Dietary supplementation with nuts has shown a variety of health benefits. Among the tree nuts, walnuts (Juglans regia
) contain the highest levels of the omega-3 fatty acid, namely alpha-linoleic acid (ALA), with the most favorable ratio of omega-3:omega-6 fatty acids (1:4.2) [1
]. Walnuts also contain a large number of phytochemicals, including phenolic antioxidants, and high levels of nutrients with beneficial properties to guard against a variety of diseases, including heart disease, diabetes, neurological disorders, inflammation and cancer [1
]. Moreover, walnuts are a rich source of fiber (up to 6.4%), which has been shown to support the maintenance of disease remission in patients suffering from inflammatory bowel disease (IBD) [4
IBD, encompassing both Crohn’s disease (CD) and ulcerative colitis (UC), are chronic intestinal inflammatory disorders of largely unknown etiology [5
]. IBD is typically characterized by alternating periods of clinical remission and disease flare-ups [6
]. As recently reviewed [7
], diet clearly plays an important role in IBD progression. A variety of dietary strategies have been proposed for IBD patients, many of them considered ’fad’ diets and often not sufficiently controlled for affording clinical efficacy [8
]. Furthermore, many of these restricted diets that are given during remission, if not properly maintained, may ultimately contribute to nutritional deficiencies in carbohydrates, monounsaturated fatty acid (MUFAs), fiber, calcium and vitamins C, D, E and K [9
]. However, epidemiological data have demonstrated that a high intake of total fats, polyunsaturated fatty acids (PUFAs) and red meat are associated with increased risk of developing active CD and UC [10
]. Alternatively, diets enriched in fiber and plant-based foods, including fruits, vegetables and nuts, have shown a preventive benefit during remission [7
The following study design is based upon the reported anti-inflammatory properties of walnuts [11
]. Using a preclinical mouse model of UC, we have examined the potential health benefits of whole walnuts added to a Total Western Diet (TWD) [14
] on the extent of intestinal injury following exposure of mice to the ulcerogenic agent, DSS. We have examined how walnut consumption, starting prior to DSS exposure and continuing throughout the entire disease course, may influence the extent of intestinal inflammation during both the acute and recovery phases of the experimentally-induced disease. To gain further insight into how walnut consumption may ’condition’ the colonic mucosa towards a protective state, we have conducted a discovery-based metabolomic profiling analysis on both fecal samples and colonic mucosa obtained from mice maintained for two weeks on a walnut-supplemented diet. This analysis has identified significant increases in a number of lumenal and tissue metabolites, several of which may contribute to the observed protection from ulcerogenic injury observed in this study.
Walnuts contain a complex array of natural compounds and phytochemicals with a broad range of health benefits, including protection against some forms of cancer and inflammation [1
]. In this study, we demonstrate for the first time that dietary supplementation with walnuts protects the colonic epithelium against mucosal injury induced by the ulcerogenic agent, DSS. While even the lowest concentration of walnuts (3.5%) present in the diet showed a moderate reduction in the extent of colonic ulceration, the protection by walnuts was most pronounced during the recovery phase. As shown in Figure 2
h, at ten days after DSS treatment, mice consuming a diet containing 14% walnuts for four weeks showed almost no evidence of remaining colonic ulceration. Given the likelihood that adjacent undamaged epithelial cells are critical for initiating the wound-healing process [29
], we believe that walnut-fed mice have sustained less initial mucosal damage. As a consequence of the attenuated destruction of the colonic mucosa, a more rapid and robust recovery from the initial damage is possible. Based upon these observations, we believe that walnut supplementation given prior to DSS treatment may somehow adapt the colonic mucosa towards an inflammation-suppressive microenvironment that affords significant protection against DSS-induced inflammatory insult.
The anti-inflammatory properties of whole walnuts and walnut extracts have been demonstrated in several preclinical inflammatory disease models. For example, dietary supplementation with whole walnuts resulted in a significant decrease in macrophage infiltration and suppression of pro-inflammatory gene expression (tumor necrosis factor; TNF-α, interleukin-6; IL-6 and IL-10) in a mouse model of high-fat diet (HFD)-induced fatty liver disease [12
]. Similarly, lung injury induced by bleomycin (BLM) was significantly reduced with dietary walnuts, accompanied by a decline in tissue biomarkers of oxidative stress and the alveolar macrophage inflammatory response [30
]. More recently, Koh et al. [31
] have shown that treatment with a walnut phenolic extract (WPE) attenuated DSS-induced acute colitis, while inhibiting the production of several key pro-inflammatory cytokines, including IL-8 and IL-1α. In addition, WPE inhibited NF-κB DNA binding activity. Our results are consistent with these earlier studies indicating that walnuts can exert a positive effect on lipid metabolism. As a consequence, walnuts enhance antioxidant activity in the tissue, which will ultimately contribute to the observed suppression against mucosal ulceration.
The beneficial effects of walnuts may be attributed in part to the actions of the omega-3 fatty acids. It is well-established that the primary omega-3 fatty acid found in walnuts, alpha-linoleic acid (ALA), and its metabolites, EPA and docosahexaenoic acid (DHA), possess potent anti-inflammatory properties [32
]. In fact, in the present study we found a significant increase in the levels of DHA in fecal samples collected from mice ingesting walnuts (Table 1
). Among its many actions, DHA provides an important substrate for the pro-resolving mediators, (SPMs), including the resolvins, protectins and maresins, that are synthesized in the tissue during the acute phase of the inflammatory response [33
]. These bioactive lipids facilitate the clearing of inflammatory cells and mediators, thereby enhancing resolution of tissue injury [33
]. Although this class of bioactive lipid was not identified at significant levels within the tissue, it is possible that biologically relevant changes to these important lipid mediators have occurred at levels that are below our ability to detect. Thus, future studies with more sensitive analytical capabilities are warranted to evaluate these possibilities.
In addition to the omega-3 metabolites, our data suggest that omega-6 fatty acids present in walnuts may also contribute to protection. In fact, our study is the first to show a significant increase in the levels of the omega-6 metabolite, 9-oxoODA, upon walnut supplementation (Table 1
). 9-oxoODA is abundantly present in tomatoes, a food that is known to improve overall lipid metabolism [34
]. It has also been shown that 9-oxoODA under some conditions can increase PPARα expression in mouse primary hepatocytes, which in turn causes a decrease in triglyceride accumulation [20
]. PPARα is a potent metabolic regulator that can directly modulate inflammatory signaling [35
]. In fact, one of the most important roles of PPARα is its ability to inhibit NF-κB activity [36
], a potential mechanism associated with the property of walnuts extract as an anti-inflammatory agent [31
Another important property of PPARα is its ability to transcriptionally regulate the expression of a panel of genes involved in fatty acid metabolism, including carnitine-palmitoyl transferase-1 (CPT1) and acyl-CoA synthase (ACS) [37
]. Interestingly, we found that several key metabolites related to fatty acid β-oxidation were increased within the colonic mucosa, including SAH and betaine (Table 2
). Fatty acid β-oxidation is a multistep enzymatic process responsible for the catabolism of fatty acids, generating cellular energy in the form of ATP [38
]. As reviewed by Rinaldo et al. [39
], disorders of fatty-acid metabolism caused by loss of function mutations to key pathway components, such as the fatty acid transporter (organic cation transporters novels; OCTNs and carnitine-acylcarnitine translocases; CACTs) and the acyl-coenzyme A dehydrogenases (very long-chain acyl-CoA dehydrogenases; VLCADs and short-chain acyl-CoA dehydrogenases SCADs), result in pathological conditions, including liver failure and cardiac and skeletal myopathy. In the intestines, mutations in the carnitine transporter genes, OCTN1 and OCTN2, have been shown to be associated with Crohn’s disease and mice deficient in OCTN2 have been reported to develop spontaneous colonic atrophy and inflammatory changes [40
]. Moreover, a four-week treatment with propionyl-L-carnitine (PLC), an ester of L-carnitine required for the mitochondrial transport of fatty acids, restores endothelial β-oxidation and function, as well as reducing intestinal mucosal inflammation in patients with mild-to-moderate UC [41
]. In fact, omega-3 fatty acids have been shown to induce fatty acid β-oxidation, causing reduced levels of triglycerides, providing a clinical treatment option for hypertriglyceridemia [42
]. There is also a study by Shimoda et al. [43
], which showed that a polyphenol-rich extract from walnuts exhibited hypotriglyceridemic effects by enhancing peroxisomal fatty acid β-oxidation in the liver. Based on our data and others, we speculate that regular intake of walnuts increases the levels of DHA and 9-oxoODA, thereby enhancing lipid metabolism, an effect that may prevent lipid-mediated inflammation associated with IBD.
The significant increase in the tryptophan metabolite, kynurenic acid, may have important implications regarding the protection to the colon afforded by walnuts. In a recent comprehensive review by Wirthgen et al. [21
], the remarkably broad scope of biological activity exerted by kynurenic acid is discussed. As a product of the kynurenine pathway of tryptophan metabolism, kynurenic acid exerts potent anti-inflammatory and immunosuppressive properties via its ability to act as a ligand of G protein-coupled receptor 35 (GPR35) and the aryl hydrocarbon receptor (AhR) [21
]. We speculate that the immunomodulatory properties of kynurenic acid may contribute, in part, to the resolution of inflammation observed in the present study.
The conditioning of the colon afforded by walnut supplementation may also be associated with the ellagitannins, a group of complex phytochemicals commonly found in walnuts and other natural products that give rise within the gut to ellagic acid and the urolithins, a potent family of antioxidants [1
]. In fact, it has been shown that ellagic acid exhibits more potent radical scavenging and metal chelating activities than other well-known antioxidant compounds, including the tocopherols and ascorbic acid [44
]. In addition, several studies have shown that administration of ellagic acids or pomegranate polyphenolics significantly attenuated DSS-induced colitis [45
]. Furthermore, free ellagic acid is metabolized by the gut microbiome to the urolithins [48
], a large group of molecules that have been extensively studied for their antioxidant, anti-inflammatory and anti-cancer activities [49
]. In fact, a recent study by Singh et al. [53
] showed that urolithin A and its potent synthetic analogue protected from 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced or DSS-induced colitis through enhancement of gut barrier function by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. In the present study, we found a modest, although non-significant trend towards increased levels of urolithin A in the colons of mice treated with 14% walnuts (C13H8O4; 1.77-fold; p
= 0.630; data not shown). Based upon these results, we believe that further targeted analysis of ellagic acid-derived metabolites may be warranted in order to better define the potential role of the urolithins in the maintenance of colon health, particularly with respect to ulcerative colitis.