Maternal Diet Supplemented with Methyl-Donors Protects against Atherosclerosis in F1 ApoE?/? Mice

Atherosclerosis is an inflammatory condition of the arterial wall mediated by cells of both innate and adaptive immunity. T lymphocytes play an important role in orchestrating the pathogenic immune response involved in the acceleration of atherosclerosis. Previously, we have shown that a prenatal methyl-donor supplementation diet (MS), when fed to dams during pregnancy and lactation, decreased the T cell-mediated pro-inflammatory cytokine and chemokine response in F1 mice. In the current study, we report feeding Apolipoprotein E (ApoE^−/−) deficient dams with the MS diet during pregnancy reduces atherosclerotic plaques in F1 mice that were fed high fat diet (HFD) after weaning. F1 mice from dams on the MS diet exhibited increased global T cell DNA methylation. T-cell chemokines and their receptors (in particular CCR2, CCR5, and CXCR3) play important roles in the inflammatory cell recruitment to vascular lesions. MS diet significantly reduced Ccr2 mRNA and protein expression in CD3+ T cells but not in CD11b+ monocytes in MS F1 mice relative to controls. F1 litter size, HFD consumption, body weight, and body fat were similar between control and MS diet groups. Moreover, serum thiol metabolite levels were similar between the two groups. However, MS diet is associated with significantly higher serum HDL and lower LDL+VLDL levels in comparison to F1 mice from dams on the control diet. Inflammatory cytokines (IL-17, TNF-α, IL-6) were also lower in MS F1 mice serum and conditioned media from T-cell culture. Altogether, these data suggest that the MS diet ameliorates development of atherosclerosis by inhibiting the T-cell Ccr2 expression, reducing inflammatory cytokines production and increasing serum HDL:LDL ratio.     

BAFF Receptor mAb Treatment Ameliorates Development and Progression of Atherosclerosis in Hyperlipidemic ApoE?/? Mice

Aims

Option to attenuate atherosclerosis by depleting B2 cells is currently limited to anti-CD20 antibodies which deplete all B-cell subtypes. In the present study we evaluated the capacity of a monoclonal antibody to B cell activating factor-receptor (BAFFR) to selectively deplete atherogenic B2 cells to prevent both development and progression of atherosclerosis in the ApoE^−/− mouse.

Methods and Results

To determine whether the BAFFR antibody prevents atherosclerosis development, we treated ApoE^−/− mice with the antibody while feeding them a high fat diet (HFD) for 8 weeks. Mature CD93⁻ CD19⁺ B2 cells were reduced by treatment, spleen B-cell zones disrupted and spleen CD20 mRNA expression decreased while B1a cells and non-B cells were spared. Atherosclerosis was ameliorated in the hyperlipidemic mice and CD19⁺ B cells, CD4⁺ and CD8⁺ T cells were reduced in atherosclerotic lesions. Expressions of proinflammatory cytokines, IL1β, TNFα, and IFNγ in the lesions were also reduced, while MCP1, MIF and VCAM-1 expressions were unaffected. Plasma immunoglobulins were reduced, but MDA-oxLDL specific antibodies were unaffected. To determine whether anti-BAFFR antibody ameliorates progression of atherosclerosis, we first fed ApoE^−/− mice a HFD for 6 weeks, and then instigated anti-BAFFR antibody treatment for a further 6 week-HFD. CD93⁻ CD19⁺ B2 cells were selectively decreased and atherosclerotic lesions were reduced by this treatment.

Conclusion

Anti-BAFFR monoclonal antibody selectively depletes mature B2 cells while sparing B1a cells, disrupts spleen B-cell zones and ameliorates atherosclerosis development and progression in hyperlipidemic ApoE^−/− mice. Our findings have potential for clinical translation to manage atherosclerosis-based cardiovascular diseases.     

Valsartan Attenuates Atherosclerosis via Upregulating the Th2 Immune Response in Prolonged Angiotensin II–Treated ApoE?/? Mice

Valsartan has a protective effect against hypertension and atherosclerosis in humans and experimental animal models. This study aimed to determine the effect of prolonged treatment with angiotensin II (Ang II) on atherosclerosis and the effect of valsartan on the activity of CD4⁺ T lymphocyte subsets. The results showed that prolonged treatment (8 wks) with exogenous Ang II resulted in an increased atherosclerotic plaque size and a switch of stable-to-unstable plaque via modulating on CD4⁺ T lymphocyte activity, including an increase in the T helper cell type 1 (Th1) and Th17 cells and a decrease in Th2 and regulatory T (Treg) cells. In contrast, valsartan treatment efficiently reversed the imbalance in CD4⁺ T lymphocyte activity, ameliorated atherosclerosis and elicited a stable plaque phenotype in addition to controlling blood pressure. In addition, treatment with anti-interleukin (IL)-5 monoclonal antibodies weakened the antiatherosclerotic effects of valsartan without affecting blood pressure.     

Suppressive Effects of Genistein on Oxidative Stress and NFκB Activation in RAW 264.7 Macrophages

This study was designed to investigate whether genistein may ameliorate oxidative stress and nuclear factor κB (NFκB) activation in the lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cell line. Treatment of RAW 264.7 cells with genistein significantly reduced lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production in a dose-dependent manner with an IC₅₀ of 69.4 μM. Genistein at 50 μM and 100 μM concentrations reduced thiobarbituric acid-reactive substances (TBARS) accumulation, increasing the GSH level and antioxidant enzyme activities, such as superoxide dismutase (SOD) and catalase. The specific DNA-binding activities of nuclear factor κB (NFκB) on nuclear extracts from 50 μM and 100 μM genistein treatments were significanly suppressed. These results suggest that genistein has mild antioxidant activity to suppress intracellular oxidative stress and NFκB activation.     

Chronic Cadmium Exposure Accelerates the Development of Atherosclerosis and Induces Vascular Dysfunction in the Aorta of ApoE−/− Mice

Biological Trace Element Research - Cadmium exposure is related to cardiovascular diseases, including hypertension, atherosclerosis, increased oxidative stress, endothelial dysfunction, and...     

Stimulation of the α7 Nicotinic Acetylcholine Receptor Protects against Neuroinflammation after Tibia Fracture and Endotoxemia in Mice

Surgery and critical illness often associate with cognitive decline. Surgical trauma or infection can lead independently to learning and memory impairments via similar, but not identical, cellular signaling of the innate immune system that promotes neuroinflammation. In this study we explored the putative synergism between aseptic orthopedic surgery and infection, the latter reproduced by postoperative lipopolysaccharide (LPS) administration. We observed that surgery and LPS augmented systemic inflammation up to postoperative d 3 and this was associated with further neuroinflammation (CD11b and CD68 immunoreactivity) in the hippocampus in mice compared with those receiving surgery or LPS alone. Administration of a selective α7 subtype nicotinic acetylcholine receptor (α7 nAChR) agonist 2 h after LPS significantly improved neuroinflammation and hippocampal-dependent memory dysfunction. Modulation of nuclear factor-kappa B (NF-κB) activation in monocytes and regulation of the oxidative stress response through nicotinamide adenine dinucleotide phosphate (NADPH) signaling appear to be key targets in modulating this response. Overall, these results suggest that it may be conceivable to limit and possibly prevent postoperative complications, including cognitive decline and/or infections, through stimulation of the cholinergic antiinflammatory pathway.     

Punicic Acid a Conjugated Linolenic Acid Inhibits TNFα-Induced Neutrophil Hyperactivation and Protects from Experimental Colon Inflammation in Rats

Background

Neutrophils play a major role in inflammation by releasing large amounts of ROS produced by NADPH-oxidase and myeloperoxidase (MPO). The proinflammatory cytokine TNFα primes ROS production through phosphorylation of the NADPH-oxidase subunit p47phox on Ser345. Conventional anti-inflammatory therapies remain partially successful and may have side effects. Therefore, regulation of neutrophil activation by natural dietary components represents an alternative therapeutic strategy in inflammatory diseases such as inflammatory bowel diseases. The aim of this study was to assess the effect of punicic acid, a conjugated linolenic fatty acid from pomegranate seed oil on TNFα-induced neutrophil hyperactivation in vitro and on colon inflammation in vivo.

Methodology and Principal Findings

We analyzed the effect of punicic acid on TNFα-induced neutrophil upregulation of ROS production in vitro and on TNBS-induced rat colon inflammation. Results show that punicic acid inhibited TNFα-induced priming of ROS production in vitro while preserving formyl-methionyl-leucyl-phenylalanine (fMLP)-induced response. This effect was mediated by the inhibition of Ser345-p47phox phosphorylation and upstream kinase p38MAPK. Punicic acid also inhibited fMLP- and TNFα+fMLP-induced MPO extracellular release from neutrophils. In vivo experiments showed that punicic acid and pomegranate seed oil intake decreased neutrophil-activation and ROS/MPO-mediated tissue damage as measured by F2-isoprostane release and protected rats from TNBS-induced colon inflammation.

Conclusions/Significance

These data show that punicic acid exerts a potent anti-inflammatory effect through inhibition of TNFα-induced priming of NADPH oxidase by targeting the p38MAPKinase/Ser345-p47phox-axis and MPO release. This natural dietary compound may provide a novel alternative therapeutic strategy in inflammatory diseases such as inflammatory bowel diseases.     

Oral Supplementation with Non-Absorbable Antibiotics or Curcumin Attenuates Western Diet-Induced Atherosclerosis and Glucose Intolerance in LDLR?/? Mice – Role of Intestinal Permeability and Macrophage Activation

Association between circulating lipopolysaccharide (LPS) and metabolic diseases (such as Type 2 Diabetes and atherosclerosis) has shifted the focus from Western diet-induced changes in gut microbiota per se to release of gut bacteria-derived products into circulation as the possible mechanism for the chronic inflammatory state underlying the development of these diseases. Under physiological conditions, an intact intestinal barrier prevents this release of LPS underscoring the importance of examining and modulating the direct effects of Western diet on intestinal barrier function. In the present study we evaluated two strategies, namely selective gut decontamination and supplementation with oral curcumin, to modulate Western-diet (WD) induced changes in intestinal barrier function and subsequent development of glucose intolerance and atherosclerosis. LDLR−/− mice were fed WD for 16 weeks and either received non-absorbable antibiotics (Neomycin and polymyxin) in drinking water for selective gut decontamination or gavaged daily with curcumin. WD significantly increased intestinal permeability as assessed by in vivo translocation of FITC-dextran and plasma LPS levels. Selective gut decontamination and supplementation with curcumin significantly attenuated the WD-induced increase in plasma LPS levels (3.32 vs 1.90 or 1.51 EU/ml, respectively) and improved intestinal barrier function at multiple levels (restoring intestinal alkaline phosphatase activity and expression of tight junction proteins, ZO-1 and Claudin-1). Consequently, both these interventions significantly reduced WD-induced glucose intolerance and atherosclerosis in LDLR−/− mice. Activation of macrophages by low levels of LPS (50 ng/ml) and its exacerbation by fatty acids is likely the mechanism by which release of trace amounts of LPS into circulation due to disruption of intestinal barrier function induces the development of these diseases. These studies not only establish the important role of intestinal barrier function, but also identify oral supplementation with curcumin as a potential therapeutic strategy to improve intestinal barrier function and prevent the development of metabolic diseases.     

Elsholblanosides A−D,Four New Oleuropeic Acid Derivatives Isolated from Elsholtzia blanda and Their Inhibition of NO Production in LPS-activated RAW264.7 Cells

Phytochemical investigation on the aerial parts of Elsholtzia blanda Benth. afforded four new oleuropeic acid derivatives ( 1 – 4 ), named as elsholblanosides A−D, respectively, together with 11 known compounds ( 5 – 15 ). Their structures were determined based on extensive analyses of HR-ESI-MS, 1D and 2D NMR, and ECD spectra. Compounds 1 – 4 and 14 showed moderate NO production inhibition in LPS-activated RAW264.7 cells with their IC₅₀ values ranging from 23.2 to 86.33 μM, compared to that of the positive control compound, dexamethasone, IC₅₀ value of 16.9 μM.     

Valproic Acid Treatment Inhibits Hypoxia-Inducible Factor 1α Accumulation and Protects against Burn-Induced Gut Barrier Dysfunction in a Rodent Model

Objective

Burn-induced gut dysfunction plays an important role in the development of sepsis and multiple organ dysfunction. Emerging evidence suggests that hypoxia-inducible factor-1α (HIF-1α) is critical in paracelluar barrier functions via regulating vascular endothelial growth factor (VEGF) and myosin light chain kinase (MLCK) expression. Previous studies have also demonstrated that histone deacetylase inhibitors (HDACIs) can repress HIF-1α. This study aims to examine whether valproic acid (VPA), a HDACI, protects against burn-induced gut barrier dysfunction via repressing HIF-1α-dependent upregulation of VEGF and MLCK expression.

Methods

Rats were subjected to third degree 55% TBSA burns and treated with/ without VPA (300mg/kg). Intestinal barrier dysfunction was evaluated by permeability of intestinal mucosa to fluorescein isothiocyanate (FITC)-dextran and histologic evaluation. Histone acetylation, tight junction protein zonula occludens 1 (ZO-1), VEGF, MLCK and HIF-1α were measured. In addition, CaCO₂ cells were transfected with siRNA directed against HIF-1α and were stimulated with CoCl2 (1mM) for 24 hours with/without VPA (2mM) followed by analysis of HIF-1α, MLCK, VEGF and ZO-1.

Results

Burn insults resulted in a significant increase in intestinal permeability and mucosal damage, accompanied by a significant reduction in histone acetylation, ZO-1, upregulation of VEGF, MLCK expression, and an increase in HIF-1α accumulation. VPA significantly attenuated the increase in intestinal permeability, mucosa damage, histone deacetylation and changes in ZO-1 expression. VPA also attenuated the increased VEGF, MLCK and HIF-1α protein levels. VPA reduced HIF-1α, MLCK and VEGF production and prevented ZO-1 loss in CoCl2-stimulated Caco-2 cells. Moreover, transfection of siRNA directed against HIF-1α led to inhibition of MLCK and VEGF production, accompanied by upregulation of ZO-1.

Conclusions

These results indicate that VPA can protect against burn-induced gut barrier dysfunction. These protective effects may be due to its inhibitory action on HIF-1α, leading to a reduction in intestinal VEGF and MLCK expression and minimizing ZO-1 degradation.     

Oral Administration of a Fusion Protein between the Cholera Toxin B Subunit and the 42-Amino Acid Isoform of Amyloid-β Peptide Produced in Silkworm Pupae Protects against Alzheimer's Disease in Mice

A key molecule in the pathogenesis of Alzheimer''s disease (AD) is a 42-amino acid isoform of the amyloid-β peptide (Aβ42), which is the most toxic element of senile plaques. In this study, to develop an edible, safe, low-cost vaccine for AD, a cholera toxin B subunit (CTB)-Aβ42 fusion protein was successfully expressed in silkworm pupae. We tested the silkworm pupae-derived oral vaccination containing CTB-Aβ42 in a transgenic mouse model of AD. Anti-Aβ42 antibodies were induced in these mice, leading to a decreased Aβ deposition in the brain. We also found that the oral administration of the silk worm pupae vaccine improved the memory and cognition of mice, as assessed using a water maze test. These results suggest that the new edible CTB-Aβ42 silkworm pupae-derived vaccine has potential clinical application in the prevention of AD.     

Combinatorial Effects of Nonsteroidal Anti-inflammatory Drugs and Food Constituents on Production of Prostaglandin E2 and Tumor Necrosis Factor-α in RAW264.7 Murine Macrophages

Combinatorial chemopreventive strategies, in contrast to those with individual agents, show potential in terms of potentially lower toxicity and higher efficacy. In this study, we combined several agents and examined their suppressive effects on the combined lipopolysaccharide (LPS)- and interferon(IFN)-γ-induced formation of proinflammatory mediators, including prostaglandin (PG) E₂ and tumor necrosis factor (TNF)-α, in RAW264.7 murine macrophages. The combinatorial effects of indomethacin/genistein (GEN) and aspirin/GEN were found to be synergistic for PGE₂ suppression, while the nimesulide/GEN combination was antagonistic. Further, while (?)-epigallocatechin gallate (EGCG) alone increased LPS/IFM-γ-induced production of PGE₂ and TNF-α as well as cyclooxygenase-2 expression, the EGCG/GEN combination markedly suppressed these parameters. Our results suggest that certain chemopreventive agents act complexly and that, when used in combination, they affect the intracellular signaling pathways of the paired agents to exert additive, synergistic, or antagonistic effects.     

α-Lipoic Acid Inhibits Endotoxin-stimulated Expression of iNOS and Nitric Oxide Independent of the Heat Shock Response in RAW 264.7 Cells

The heat shock response protects against sepsis-induced mortality, organ injury, cardiovascular dysfunction, and apoptosis. Several inducers of the heat shock response, such as hyperthermia, sodium arsenite, and pyrollidine dithiocarbonate, inhibit NF-κB activation and nitric oxide formation. The antioxidant lipoic acid (LA) has recently been found to inhibit NF-κB activation and nitric oxide formation. We therefore tested the hypothesis that LA induces a heat shock response. To test this hypothesis, we determined whether exposure to LA affects expression of both heat shock protein 70 (HSP-70) and nuclear heat shock factor-1 (HSF-1) in lipopolysaccharide (LPS) stimulated macrophages. LA and hyperthermia attenuated LPS-induced increases in nuclear NF-κB, iNOS protein, and media nitrite concentrations. LPS and hyperthermia increased HSP-70 concentrations 8-fold and 20-fold, respectively. No effect of LA treatment alone on HSP-70 protein expression was detected. Likewise, no effect of LA on HSF-1 protein expression was detected. These data suggest that LA inhibits LPS-induced activation of iNOS in macrophages independent of the heat shock response.     

The Effect of Dietary Prebiotics and Probiotics on Body Weight,Large Intestine Indices,and Fecal Bile Acid Profile in Wild Type and IL10?/? Mice

Previous studies have suggested roles of probiotics and prebiotics on body weight management and intestinal function. Here, the effects of a dietary prebiotic, inulin (50 mg/g diet), and probiotic, Bfidobacterium animalis subsp. lactis (Bb12) (final dose verified at 10⁵ colony forming unit (cfu)/g diet, comparable to human consumption), were determined separately and in combination in mice using cellulose-based AIN-93G diets under conditions allowed for the growth of commensal bacteria. Continuous consumption of Bb12 and/or inulin did not affect food intake or body, liver, and spleen weights of young and adult mice. Fecal bile acid profiles were determined by nanoESI-MS/MS tandem mass spectrometry. In the presence of inulin, more bacterial deconjugation of taurine from primary bile acids was observed along with an increased cecal weight. Consumption of inulin in the absence or presence of Bb12 also increased the villus cell height in the proximal colon along with a trend of higher bile acid sulfation by intestinal cells. Feeding Bb12 alone at the physiological dose did not affect bile acid deconjugation and had little effect on other intestinal indices. Although interleukin (IL)10-null mice are susceptible to enterocolitis, they maintained the same body weight as the wild type mice under our specific pathogen-free housing condition and showed no signs of inflammation. Nevertheless, they had smaller cecum suggesting a mildly compromised intestinal development even before the disease manifestation. Our results are consistent with the notion that dietary factors such as prebiotics play important roles in the growth of intestinal microbiota and may impact on the intestinal health. In addition, fecal bile acid profiling could potentially be a non-invasive tool in monitoring the intestinal environment.     

Aqueous Extract of Gracilaria tenuistipitata Suppresses LPS-Induced NF-κB and MAPK Activation in RAW 264.7 and Rat Peritoneal Macrophages and Exerts Hepatoprotective Effects on Carbon Tetrachloride-Treated Rat

In addition to the previous investigations of bioactivity of aqueous extract of the edible Gracilaria tenuistipitata (AEGT) against H₂O₂-induced DNA damage and hepatitis C virus replication, the purpose of this study is to evaluate the potential therapeutic properties of AEGT against inflammation and hepatotoxicity using lipopolysaccharide (LPS)-stimulated mouse RAW 264.7 cells, primary rat peritoneal macrophages and carbon tetrachloride (CCl₄)-induced acute hepatitis model in rats. AEGT concentration-dependently inhibited the elevated RNA and protein levels of inducible nitric oxide synthase and cyclooxygenase-2, thereby reducing nitric oxide and prostaglandin E2 levels, respectively. Moreover, AEGT significantly suppressed the production of LPS-induced proinflammatory cytokines, including interleukin (IL)-1β, IL-6 and tumor necrosis factor-α. These inhibitory effects were associated with the suppression of nuclear factor-kappa B activation and mitogen-activated protein kinase phosphorylation by AEGT in LPS-stimulated cells. In addition, we highlighted the hepatoprotective and curative effects of AEGT in a rat model of CCl₄-intoxicated acute liver injury, which was evident from reduction in the elevated serum aspartate aminotransferase and alanine aminotransferase levels as well as amelioration of histological damage by pre-treatment or post-treatment of AEGT. In conclusion, the results demonstrate that AEGT may serve as a potential supplement in the prevention or amelioration of inflammatory diseases.     

Dammarane-type saponins from Ziziphus jujube and their inhibitory effects against TNF-α release in LPS-induced RAW 246.7 macrophages

Four new dammarane-type saponins jujubosides F–J, together with six known compounds were isolated from the seeds of Ziziphus jujube. Their structures were elucidated on the basis of chemical and spectroscopic evidences. Compounds 1–10 showed moderate inhibitory effects against pro-inflammatory cytokine TNF-α release in LPS-induced RAW 246.7 macrophages.