Cashew (Anacardium occidentale L.) Nuts Modulate the Nrf2 and NLRP3 Pathways in Pancreas and Lung after Induction of Acute Pancreatitis by Cerulein

Background: One of the most common co-morbidities, that often leads to death, associated with acute pancreatitis (AP) is represented by acute lung injury (ALI). While many aspects of AP-induced lung inflammation have been investigated, the involvement of specific pathways, such as those centered on nuclear factor E2-related factor 2 (Nrf2) and nucleotide-binding domain leucine-rich repeat (NLR) and pyrin domain containing receptor 3 (NLRP3), has not been fully elucidated. Methods: To investigate the effect of cashew (Anacardium occidentale L.) nuts on pancreatic and lung injury induced by cerulein injection, cerulein (50 μg/kg) was administered to CD1 mice for 10 h. Oral treatment with cashew nuts at a dose of 100 mg/kg was given 30 min and 2 h after the first cerulein injection. One hour after the final cerulein injection, mice were euthanized and blood, lung and pancreatic tissue samples were collected. Results: Cashew nuts were able to (1) reduce histological damage; (2) mitigate the induction of mast cell degranulation as well as the activity of myeloperoxidase and malondialdehyde; (3) decrease the activity levels of amylase and lipase as well as the levels of pro-inflammatory cytokines; and (4) enhance the activation of the Nrf2 pathway and suppress the activation of the NLRP3 pathway in response to cerulein in both pancreas and lung. Conclusions: Cashew nuts could have a beneficial effect not only on pancreatitis but also on lung injury induced by cerulein.


Introduction
Acute pancreatitis (AP) is a multifactorial disease, with a mortality rate that can be as high as 15-20%; it initiates in the pancreas in response to an inflammatory event and leads to deleterious local and systemic effects [1,2].

Experimental Protocol
AP was induced by cerulein hyperstimulation through 10 intraperitoneal (i.p.) injections (one injection every hour for 10 h at a dose of 50 µg/kg). Animals were euthanized one hour after the last injection, and samples of blood, lung, and pancreatic tissue were collected for further study [39].

Experimental Groups
Mice were randomly distributed into the following groups: (1) Sham: Animals were subjected to injections of saline and were treated by oral gavage with saline.
(2) Sham + cashew nuts (100 mg/kg): Animals were subjected to injections of saline and were treated by oral gavage with cashew nuts at the dose of 100 mg/kg (data not shown because there were no differences between the sham+saline and sham+cashew nuts groups.). (3) Cerulein: Mice were subjected to cerulein injections as described above and treated by oral gavage with saline. (4) Cashew nuts (100 mg/kg): Mice were subjected to cerulein injections a described above and treated by oral gavage with cashew nuts (100 mg/kg).
The cashew nuts were given 30 min and 2 h after the first cerulein injection [1] (experimental timeline in Supplementary Figure S1). The dose used was chosen based on previous studies [31,32,36].

Pancreatic and Lung Oedema
Pancreatic and lung oedema was quantified as previously described by calculating the ratio between the water content of the tissue and its dry weight [40,41].

Histological Evaluation and Detection of Mast Cells
At the end of experiments, pancreas and lung tissues were fixed in 10% (w/v) PBS-buffered formaldehyde at room temperature. Seven micrometer sections were prepared from paraffin embedded tissues and stained with hematoxylin and eosin (H&E) for histological evaluation and with toluidine blue for detection of mast cells. After staining, they were evaluated using a Leica DM6 microscope (Leica Microsystems SpA, Milan, Italy) with Leica LAS X Navigator software (Leica Microsystems SpA). The injury score for both pancreas and lung was calculated as previously described [40,42].

Evaluation of Myeloperoxidas and Malonaldehyde
Myeloperoxidase (MPO) and malonaldehyde (MDA) levels were assessed as previously described in both pancreas and lung tissue. Briefly, after homogenization in respective specific buffers, absorbance was measured at 650 nm, using a spectrophotometer. Levels were expressed in milli-units per 100 milligram (mU/100 mg) of tissue [44][45][46].

Cashew Nuts Nutritional Composition
The cashew kernel samples (Anacardium occidentale L.) used were obtained from Ivory Coast; per 100 g they contained 5.40 g moisture, 22.46 g protein, 44.19 g total lipids, 4.48 g total dietary fibre, 30.95 g total sugars, 2.68 g ash, and 80.01 mg total phenols. The nutritional composition was analyzed according to the Association of Official Analytical Chemists (AOAC) Official Method as previously reported [50][51][52][53].

Reagents
All other materials were purchased from Sigma-Aldrich Co. Stock solutions were prepared in nonpyrogenic saline (0.9% NaCl, Baxter Healthcare Ltd., Thetford, Norfolk, UK).

Data Analysis
All values are expressed as mean ± standard error of the mean (SEM). For in vivo experiments, each group comprised 6 animals. For experiments involving histology, images shown are representative at least 3 independent experiments on tissue sections collected from all animals in each group. The results were analyzed by one-way ANOVA followed by a Bonferroni post-hoc test for multiple comparisons. A p value < 0.05 was considered significant. # p < 0.05 vs. cerulein; ## p < 0.01 vs. cerulein; ### p < 0.001 vs. cerulein; * p < 0.05 vs. sham; ** p < 0.01 vs. sham; *** p < 0.001 vs. sham.

Effect of Cashew Nuts on Cerulein-Induced Oedema and Tissue Damage
Histological analysis of the pancreas of cerulein-treated mice showed tissue damage characterized by interstitial edema and inflammatory cell infiltrates ( Figure 1B,D). These inflammatory signs were significantly reduced in the group of mice orally administered 100 mg/kg cashew nuts ( Figure 1C,D). The histological analysis of the lung yielded similar findings. Lung injury during AP was characterized by alveolar thickening and abundance of inflammatory cell infiltrates ( Figure 1F,H). Lung inflammation was significantly reduced by administration of cashew nuts ( Figure 1G,H). Cerulein-induced AP is accompanied by tissue oedema in both the pancreas and the lung, which was quantified by determining the water content of the tissue. The oedema was significantly decreased after cashew nuts treatment in both pancreas ( Figure 1I) and lung ( Figure 1J).

Effects of Cashew Nuts on Cerulein-Induced Mast Cell Degranulation and on Myeloperoxidase and Malondialdehyde Activity
Mast cells are well known to play a significant role under inflammatory conditions, and there are remarkable overlaps between factors that cause mast cell degranulation and the progression of AP. We therefore evaluated whether administration of cashew nuts could have a beneficial effect on mast cell degranulation during cerulein-induced AP, as assessed by toluidine blue staining. In both pancreas ( Figure 2B) and lung ( Figure 2F), a significant increase in mast cell degranulation was observed after cerulein injection as compared to the sham group (Figure 2A,E). Treatment with cashew nuts significantly decreased the number of degranulated mast cells in pancreas ( Figure 2C) and lung ( Figure 2G). Moreover, oral treatment with cashew nuts significantly mitigated the cerulein-induced increase in the activity of malondialdehyde (MDA, a marker of lipid peroxidation) and myeloperoxidase (MPO, a marker of neutrophilic infiltration) in pancreas ( Figure 2D,I) and lung tissue ( Figure 2H,J).

Effects of Cashew Nuts on the Levels of Amylase, Lipase, and Pro-Inflammatory Cytokines
Administration of cerulein is well-known to cause an increase in the serum levels of amylase and lipase, as well as to promote the release of different pro-inflammatory cytokines into the blood. Indeed, in cerulein-induced AP, a significant increase in serum levels of amylase ( Figure 3A), lipase ( Figure 3B), IL-1β ( Figure 3C), IL-6 ( Figure 3D), and TNF-α ( Figure 3E) was observed compared to the sham group; administration of cashew nuts significantly ameliorated all the above inflammatory markers.

Effects of Cashew Nuts on the Nrf2 Pathway in Cerulein-Induced AP
Considering the key role of oxidative stress during AP, we investigated the effect of cashew nuts on the Nrf2 pathway in pancreas and lung by Western blotting. Administration of cashew nuts following cerulein injection significantly increased the nuclear protein abundance of Nrf2 in both pancreas ( Figure 4C,C1) and lung ( Figure 4H,H1). The gene encoding Mn-SOD is known to be upregulated by Nrf2, and cashew nuts significantly increased the protein abundance of Mn-SOD in both pancreas ( Figure 4B,B1) and lung ( Figure 4G,G1). The same pattern was observed for HO-1 in both pancreas ( Figure 4A,A1) and lung ( Figure 4F,F1).  These results were further confirmed by immunohistochemical staining for Nrf2 in pancreas ( Figure 5A-C,M) and lung ( Figure 5D-F,O), as well as for HO-1 in pancreas ( Figure 5G-I,N) and lung ( Figure 5J-L,P).

Effects of Cashew Nuts on the NLRP3 Pathway in Cerulein-Induced AP
Finally, we evaluated whether cashew nuts could reduce inflammasome activation in cerulean-induced AP. Analysis of inflammasome components by Western blotting showed that the protein abundance of NLRP3 increased significantly in pancreas ( Figure 6A,A1) and lung ( Figure 6E,E1).
The same was observed for the protein abundance of ASC in pancreas ( Figure 6C,C1) and lung ( Figure 6G,G1), as well as for the protein abundance of Caspase-1 in pancreas ( Figure 6B,B1) and lung ( Figure 6F,F1); administration of cashew nuts significantly ameliorated all of the above parameters.

Discussion
AP is a common disease whose severity can vary mild disease to sepsis and multiple organ failure (MOF) [54]. Though AP can affect various distant organs, such as the colon, ALI is considered the most frequent possible complication of AP [41,55]. The relationship between AP and ALI is most probably due to an increase in the number of neutrophils in the lungs that lead to ROS generation with a consequent increase in the production of proinflammatory cytokines [56]. Human studies have indeed demonstrated very high concentrations of IL-1β, IL-6, TNF-α, neutrophil enzymes, and pancreatic enzymes including amylase and lipase in plasma, but the exact pathogenesis of AP-associated ALI remains unclear [56].
Even though there is still no specific drug therapy for AP, with treatment being generally supportive, it has been hypothesized that targeting inflammatory cascade molecules and oxidative stress could be a promising strategy to counteract the development of AP and ALI [57]. Some antioxidants, mainly naturally occurring ones, have been tested as potential beneficial agents in patients with AP. However, results to date have been inconsistent, and there are insufficient clinical data to support their routine use in humans. For example, resveratrol has been shown to be effective in the treatment of AP in rodent models, but clinical studies have not yet been conducted using this compound as an activator of Nrf2 [6]. In contrast, selenium, if given early, has been shown to reduce mortality, complications and need for surgery [58]. Intravenous administration of ascorbic acid (vitamin C) also significantly reduced markers of oxidative stress such as superoxide dismutase and catalase, and led to a faster normalization of the leukocyte count and of amylase levels, as well as to a significant reduction of TNF-α, IL-6, and IL-8 levels [59]. Melatonin has also been shown to be able to neutralize oxygen radicals, activate enzymes involved in the antioxidant response, and suppress the release of pro-inflammatory cytokines [60].
Despite such promising results, the data currently available are not sufficient to support the clinical use of antioxidants for AP. Therefore, further studies are needed to understand the precise mechanisms underlying this serious disease and to optimize its treatment by counteracting both the inflammatory and oxidative processes implicated in its pathogenesis. In this regard, several trials in recent years have focused on the use of nutritional support to traditional treatment [61].
Cashew nuts, fruits of Anacardium occidentale L., an original plant from Brazil, have shown a good capacity to counteract oxidative damage, primarily thanks to the abundance of secondary metabolites such as polyphenols, flavonoids and others [62][63][64][65][66]. The most plausible hypothesis is that polyphenolic components of dietary plants modulate the cellular redox state by boosting the endogenous antioxidant defense [66,67]. Recently, cashew nuts were used for their antioxidant, anti-genotoxic, anti-mutagenic, anti-inflammatory, and other protective properties [31,[68][69][70][71][72][73][74][75]. In previous studies, we demonstrated that cashew nuts treatment, was able to alleviate oxidative stress and inflammation in different in vivo models, such as dinitrobenzene sulfonic acid (DNBS)-induced colitis, carrageenan-induced paw edema, and monosodium iodoacetate (MIA)-induced osteoarthritis. These effects are likely exerted through a reduction of various pro-inflammatory pathways and mediators, including MPO and MDA levels, mast cell degranulation and neutrophil infiltration, release of pro-inflammatory cytokines, modulation of NF-κB signaling, modulation of ROS production, etc. [31,32,36].
Among several different in vivo experimental models of AP that exhibit the same pathophysiological development of human pancreatitis, the use of cerulein, an analog of cholecystokinin (CCK), is one of most frequently used. We thus used this model to induce AP in mice and to investigate for the first time the effect of cashew nuts treatment on inflammation and oxidative stress in the pancreas and lung during AP.
Cashew nuts treatment had beneficial effects on cerulein-induced histological alterations in both pancreas and lung. Cerulein treatment led to severe alterations of tissue architecture with oedema formation and inflammatory cells infiltration. These modifications were significantly attenuated by oral treatment with cashew nuts at a dose of 100 mg/kg. Mast cells have been reported to play a pivotal role during pancreatitis-associated ALI [76]. Previous studies have shown that mast cells are usually located in the pancreatic interstitial and periacinar space as well as in the mesentery, but during AP they were highly correlated with neutrophil infiltration and oedema formation in both pancreas and lung [76,77]. Neutrophil infiltration is also important in acute pancreatitis. MPO activity is a useful indicator of neutrophil activation and inflammation, since the enzyme is stored in the neutrophils' granules [78]. Additionally, because while one of the most dangerous consequences of oxidative stress is cellular injury triggered by ROS, it is informative to assess the levels of oxidation products as markers of oxidative stress [79,80]. Considering that lipid peroxides are extremely reactive compounds, they degrade rapidly into a range of metabolites. MDA is one of the best known secondary metabolites of lipid peroxidation, and it is used as a marker of cell membrane damage [80]. In the present study, oral treatment with cashew nuts was able to partially suppress mast cell degranulation, neutrophil infiltration, and lipid peroxidation.
Cerulein administration is well known to induce a dysregulation of the production and secretion of digestive enzymes, such as amylase and lipase, specifically inhibiting their secretion by the exocrine pancreas into the digestive tract, and leading to elevation in their respective levels in the blood circulation [81]. In parallel, pro-inflammatory cytokines play a fundamental role in the inflammatory response associated with AP. Different clinical studies have, in fact, documented a pro-inflammatory cytokine profile in the sera of patients with AP, including increased levels of IL-1β, IL-6 and TNF-α [82,83]. This profile was also observed in the present study. Importantly, cashew nuts were able to decrease the cerulein-induced levels of amylase and lipase as well as the levels of IL-1β, IL-6 and TNF-α.
AP being an oxidative stress condition, the Nrf2/Keap1 signaling pathway is activated in the pancreas, but this not sufficient to prevent the disease [12]. Several studies have shown that the hyper-stimulation of Nrf2 via plant-derived natural compounds such as visnagin or hydroxytyrosol could be a promising strategy against the excessive oxidative stress that characterizes AP [41,84,85]. In the present study, we found that cashew nuts treatment was able to promote Nrf2 nuclear translocation and to induce the expression of the Nrf2-regulated factors HO-1 and Mn-SOD in both pancreas and lung.
Another pathway that recently attracted attention is NRLP3, and it has been recently demonstrated that NLRP3 modulation may be a promising strategy to alleviate AP and ALI [86,87]. Inflammasome formation starts with the interaction of NRLP3 with ASC, which in turn recruits and activates procaspase-1 to active caspase-1, converting the cytokine precursors pro-IL-1β and pro-IL-18 into mature and active IL-1β and IL-18, respectively. The activation of these cytokines leads to a series of cellular responses that induce a very strong inflammatory response in the cell which can culminate in its death [88][89][90][91][92][93]. Researchers have focused their attention on the role of the inflammasome in the initiation or evolution of disorders with a high impact on public health, such as metabolic pathologies, cardiovascular diseases, inflammatory issues, and neurologic disorders [20]. It has been shown that the NLP3-induced caspase-1-mediated activation and secretion of IL-1β and IL-18 plays a key role during the development of AP [11]. In the present study, we found that the levels of NLRP3, ASC and caspase-1 were significantly increased after cerulein induction, and that cashew nuts considerably diminished this increase in both pancreas and lung.

Conclusions
Considering the key role played by inflammation and oxidative stress in several diseases, antioxidant and anti-inflammatory dietary compounds are a main research attention is focus. Antioxidant treatment is believed to have great prospects, since its therapeutic efficacy has already been demonstrated in several experimental settings of AP. Nuts are one of the main sources of polyphenols in the diet worldwide. The present work adds further support to the concept that natural-based compounds can be useful for the treatment not only of pancreatitis but also of the lung complications associated with it. Specifically, compounds present in cashew nuts could be a useful adjunct to mitigate the inflammation and oxidative stress that underlie these conditions. Supplementary Materials: The following are available online at http://www.mdpi.com/2076-3921/9/10/992/s1, Figure S1: Experimental protocol of cerulein-induced acute pancreatitis (AP). AP was induced by cerulein hyperstimulation through ten hourly intraperitoneal (i.p) injection at the dose of 50 µg/kg. Cashew nuts were given 30 min and 2 h after the first cerulein injection. Animals were euthanized 1 h after the last injection, and samples of blood, lung and pancreatic tissue were preserved for further study.