Inhibition of nitric oxide and inflammatory cytokines in LPS-stimulated murine macrophages by resveratrol, a potent proteasome inhibitor

Qureshi AA, Guan XQ, Reis JC, Papasian CJ, Jabre S, Morrison DC, Qureshi N.

Lipids Health Dis. 2012 Jul 10;11:76.

Background: Altered immune function during ageing results in increased production of nitric oxide (NO) and other inflammatory mediators. Recently, we have reported that NO production was inhibited by naturally-occurring proteasome inhibitors (quercetin, δ-tocotrienol, and riboflavin) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, and thioglycolate-elicited peritoneal macrophages from C57BL/6 mice. In a continuous effort to find more potent, non-toxic, commercially available, naturally-occurring proteasome inhibitors that suppress inflammation, the present study was carried out to describe the inhibition of NF-κB activation and NO, TNF-α, IL-6, IL-1β, and iNOS expression by trans-resveratrol, trans-pterostilbene, morin hydrate, and nicotinic acid in LPS-induced RAW 264.7 cells and thioglycolate-elicited peritoneal macrophages from C57BL/6 and BALB/c mice.

Results: The present results indicate that resveratrol, pterostilbene, and morin hydrate caused significant inhibition (>70% to 90%; P < 0.02) in the activities of chymotrypsin-like, trypsin-like, and post-acidic (post-glutamase) proteasome sites in RAW 264.7 cells at a dose of only 20 μM. These compounds also inhibited the production of NO by RAW-264.7 cells stimulated with LPS alone (>40%; P < 0.05), or LPS + interferon-γ (IFN-γ; >60%; P < 0.02). Furthermore, resveratrol, pterostilbene, morin hydrate, and quercetin suppressed secretion of TNF-α (>40%; P < 0.05) in LPS-stimulated RAW 264.7 cells, and suppressed NF-κB activation (22% to 45%; P < 0.05) in LPS-stimulated HEK293T cells. These compounds also significantly suppressed LPS-induced expression of TNF-α, IL-1β, IL-6, and iNOS genes in RAW 264.7 cells, and also in thioglycolate-elicited peritoneal macrophages from C57BL/6 and BALB/c mice.

Conclusions: The present results clearly demonstrate that resveratrol and pterostilbene are particularly potent proteasome inhibitors that suppress expression of genes, and production of inflammatory products in LPS-stimulated RAW 264.7 cells, and macrophages from C57BL/6 and BALB/c mice. Resveratrol and pterostilbene which are present in grapes, blueberries, and red wine, have been implicated as contributing factors to the lower incidence of cardiovascular disease in the French population, despite their relatively high dietary fat intake. Consequently, it appears likely that the beneficial nutritional effects of resveratrol and pterostilbene are due at least in part, to their ability to inhibit NF-κB activation by the proteasome, thereby suppressing activation of pro-inflammatory cytokines and iNOS genes, resulting in decreased secretion of TNF-α, IL-1β, IL-6, and NO levels, in response to inflammatory stimuli. This is the first report demonstrating that resveratrol and pterostilbene act as proteasome inhibitors, thus providing a mechanism for their anti-inflammatory effects.


Suppression of Nitric Oxide Production and Cardiovascular Risk Factors in Healthy Seniors and Hypercholesterolemic Subjects by a Combination of Polyphenols and Vitamins

Qureshi AA, Khan DA, Mahjabeen W, Papasian CJ, Qureshi N.

J Clin Exp Cardiolog. 2012 Jun 7;S5:8.

Background: Dysregulated immune function associated with ageing has been implicated in a variety of human diseases. We have demonstrated the anti-inflammatory properties of resveratrol, pterostilbene, morin hydrate, quercetin, δ-tocotrienol, riboflavinin a variety of experimental animal models, and determined that these compounds act by inhibiting proteasome activity.

Aims: To determine whether serum nitric oxide (NO) levels increase with age in humans, and whether the combined cholesterol-lowering and inflammation-reducing properties of resveratrol, pterostilbene, Morin hydrate, quercetin, δ-tocotrienol, riboflavin, and nicotinic acid would reduce cardiovascular risk factors in humans when used as nutritional supplements with, or without, other dietary changes.

Methods: Elderly human subjects were stratified into two groups based on total serum cholesterol levels. Initial total serum cholesterol levels were normal and elevated in Group 1 and 2 subjects, respectively. Baseline serum NO, C-reactive protein (CRP), γ-glutamyltransferase (γ-GT) activity, uric acid, total antioxidant status (TAS), total cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides levels were established over a four week period. Group 1 subjects subsequently received nutritional supplementation with one of two different combinations (NS-7 = 25 mg of each, resveratrol, pterostilbene, quercetin, δ-tocotrienol, nicotinic acid, morin hydrate or NS-6 = morin hydrate replaced with quercetin, 50 mg/capsule). Group 2 subjects also received these nutritional supplements (two capsules/d), but an AHA Step-1 diet was also implemented. After these interventions were administered for four weeks, the above parameters were re-measured and changes from baseline levels determined. Nitric acid (NO) levels in children, young adults, and seniors were also compared.

Results: The key results of the current study were: 1) that serum NO levels were significantly increased in seniors compared to both children (~80%) and young adults (~65%); 2) that the intake of two capsules/d of NS-7 or NS-6 for four weeks significantly (P < 0.05) decreased serum NO (39%, 24%), CRP (19%, 21%), uric acid (6%, 12%) levels, and γ-GT activity (8%, 6%), respectively in free-living healthy seniors; 3) that serum NO (36%, 29%), CRP (29%, 20%), uric acid (6%, 9%) γ-GT activity (9%, 18%), total cholesterol (8%, 11%), LDL-cholesterol (10%, 13%), and triglycerides (16%, 23%) levels were significantly (P < 0.02) decreased in hypercholesterolemic subjects restricted to AHA Step-1 diet plus intake of SN-7 or SN-6 (two capsules/d), respectively; 4) that TAS was increased (3%, 9%; P < 0.05) in free-living healthy seniors receiving NS-7 or NS-6 alone, and in hypercholesterolemic subjects plus AHA Step-1 diet (20%, 12%; P < 0.02) with either of the combinations tested.

Conclusion: Serum NO levels are elevated in elderly humans compared to children or young adults. Diet supplementation with combinations of resveratrol, pterostilbene, morin hydrate, quercetin, δ-tocotrienol, riboflavin, and nicotinic acid reduce cardiovascular risk factors in humans when used as nutritional supplements with, or without, other dietary changes.

γ-Tocotrienol attenuates TNF-α-induced changes in secretion and gene expression of MCP-1, IL-6 and adiponectin in 3T3-L1 adipocytes

Matsunaga T, Shoji A, Gu N, Joo E, Li S, Adachi T, Yamazaki H, Yasuda K, Kondoh T, Tsuda K.

Mol Med Report. 2012 Apr;5(4):905-9. Epub 2012 Jan 30.

Tocotrienols, members of the vitamin E family, have been shown to possess anti-inflammatory properties and display activity against a variety of chronic diseases, such as cancer, cardiovascular and neurological diseases. However, whether tocotrienols contribute to the prevention of inflammatory responses in adipose tissue remains to be elucidated. In this study, we examined the effects of γ-tocotrienol, the most commontocotrienol isomer, on tumor necrosis factor-α (TNF-α)-induced inflammatory responses by measuring the expression of the adipokines, monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6) and adiponectin in 3T3-L1 adipocytes. Exposure to TNF-α (10 ng/ml) for 24 h increased MCP-1 and IL-6 secretion, and decreased adiponectin secretion and peroxisome proliferator-activated receptor-γ (PPARγ) mRNA expression. γ-tocotrienoleffectively improved the TNF-α-induced adverse changes in MCP-1, IL-6 and adiponectin secretion, and in MCP-1, IL-6, adiponectin and PPARγ mRNA expression. Furthermore, TNF-α-mediated IκB-α phosphorylation and nuclear factor-κB (NF-κB) activation were significantly suppressed by the γ-tocotrienol treatment. Our results suggest that γ-tocotrienol may improve obesity-related functional abnormalities in adipocytes by attenuating NF-κB activation and the expression of inflammatory adipokines.

Comparative effect of TRF aTP and aTPA on inflammatory mediators and NF-KB expression in mouse peritoneal macrophages

Lean-Teik Ng, Huey-Jiun Ko

Food Chemistry 134 (2012) 920–925 Epub 16 March 2012

The effects of tocotrienol-rich fraction (TRF), a-tocopherol (T) and a-tocopheryl acetate (TA) on lipopolysaccharide (LPS)-induced inflammatory responses in mouse peritoneal macrophages were examined. Results showed that at 5–30 lg/ml, all test compounds plus 1 lg/ml LPS exhibited no cytotoxic effects on macrophage cells. Compared with T and TA, TRF showed the strongest anti-inflammatory activity as demonstrated by its potency in inhibiting the LPS-induced nitric oxide (NO), prostaglandin E2 (PGE2), and proinflammatory cytokine (TNF-a, IFN-c, IL-1b and IL-6) production. At 10 lg/ml, it signifi-
cantly blocked the LPS induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression, but has no effect on cyclooxygenase-1 (COX-1). Furthermore, TRF also showed a greater inhibition on the nuclear factor kappa B (NF-jB) expression than T and TA. These results suggest that TRF could be a better agent than T and TA for use in the prevention of chronic inflammatory diseases.

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

Qureshi AA, Tan X, Reis JC, Badr MZ, Papasian CJ, Morrison DC, Qureshi N.

Lipids Health Dis. 2011 Dec 20;10:239.

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, δ-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-α(-/-) knockout mice). The present study was carried out in order to further explore the potential effects of diet supplementation with naturally-occurring inhibitors (δ-tocotrienol and quercetin) on LPS-stimulated production of NO, TNF-α, 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), δ-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-β (IFN-β), or LPS plus interferon-γ (IFN-γ), and inflammatory responses assessed as measured by production of NO and TNF-α, mRNA reduction for TNF-α, 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-α among these two groups. However, macrophages obtained after feeding with quercetin, δ-tocotrienol, and dexamethasone significantly inhibited (30% to 60%; P < 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, δ-tocotrienol, or dexamethasone were also treated with IFN-β or IFN-γ 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 < 0.05), compared to young mice. In contrast, age did not appear to impact levels of TNF-α 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 δ-tocotrienols inhibit the LPS-induced NO production in vivo. The microarray DNA analyses, followed by pathway analyses indicated that quercetin or δ-tocotrienol inhibit several LPS-induced expression of several ageing and pro-inflammatory genes (IL-1β, IL-1α, IL-6, TNF-α, IL-12, iNOS, VCAM1, ICAM1, COX2, IL-1RA, TRAF1 and CD40). The NF-κB 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 δ-tocotrienol may be of clinical significance treating several inflammatory diseases, including ageing process.

Protective effect of dietary tocotrienols against infection and inflammation-induced hyperlipidemia: An in vivo and in silico study

Salman Khan M, Akhtar S, Al-Sagair OA, Arif JM.

Phytother Res. 2011 Nov;25(11):1586-95. Epub 2011 Mar 11.

Currently used hypolipidemic drugs, Fluvastatin and Atorvastatin, act via inhibiting the rate-limiting enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase of the mevalonate pathway. The associated severe side-effects of these statins led us to explore the therapeutic potentials of naturally occurring Tocomin (mixture of dietary α-, β-, γ- and δ-tocotrienols). Tocomin (10 mg) was orally administered daily for 10 days before and 12 h after bacterial lipopolysaccharide (200 μg) or 24 h after zymosan (20 mg) or turpentine (0.5 mL) to Syrian hamsters. The data showed that Tocomin significantly reduced the levels of plasma and lipoprotein lipids, cholesterol, apoB, small dense (sd)-LDL as well as LDL in the hyperlipidemia-induced hamsters. Further, the mechanism of action of α-, β-, γ- and δ-tocotrienols was validated by docking studies with HMG-CoA reductase enzyme using the Molegro Virtual Docker. The inhibition of HMG-CoA reductase predicted in terms of MolDockScore and interaction energy suggest the comparative potential in the descending order: Atorvastatin > Fluvastatin ~ δ > γ > β > α. The results favor the daily intake of naturally occurring tocotrienols as dietary supplement in the prevention and treatment of infection/inflammation induced dyslipidemia compared with the hypolipidemic drugs.

Assessment of the protective effects of oral tocotrienols in arginine chronic-like pancreatitis

González AM, Garcia T, Samper E, Rickmann M, Vaquero EC, Molero X.

Am J Physiol Gastrointest Liver Physiol. 2011 Nov;301(5):G846-55. Epub 2011 Aug 18.

Tocotrienols exhibit anti-inflammatory properties over macrophages and promote cytotoxicity in activated pancreatic stellate cells, suggesting that they may limit chronic pancreatitis progression. We aimed to quantitate the effect of oral tocotrienols on a rat model of chronic pancreatic injury. Chronic-like pancreatitis was induced by repeated arginine pancreatitis. Palm oil tocotrienol-rich fraction (TRF) was given by gavage before and after pancreatitis inductions. Amylase and hydroxyproline were determined in pancreatic homogenates; collagen, fibronectin, α-smooth muscle actin (SMA), glial fibrillary acidic protein (GFAP), and phosphorylated Smad3 were assessed by Western blotting. Transforming growth factor (TGF)-β1 was measured in plasma. Morphological assessment included light microscopy, fibrosis area fraction, and collagen network fractal analysis. Arginine pancreatitis induced pancreatic atrophy and increased hydroxyproline that ameliorated after TRF. Arginine increased TGF-β1 (185 ± 40 vs. 15 ± 2 ng/ml; P <0.01) that was blunted by TRF (53 ± 19; P < 0.01). TRF reduced protease and Smad3 activation, collagen, and fibronectin. α-SMA increased and GFAP diminished in arginine pancreatitis, consistent with long-term stellate cell activation, and TRF reverted these changes to basal. Arginine pancreatitis increased fibrosis area fraction (4.5 ± 0.3% vs. 0.2 ± 0.2%), collagen network complexity (fractal dimension 1.52 ± 0.03 vs. 1.42 ± 0.01; P < 0.001), and inhomogeneity (lacunarity 0.63 ± 0.03 vs. 0.40 ± 0.02; P < 0.001), which were all reduced by TRF (1.3 ± 0.4%, 1.43 ± 0.02%, and 0.51 ± 0.03%, respectively; P < 0.01). Best correlation coefficients were obtained when comparing fibrosis area fraction with lacunarity (r = 0.88) and both parameters with pancreatic weight (r = -0.91 and -0.79, respectively). TRF administered only before pancreatitis best, but not fully, recapitulated the beneficial effects of TRF. Tocotrienols improve quantitative measures of chronic pancreatic damage. They may be of benefit in human chronic pancreatitis.

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Comparison of antioxidative and antifibrotic effects of α-tocopherol with those of tocotrienol-rich fraction in a rat model of chronic pancreatitis

Jiang F, Liao Z, Hu LH, Du YQ, Man XH, Gu JJ, Gao J, Gong YF, Li ZS.

Pancreas. 2011 Oct;40(7):1091-6.

Objectives: The α-tocopherol and tocotrienol-rich fraction (TRF) are considered effective antioxidants. This study aimed to compare the antioxidative and antifibrotic effects of α-tocopherol and TFR in dibutylin dichloride (DBTC)-induced chronic pancreatitis (CP) rats.

Methods: Oral administration of α-tocopherol and TFR (both 800 mg/kg per day) started the next day after DBTC (8 mg/kg) infusion into the tail vein for 4 weeks. Histological examination, Sirius red staining, and measurement of the contents of hydroxyproline and malondialdehyde of the pancreas were performed to evaluate pancreatic damage and fibrosis. Immunohistochemical analysis of α-smooth muscle actin and real-time reverse transcription polymerase chain reaction for transforming growth factor-β1 (TGF-β1) and collagen-α1(I) were performed to evaluate the activation of pancreatic stellate cells and the mRNA levels of fibrosis-related genes, respectively.

Results: Both α-tocopherol and TRF reduced oxidative stress, ameliorated inflammation and fibrosis, and down-regulated the mRNA expression of TGF-β1 and collagen-α1(I) in DBTC-induced CP. The TRF was superior to α-tocopherol in alleviating inflammation and fibrosis and down-regulating TGF-β1 mRNA expression.

Conclusion: Oral administration of α-tocopherol and TRF improves pancreatic inflammation and fibrosis in DBTC-induced CP rats, with TRF being more effective than α-tocopherol. Therefore, TRF may be a novel option for alleviating inflammation and, particularly, the fibrotic process in CP.

The anti-inflammatory role of vitamin E in prevention of osteoporosis

Nazrun AS, Norazlina M, Norliza M, Nirwana SI.

Adv Pharmacol Sci. 2012;2012:142702. Epub 2011 Nov 17.

There is growing evidence that inflammation may be one of the causal factors of osteoporosis. Several cytokines such as IL-1, IL-6, RANKL, OPG, and M-CSF were implicated in the pathogenesis of osteoporosis. These cytokines are important determinants of osteoclast differentiation and its bone resorptive activity. Anticytokine therapy using cytokine antagonists such as IL-receptor antagonist and TNF-binding protein was able to suppress the activity of the respective cytokines and prevent bone loss. Several animal studies have shown that vitamin E in the forms of palm-derived tocotrienol and α-tocopherol may prevent osteoporosis in rat models by suppressing IL-1 and IL-6. Free radicals are known to activate transcription factor NFκB which leads to the production of bone resorbing cytokines. Vitamin E, a potent antioxidant, may be able to neutralise free radicals before they could activate NFκB, therefore suppressing cytokine production and osteoporosis. Vitamin E has also been shown to inhibit COX-2, the enzyme involved in inflammatory reactions. Of the two types of vitamin E studied, tocotrienol seemed to be better than tocopherol in terms of its ability to suppress bone-resorbing cytokines.

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

Qureshi AA, Tan X, Reis JC, Badr MZ, Papasian CJ, Morrison DC, Qureshi N.

Lipids Health Dis. 2011 Oct 12;10:177.

Background: 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.

Results: 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%; P < 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.

Conclusions: 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.