No changes were identified in the total lipid content and the FA profile between the treatments, thus demonstrating that brain tissue is not affected by diet in the short-term. The results found in the functional enrichment, demonstrated that the use of different levels of soybean oil alters the transcriptomic profile of pig brain, affecting key processes for the well-functioning of this tissue. For the enriched pathways in Figs. 1 and 2, the DEG ALOX15B participates in lipid oxidation and peroxidation reactions. According to Stelzer et al. , among the pathways associated with this gene there are “eicosanoid synthesis” and “arachidonic acid metabolism” and the related Gene Ontology (GO) annotations include “calcium ion binding” and “lipid binding”. In our study we observed that ALOX15B has a lower expression in the SOY1.5 group compared to SOY3.0.
Lipoxygenases (LOX) are a family of enzymes responsible for the oxidation of lipids and the generation of a range of metabolites such as eicosanoids and PUFA-related compounds. These metabolites play diverse physiological and pathological roles in inflammatory, neurodegenerative, and cardiovascular diseases, as well as, in defense mechanisms [32, 33]. The LOX have also been reported in cell differentiation [34, 35], apoptosis , and play an important role in the immune response by helping to regulate cytokine secretion .
Among the LOX reported in mammals, the ALOX15 isoform my oxygenate complex lipid-protein assemblies found in biomembranes and lipoproteins . The ALOX15 also binds to membranes, with intracellular calcium as a main cofactor for this interaction [39, 40]. It is reported that ALOX15 is expressed at higher levels in in human airway epithelial cells, in eosinophils and immature red blood cells . Furthermore, according to van Leyen et al.  and Han et al. , expression and regulation of ALOX15 transcription also occurs in various areas of the brain, but at lower levels. In the study of Shalini et al. , a higher expression of ALOX15 mRNA was found in the prefrontal cortex.
The main product of AA oxygenation by ALOX15 and ALOX15B is 15-hydroxyeicosatetraenoic (15-HETE) . The 15-HETE is considered an important precursor of specialized pro-resolving lipid mediators (SPM) and is associated with pro- and anti-inflammatory effects [46, 47]. It has also been reported that 15-HETE is a ligand and activator of peroxisome proliferator-activated receptor gamma (PPAR-γ), which at high concentrations may generate reactive oxygen species (ROS) in cells [48, 49] and may induce the production of the pro-inflammatory cytokine Interleukin-12 (IL-12) [47, 50].
Among the results of DHA oxidation by ALOX15, are the SPM resolvin D5, a mediator that may be associated in the resolution of inflammation and in regulating the immune response . Another important mediator related to the resolution of inflammation, reduction of leukocyte trafficking, and negative regulation of cytokine expression is neuroprotectin D1 (NPD1) [52, 53]. NPD1 is reported as an anti-inflammatory molecule, which acts in neuroplasticity and brain signaling and when in altered conditions, may be found in neuroinflammatory disorders and chronic neurodegeneration .
The ALOX15 was found to have increased expression in the brains of Alzheimer's patients [38, 54, 55]. Praticò et al. , reported higher levels of 12/15-LOX and its metabolites 12/15(S)-HETE in the temporal and frontal brain regions of Alzheimer's patients. It was further found in in vitro studies using neuronal cells with Alzheimer's mutation that 12/15-LOX is associated with regulation of tau phosphorylation and Aβ plaque production, and regulates synaptic pathology associated with behavioral deficiencies [56, 57].
In addition, 12/15-LOX has been shown to play an important role in Parkinson's disease. In the study of Li et al.  and Canals et al. , the authors showed that the activation of these isoforms was associated with a decrease in glutathione concentration (a marker of Parkinson's disease) in neurons, which may lead to nitric oxide neurotoxicity and damage dopaminergic neurons. In the study of Zhang et al. , the inhibition of 12/15-LOX assisted in reducing the generation of ROS induced neuronal cell death. Thus, it is commonly found in the literature that 12/15-LOX and its metabolites possess both pro-inflammatory and anti-inflammatory effects. This controversial nature is dependent on the metabolites generated, the site of inflammation, as well as, the levels generated of these metabolites . Thus, with a down-regulation of ALOX15B in the SOY1.5 group, our study demonstrates that diet may have positively or negatively influenced various metabolic and oxidative processes in brain tissue. Further studies are needed to verify this action.
For the enriched pathway in Fig. 3, the CALB1 gene binds to intracellular calcium transported via the epithelial calcium channel and transports it across the cytosol toward the basolateral membrane . The CALB1 is a protein-encoding gene that acts in calcium transport. The GO annotations related to this gene include “calcium ion binding” and “vitamin D binding” . In our study we observed that CALB1 also showed lower expression for the SOY1.5 group compared to SOY3.0.
The CALB1 is highly conserved in evolution and belongs to a family of high-affinity calcium-binding proteins [62, 63]. The CALB1 is found highly expressed in brain tissue, is present in most neuronal cell groups, and is not vitamin D dependent [62–64].
Calcium is one of the most important signaling factors and acts to regulate several important cellular functions such as growth, differentiation, proliferation, cell survival and apoptosis, membrane excitability, and gene transcription . Calcium is also essential for maintaining normal brain function . Thus, the dysregulation of calcium homeostasis and endoplasmic reticulum stress is associated with several pathological conditions such as Parkinson's, Huntington's, and Alzheimer's diseases, and affects numerous signaling pathways [65, 66]. This pathogenic event may also cause amyloidogenesis, energy deficits in neurons, protein aggregation and oxidative stress, and changes in mitochondrial dysfunction, plasticity and synaptic transmission .
Disturbed mitochondrial calcium regulation may also be associated with the link between neuronal dysfunction and disruption of the mitochondria-associated membrane (MAM) contact site of the endoplasmic reticulum and mitochondria, since calcium acts to modulate neurotransmitter release during the synapse . This dysregulation of the MAM-mitochondria linkage dysfunction may also be associated with neurodegenerative diseases such as Alzheimer's disease . The MAMs are regions of the endoplasmic reticulum that mediate communication between the reticulum and the mitochondria [68, 69]. They are regions that are involved in calcium transport, are responsible for several lipid biosynthetic enzymatic activities, and are also a strategic site for lipid metabolism [68, 70, 71]. According to Vance , defects associated with these regions have been identified in neurodegenerative diseases and insulin resistance/type 2 diabetes.
The CALB1 helps maintain calcium homeostasis, helps regulate intracellular calcium responses to physiological stimuli, and assists in modulating synaptic transmission . Another important role of CALB1, is its action in the prevention of neuronal death [62, 72]. The CALB1 also plays an important role in buffering cytosolic calcium and helps prevent lipid peroxidation, through its expression in pancreatic-β cells, by eliminating the production of lipid hydroperoxide, which is induced by proinflammatory cytokines . There is evidence that CALB1 acts to protect neurons against calcium-mediated neurotoxicity and may be considered a cytochemical marker for neuronal plasticity .
Decreases in CALB1 expression/concentration in brain tissue has been associated with neurodegeneration in Alzheimer's, Parkinson's, and Huntington's diseases [31, 74] and in ischemic injury studies [75, 76]. Lower CALB1 expression has also been associated with a higher rate of neuronal death . Increased expression of CALB1, on the other hand, has been reported to induce neurite growth in dopaminergic neuronal cells, demonstrating its protective role, especially in neurological diseases, such as Parkinson's disease [72, 78].
For Alzheimer's disease, it has been reported that CALB1 has protective effects against the pro-apoptotic action of mutant presenilin 1 (PS-1), attenuating the increase in intracellular calcium and aiding in the prevention of impaired mitochondrial function . PS-1 acts by sensitizing cells to apoptosis induced by Aβ peptide, which damages neurons through a mechanism involving disruption of calcium homeostasis and generation of oxidative stress . Thus, with a down-expression of CALB1, we observed that with a lower percentage of soybean oil, the CALB1 gene was less expressed, showing a negative relationship with this diet.
For the enriched pathway in Fig. 4, IL-5 activates and elevates the expression of CAST. The DEG CAST binds to and inhibits calpain 1 (mu) in the presence of calcium, which activates and cleaves the apoptosis regulatory protein Bax. The Bax will act by preventing or reducing the frequency, rate, or extent of cell death by apoptotic process [80, 81]. In our study, CAST had a higher expression (up-regulation) in the SOY1.5 group compared to SOY3.0. The protein encoded by CAST is an endogenous calpain inhibitor and is also related to the proteolysis of amyloid precursor protein. Moreover, this protein likewise is thought to affect the expression levels of genes that are responsible for encoding structural or regulatory proteins . Among the related pathways associated with this gene are “neuroscience” and “neurodegenerative diseases”. Related GO annotations include “RNA binding” and “cysteine-type endopeptidase inhibitor activity” .
The CAST is a cell-permeable peptide that acts as an endogenous inhibitor of calpain in the central nervous system [82, 83]. Calpains are cysteine proteases that are activated by calcium, that is, they are positively regulated by calcium and negatively regulated by CAST [84, 85]. These proteases, when in dysregulation of calcium homeostasis, have been implicated in neuronal cell dysfunction and death , as well as, neurodegenerative diseases [86–88].
Calpains have several important roles such as differentiation, cell attachment motility, signal transduction covering cell signaling pathways, regulation of gene expression and membrane fusion [82, 85]. Furthermore, calpains are reported to play important roles in neuronal functions, implying that the activation of this protease needs to be under a rigid control, which is performed by CAST. Thus, the well-known calpain-calpastatin system may be an important target for therapeutic approaches related to neurodegenerative diseases .
According to Goll et al. , CAST is further associated in the regulation of kinases, receptors and transcription factors. Increased expression of CAST has been reported to have a neuroprotective effect in cerebral ischemia . In the study of Rao et al. , higher expression of CAST in JNPL3 (mutant tau P301L) mouse models for aided in the attenuation of calpain, which has been reported in the development of tauopathy (neurotoxicity caused by tau protein) and neurodegeneration reported in Alzheimer's disease. Higher expression of CAST was also associated with neuroprotective results in an Amyotrophic lateral sclerosis (ALS) mouse model. According to Rao et al. , the CAST gene acts by reducing calpain activation, decreasing abnormal breakdown of cytoskeletal proteins, increasing survival time, inhibiting tau production and CDK5 activation, and reducing SOD1 .
The calpain-calpastatin system is also reported in excitotoxicity, a pathological or neurodegenerative process that is initiated by overactivation of neurotransmitters such as glutamate and it is like. Excitotoxicity, leads to increased cellular calcium levels, which causes activation of various proteases, including calpains . Furthermore, missing CAST may impair early stages of neurogenesis . Thus, we observed a higher expression of CAST in SOY1.5, that suggests a positive relationship between the gene and the metabolic and oxidative processes found for this group.
The identified network together with the illustrated genes corroborate the results found in the pathway maps, and thus, shows us that altering the level of soybean oil in the diet of immunocastrated male pigs has an effect on gene expression in brain tissue. Moreover, it is noteworthy the importance of the detected DEG and their association with intracellular calcium.
Thereby, the network of processes "Calcium transport" identified and the genes present, corroborate the results found in the pathway maps, and thus, shows us that altering the level of soybean oil in the diet of pigs have an effect on gene expression in brain tissue. Moreover, it is noteworthy the importance of the detected DEG and their association with intracellular calcium. Thus, the results found in our study represent an important direction for the understanding of pathways and networks associated with calcium-dependent metabolic processes involved in lipid metabolism and oxidative processes in brain tissue. Further, more studies are needed to better understand the mechanisms by which dietary factors such as FA may influence important physiological processes and gene expression in the brain tissue. In addition, understanding the mechanisms involved in calcium homeostasis and energy metabolism involved in the initiation and progression of neurodegenerative diseases and oxidative/inflammatory processes is also quite relevant.