Diet and Inflammation in Alzheimer’s Disease and Related Chronic Diseases: A Review

Flavonoids and markers of inflammation in vitro

Abundant in vitro evidence suggests flavonoids have effective anti-inflammatory activity. The volume of published literature is too great to discuss within the context of this review. Consequently, a few key papers are described below, while additional studies of interest are summarized in Table 4.

Hämäläinen et al. [35] investigated the effects of flavonoids on nitric oxide (NO) production in macrophages exposed to the inflammatory stimulus LPS. Flavones, the isoflavones daidzein and genistein;the flavonols isorhamnetin, kaempferol, and quercetin; the flavanone naringenin; and the anthocyanin pelargonidin, inhibited iNOS protein and messengerribonucleic acid (mRNA) expression and also NO production in a dose-dependent manner. All the flavonoids investigated inhibited the activation of NF-κB, a significant transcription factor for iNOS. Genistein, daidzein, kaempferol, and quercetin also inhibited activation of the signal transducer and activator of transcription-1 (STAT-1), another important transcription factor for iNOS. The mechanism by which the above four flavonoids inhibit STAT-1 activation is not known, but it may be associated with inhibition of phosphorylation of STAT-1 or its upstream kinase, janus kinase-2 (JAK-2). Those flavonoids that inhibited NF-κB and STAT-1 as well as iNOS expression and NO production, in comparison to those that just affected iNOS expression and NO production, had a larger effect on iNOS expression, and therefore are likely to downregulate production of an array of inflammatory mediators in addition to iNOS, as NF-κB and STAT-1 are involved in the activation of several inflammatory genes.

Valles et al. [42] pre-treated astrocytes with genistein, and 48 h later 5 micromole ( μM) Aβ for 24 h. Aβ induces inflammatory mediators including COX-2, iNOS, IL-1β and TNF-α, which was prevented in the pre-treated cells. Aβ-stimulated expression of pro-inflammatory genes is antagonized by the action of PPARs, and the authors observed an increase in PPAR-γ expression in astrocytes treated with Aβ and genistein. Thus, some of the anti-inflammatory effects of genistein may be mediated and activated by PPARs suppressing a diverse array of inflammatory responses caused by Aβ.

The pro-inflammatory cytokine IL-1β contributes to inflammation and neuronal death in central nervous system injuries and neurodegenerative diseases. Sharma et al. [43] investigated the effect of quercetin (a flavonol found in green tea, grape seed, and bilberries) and luteolin (a flavone found in celery and green pepper) in modulating the response of cultured human astrocytes to IL-1β. Both quercetin and luteolin significantly decreased the release of reactive oxygen species (ROS) from astrocytes stimulated with IL-1β, and also decreased the elevated levels of pro-inflammatory cytokine IL-6 and chemokines IL-8, IFN-γ, inducible protein-10 (IP-10), monocyte-chemoattractant protein-1 (MCP-1) and RANTES (regulated on activation, normal T cell expressed and secreted, also called chemokine (C-C motif) ligand 5) from IL-1β activated astrocytes. The authors found a significant decrease in neuronal apoptosis in neurons cultured in conditioned medium obtained from astrocytes treated with a combination of Il-1β and flavonoids compared to those treated with IL-1β alone. The flavonoids quercetin and luteolin therefore appear to confer protection against IL-1β induced astrocyte-mediated neuronal damage by enhancing the potential of activated astrocytes to detoxify free radicals, reducing the expression of pro-inflammatory cytokines and chemokines, and by modulating expression of mediators associated with enhanced physiological activity of astrocytes in response to injury [43].

Flavonoids and markers of inflammation in humans

In a randomized human intervention trial with healthy normal-weight adults, supplementation with a bilberry extract providing 300 milligrams per day (mg/d) anthocyanins (equal to 100 g of fresh bilberries) reduced the plasma concentrations of the NF-κB-induced pro-inflammatory cytokines IL-8, RANTES, and interferon-alpha (IFN-α) [44]. Eighteen healthy men and women supplemented their diet with Bing sweet cherries (280 grams per day (g/d); containing anthocyanins, catechins, and flavonal glycosides) for 28 days. Following cherry consumption, CRP, RANTES and NO concentrations decreased by 25, 21, and 18% respectively. Twenty-eight days after cherry consumption had finished, RANTES concentrations continued to decrease, whereas CRP and NO levels had returned to baseline [45].

In a population survey conducted in the United States of America, consumption of quercetin, kaempferol, malvidin, peonidin, daidzein, and genistein each had inverse associations with serum hs-CRP concentration, after adjustment for total fruit and vegetableconsumption [46].

Quercetin supplements (1 g/d) were ingested by 20 trained male cyclists for three weeks before and during a three day period in which participants cycled for 3 h a day at 57% maximal work rate. Post-exercise increases in plasma cytokines did not differ between the treatment and placebo groups, however, leukocyte IL-8 (p = 0.019) and IL-10 (p = 0.012) mRNA were significantly reduced in the group consuming the quercetin supplements [47].

Genistein is an isoflavone which primarily occurs in soya beans. In two intervention studies with healthy postmenopausal women, the intake of either 54 or 40 mg/d of genistein for six months did not significantly affect plasma CRP levels [48, 49]. Consumption of a soya isoflavone-enriched cereal bar (50 mg/d) for eight weeks in postmenopausal women also had no effect on the level of serum CRP or other plasma markers of inflammation [50]. Further, a higher intake of soya isoflavones, 114 mg/d for three months, did not reduce serum CRP level or soluble endothelial-leukocyte adhesion molecule 1 (sELAM-1) concentrations in postmenopausal women [51], and isoflavone-rich soya consumption for six weeks did not affect soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular adhesion molecule-1 (sVCAM-1) or sELAM-1 concentrations in healthy postmenopausal women compared with isoflavone-poor soya [52].

Landberg et al. [53] collected dietary data in 1990 from 2115 women in the Nurses’ Health Study and measured plasma CRP, IL-6, IL-8, soluble tumor necrosis factor receptor 2 (TNFR-2), sICAM-1, sVCAM-1, and ELAM-1 in blood samples. Six flavonoid subclasses (flavonols, flavones, flavanones, flavnan-3-ols, anthocyanidins, and polymeric flavonoids) and the main food sources of these flavonoids were examined. After multivariate-adjustments, IL-18 levels were lower for women in the highest intake quintile of flavones, flavanones, and total flavonoids compared with those in the lowest intake quintile (p = 0.019; p = 0.011; p = 0.012, respectively). sVCAM-1 levels were lower for women in the highest intake quintile of flavonols compared to the lowest (p = 0.012). Among the flavonoid rich foods, grapefruit was significantly negatively associated with concentrations of CRP and sTNFR-2.

Dietary flavonoids, AD, and related chronic diseases in animal and in vitro models

Evidence of the neuro- and cardio-protective effects of flavonoids has increased significantly in recent years [reviewed in 54]. In a rodent study of cognition, supplementation with pure anthocyanins (179 micrograms ( μg)) or pure flavonols (14.8 μg (-)-epicatechin and 59.3 μg (+)-catechin) for six weeks, resulted in enhanced spatial memory in 18 month old rats [55]. Blueberry supplementation has been shown to be effective in reversing age-related deficits in learning and memory in a number of studies [56–59]. Supplementation with a blueberry diet for 12 weeks improved the performance of aged animals in spatial working memory tasks. This improvement emerged within three weeks and persisted for the remainder of the testing period [59]. Eight week supplementation of strawberries, spinach, or blueberries to 19-month-old Fischer rats was also effective in reversing age-related deficits in several neuronal and behavioral parameters including motor behavior performance on the rod walking and accelerod tasks, and Morris water maze performance [57].

Maher et al. [60] have also shown older rats fed a diet enriched in strawberry extract (which contains the flavonoid fisetin) for eight weeks had enhanced cognitive performance in the Morris water maze relative to rats fed a control diet. Significantly, the flavonoid naringin has demonstrated beneficial effects in amyloid-β protein precursor (AβPP) transgenic mice (AβPP^swe/PS^ΔE9) after 16 weeks (dose 100 milligrams per kilogram (mg/kg) of body weight per day), including; lessening learning and memory deficits, improving locomotor activity, reducing scattered senile plaques, and ameliorating disturbances in brain energy metabolism. Although naringin has previously been shown to repress COX-2 and iNOS expression in vitro and in vivo [62], the authors of the feeding study could not confirm the beneficial effects observed were due to the anti-inflammatory effect of naringin, with an increase in glucose uptake through naringin-mediated inhibition of glycogen synthase kinase 3 activity suggested as another potential protective mechanism [63].

Pre-treatment with quercetin conferred neuroprotection against 6-hydroxydopamine (6-OHDA)-stimulated dopaminergic neuron loss in zebrafish. Quercetin was able to protect but not rescue the dopaminergic neuron damage when the fish were treated with quercetin at different maturation stages of the blood-brain barrier (BBB). A mechanistic study showed that quercetin could downregulate the overexpression of pro-inflammatory genes including IL-1β, TNF-α, and COX-2, suggesting these genes play a role in the neuroprotective effect of quercetin [64].

Nobiletin, a citrus flavonoid, has been shown to ameliorate Aβ-induced memory impairments in AD rat models, and improve memory deficits in AβPP transgenic mice that overexpress human AβPP695 harboring the double Swedish and London mutations (AβPP-SL 7-5 transgenic mice). Enzyme-linked immunosorbent assays showed administration of nobiletin to these transgenic mice for four months markedly reduced the quantity of Aβ_1 - 40 and Aβ_1 - 42 in the brain. Administration of nobiletin also decreased Aβ burden and plaques in the hippocampus of AβPP-SL 7-5 transgenic mice. The focal deposits of Aβ elicit a significant microglial mediated inflammatory response in the brain, and it has been observed that nobiletin suppresses LPS-induced COX-2 expression in C6 rat glioma cells [65]; therefore, it is possible that nobiletin may inhibit an inflammatory response produced by extensive Aβ deposition in the brains of the transgenic mice, providing a possible mechanism for the decreased Aβ burden observed [65].

The effect of exogenously administering the flavonoids apigenin-7-glucoside and quercetin on cognitive performance in aged and LPS-treated mice using passive avoidance and elevated plus-maze tasks was investigated by Patil et al. [66]. The mice showed poor retention of memory in step-through passive avoidance and in plus-maze tasks, which was ameliorated following chronic administration of the flavonoids in a dose-dependent fashion (apigenin-7-glucoside 5–20 mg/kg and quercetin 25–100 mg/kg). Apigenin-7-glucoside demonstrated higher efficacy, likely due to its greater ability to inhibit COX-2 and iNOS. Chronic treatment did not alter locomotor activity in both young and aged mice; however, aged mice showed improvement of performance in the Rota-Rod test.

An important factor in the increased vulnerability to inflammation seen in aging may be decreases in inducible shock protein 70 (HSP70). Both in vitro and in vivo experiments have demonstrated that increasing the level of inducible HSP70 can protect neuronal cells from a wide range of damaging agents and processes [67]. Galli et al. [67] examined whether short-term dietary supplementation with blueberries might enhance the brain’s ability to generate a HSP70-mediated neuroprotective response. Young and old Fischer rats were fed either a control or supplemented diet for ten weeks prior to hippocampal regions being removed, washed with LPS, and examined for levels of HSP70 at 30, 90, and 240 min post LPS administration. Increases in HSP70 levels were significantly less in old rats compared to young control rats; however, blueberry diet supplementation fully restored the HSP70 response to LPS in the old rats at 90 and 240 min, providing evidence that short-term blueberry supplementation may result in improved HSP70-mediated protection against a number of neurodegenerative processes in the brain.

Adipocytes are the main cellular component of adipose tissues, which is the largest endocrine organ in the body and secretes numerous cytokines and adipokines into the circulation. In obesity, adipocytes grow in size until they reach non-physiological limits, becoming incapable of functioning, rapidly developing apoptosis and changing their endocrine function, becoming resistant to insulin, and developing adipose tissue inflammation. The enlargement of the adipose tissue is also associated with the release of chemoattractant substances that set off an inflammatory process via activation of macrophages. While the etiology of obesity is multi-faceted, low-grade inflammation is common and represents a potentially useful and general therapeutic target. Rivera et al. [68] analyzed the effects of quercetin supplementation in rats on metabolic syndrome abnormalities including obesity, dyslipidemia, hypertension, and insulin resistance. Obese Zucker rats received a daily dose of quercetin (2 or 10 mg/kg of body weight) or control for ten weeks. The raised systolic blood pressure, and high plasma concentrations of triglycerides, total cholesterol, free-fatty acids, and insulin found in obese rats were reduced in those rats receiving either quercetin dose. The higher quercetin dose also improved inflammatory status by lowering visceral adipose tissue TNF-α, enhancing visceral adipose tissue endothelial NOS, and downregulating visceral adipose tissue iNOS.

Terra et al. [69] tested the hypothesis that grape-seed procyanidin extract (PE) would improve local and systemic inflammation in diet-induced obese rats. Initially the authors analyzed the preventative effects of procyanidins (30 mg/kg of body weight per day) on rats fed a 60% kilocalories (kcal) fat diet for 19 weeks. The PE group had reduced body weight as well as reduced levels of the plasma markers of inflammation TNF-α and CRP. The PE treated group exhibited significantly increased adiponectin expression and decreasedTNF-α, IL-6, and CRP expression in white adipose tissue and muscle TNF-α. Reduced NF-κB activity in liver was also observed in the PE group, which could be related to low expression rates of hepatic inflammatory markers. The authors then induced cafeteria diet obesity for 13 weeks in the rats post completion of the first feeding study, to investigate the corrective effects of two PE doses (25 and 50 mg/kg of body weight per day) administered for ten and 30 days. Only the high PE dose reduced CRP plasma levels in the ten-day treatment group, no changes in plasma TNF-α were seen. Although PE helps prevent imbalanced obesity-induced cytokine expression pattern, its corrective effects need to be further investigated.

In a 12-week feeding study, male C57BL/6 mice (age four weeks) were divided into four groups: control diet, control + C3-G-rich purple corn color (PCC; an extract from purple corn which contains anthocyanins at 0.2% concentration), high-fat diet (HF; containing 30% lard), or HF + PCC. At the end of the feeding study, the body weight of the HF group was significantly higher than that of the control, control + PCC, and HF + PCC groups: interestingly the control, and HF + PCC groups did not differ in body weight. PCC did not affect food intake, and energy and fecal lipid content did not differ among the groups, suggesting that the suppression of body weight was not due to inhibition of dietary fat digestion or reduction of energy intake. All of the adipose tissue weights were significantly greater in the HF group compared to the control group; PCC clearly suppressed the HF-diet-induced increase in tissue weight deposits, suggesting PCC has a significant potency for anti-obesity. PCC also downregulated plasminogen activator inhibitor-1 and IL-6 (both of which are elevated in obesity and in those with type-2 diabetes), and induced adiponectin gene expression due to a PPAR-γ-independentmechanism [70].

The health-promoting effects of green tea consumption are mainly attributed to the presence of catechins, notably epigallocatechin gallate (EGCG), epicatechin-3-gallate, epigallocatechin, and epicatechin. Research showing the health benefits of green tea is based on the amount of green tea typically consumed in Asian countries, i.e., on average three cups per day, which would provide 240–320 milligrams per day of polyphenols. EGCG is the most active and abundant compound in green tea and has been shown to possess anti-inflammatory properties including the capacity to reduce NO production, as measured by nitrite accumulation in culture medium [71]. The same authors went on to show that this reduction in NO production may occur via two mechanisms: reduction of iNOS gene expression and inhibition of enzyme activity. In mice fed a high-fat diet (60% energy as fat), supplementation with dietary EGCG (3.2 grams per kilogram (g/kg) of body weight) for 16 weeks reduced body weight gain, percent body fat and visceral fat weight compared to mice receiving the high-fat diet only. EGCG supplementation attenuated insulin resistance, plasma cholesterol, and MCP-1 concentrations, and decreased liver weight, liver triglycerides, and plasma alanine aminotransferase concentrations. Histological analyses of liver samples revealed decreased lipid accumulation in hepatocytes of the mice fed EGCG [72]. In a second experiment conducted by the same authors, 3-month-old high fat-induced obese mice received short-term EGCG treatment (3.2 g/kg of body weight for four weeks) which resulted in decreased mesenteric fat weight and blood-glucose compared to high-fat-fed control mice [72]. These results indicate that long-term EGCG treatment attenuates the development of obesity, symptoms associated with metabolic syndrome and fatty liver, while short-term treatment appears to reverse pre-existing high-fat-induced metabolic pathologies in obese mice. The authors concluded that these effects may be mediated by decreased inflammation, decreased lipid absorption and/or other mechanisms [72].

Mahmoud et al. [73] explored the effect of quercetin on diabetes-induced exaggerated vasoconstriction in insulin deficient and insulin resistant rat models. Insulin deficiency and insulin resistance were induced by streptozotocin and fructose respectively, after which the rats were left untreated for eight or 12 weeks, with quercetin administered daily in the last six weeks. Quercetin protected against diabetes-induced exaggerated vasoconstriction and reduced the elevated blood pressure, while also inhibiting diabetes associated adventitial leukocyte infiltration, endothelia pyknosis,and increased collagen deposition. These effects were accompanied by a reduction in serum TNF-α and CRP levels and inhibition of aortic NF-κB. Quercetin did not affect glucose levels in these diabetic models, suggesting the protective effect is mediated by anti-inflammatory effects rather than metabolic effects.

Diabetic renal injury, also referred to as diabetic nephropathy, is one of many diabetic complications. Renal protective effects of naringenin supplementation at 0.5, 1, and 2% of the diet were explored in diabetic mice. Compared with the diabetic control group, naringenin treatments at 1 and 2% lowered plasma glucose levels and blood urea nitrogen, and increased insulin level and creatinine clearance. Naringenin treatment dose-dependently reduced renal TNF-α level and expression, but only the 1 and 2% dose significantly decreased production and expression of IL-6, IL-1β, and MCP-1 decreased protein kinase C activity and suppressed NF-κB p65 activity, mRNA expression, and protein production in the kidney. Only with the 2% dose, were NF-κB p50 activity, mRNA expression, and protein production diminished, and renal formation and expression of type IV collagen, fibronectin, and TGF-β decreased. These results indicate that naringenin could attenuate diabetic nephropathy via its anti-inflammatory and anti-fibrotic activities [74].

Dietary flavonoids, AD, and related chronic diseases in humans

Beking and Vieira [75] generated databases for; 1) flavonoid content of foods; 2) per capita national dietary intakes of flavonoids and other dietary factors; and 3) disability-adjusted life years due to dementia (a measure of burden and death). These databases were constructed using dietary data obtained from Food and Agriculture Food Balance Sheets for grams per capita per day for flavonoid-containing foods, and information from the United States Department of Agriculture on the flavonoid content of selected foods, with additional literature sources on food flavonoid content also used. For the measure of burden and death, global statistics for the incidence of dementia were obtained from the World Health Organization Burden of Disease Statistics. The authors examined five major flavonoid subclasses (anthocyanidins, flavanols, flavanones, flavones, and flavonols) in 23 developed countries. Flavonols and combined flavonoid (all five subclasses combined) intakes were significantly negatively correlated with dementia incidence, suggesting that higher consumption of dietary flavonoids, especially flavonols, is associated with lower population rates of dementia in those countries. Further, in nine older adults with early memory changes, daily consumption of wild blueberry juice was associated with improved paired association learning and word list recall, and trends suggesting reduced depressive symptoms and lower glucose levels after 12 weeks [58]. Moreover, regular consumption of flavonoid-rich foods such as tea and wine has been associated with better performance on cognitive tests and decreased risk of cognitive decline in elderly populations in both Asia and Europe [76, 77]. Macready et al. [54] reviewed 15 human dietary intervention studies that examined the effects of particular types of flavonoids on cognitive performance. Nine of these studies reported significant improvement in executive function or working memory performance due to flavonoid supplementation. The authors also noted several cognitive domains were overlooked in all studies, including implicit memory and prospective memory, and comparisons across studies were difficult due to inconsistency in the particular cognitive tests administered. The authors concluded future studies need to ensure cognitive tests are adequate in their sensitivity to detect treatment effects.

The association between flavonoid intake and CVD has been reported in several studies, McCullough et al. [78] followed 38,180 men and 60,289 women for seven years and found those with total flavonoid intake level in the highest quintile compared with the lowest quintile had a lower risk of fatal CVD; additionally, five flavonoid classes were individually associated with lower fatal risk of CVD (anthocyanidins, flavan-3-ols, flavones, flavonols and proanthocyanidins). In a Finnish cohort of 10,054 men and women, those with higher kaempferol, naringenin, and hesperetin intakes were associated with lower incidence of cerebrovascular disease, and there was a trend toward a reduction in risk of type-2 diabetes with higher quercetin and myricetin intakes (disease incidence was collected 28 years after baseline assessment) [79]. In the Iowa Women’s Health Study, 34,489 postmenopausal women free of CVD had their dietary data collected and disease incidence assessed 16 years later. Significant inverse associations were observed between anthocyanidins and coronary heart disease, CVD, and total mortality for “any” verses “no intake”; interestingly strawberry and chocolate intake were individually inversely associated with CVD risk [80]. In contrast to these results, Sesso et al. [81] investigated 38,445 women free of CVD over a mean of 6.9 years and found no significant associations for both CVD and important vascular events across quintiles of flavonoid intake. While these studies were not investigating the mechanism underlying the protective role of flavonoids, several authors concluded that the observed effects likely involve anti-inflammatory and antioxidant functions, and vascular effects.

Thirty-two type-2 diabetes patients received grape seed extract (GSE; 600 mg/day; a flavonoid-rich product) or placebo for four weeks in a double-blinded randomized crossover trial. Markers of endothelial function, oxidative stress, inflammation, insulin resistance, and metabolism were measured at baseline and after intervention. Following GSE treatment, significant changes were seen in fructosamine (marker of metabolism; decreased), whole blood reduced glutathione (anti-oxidative stress enzyme; increased), and hs-CRP (marker of inflammation; decreased) [82].

The majority of human studies report that green tea consumption has no significant effect on markers of inflammation, which is surprising in view of the positive in vitro results. This may be due to the fact that tea catechins undergo methylation, glucuronidation and sulfation during uptake which may limit bioavailability. Further, in vitro studies often use catechin concentrations greater than 10 μg, whereas plasma concentrations after ingestion of tea rarely exceed 1 μg [83]. Consistent with the statements above, Laurin et al. [84] found consumption of green and black tea was not associated with risk of dementia or its subtypes. This analysis was undertaken in 2459 men (aged 45–68 years) who were participants of the Honolulu-Asia Aging Study. Tea consumption was measured in 1965–1968, with the study participants assessed for dementia between 1991 and 1999; 235 incident cases of dementia were recorded in this period. The results of this particular study suggest that mid-life dietary intake of tea flavonoids does not modify risk of late-life dementia. Whether tea consumption remained consistent beyond mid-life, however, was not determined. In contrast to these negative results, a study of Japanese people with visceral fat-type obesity found that ingesting green tea containing 583 mg of catechins per day for 12 weeks led to a reduction in body fat, systolic blood pressure, and low-density lipoprotein cholesterol level (compared to a control group ingesting 96 mg catechins per day), suggesting the green tea extract contributes to a decrease in obesity and CVD risks [85]. However, a randomized controlled trial of green tea consumption conducted in 35 obese participants demonstrated no significantly altered features of metabolic syndrome or biomarkers of inflammation. This was an eight-week study where participants consumed either four cups of green tea a day, green tea extract (two capsules, providing a similar dosing as four cups of tea), or no green tea/extract [86]. Furthermore, Ryu et al. [87] observed no change in levels of the inflammatory markers hs-CRP and IL-6, and unchanged blood glucose, lipid profiles, insulin resistance, and serum adiponectin levels in 55 people with type-2 diabetes (31 males; mean age 53.9±7.7 years) participating in a green tea study with a cross-over design. Effects were examined after consuming 900 ml water containing 9 g of green tea daily for four weeks. The authors concluded that green tea consumption did not influence inflammation, insulin resistance, or adiponectin levels in their type-2 diabetic cohort, and that these mechanisms are unlikely to explain the reduction in the risk of cardiovascular events induced by green tea drinking observed in epidemiological studies. It is worthy of note that inflammation is very high in diabetics and it is conceivable that a higher dose of green tea may be necessary in these individuals to produce positive effects, alternatively, a longer period of follow-up may be required.

Several studies have investigated consumption of flavonoids other than EGCG in the diet and risk of type-2 diabetes [88, 89]. In 1986, 35,816 postmenopausal women free of diabetes completed a food frequency questionnaire. Self-reported incident diabetes was ascertained in 1987, 1989, 1992, 1997, and 2004. Hazard ratios for diabetes were calculated according to categories of total flavonoids and anthocyanidins, flavones, flavanones, flavonols, flavan-3-ols, isoflavones, and proanthocyanidins, as well as intake of flavonoid-rich foods and beverages (apples, pears, broccoli, bran, citrus, tea, and red wine). After multivariate adjustment, red wine was the only category inversely associated with diabetes (women drinking red wine once or more a week had a 16% reduced risk of diabetes compared to those drinking red wine less than once a week). However, there was also an inverse association between alcoholic drinks in general and diabetes risk, and this association may reflect the effects of non-flavonoid constituents common to all alcoholic drinks, and the authors concluded their data does not support a diabetes-protective effect of flavonoids [89]. In 38,018 women (from the Women’s Health Study, aged over 45 years) free of CVD, cancer, and diabetes at baseline, with an average follow-up of 8.8 years, total flavonols and flavones, quercetin, kaempferol, myricetin, apigenin, and luteolin were not associated with risk of type-2 diabetes; however, the flavonoid-rich dietary constituents of apples and tea were significantly associated with decreased risk of type-2 diabetes. Plasma concentrations of insulin,glycated hemoglobin, CRP, and IL-6 were measured in 344 of these women, and flavonoid intake was not associated with these blood measures [88]. Assessment of dietary recall at one time-point only may in part be an explanation for these non-significant results, as this cross-sectional analysis may have been a poor reflection of lifetime dietary exposures of importance to disease risk. An additional consideration with all studies discussed, is the fact that accuracy of dietary data may vary between studies due to the use of FFQs which may be less comprehensive and exclude specific sources of flavonoids. Furthermore, differences in the comprehensiveness of flavonoid databases utilized can influence results.

Curcumin

Curcumin is extracted from the rhizome of Curcuma longa, and is the substance that gives the bright yellow color to the spice turmeric. The average intake of curcumin in the diet in India is 60–100 mg daily, toxicity studies have shown that curcumin is safe even at high doses in rats, guinea pigs, and monkeys; however, some species are susceptible to curcumin-induced hepatotoxicity (e.g., mice and rats with prolonged high-dose intake) and the spice can also cause some gastric irritation in humans [90]. Curcumin is a polyphenolic substance that has the potential to effectively scavenge and neutralize ROS and NO-based free radicals [91]. Of particular interest is the ability of curcumin to inhibit COX enzymes [25] and to reduce the activation of NF-κB [24]. Through the inhibition of NF-κB, curcumin has been shown to effectively inhibit the expression of inflammatory cytokines, COX-2, and iNOS [22, 23]. Singh and Aggarwal [24] demonstrated that curcumin is a potent inhibitor of NF-κB activation by treating human myeloid ML-1a cells withTNF-α which rapidly activates NF-κB, and subsequently observing that this NF-κB activation is inhibited in the presence of curcumin. Furthermore, curcumin (5 μM) inhibited LPS-induced production of TNF-α and IL-1 in a human monocytic macrophage cell line, and inhibited LPS-induced activation of NF-κB and reduced the biological activity of TNF-α in a fibroblast lytic assay [22]. Chan et al. [92] reported that 1–20 μM of curcumin reduced the production of iNOS mRNA in a concentration-dependent manner in ex vivo cultured BALB/c mouse peritoneal macrophages. The authors also demonstrated that, in vivo, two oral treatments of 0.5 millilitre (mL) of a 10 μM solution of curcumin reduced iNOS mRNA expression in the livers of LPS-injected mice by 50–70% . Both a low dose (160 parts per million (ppm)) and a high dose of dietary curcumin (5000 ppm) significantly lowered the pro-inflammatory cytokine IL-1β in AβPPsw mice, and at the low dose, also decreased expression of glial fibrillary acidic protein (GFAP), an intermediate filament protein that is expressed by numerous cell types of the central nervous system [93].

A review on the safety and activity of curcumin has found curcumin acts at various levels of the AA inflammatory cascade, and through effects on various enzymes and cytokines in inflammation pathways. The in vitro studies included in this safety review show curcumin may act in the following ways by decreasing the catalytic activities of phospholipase A₂ and phospholipase C γ1, thereby decreasing AA release from cellular phospholipid; by inhibiting phospholipase D activity; through inhibition of COX-2 expression, lipooxygenase and TNF-α; by inhibition of LPS and interferon-γ-induced production of NO; by inhibition of T_H1 (a type of T helper cell) cytokine profile in CD4 ⁺ T cells (white blood cells that are an essential part of the human immune system), and by suppressing IL-12 production in macrophages [90]. Figure 2 depicts some of the sites of action of curcumin along the inflammation pathway.

Dietary curcumin administered both chronically to aged Tg2576 AβPPsw mice and acutely to LPS-injected wild-type mice reduced IL-1β and insoluble Aβ levels, as well as preventing Aβ aggregation, and reducing Aβ plaque burden [94]. In another mouse model of AD, curcumin treatment at a dose of 7.5 mg/kg per day for seven days reduced existing senile plaques [95], and Lim et al. [93] found low-dose but not high-dose curcumin treatment significantly decreased insoluble Aβ, soluble Aβ and plaque burden by 43–50% . Human AD studies have, however, yielded less consistent results.

An epidemiological study reported that Asians who “occasionally” (less than once a month) and “often” (more than once a month) ate curry performed better on a standard test of cognitive function than those who “never” or “rarely” ate curry (n = 1010; 60–93 years of age) [96]. Ringman et al. [97] performed a 24 week randomized, double blind, placebo-controlled study of curcumin. Thirty-six people with mild-moderate AD were randomized to consume placebo, 2 g/d or 4 g/d of oral curcumin (1 participant on placebo and 5 in the curcumin group withdrew). Curcumin significantly lowered hematocrit and increased glucose levels;however, there were no differences between the 2 g/d or 4 g/d consumption treatment groups in clinical or biomarker efficacy measures. The authors concluded the small sample size and short duration of the study may have impacted the results, as could have baseline disease severity. The authors also suggested that limited bioavailability of the curcumin compound utilized (Curcumin C3 Complex ^®) likely impacted the results as low levels of native curcumin were measured in participants’ plasma. Curcumin has poor bioavailability, and absorption following ingestion is low. Co-supplementation with piperine (from black pepper) significantly increases the bioavailability of curcumin. Production of formulations with improved bioavailability is an area of active research.

A prior six-month randomized, double blind, placebo-controlled study of curcumin study in older adults also found no change in cognitive function or plasma Aβ levels [98]. Enrolled participants either demonstrated progressive decline in memory and cognitive function for six months preceding the trial or had a diagnosis of AD, and were randomized to 0, 1, or 4 g/d curcumin (there were 7 withdrawals in total, 4 on 0 g/d and 3 on 1 g/d). There was a lack of cognitive decline in the placebo group, which may have precluded any ability to detect a protective effect of curcumin, which would have likely manifest as a slower decline rather than an improvement in cognition. Thus, a study of longer duration, incorporating more sensitive cognitive testing and reduced participant treatment by other AD drugs may show greater benefit of curcumin [98].

Obesity-induced inflammation involves enhanced recruitment of macrophages into adipose tissue and the release of pro-inflammatory proteins from fat tissue. Mesenteric adipose tissue was isolated from obese mice fed a high-fat diet, and cultured to produce an adipose tissue-conditioning medium. RAW 264.7 macrophages were treated with the tissue conditioning medium and curcumin. Migration of macrophages induced by the tissue-conditioning medium was markedly suppressed following curcumin treatment, and the production of pro-inflammatory mediators including TNF-α and NO was significantly inhibited. The release of MCP-1 from 3T3-L1 adipocytes was also significantly inhibited when treated with the tissue-conditioning medium and curcumin [99]. Weisberg et al. [100] tested the hypothesis that curcumin would ameliorate diabetes and inflammation in murine models of insulin-resistance obesity. As expected, curcumin treatment ameliorated diabetes in high-fat diet-induced obese and leptin-deficient ob/ob male C57BL/6J mice, as shown by glucose and insulin tolerance testing. Curcumin treatment additionally reduced macrophage infiltration of white adipose tissue, increased adipose tissue adiponectin production, and decreased hepatic NF-κB activity and markers of hepatic inflammation. The authors concluded that orally ingested curcumin is able to reverse many of the inflammatory and metabolic changes associated with obesity and can improve glycemic control in mouse models of type-2 diabetes [100]. Jain et al. [101] examined curcumin supplementation and blood levels of inflammatory markers using a cell-culture model and a diabetic rat model. Monocytes were cultured with control or high glucose in the absence or presence of curcumin for 24 h, while diabetes was induced in Sprague-Dawley rats by injection of streptozotocin, and either control or curcumin supplementation was administered daily for seven weeks. In the monocytes, the effect of high glucose on IL-6, IL-8, MCP-1, and TNF-α levels was inhibited by curcumin. In the rats, diabetes caused a significant increase in blood levels of IL-6, MCP-1, TNF-α, and glucose, which was significantly decreased by curcumin supplementation [101].

Cinnamon

Anti-inflammatory properties of cinnamon have been demonstrated for Cinnamomum osmophloeum [102–104], but less is known about “true cinnamon” Cinnamomum zeylanicum (most commonly used cinnamon is Cinnamomum osmophloeum, also referred to as “cassia” to distinguish from “true cinnamon”). Benzoic acid occurs in low levels in several foods including cinnamon, and has been shown to inhibit LPS-induced expression of iNOS, pro-inflammatory cytokines TNF-α and IL-1β, and surface markers for inflammatory activation such as cluster of differentiation molecule 11b (CD11b), cluster of differentiation molecule 11c (CD11c), and cluster of differentiation molecule 68 (CD68) in mouse microglia [105]. Bioactive compounds in cinnamon act through PPARs to efficiently suppress pro-inflammatory cytokines.

Previous studies of the effect of cinnamon extract on AD pathology have shown cinnamon extract to markedly inhibit the formation of toxic Aβ oligomers and prevent the toxicity of Aβ on neuronal PC12 cells [106]. In AD drosophila models, cinnamon extract rectified reduced longevity, recovered locomotion defects, and abolished tetrameric species of Aβ in the drosophila brains [106]. Further, oral administrationof cinnamon extract to an AD transgenic mouse model led to marked decrease in 56 kDa Aβ oligomers (specifically shown to be a species correlated with impaired cognitive function in AD mice), reduction of plaques and improvement in cognitive behavior [106]. While these studies did not directly investigate the mechanism underlying the positive associations observed with cinnamon and AD pathology, inflammation was suggested as a potential candidate.

The ability of cinnamon to inhibit neuroinflammation was evaluated using LPS-activated BV-2 microglia, whereby 50 μg/mL cinnamon extract significantly decreased the production and expression of NO, IL-1β, IL-6 and TNF-α in the activated microglia. The authors concluded that blocking of NF-κB activation was the most likely mechanism responsible for cinnamon-mediated inhibition of neuroinflammation [107]. Tristetraprolin (TTP) family proteins have anti-inflammatory effects, are induced by cinnamon polyphenol extract in adipocytes and macrophages, and are reduced in fats of obese people with metabolic syndrome. In mouse RAW 264.7 macrophages, Cao et al. [108] observed cinnamon polyphenol extract increased TTP mRNA and protein levels; thereby representing another possible pathway for the anti-inflammatory activity of cinnamon.

Ginger

The anti-inflammatory properties of ginger (Zingiber officinale Roscoe) have been known and valued for centuries. Ginger extracts are a complex, multicomponent mixture of biologically active constituents. More than 400 chemical compounds have been isolated and identified in extracts of ginger rhizomes. Current evidence suggests that a sub-fraction containing the structurally related compounds gingerols, shogaols, and paradols accounts for a major portion of ginger’s anti-inflammatory properties [109]. Kiuchi et al. [110] were the first to show that ginger inhibits prostaglandin synthesis in vitro, via inhibition of AA metabolism by COX-2 [111]; prostaglandins are a product of the metabolism of AA via the COX-2 pathway, as depicted in Fig. 2. Some ginger constituents also inhibit leukotriene synthesis, the second pathway responsible for the metabolism of AA by LOX-1 [112], also depicted in Fig. 2. Grzanna et al. [113] tested the effect of the ginger extract EV.EXT.77 on THP-1 cells to determine whether ginger can block the induction of pro-inflammatory cytokines. THP-1 cells were exposed to an activator (TNF-α, IL-1β, or LPS) in the presence or absence of the ginger extract. Pre-treatment of these cells with the ginger extract reduced or completely prevented gene induction, providing evidence that ginger may provide beneficial effects similar to those of currently used COX inhibitors for the treatment of AD such as celecoxib and valdecoxib. In agreement with this evidence, Frondoza et al. [114] showed that the same ginger extract significantly inhibits NF-κB expression in activated synoviocytes, offering an explanation for the broad effect of ginger on inflammatory processes in various cell types and tissues. Furthermore, in murine peritoneal macrophages, ginger extract inhibited IL-12, TNF-α, IL-1β, RANTES, and MCP-1 production in the LPS-stimulated macrophages [115]. Numerous studies suggest that ginger extracts influence characteristics of AD [116–118]. For example, ginger extract has been shown to effectively protect cells from Aβ_1 - 42 insult, as has Ginkgo biloba and Chinese cinnamon, which are potentially important resources in the quest to discover novel therapeutic candidates against AD [119]. Zeng et al. [116] assessed the ability of a traditional Chinese medicinal ginger root extract (GRE) to prevent behavioral dysfunction in an AD rat model. Rats had GRE administered intra-gastrically at one of three doses (four, two, and one gram per kilogram (g/kg) of body weight) for 35 days, prior to learning and memory assessment and processing of brain sections for immunohistochemistry. The Morris water maze test was utilized to assess spatial learning and memory. The rats given the higher dose of GRE demonstrated shorter latency of platform location in water. There were also decreases in latency with low and moderate doses of GRE, although smaller effects than with the high dose group. The high dose rats also had higher levels of the antioxidant markers superoxide dismutase (SOD) and catalase (CAT) and lower levels of the inflammatory markers NF-κB and Il-1β. Again, the low and moderate dose groups also exhibited these responses but to a lesser extent. The neurons in the brains of the high dose rats were significantly more organized than the neurons in the low and moderate dose group. The authors concluded that 35 days of daily treatment with four g/kg of GRE protects rats from behavioral dysfunction, and this was sufficient to exhibit improvements in the number of neurons and neuronal activity in the hippocampus [116]. Wattanathorn et al. [118] conducted a study whereby three groups of six male adult Wistar rats were orally administered an alcoholic extract of ginger rhizome at either 100, 200, or 300 mg/kg of body weight, 14 days before and 21 days after permanent occlusion of the right middle cerebral artery (reported to induce brain damage and neuronal death in the hippocampus). The authors observed significantly decreased latency in the Morris maze tests with all dose groups. The low dose increased neuronal density in the hippocampal subfield Cornu Ammonis region 3, whereas the moderate dose increased neuronal density in the dentate gyrus as well as the Cornu Ammonis region 3. The moderate dose was the most effective at reducing brain infarct volume, both in cortical and subcortical areas [118]. Mathew and Subramanian [117] found that a methanolic extract of dry ginger (GE) increased cell survival against Aβ-induced toxicity in primary adult rat hippocampal cell culture. Aggregation experiments using thioflavin T binding showed that GE effectively prevented the formation of Aβ oligomers and dissociated the preformed oligomers [117].

Pepper

Hot red and chili peppers are among the most heavily and frequently consumed spices. Their principal pungent constituent is the phenolic substance capsaicin. Capsaicin has the ability to inhibit platelet aggregation possibly through blockage of phospholipase A₂ [120], a key enzyme responsible for formation of AA from the membrane lipid. Capsaicin has also been shown to repress calcium-ionophore stimulated pro-inflammatory responses, such as generation of superoxide anion, phospholipase A₂ activity, and membrane lipid peroxidation in macrophages. Chili peppers have been shown to reduce the activity of pro-inflammatory cytokines and enhance the activity of anti-inflammatory cytokines, partly through inhibiting COX-2 and iNOS [121]. Capsaicin has been shown to have an effect on obesity. The effect of capsaicin on body weight in rabbits was investigated by Yu et al. [122] who found that rabbits fed a high-fat diet containing 1% hot pepper for 16 weeks gained significantly less body mass than the control group fed a high-fat diet without the hot pepper (amount of food consumed was monitored and both groups consumed a similar amount of food). Capsaicin has been shown to attenuate obesity-induced inflammation by reducing levels of TNF-α, IL-6, and MCP-1 and to enhance adiponectin levels in adipose tissue and liver, which are important for insulin response. These beneficial effects are associated with the dual action of capsaicin on PPAR-γ/PPAR-α and transient receptor potential cation channel subfamily V member 1 (TRPV-1) expression and/or activation associated with NF-κB inactivation [123, 124]. Black pepper is also an anti-inflammatory spice with a similarpattern of biological activity as chili peppers but with a lower potency as it does not enhance the expression of anti-inflammatory cytokines, only suppresses pro-inflammatory cytokines [121]. Results from a feeding study of adult male rats showed that piperine (an alkaloid present in black pepper) significantly improved spatial memory impairment and neurodegeneration in the hippocampus following exposure to a cholinergic neuron-specific neurotoxin [125].

Saturated and monounsaturated fatty acids and markers of inflammation

Zhang et al. [126] placed male rats on either a high-fat or low-fat diet for five months commencing at one month of age. The results demonstrated the pro-inflammatory actions of a high-fat diet in cerebral cortical tissue, which is in agreement with previous studies. The authors found COX-1, and to a greater degree COX-2 were upregulated in rats fed the high-fat diet. Saturated fatty acids, but not unsaturated fatty acids were reported to induce the expression of COX-2 in macrophages, suggesting that the saturated fatty acids in the high-fat diet might be the dominant contributors to the increased inflammatory profile. The authors additionally reported that saturated fatty acids induced COX-2 expression through the activation of NF-κB. In vitro studies have suggested that saturated fatty acids may promote inflammatory processes; for example, exposure of myotubes or adipocytes to the saturated fatty acid palmitic acid increased IL-6 mRNA expression and subsequent protein production, possibly via activation of NF-κB [127, 128]. Monocytes are directly activated by saturated fatty acids, in particular lauric acid, via toll-like receptor 4 (TLR-4) and through this mechanism induce NF-κB activity [129, 130]. Fernandez-Real et al. [131] saw no association between serum saturated fatty acid level and CRP or IL-6 levels in lean individuals; however, in overweight individuals, serum saturated fatty acid level was positively associated with IL-6 concentration, and the ratio of saturated fatty acid to omega-6 polyunsaturated fatty acid (PUFA) oromega-3 PUFA was positively associated with IL-6 and CRP concentrations, suggesting that decreasing saturated fatty acid status while increasing omega-6 PUFA status might reduce low-grade inflammation. In a double-blind randomized, controlled trial of 30 healthy middle-aged men (and 30 controls), participants consuming a monounsaturated fatty acid (MUFA) diet for two months had a significant decrease in the expression of ICAM-1 by peripheral blood mononuclear cells. ICAM-1 is thought to play an important role in the recruitment of mononuclear cells to atherosclerotic plaques [132]. Among 4,900 American adults who participated in the 1999 to 2000 National Health and Nutrition Examination Survey, saturated fat consumption was shown to be modestly associated with elevated CRP levels, after controlling for a range of variables [133]. In another study investigating the effects of dietary fat on postprandial (normal metabolic condition throughout the day) expression of pro-inflammatory genes in peripheral blood mononuclear cells, 20 healthy men followed three diets for four weeks each in a randomized, cross-over design. The diets were a western diet (38% fat, 22% of which was saturated fat), Mediterranean diet (38% fat, 24% of which was MUFA), and a carbohydrate rich and omega-3 diet (less than 30% fat, 8% of which was PUFA). After a 12-h fast, participants were given a breakfast with a fat composition similar to that consumed in each of the diets; a butter breakfast, an olive oil breakfast, and a walnut breakfast. The butter breakfast resulted in increased TNF-α mRNA expression compared to the olive oil or walnut breakfast. There was a higher postprandial response in the mRNA of IL-6 with the butter and olive oil breakfasts, however, the plasma concentrations of these pro-inflammatory markers showed no significant differences. The authors reported that these three diets did not activate NF-κB or affect plasma or expression levels of MCP-1 [134].

Eicosapentaenoic acid and docosahexaenoic acid and markers of inflammation

EPA and DHA, long chain omega-3 PUFAs, are found in seafood, especially oily fish. Consumption of EPA and DHA has been shown to decrease concentrations of CRP, sTNFR-1, sTNFR-2, sICAM-1, sVCAM-1, and sELAM-1. Numerous experimental and observational studies in humans have found an inverse association between dietary consumption of omega-3 PUFA and systemic markers of inflammation [135]. For example in the Multi-Ethnic Study of Atherosclerosis, omega-3 PUFA intake was inversely associated with plasma concentrations of IL-6 and the inflammatory marker matrix metalloproteinase (MMP) 3 [136]. In young Japanese women, plasma hs-CRP concentration was negatively associated with omega-3 PUFA intake, only when omega-3 PUFA intake was greater than 1.1% of dietary calories [137]. In an in vitro study, RAW 264.7 cells (a mouse leukemia monocyte macrophage cell line), grown in EPA-rich media for 24–48 h before being exposed to LPS for 2 h, exhibited decreased TNF-α mRNA and NF-κB activity, compared to cells without EPA in their media. Moreover, the cells grown in the EPA-rich media also demonstrated altered composition of dimers of the NF-κB subunits P65/P50 [138]. Novak et al. [139] demonstrated that omega-3 fatty acid inhibition of macrophage TNF-α following LPS stimulation is mediated, in part, through inactivation of the NF-κB signal transduction pathway secondary to inhibition of inhibitor of kappa B (IκB) phosphorylation. The authors utilized RAW 264.7 cells pre-treated with isocaloric emulsions of omega-3 fatty acids, prior to LPS exposure. The omega-3 pre-treated cells inhibited IκB phosphorylation and significantly decreased NF-κB activity, while additionally demonstrating significant decreases in both TNF-α mRNA and protein expression.

There have been several supplementation studies of EPA and DHA conducted in both humans and animals which have shown reduced concentration of several inflammatory markers including Il-6, IL-18, TNF-α, CRP, sICAM-1, sVCAM-1, and sELAM-1 [140–151]. Feeding fish oil to mice decreased production of TNF-α, IL-1β, and IL-6 by endotoxin-stimulated macrophages [28, 153], while several human studies have demonstrated decreased production of TNF-α, IL-1β, and IL-6 by endotoxin-stimulated monocytes following dietary supplementation with fish oil [30, 154–156]. In 1990, blood samples were taken from 727 healthy women (aged 43 –68 y) in the Nurses’ Health Study from whom dietary data had also been collected between 1986 and 1990. CRP, IL-6, ELAM-1,sICAM-1, and sVCAM-1 levels were shown to be inversely related to total fatty acid consumption. Specifically, the long chain fatty acids EPA and DHA were inversely related to sICAM-1 and sVCAM-1 levels. α-linolenic acid was also inversely related to plasma concentrations of CRP, IL-6 and ELAM-1; all associations were observed after controlling for age, BMI, physical activity level, smoking status, and alcohol consumption [157]. Flaxseed oil (one of the most concentrated plant sources of omega-3 fats) incorporated into the diet of 28 healthy participants for four weeks resulted in inhibition of TNF-α and IL-1β production by approximately 30% . When fish oil supplements totaling 9 g/d were subsequently consumed for another four weeks, levels of these cytokines were inhibited by 74% and 80% respectively. A significant inverse exponential relationship between TNF-α and IL-1β synthesis and the mononuclear cell content of EPA was also observed. TNF-α and IL-1β decreased as mononuclear cellular EPA content increased to approximately 1% , after which further increases in EPA content did not result in a further decrease in cytokine production [154]. Sadeghi et al. [158] fed mice for five weeks on a low-fat diet or one of four high-fat diets (containing 20% by weight of coconut oil (CO), olive oil (OO), safflower oil (SO), or fish oil (FO)). At the end of the feeding study, the mice were injected with LPS before sacrifice either 90 or 180 min later. Peak plasma TNF-α, IL-1β, and IL-6 concentrations were lower in the CO- and FO-fed mice than in the SO-fed mice, while peak plasma IL-10 concentrations were highest in the CO-fed mice. Relative to the omega-6 PUFA-rich SO diet, the CO and FO diets diminished production of pro-inflammatory cytokines. However, there are a number of human intervention studies that have failed to replicate these positive results with some studies even showing an enhancement of inflammatory markers; for example several studies investigating hyperlipidemia and coronary heart disease [159–165]. The effects of 1 y dietary supplementation with 0, 3, 6, or 9 g of fish oil was investigated in 58 monks with a mean age of 56 y. All doses had no effect on circulating cytokine concentrations relative to placebo [165]. Kew et al. found supplementation with EPA or DHA for four weeks had no significant effect on cytokine production in 42 healthy subjects [163], and the authors came to the same conclusion in 150 healthy men and women who had ALA, or EPA and DHA supplementation at two concentrations for six months [164]. The lack of consistency in results may be due to differences in duration of treatment between studies, varying sample-sizes, heterogeneous study populations (age, health, smoking status, etc.), participants’ background diet, dose of EPA and DHA supplement administered, different chemical formulation of EPA and DHA supplements used, and/or genetic differences between individuals which may impact the ability of EPA and DHA to have an anti-inflammatory effect [166, 167]. For example, the effect of dietary fish oil on cytokine production by human mononuclear cells has been shown to be dependent on the nature of the -308 TNF-α and the +252 TNF-β polymorphisms [167].

Enrichment of EPA and DHA in the diet competitively inhibits the oxygenation of AA by COX, thus suppressing the production of pro-inflammatory eicosanoids and cytokines. The metabolism of EPA and DHA produces 3-series prostaglandins and thromboxanes, and 5-series leukotrienes as opposed to 3-series prostaglandins and thromboxanes, and 4-series leukotrienes produced via metabolism of AA; these metabolites have different biological properties. The metabolism of EPA and DHA also produces resolvins and neuroprotectins that downregulate expression of cytokines. Resolvins and neuroprotectins inhibit both IL-1β mediated NF-κB activation and COX activation [168]. Increased generation of 5-series leukotrienes from macrophages in fish oil-fed mice [27], and from neutrophils in humans taking fish oil supplements for several weeks has been observed [29–31]. Resolvin synthesis is increased by feeding fish oil-rich diets to laboratory animals [169]. In addition, animal studies have shown that production of AA-derived eicosanoids like prostaglandin E₂ (PGE₂) is decreased by EPA or DHA feeding [27, 170], and in humans there has been decreased production of PGE₂ and the 4-series leukotrienes following use of fish oil supplements for a period of weeks to months [29–31].

Mechanisms underlying the anti-inflammatory actions of EPA and DHA include altered cell membrane phospholipid fatty acid composition, disruption of lipid rafts, inhibition of activation of the pro-inflammatory transcription factor NF-κB thereby reducing expression of inflammatory genes, activation of the anti-inflammatory PPAR-γ, and binding to the G protein coupled receptor 120 [171]. A review by Farooqui et al. [168] concluded that EPA and DHA decrease the activation of NF-κB, possibly by decreasing phosphorylation of IκB, which is required to allow NF-κB to dissociate from IκB and translocate to the nucleus (see Fig. 1 for pathway). Following this translocation, NF-κB binds to cis-acting κB element of COX-2, controlling the expression of pro-inflammatory genes including COX-2, ICAM-1, VCAM-1, ELAM-1, TNF-α, IL-1β, IL-6, iNOS, and MMP.

Linoleic and alpha-linolenic acids and markers of inflammation

Linoleic and alpha-linolenic (α-linolenic) acids are the parent omega-6 and omega-3 PUFAs respectively, and constitute over 95% of the PUFA in most “western diets.” Linoleic acid is the precursor of AA, which is the substrate for the synthesis of pro-inflammatory eicosanoids including PGE₂ and 4-seriesleukotrienes [83]. In a large population-based Swedish study of 767 men followed for 20 y, the concentrations of linoleic and α-linolenic acid in blood lipids [172, 173] and granulocytes [174] were not associated with CRP or Il-6 level, although linoleic concentration in cholesterol esters was negatively associated with CRP concentration [175]. In an Italian community-based study of 1123 people aged 20–98 y of age (55% women), alpha-linolenic concentration in plasma fatty acids was not associated with a range of cytokines including IL-6 and TNF-α, however, was inversely associated with CRP level [173]. Dietary intakes of linoleic and α-linolenic acid were not associated with CRP, IL-6, sTNFR-1 or sTNFR-2 concentrations in the Physicians’ Health Study and the Nurses’ Health Study [176]. However, within a subgroup of the Nurses’ Health Study, α-linolenic acid intake was associated with lower IL-6 and sVCAM-1 concentrations, but not with CRP, sICAM-1, sELAM-1, or sTNFR-2 concentrations [157]. These findings suggest a modest anti-inflammatory effect of linoleic acid and α-linolenic acid. Conjugated linoleic acid, naturally found in milk fat, has been shown to increase lipid peroxidation but not affect risk markers of CVD, inflammation (including CRP), fasting insulin, or glucose concentrations. This analysis was conducted in 38 healthy young males fed either a diet containing 115 g/d of conjugated linoleic acid-rich fat, or a control diet with a low conjugated linoleic acid content, as part of a five-week double-blind, randomized, parallel intervention study [177].

Trans-fatty acid and markers of inflammation

Trans-fatty acids are unsaturated fats with at least one double bond in the trans configuration which is formed during the industrial hydrogenation of vegetable oils for food manufacturing. Mozaffarian et al. [32] found trans-fatty acid intake was positively associated with concentrations of sTNFR-1 and sTNFR-2 in 823 generally healthy women (aged 32 - 70 y) in the Nurses’ Health Study. sTNFR-1 and sTNFR-2 concentrations were 10% and 12% higher respectively, in the highest intake quintile compared to the lowest intake quintile. Trans-fatty acid intake was not associated with IL-6 or CRP concentrations overall; however, it was positively associated in women with a higher BMI. In a separate publication also reporting data from the Nurses’ Health Study, levels of CRP were 73% higher, IL-6 levels were 17% higher, sICAM-1 levels were 10% higher, sVCAM-1 levels were 10% higher, ELAM-1 levels were 20% higher, and sTNFR-2 levels were 5% higher in those individuals within the highest quintile of trans-fatty acid intake compared with the lowest quintile. Trans-fatty acid intake was also positively associated with plasma concentrations of CRP, ELAM-1, sICAM-1, sVCAM-1, and sTNFR-2 in linear regression models after controlling for a range of variables [178]. Furthermore, a five-week intervention in healthy men consuming a trans-fatty acid-enriched diet resulted in higher CRP and IL-6 concentrations compared to diets rich in oleic acid, stearic acid or a combination of lauric, myristic, and palmitic acids [179].

Dietary fats, AD, and related chronic diseases

Higher intakes of saturated and trans fat since midlife, and lower PUFA to saturated fat ratio have been associated with increased cognitive decline in 1486 females age 70 y and older, with type-2 diabetes [180]. In 5386 participants (age 55 y and older) without dementia at baseline, total fat, saturated fat, and cholesterol were positively associated with an increased risk of dementia, over an average of 2.1 y of follow-up [181]. Morris et al. [182] related fat consumption to change in cognitive function over 6 y in 2560 participants aged 65 y and older, with no history of heart attack, stroke, or diabetes at baseline. Higher intakes of saturated fat and trans fat were linearly associated with greater decline in cognitive score. Higher intake of monounsaturated fat, and a higher ratio of PUFA to saturated fat were inversely associated with cognitive decline, only however when people who had changed their fat intake in recent years or whose baseline cognitive scores were in the lowest 15% were excluded. Intakes of total fat, vegetable and animal fats, and cholesterol were not found to be associated with cognitive change [182]. Clinical evaluations were performed on a random sample of 815 community residents age 65 y and older, who were unaffected by AD at baseline, and who completed a food frequency questionnaire a mean of 2.3 y before baseline clinical evaluation. After a mean of 3.9 y, 131 of these participants developed AD. Intakes of saturated, trans and vegetable fat were positively associated with risk of AD, and intakes of omega-6 PUFA and MUFA were inversely associated. Intakes of total fat, animal fat and dietary cholesterol were not associated with AD incidence [183]. A cross-sectional population-based study of 1613 participants ranging from 45 to 70 y, assessed the association of fatty acid and fish intake with cognitive function. Participants completed an extensive cognitive battery and compound scores were constructed for memory, psychomotor speed, cognitive flexibility and overall cognition. EPA and DHA intake were inversely related to risk of impaired overall cognitive function and speed; results for fatty fish consumption were similarly inverse. By contrast, dietary cholesterol intake was observed to be significantly positively associated with increased risk of impaired memory and flexibility. Per standard deviation increase in saturated fat intake, the risk of impaired memory, speed and flexibility was also increased, although not significantly [184]. Numerous other studies have also shown a positive association between fish and omega-3 PUFA consumption and reduced risk for dementia or slower cognitive decline [181, 186]; for example, participants who consumed fish “once per week,” or more, demonstrated 60% less risk of AD compared with those who “rarely” or “never” ate fish [185]. Pistell et al. [187] showed that while consumption of different fats in the diet can increase body weight, the ability of high-fat diets to disrupt cognition is linked to brain inflammation. The authors used mice fed either a high-fat western diet (with 41% calories from fat) or a very-high-fat lard diet (with 60% calories from fat). Western diet consumption resulted in significantly increased body weight and astrocyte reactivity, with unchanged cognition, microglial reactivity, and cytokine levels, while the very-high-fat lard diet increased body weight and reactive astrocytosis, as well as impairing cognition and increasing brain inflammation (by significantly increasing expression of the cytokines TNF-α, IL-6, and the chemokine MCP-1).

Tanasescu et al. [188] examined the relationship between types of dietary fat and cholesterol and risk of CVD among women with type-2 diabetes in the Nurses’ Health Study. The authors found a higher intake of cholesterol and saturated fat, and a low PUFA to saturated fat ratio, were related to increased CVD risk. Tsitouras et al. [189] investigated whether a high omega-3 PUFA diet would improve endocrine function in six males and six females aged over 60 y. Participants ate an isocaloric control diet for six weeks before eight weeks of a diet including 720 g of fatty fish a week and 15 ml of sardine oil a day. Insulin sensitivity increased significantly after eight weeks on the PUFA diet, and serum CRP levels were significantly reduced with a trend towards lower IL-6 levels also observed. No differences were found in other metabolic parameters, adiponectin levels, or hormone responses. To investigate how PUFA affects white adipose tissue inflammation and gene expression in obese diabetic animals, diabetic mice were treated with either a low-fat standard diet (LF), or high-fat diets rich in: saturated and MUFA (HF/S); omega-6 PUFA (HF/6); or omega-6 PUFA and omega-3 PUFA (HF/3). Many genes involved in inflammatory alterations were upregulated in the mice on the HF/S diet compared with the LF diet, with phosphorylation of c-Jun N-terminal kinase (JNK) also increased within the HF/S group. Adipose tissue infiltration with macrophages was markedly enhanced by the HF/S diet. Compared with HF/S, the HF/6 diet showed marginal effects on adipose tissue inflammation; however, inclusion of omega-3 (HF/3) completely prevented macrophage infiltration and upregulation of the inflammatory gene expression, as well as reducing JNK phosphorylation, despite no change in the body weight of the mice. To summarize, omega-3 PUFA prevented adipose tissue inflammation induced by high-fat diets in obese diabetic mice, suggesting beneficial effects of omega-3 PUFA on diabetes development could be mediated by their effect on adipose tissue inflammation [147]. The concentration of plasma hs-CRP, IL-6, and TNF-α were compared in 48 obese individuals and 10 lean normolipidemic men. The obese individuals were then assigned to six weeks of treatment with either atorvastatin (40 mg/day; a statin), fish oil (4 g/day), atorvastatin and fish oil, or placebo. Compared with controls, obese individuals had increased hs-CRP and IL-6 levels, and similar TNF-α levels. Atorvastatin treatment (either with or without fish oil) significantly decreased plasma hs-CRPand IL-6 levels, an effect which was not seen in the group treated with fish oil alone. TNF-α levels were not affected by any of the treatments. The reductions in hs-CRP with atorvastatin were not significantly correlated with changes in plasma lipids, insulin resistance, or cholesterogenesis. This study shows that visceral obesity is associated with increased plasma hs-CRP and IL-6, and therefore a low-grade chronic inflammatory state which treatment with atorvastatin, or atorvastatin and fish oil, reversed, but not fish oil alone [190].

Browning et al. [191] investigated the impact of omega-3 PUFA on CVD risk in high and low inflammatory status groups utilizing a controlled dietary intervention study conducted on 32 female participants. Participants were between the ages of 26 and 51 y, had a BMI ranging from 24–44 kg/m² (overweight or obese), and all were non-diabetic. Serum sialic acid levels were used to divide participants into high and low inflammatory status. Participants consumed capsules daily which provided either omega-3 PUFA (1.3 g EPA and 2.9 g DHA) or placebo in a randomized cross-over design for a period of 12 weeks for each treatment with a four-week washout period between treatments. At baseline, the higher inflammatory status group had higher BMI, and area under the insulin curve (calculated from an oral glucose tolerance test, to provide a dynamic measure of insulin sensitivity), but no significant difference in fasting insulin, glucose, or area under the glucose curve (index of whole body, insulin-mediated glucose disposal); supporting previous evidence of the association between inflammatory status and obesity-related disease. PUFA supplementation significantly improved area under the insulin curve in the higher inflammatory status group, with no change in the placebo group, and no change in any other markers observed. There were no changes in the low inflammatory status group regardless of whether they were taking placebo or the PUFA supplement. This study suggests that overweight individuals with raised inflammatory levels derive significant benefits from omega-3 PUFA supplementation. Browning et al. then went on to hypothesize the role of inflammation in obesity-related diseases and the inverse role PUFA likely plays in this pathway (shown in Fig. 3).