by cell adhesion assay. The oxidized nucleoside 8-hydroxydeoxyguanosine (8-OHdG) (an indicator of oxidative damage to DNA), ICAM-1, and PG E(2) were measured by ELISA. IκB and COX-2 expression were evaluated by western blot analysis. RESULTS: TNF-α (10, 20, and 50 ng/mL), GSE (50 and 200 µg/mL), or vitamin C (100 µM) did not affect cell viability. GSE (50-100 µg/mL) attenuated TNF-α (20 ng/mL)-induced 8-OHdG production, THP-1 adhesion, the expression of IκB degradation, ICAM-1 and COX-2, and the production of PGE(2) in a dose-dependent manner. Vitamin C (100 µM) also showed significant antioxidative and anti-inflammatory effects. CONCLUSIONS: GSE effectively ameliorates TNF-α-induced inflammatory status of HUVECs. The findings of the present study suggest that consumption of GSE may be beneficial to inflammatory atherosclerosis.