Tocotrienols inhibit lipopolysaccharide-induced pro-inflammatory cytokines in macrophages of female mice

<p>Abstract</p> <p>Background</p> <p>Inflammation has been implicated in cardiovascular disease, and the important role of proteasomes in the development of inflammation and other macrophage functions has been demonstrated. Tocotrienols are potent hypocholesterolemic agents that inhibit β-hydroxy-β-methylglutaryl coenzyme A reductase activity, which is degraded via the ubiquitin-proteasome pathway. Our objective was to evaluate the effect of tocotrienols in reducing inflammation. Lipopolysaccharide (LPS) was used as a prototype for inflammation in murine RAW 264.7 cells and BALB/c female mice.</p> <p>Results</p> <p>The present results clearly demonstrate that α-, γ-, or δ-tocotrienol treatments inhibit the chymotrypsin-like activity of 20 S rabbit muscle proteasomes (> 50%; <it>P </it>< 0.05). Chymotrypsin, trypsin, and post-glutamase activities were decreased > 40% (<it>P </it>< 0.05) with low concentrations (< 80 μM), and then increased gradually with concentrations of (80 - 640 μM) in RAW 264.7 whole cells. Tocotrienols showed 9 - 33% (<it>P </it>< 0.05) inhibitions in TNF-α secretion in LPS-stimulated RAW 264.7 cells. Results of experiments carried out in BALB/c mice demonstrated that serum levels of TNF-α after LPS treatment were also reduced (20 - 48%; <it>P </it>< 0.05) by tocotrienols with doses of 1 and 10 μg/kg, and a corresponding rise in serum levels of corticosterone (19 - 41%; <it>P </it>< 0.05) and adrenocorticotropic hormone (81 - 145%; <it>P </it>< 0.02) was observed at higher concentrations (40 μM). Maximal inhibition of LPS-induced TNF-α was obtained with δ-tocotrienol (10 μg/kg). Low concentrations of δ-Tocotrienols (< 20 μM) blocked LPS-induced gene expression of TNF-α, IL-1β, IL-6 and iNOS (> 40%), while higher concentrations (40 μM) increased gene expression of the latter in peritoneal macrophages (prepared from BALB/c mice) as compared to control group.</p> <p>Conclusions</p> <p>These results represent a novel approach by using natural products, such as tocotrienols as proteasome modulators, which may lead to the development of new dietary supplements of tocotrienols for cardiovascular diseases, as well as others that are based on inflammation.</p

Lipopolysaccharide Increases the Expression of Multidrug Resistance-Associated Protein 1 (ABCC1) in Macrophages

Multidrug resistance-associated protein 1 (MRP-1) is a ubiquitously expressed member of the ATP-binding cassette transporter family. MRP-1 is one of the primary transporters of glutathione and glutathione conjugates. This protein also transports antiretroviral therapeutics, such as HIV-1 protease inhibitors (PI). We hypothesized that inflammatory mediators that activate macrophages would modify the expression and activity of MRP-1 in macrophages. Real time PCR assays western blots and calcein efflux assays were used to show that exposure of macrophage cell line RAW 264.7 to LPS increased expression of MRP-1 at the level of mRNA and protein, as well as at the level of functional activity. Treatment of macrophages with LPS resulted in 2-fold increases of MRP-1 expression or functional activity. LPS-mediated increases in calcein efflux were repressed by the MRP-specific inhibitor MK-571. These results suggest that the effectiveness of HIV-1 PI therapy may be compromised by the presence of opportunistic infections

Treatment of cardiovascular risk factors, nitric oxide synthesis, and c-reactive protein using a combination therapy of vitamins and flavanoids [abstract]

Researchers at the University of Missouri Kansas City have studied the synergistic effects of a novel combination of vitamins and flavanoids on cardiovascular risk factors, C-reactive protein levels, and nitric oxide production in the human body. Clinical trial results showed a marked decrease in the patients' serum nitric oxide and C-reactive protein levels. In addition, Hypercholesterolemic human subjects showed significant reductions in their lipid parameters (except for HDL cholesterol), and all subjects showed increases in total antioxidant status. Potential Areas of Applications: * Use as a nutritional supplemen

Tocotrienols-induced inhibition of platelet thrombus formation and platelet aggregation in stenosed canine coronary arteries

<p>Abstract</p> <p>Background</p> <p>Dietary supplementation with tocotrienols has been shown to decrease the risk of coronary artery disease. Tocotrienols are plant-derived forms of vitamin E, which have potent anti-inflammatory, antioxidant, anticancer, hypocholesterolemic, and neuroprotective properties. Our objective in this study was to determine the extent to which tocotrienols inhibit platelet aggregation and reduce coronary thrombosis, a major risk factor for stroke in humans. The present study was carried out to determine the comparative effects of α-tocopherol, α-tocotrienol, or tocotrienol rich fraction (TRF; a mixture of α- + γ- + δ-tocotrienols) on <it>in vivo platelet thrombosis </it>and <it>ex vivo </it>platelet aggregation (PA) after intravenous injection in anesthetized dogs, by using a mechanically stenosed circumflex coronary artery model (Folts' cyclic flow model).</p> <p>Results</p> <p>Collagen-induced platelet aggregation (PA) in platelet rich plasma (PRP) was decreased markedly after treatment with α-tocotrienol (59%; <b><it>P </it></b>< 0.001) and TRF (92%; <b><it>P </it></b>< 0.001). α-Tocopherol treatment was less effective, producing only a 22% (<b><it>P </it></b>< 0.05) decrease in PA. Adenosine diphosphate-induced (ADP) PA was also decreased after treatment with α-tocotrienol (34%; <b><it>P </it></b>< 0.05) and TRF (42%; <b><it>P </it></b>< 0.025). These results also indicate that intravenously administered tocotrienols were significantly better than tocopherols in inhibiting cyclic flow reductions (CFRs), a measure of the acute platelet-mediated thrombus formation. Tocotrienols (TRF) given intravenously (10 mg/kg), abolished CFRs after a mean of 68 min (range 22 -130 min), and this abolition of CFRs was sustained throughout the monitoring period (50 - 160 min).</p> <p>Next, pharmacokinetic studies were carried out and tocol levels in canine plasma and platelets were measured. As expected, α-Tocopherol treatment increased levels of total tocopherols in post- vs pre-treatment specimens (57 vs 18 μg/mL in plasma, and 42 vs 10 μg/mL in platelets). However, treatment with α-tocopherol resulted in slightly decreased levels of tocotrienols in post- vs pre-treatment samples (1.4 vs 2.9 μg/mL in plasma and 2.3 vs 2.8 μg/mL in platelets). α-Tocotrienol treatment increased levels of both tocopherols and tocotrienols in post- vs pre-treatment samples (tocopherols, 45 vs 10 μg/mL in plasma and 28 vs 5 μg/mL in platelets; tocotrienols, 2.8 vs 0.9 μg/mL in plasma and 1.28 vs 1.02 μg/mL in platelets). Treatment with tocotrienols (TRF) also increased levels of tocopherols and tocotrienols in post- vs pre-treatment samples (tocopherols, 68 vs 20 μg/mL in plasma and 31.4 vs 7.9 μg/mL in platelets; tocotrienols, 8.6 vs 1.7 μg/mL in plasma and 3.8 vs 3.9 μg/mL in platelets).</p> <p>Conclusions</p> <p>The present results indicate that intravenously administered tocotrienols inhibited acute platelet-mediated thrombus formation, and collagen and ADP-induced platelet aggregation. α-Tocotrienols treatment induced increases in α-tocopherol levels of 4-fold and 6-fold in plasma and platelets, respectively. Interestingly, tocotrienols (TRF) treatment induced a less pronounced increase in the levels of tocotrienols in plasma and platelets, suggesting that intravenously administered tocotrienols may be converted to tocopherols. Tocotrienols, given intravenously, could potentially prevent pathological platelet thrombus formation and thus provide a therapeutic benefit in conditions such as stroke and myocardial infarction.</p

δ-Tocotrienol and quercetin reduce serum levels of nitric oxide and lipid parameters in female chickens

<p>Abstract</p> <p>Background</p> <p>Chronic, low-grade inflammation provides a link between normal ageing and the pathogenesis of age-related diseases. A series of <it>in vitro </it>tests confirmed the strong anti-inflammatory activities of known inhibitors of NF-κB activation (δ-tocotrienol, quercetin, riboflavin, (-) Corey lactone, amiloride, and dexamethasone). δ-Tocotrienol also suppresses β-hydroxy-β-methylglutaryl coenzyme A (HMG-CoA) reductase activity (the rate-limiting step in <it>de novo </it>cholesterol synthesis), and concomitantly lowers serum total and LDL cholesterol levels. We evaluated these compounds in an avian model anticipating that a dietary additive combining δ-tocotrienol with quercetin, riboflavin, (-) Corey lactone, amiloride, or dexamethasone would yield greater reductions in serum levels of total cholesterol, LDL-cholesterol and inflammatory markers (tumor necrosis factor-α [TNF-α], and nitric oxide [NO]), than that attained with the individual compounds.</p> <p>Results</p> <p>The present results showed that supplementation of control diets with all compounds tested except riboflavin, (-) Corey lactone, and dexamethasone produced small but significant reductions in body weight gains as compared to control. (-) Corey lactone and riboflavin did not significantly impact body weight gains. Dexamethasone significantly and markedly reduced weight gain (>75%) compared to control. The serum levels of TNF-α and NO were decreased 61% - 84% (<it>P </it>< 0.001), and 14% - 67%, respectively, in chickens fed diets supplemented with δ-tocotrienol, quercetin, riboflavin, (-) Corey lactone, amiloride, or dexamethasone as compared to controls. Significant decreases in the levels of serum total and LDL-cholesterol were attained with δ-tocotrienol, quercetin, riboflavin and (-) Corey lactone (13% - 57%; <it>P </it>< 0.05), whereas, these levels were 2-fold higher in dexamethasone treated chickens as compared to controls. Parallel responses on hepatic lipid infiltration were confirmed by histological analyses. Treatments combining δ-tocotrienol with the other compounds yielded values that were lower than individual values attained with either δ-tocotrienol or the second compound. Exceptions were the significantly lower total and LDL cholesterol and triglyceride values attained with the δ-tocotrienol/(-) Corey lactone treatment and the significantly lower triglyceride value attained with the δ-tocotrienol/riboflavin treatment. δ-Tocotrienol attenuated the lipid-elevating impact of dexamethasone and potentiated the triglyceride lowering impact of riboflavin. Microarray analyses of liver samples identified 62 genes whose expressions were either up-regulated or down-regulated by all compounds suggesting common impact on serum TNF-α and NO levels. The microarray analyses further identified 41 genes whose expression was differentially impacted by the compounds shown to lower serum lipid levels and dexamethasone, associated with markedly elevated serum lipids.</p> <p>Conclusions</p> <p>This is the first report describing the anti-inflammatory effects of δ-tocotrienol, quercetin, riboflavin, (-) Corey lactone, amiloride, and dexamethasone on serum TNF-δ and NO levels. Serum TNF-δ levels were decreased by >60% by each of the experimental compounds. Additionally, all the treatments except with dexamethasone, resulted in lower serum total cholesterol, LDL-cholesterol and triglyceride levels. The impact of above mentioned compounds on the factors evaluated herein was increased when combined with δ-tocotrienol.</p

Impact of China Pakistan Economic Corridor on Local Economy and Tourism Development: Case of Hunza, Gilgit-Baltistan, Pakistan

This study is aimed to analyze the role of tourism in affecting the local economy of Hunza. The Structural Equation Modeling was applied for knowing the local people perception about CPEC projects and their effect on tourism economy development. In addition, descriptive statistics tools using STAT 12 have been used for the data analysis and prior to analysis, Cronbach’s alpha test was used to check reliability of various items of the questionnaire. The data werecollected from students, retailers, hotel owners, tour operators and travel agents. Results reveal that according to the respondents, CPEC is a game changer for the economy. In addition, there is significant effect of CPEC projects on the tourism development in Hunza. Results of the study also revealed that CPEC projects have negative effect on environment and local culture

Inhibition of nitric oxide in LPS-stimulated macrophages of young and senescent mice by δ-tocotrienol and quercetin

Abstract Background Changes in immune function believed to contribute to a variety of age-related diseases have been associated with increased production of nitric oxide (NO). We have recently reported that proteasome inhibitors (dexamethasone, mevinolin, quercetin, &#948;-tocotrienol, and riboflavin) can inhibit lipopolysaccharide (LPS)-induced NO production in vitro by RAW 264.7 cells and by thioglycolate-elicited peritoneal macrophages derived from four strains of mice (C57BL/6, BALB/c, LMP7/MECL-1-/- and PPAR-&#945;-/- knockout mice). The present study was carried out in order to further explore the potential effects of diet supplementation with naturally-occurring inhibitors (&#948;-tocotrienol and quercetin) on LPS-stimulated production of NO, TNF-&#945;, and other pro-inflammatory cytokines involved in the ageing process. Young (4-week-old) and senescent mice (42-week old) were fed control diet with or without quercetin (100 ppm), &#948;-tocotrienol (100 ppm), or dexamethasone (10 ppm; included as positive control for suppression of inflammation) for 4 weeks. At the end of feeding period, thioglycolate-elicited peritoneal macrophages were collected, stimulated with LPS, LPS plus interferon-&#946; (IFN-&#946;), or LPS plus interferon-&#947; (IFN-&#947;), and inflammatory responses assessed as measured by production of NO and TNF-&#945;, mRNA reduction for TNF-&#945;, and iNOS genes, and microarray analysis. Results Thioglycolate-elicited peritoneal macrophages prepared after four weeks of feeding, and then challenged with LPS (10 ng or 100 ng) resulted in increases of 55% and 73%, respectively in the production of NO of 46-week-old compared to 8-week-old mice fed control diet alone (respective control groups), without affecting the secretion of TNF-&#945; among these two groups. However, macrophages obtained after feeding with quercetin, &#948;-tocotrienol, and dexamethasone significantly inhibited (30% to 60%; P &lt; 0.02) the LPS-stimulated NO production, compared to respective control groups. There was a 2-fold increase in the production of NO, when LPS-stimulated macrophages of quercetin, &#948;-tocotrienol, or dexamethasone were also treated with IFN-&#946; or IFN-&#947; compared to respective control groups. We also demonstrated that NO levels and iNOS mRNA expression levels were significantly higher in LPS-stimulated macrophages from senescent (0.69 vs 0.41; P &lt; 0.05), compared to young mice. In contrast, age did not appear to impact levels of TNF-&#945; protein or mRNA expression levels (0.38 vs 0.35) in LPS-stimulated macrophages. The histological analyses of livers of control groups showed lesions of peliosis and microvesicular steatosis, and treated groups showed Councilman body, and small or large lymphoplasmacytic clusters. Conclusions The present results demonstrated that quercetin and &#948;-tocotrienols inhibit the LPS-induced NO production in vivo. The microarray DNA analyses, followed by pathway analyses indicated that quercetin or &#948;-tocotrienol inhibit several LPS-induced expression of several ageing and pro-inflammatory genes (IL-1&#946;, IL-1&#945;, IL-6, TNF-&#945;, IL-12, iNOS, VCAM1, ICAM1, COX2, IL-1RA, TRAF1 and CD40). The NF-&#954;B pathway regulates the production of NO and inhibits the pro-inflammatory cytokines involved in normal and ageing process. These ex vivo results confirmed the earlier in vitro findings. The present findings of inhibition of NO production by quercetin and &#948;-tocotrienol may be of clinical significance treating several inflammatory diseases, including ageing process.Peer Reviewe

Suppression of nitric oxide induction and pro-inflammatory cytokines by novel proteasome inhibitors in various experimental models

<p>Abstract</p> <p>Background</p> <p>Inflammation has been implicated in a variety of diseases associated with ageing, including cancer, cardiovascular, and neurologic diseases. We have recently established that the proteasome is a pivotal regulator of inflammation, which modulates the induction of inflammatory mediators such as TNF-α, IL-1, IL-6, and nitric oxide (NO) in response to a variety of stimuli. The present study was undertaken to identify non-toxic proteasome inhibitors with the expectation that these compounds could potentially suppress the production of inflammatory mediators in ageing humans, thereby decreasing the risk of developing ageing related diseases. We evaluated the capacity of various proteasome inhibitors to suppress TNF-α, NO and gene suppression of TNF-α, and iNOS mRNA, by LPS-stimulated macrophages from several sources. Further, we evaluated the mechanisms by which these agents suppress secretion of TNF-α, and NO production. Over the course of these studies, we measured the effects of various proteasome inhibitors on the RAW 264.7 cells, and peritoneal macrophages from four different strains of mice (C57BL/6, BALB/c, proteasome double subunits knockout LMP7/MECL-1^-/-, and peroxisome proliferator-activated receptor-α,^-/-(PPAR-α,^-/-) knockout mice. We also directly measured the effect of these proteasome inhibitors on proteolytic activity of 20S rabbit muscle proteasomes.</p> <p>Results</p> <p>There was significant reduction of chymotrypsin-like activity of the 20S rabbit muscle proteasomes with dexamethasone (31%), mevinolin (19%), δ-tocotrienol (28%), riboflavin (34%), and quercetin (45%; <b><it>P </it></b>< 0.05). Moreover, quercetin, riboflavin, and δ-tocotrienol also inhibited chymotrypsin-like, trypsin-like and post-glutamase activities in RAW 264.7 whole cells. These compounds also inhibited LPS-stimulated NO production and TNF-α, secretion, blocked the degradation of P-IκB protein, and decreased activation of NF-κB, in RAW 264.7 cells. All proteasome inhibitors tested also significantly inhibited NO production (30% to 60% reduction) by LPS-induced thioglycolate-elicited peritoneal macrophages derived from all four strains of mice. All five compounds also suppressed LPS-induced TNF-α, secretion by macrophages from C57BL/6 and BALB/c mice. TNF-α, secretion, however, was not suppressed by any of the three proteasome inhibitors tested (δ-tocotrienol, riboflavin, and quercetin) with LPS-induced macrophages from LMP7/MECL-1^-/-and PPAR-α,^-/-knockout mice. Results of gene expression studies for TNF-α, and iNOS were generally consistent with results obtained for TNF-α, protein and NO production observed with four strains of mice.</p> <p>Conclusions</p> <p>Results of the current study demonstrate that δ-tocotrienol, riboflavin, and quercetin inhibit NO production by LPS-stimulated macrophages of all four strains of mice, and TNF-α, secretion only by LPS-stimulated macrophages of C57BL/6 and BALB/c mice. The mechanism for this inhibition appears to be decreased proteolytic degradation of P-IκB protein by the inhibited proteasome, resulting in decreased translocation of activated NF-κB to the nucleus, and depressed transcription of gene expression of TNF-α, and iNOS. Further, these naturally-occurring proteasome inhibitors tested appear to be relatively potent inhibitors of multiple proteasome subunits in inflammatory proteasomes. Consequently, these agents could potentially suppress the production of inflammatory mediators in ageing humans, thereby decreasing the risk of developing a variety of ageing related diseases.</p

Mammalian Lipopolysaccharide Receptors Incorporated into the Retroviral Envelope Augment Virus Transmission

SummaryThe orally transmitted retrovirus mouse mammary tumor virus (MMTV) requires the intestinal microbiota for persistence. Virion-associated lipopolysaccharide (LPS) activates Toll-like receptor 4 (TLR4), stimulating production of the immunosuppressive cytokine IL-10 and MMTV evasion of host immunity. However, the mechanisms by which MMTV associates with LPS remain unknown. We find that the viral envelope contains the mammalian LPS-binding factors CD14, TLR4, and MD-2, which, in conjunction with LPS-binding protein (LBP), bind LPS to the virus and augment transmission. MMTV isolated from infected mice lacking these LBPs cannot engage LPS or stimulate TLR4 and have a transmission defect. Furthermore, MMTV incorporation of a weak agonist LPS from Bacteroides, a prevalent LPS source in the gut, significantly enhances the ability of this LPS to stimulate TLR4, suggesting that MMTV intensifies these immunostimulatory properties. Thus, an orally transmitted retrovirus can capture, modify, and exploit mammalian receptors for bacterial ligands to ensure successful transmission

Methamphetamine Increases LPS-Mediated Expression of IL-8, TNF-α and IL-1β in Human Macrophages through Common Signaling Pathways

The use of methamphetamine (MA) has increased in recent years, and is a major health concern throughout the world. The use of MA has been associated with an increased risk of acquiring HIV-1, along with an increased probability of the acquisition of various sexually transmitted infections. In order to determine the potential effects of MA exposure in the context of an infectious agent, U937 macrophages were exposed to various combinations of MA and bacterial lipopolysaccharide (LPS). Treatment with MA alone caused significant increases in the levels of TNF-α, while treatment with both MA and LPS resulted in significant increases in TNF-α, IL-1β and the chemokine IL-8. The increases in cytokine or chemokine levels seen when cells were treated with both LPS and MA were generally greater than those increases observed when cells were treated with only LPS. Treatment with chemical inhibitors demonstrated that the signal transduction pathways including NF-kB, MAPK, and PI3-Akt were involved in mediating the increased inflammatory response. As discussed in the paper, these pathways appear to be utilized by both MA and LPS, in the induction of these inflammatory mediators. Since these pathways are involved in the induction of inflammation in response to other pathogens, this suggests that MA-exacerbated inflammation may be a common feature of infectious disease in MA abusers