Supplementation of polyunsaturated fatty acids (PUFAs) and aerobic exercise improve functioning, morphology, and redox balance in prostate obese rats

The high-fat diet (HFD) stimulates an increase in lipids and can be prejudicial for harmful to prostatic morphogenesis. Polyunsaturated fatty acid (PUFAs) have anti-inflammatory and antioxidant action in some types of cancer. The combination of aerobic physical exercise and PUFA can be more effective and reduce the risk of death. The study evaluates the effects of aerobic physical exercise associated with omega-3 (fish and chia oils), on the ventral prostate of Wistar rats those fed with HFD. Here, we report that HFD modified the final body weight and the weight gain, decreased the expression of the androgen receptor and increased prostatic inflammation via TNF-α produced damage prostatic like intraepithelial neoplasia. The supplementation with fish oil decreases final body weight, reduced BCL-2 and inflammation compared to chia oil; aerobic physical exercise associated with fish oil reduced lipids circulant and prostatic, increased proteins pro-apoptotic expression and reduced IL-6 (p < 0.0001) and TNF-α potentiating the CAT (p = 0.03) and SOD-1 (p = 0.001) expression. Additionally, the chia oil increased the NRF-2 (p < 0.0001) and GSS (p = 0.4) genes. PUFAs reduced the damage caused by excessive high-fat diet in the prostate so that there is greater effectiveness in omega-3 intake, it is necessary to associate with aerobic physical exercise.


Results
Aerobic physical exercise is associated with PUFA supplementation and effects on body and adiposity. As expected, the high-fat diet increased weight gain, when compared to the CT group; and the supplementation of fish and chia oil, alone or associated with physical exercise significantly reduces the weight gain when compared to the HF group (Table 1). Adipose reserves differed significantly across groups as shown by differences in epididymal fat (p = 0.004; Table 1), mesenteric fat (p = 0.0008; Table 1), retroperitoneal fat (p = 0.002; Table 1), and fat index (p = 0.0001; Table 1). Post-hoc analysis revealed that the HF group had significantly greater epididymal fat, mesenteric, and retroperitoneal fat, and a greater fat index compared to the CT and exercise groups (Table 1). Adipose tissue and fat index in animals subjected to aerobic training and HF were comparable to the CT group. Though non-significant, retroperitoneal fat and fat index were lower in animals following aerobic physical training with fish oil and chia oil supplementation compared with the HF + FO and HF + CO groups ( Table 1). The absolute prostate weight and relative prostate weights were reduced in the HF, HF + CO, and HF + FO groups, but no significant differences across groups (Table 1).
PUFA supplementation associated with aerobic training regulates food consumption, and body weight of rats fed with HFD. During the six-week induction period, significant differences in weight gain, food consumption (p = 0.0001), and energy intake (p = 0.0001) were observed across groups. Rats in the CT groups demonstrated greater weight gain at weeks three through six (Fig. 1A), and greater food consumption and energy expenditure at weeks two through six (Fig. 1C,E) compared to the HF groups. Significant differences in weight gain (p = 0.015) and feed efficiency (p = 0.03) were also observed across groups and time, with both measures greater in the CT group at weeks two and three, but greater in the HFD groups at week four and six (Fig. 1D,M).
After the diet acclimatization period, the animals given HF were divided into six subgroups (HF, HF + Ex, HF + FO, HF + FO + Ex, HF + CO, and HF + CO + Ex) and began oil supplement and aerobic physical training until week 14. Though nominal, the groups supplemented with fish oil with and without physical exercises showed the lowest weight gain relative to all other groups (Fig. 1B). The CT group showed significantly greater food consumption than all other treatment groups (p = 0.0001; Fig. 1D) but the lowest feed efficiency of all treatments (p = 0.3; Fig. 1H). Physical exercise combined with HF did not reduce the energy intake and feed efficiency compared to www.nature.com/scientificreports/ the HF group (Fig. 1F,H). On the other hand, fish oil supplementation reduced energy intake compared to HF treatment (Fig. 1F,H), and physical exercise in combination with fish oil supplementation reduced feed efficiency from 10 weeks compared fish oil supplementation alone (Fig. 1H). Similarly, chia oil with and without physical exercise reduced energy intake and feed efficiency when compared to the HF + CO, HF, and HF + Ex treatments (Fig. 1F,H). Food consumption was also lower in all HFD groups compared to the CT group (Fig. 1D), however, HFD considerably increased feed efficiency and reduced energy intake throughout the experimental period.
Effect of PUFA associated with aerobic training altering lipid profile of rats fed with HFD. The plasma lipid composition was analyzed the following sacrifice at 14 weeks of post-dietary initiation (Fig. 2). Comparisons across groups revealed a significant main effect of treatment on plasma TAG levels (p = 0.0001). A 2.24-fold reduction in plasma TAG was observed in the HF + FO + Ex treatment group when compared with CT (95% CI 23.63-107.7; p = 0.0003); similarly, a 3.09-fold reduction in TAG in the HF + FO + Ex group was observed relative to the HF treatment group (95% CI 68.50-152.6; p < 0.0001; Fig. 2A). In addition, TAG levels in serum from the HF + CO (95% CI 9.529-93.58; p = 0.002) and HF + CO + Ex (95% CI 14.97-99.03; p = 0.002) groups were also significantly lower than levels from the CT group ( Fig. 2A) (Fig. 2B).
In contrast to TAG, LDL, and VLDL levels, the CT group demonstrated significantly lower levels of HDL compared with all other groups (Fig. 2C). Nevertheless, significant changes to the ratio of TC/HDL associated with oil supplementation and aerobic physical training suggest these treatments alter plasma lipid profile (p = 0.0001) the PUFAs supplementation and PUFAs supplementation alongside exercise (HF + Ex, HF + FO, HF + FO + Ex, HF + CO, and HF + CO + Ex) groups reduced the TC/HDL ratios compared to the CT group (Fig. 2F). The TC/ HDL ratio reduced significantly in the HF + FO, HF + FO + Ex, HF + CO, HF + CO + Ex groups compared to the HF group (Fig. 2F). Table 1. Data on initial and final body weight, weight gain, absolute and relative prostate weights, absolute and relative fats, epididimal. mesenteric and retroperitoneal adipose tissues of trained animals, fish and chia oil intake, submitted to high-fat diet for 14 weeks. Data are presented as the mean ± SEM (n = 7). The significance of p < 0.05 is indicated by lowercase letters indicating a difference between the groups. a Referring to the control group. b Referring to the high-fat diet group. c Referring to a diet rich in fat + ω-3 group. d Referring to a high-fat diet + physical exercise group. e Referring to a diet rich in fat + ω-3 + aerobic physical exercise group. f Referring to a high-fat diet + chia group. g Referring to high-fat diet + chia + physical exercise group. The Two-Way ANOVA test was used to compare the means. with the Tukey post-test. www.nature.com/scientificreports/ Effect of PUFA diet composition associated with aerobic physical exercise altering lipid oxidative stresses of the prostate of rats fed with HFD. To investigate the effects of fish oil and chia supplementation on the balance of oxidative stress production and antioxidant capacity, we analyzed the gene expressions of SOD1, CAT, GSS, NRF-2, and NOS2 in the prostate of rats that consumed a high-fat diet ( Fig. 2G-K). The expression of SOD1 and CAT mRNA was lower in the HF + CO group compared to the HF + FO group, however, the group supplemented with chia oil showed higher expression of GSS, NRF2, and NOS2 mRNA to the other groups (Fig. 2). Aerobic physical exercise increased the expression of SOD1 and CAT mRNA associ- www.nature.com/scientificreports/  www.nature.com/scientificreports/ ated with fish oil and chia oil supplementation, however, the groups that practiced aerobic physical exercise showed low GSS and NRF2 mRNA expressions (Fig. 2). The groups supplemented with chia oil (HF + CO and HF + CO + Ex, respectively) significantly increased the expression of NOS2 mRNA when compared to the other groups (Fig. 2). In another perspective, the intervention with chia and physical exercise in the HF + CO + Ex group raised NOS 2 levels in relation to the HF, HF + Ex, and HF + FO + Ex groups (Fig. 2).
Changes of the histopathological, mast cells and stereological analysis in the prostate of rats submitted the aerobic physical exercise and PUFFA supplementation across feeding HFD. The ventral prostate structure in the CT group presented the prevalence of acini with simple cylindrical epithelium, polarized nuclei in the basal part of the cells, and a clear supranuclear region, the latter of which corresponds to the Golgi Apparatus area (Fig. 3A). Stereological analysis show the reduction of epithelium and connective tissue in the HF + Ex group compared with groups, but without significant differences (Fig. 3X,Y), the other groups showed no differences in the stereological analysis. It was possible to observe epithelial cell nuclei to apical areas, where they presented different heights to give a pseudo-stratified aspect to the tissue, which was frequently observed in the HF group (Fig. 3B). The presence of cells in division moving to the apical part of the epithelium indicates proliferative activity in this tissue and in some areas, the epithelium had become thick with agglomerated nuclei of various heights similar to prostatic intraepithelial neoplasia (PIN) and was showed an increase of 37% of PIN in HF group ( Table 2). The proliferative inflammatory atrophy (PIA) characterized by agglomerated epithelial cells with heterogeneous phenotypes, stratified epithelial patterns, and different compacted chromatin nuclear patterns show epithelial inflammatory reactive atypia was observed in 24% and 39% of the animals of HF and HF + CO groups respectively ( Table 2). Inflammatory foci observed in the animals of HF (39%) and HF + CO (24%) groups presented similar characteristics including a prevalence of lymphocytes and plasmatic cells ( Table 2). The HF + FO sowed lowed alteration of how PIN and inflammation foci than compared to other groups ( Table 2). On the other hand, the aerobic physical exercise showed reduced histopathological alteration in the prostate associated with supplementation or alone ( Table 2). The HF (95% CI − 4.198 to − 0.2576; p = 0.01), HF + FO (95% CI − 4.264 to − 0.3236; p = 0.01) and HF + CO (95% CI − 4.720 to − 0.7796; p = 0.0023) had a higher number of mast cells in the CT group (Fig. 3V). The aerobic physical exercise reduced mast cells in HF + CO + Ex (95% CI 0.6978 to 4.638; p = 0.003) group when compared to HF + CO (Fig. 3V).
Effect of PUFA diet composition associated aerobic physical exercise on the modulation androgenic, lipogenic, and apoptotic prostatic. We investigated the immunoreactivity of the AR, SREBP-1, IGF-1, BCL-2, BAX, and FAS/CD95 effects of fish and chia oil supplementation and physical exercise after HFD and is shown in Fig. 4. The HFD (as compared to the CT group) significantly reduced the immunoreactivity of de AR in the prostate, there was no significant difference. The HF + FO + Ex (95% CI − 47.64 to − 10.47; p = 0.0006), HF + CO (95% CI − 43.64 to − 6.474; p = 0.003) and HF + CO + Ex (95% CI − 58.01 to − 18.59; p < 0.0001) show increased AR in prostate when compared to HF group ( Fig. 4A-G and V). Likewise, the mean of AR was higher in rats fed the chia oil and submitted the aerobic physical exercise (HF + CO + Ex), were significant difference in the CT (95% CI − 46.30 to − 4.745; p = 0.009) group (Fig. 4V). To compare the effects of the aerobic physical exercise and aerobic physical exercise associated with chia oil in the expression of prostatic AR it was possible to verify the difference in AR expression stimulated by chia oil in the HF + CO + Ex group when compared to the HF + Ex group (95% CI − 40.4 − 0.985; p = 0.01).
To verify the prostatic effects of the high-fat diet and the addition of fish oil and chia in the diet associated with physical exercise, we analyzed lipogenesis through the expression of SREBP-1. There was a significant increase in the SREBP-1 expression on HF (95% CI − 3.90 to − 0.83; p = 0.001), HF + FO (95% CI − 3.92 to − 0.66; p = 0.01) and HF + CO (95% CI − 3.27 to − 0.21; p = 0.01), when compared to CT group (Fig. 4W). On the other hand, aerobic physical exercise reduced the SREBP-1 expression alone (HF + Ex, 95% CI 0.39 to 3.29; p = 0.001) and associated with chia (HF + CO + Ex 95% CI 0.84 to 3.73; p = 0.001) oil when compared to the HF group (Fig. 4W). Physical exercise reduced effects lipogenic in prostate associated chia oil vs. HF + CO group (95% CI 0.22 to 3.11; p = 0.01).
To identify the effect of the high-fat diet on epithelial progression and development of prostatic lesions and possible effects of n-3 PUFA supplementation associated with aerobic exercise, we investigated the expression of IGF-1. There was a significant increase in IGF-1 in fish and chia oil supplementation, HF + FO (95% CI − 3.43 to − 0.14; p = 0.01) and HF + CO (95% CI − 4.47 to − 0.55; p = 0.01) respectively, compared to the HF group (Fig. 5). The HF + FO + Ex increased IGF-1 compared to HF (95% CI − 3.76 to − 0.27; p = 0.05, Fig. 5).
The anti-inflammatory effects of FO and CO intake associated with physical exercise in the prostate were evaluated by the expression of cytokine IL-10. We observed that the animals that were submitted to HFD showed lower expression of prostatic IL-10 when compared to the other groups. The groups supplemented with fish oil (HF + FO) showed significantly higher expression of IL-10 when compared to the HF group (Fig. 7). The groups that were submitted to physical exercise, HF + Ex, HF + FO + Ex and HF + CO + Ex, showed no difference between them, however, all were significantly different from the HF group (Fig. 7).

Discussion
We compared the biological effects of supplementing with fish and chia oils alone or in combination with aerobic physical exercise in rats submitted to a high-fat diet. A potential mechanism by which obesity can promote severe cancer prognosis is through the induction of functional metabolic abnormalities, altering the metabolic profile, promoting inflammation and oxidative stress. The HFD exposure increased LDL-cholesterol levels, TC, and TAG, which are predominantly synthesized in the liver, are important markers of lipid metabolic disorders. Chia oil has been described as a cholesterol regulator owing to the effect of PUFA in lipid metabolism 20 . When rats were fed HFD and supplemented with fish or chia oil, HDL cholesterol levels in the plasma were not noticeably different from those not receiving oil, whereas TC levels were marginally lower relative to non-supplemented groups. Aerobic physical exercise has been reported to reduced serum levels of LDL and VLDL. Similarly, fish supplementation has been reported to reduce LDL levels 21 . The incorporation of fish and chia oils with the physical exercise clearly has more effect on lipid profile compared with fish oil supplementation or chia oil alone, as demonstrated by reduced LDL, VLDL, and TAG levels by chia oil supplementation alone. These results provide evidence that aerobic physical exercise and supplementation with fish oil may synergistically improve the amount of circulating lipoproteins and reduce lipid stocks in adipose tissue despite no apparent changes in weight during HFD consumption. Table 2. Occurrence of histopathological data of experimental animals. CT Control Group, HF high-fat diet, HF + Ex high-fat diet and aerobic physical exercise, HF + FO high-fat diet and fish oil, HF + FO + Ex high-fat diet, fish oil and aerobic physical exercise, HF + CO high-fat diet and chia oil, HF + CO + Ex high-fat diet, chia oil and aerobic physical exercise. The results were expressed in absolute values and occurrence of percentages. The significative differences were adopted based on p < 0,05. Test Mann-Whitney. www.nature.com/scientificreports/ www.nature.com/scientificreports/  www.nature.com/scientificreports/ www.nature.com/scientificreports/ PUFAs are associated with reduced risk of several types of carcinogenesis, have been evidence in the prostate, however, this depends on numerous factors, including the source of omega-3 PUFAs. The consumption of the high-fat diet and obesity cause a reduction in testosterone and even so promote prostatic changes such as prostatitis, BHP, HGPIN 22 until cancer 23 . A review study organized by Aucoin 4 showed an association between increased consumption of fish oil and reduced risk of prostate cancer, however more research is needed to demonstrate the potential effects of the treatment of omega-3 and its relationship with prostate. Fish oil has higher concentrations of EPA and DHA and exceptionally, seeds of chia (Salvia Hispanic) are abundant in ALA, and the omega-3 PUFAs are considered to be activators of cholesterol esterification, an important mechanism for cholesterol reduction 24 . We associated the increase in the expression of SREBP-1 with the consumption of HFD and increased of the BHP, HGPIN in prostate independent of the AR. SREBP-1 induced prostate cancer cell proliferation, migration and invasion in vitro and promoted prostate tumor growth through the induction of FASN expression and lipid droplet formation and accumulation in prostate cells 9 . Physical exercise aerobic alone and associated with chia oil intake, a-linolenic acid (EPA and DHA precursor) reduced the levels of prostate SREBP-1 reduced PPAR activation regulated the lipogenic effects concomitant with the increase in AR. The effects of physical training promoted an increase in AR in the prostate, thus regulating the expression of SREBP-1, exhibited different efficiencies in the inhibition of proliferation.
To determine whether supplementation of chia and fish oil would cause cellular apoptosis, we checked the intrinsic and extrinsic pathways. HFD is often accompanied by decreased levels of omega-3 PUFAs 25 and is believed to be prejudicial for the prostate. The Fas/FasL pathway is an important extrinsic apoptotic pathway and the Fas/CD95 membrane receptor initiates intracellular signaling of the apoptosis pathway by activating caspases 8 and 9. Thus, it has been shown that Fas ligand (FasL) is secreted by prostatic carcinoma cells and together with Fas/CD95 plays a key role in the development of abnormal cells 26 . Jiang 27 reported that Fas/CD95 is more expressed in a high-grade PIN. The activation of Fas/CD95 occurs by TNF-a initiating the proteolytic cleavage pathways, in the absence of TNF-α this pathway is minimized 27 . We found that omega-3 PUFAs reduced expression of Fas/CD95 and BCL-2, and increased BAX when associated with aerobic physical exercise. It is already well documented that physical exercise promotes alteration of apoptosis in the prostate cell increases the BAX reduce proliferative ratios in the ventral prostate 28 even in animals submitted to a high-fat diet 18 . Thus, it is possible to relate that supplementation of omega-3 PUFAs associated with aerobic exercise promotes prostatic cell apoptosis intrinsically.
Oxidative load is strongly implicated in the pathogenesis of age-related diseases, including the formation of prostate cancer tumor, and omega-3 fatty acids have antioxidant and anti-inflammatory properties, we investigated the component effects of fish oil with higher concentrations of EPA and DHA, and chia oil components with greater composition of ALA in reducing the effects of oxidative damage to DNA induced by obesity. The high-fat diet increases lipid peroxidation and higher lipid accumulation probably was related to increasing of omega-3 PUFAs with fish and chia oil supplementation. Such omega-3 is metabolized primarily at the peroxisome fraction, a well-known site of H2O2 generation, due to long-chain fatty acid structure. Therefore, the antioxidant effects of fish oil supplementation (concentration of EPA and DHA) were mainly at the mitochondrial compartment since, despite not recovering to control levels, such promoted decreased O2 ·− directly modifying the levels of enzymatic expression of NOS2 and increased the antioxidant capacity 29 . Physical exercise is clinically associated with a reduction in lipid peroxidation by increasing the expression of antioxidant enzymes 30 . On the other hand, supplementation with chia oil increased the levels of GSS, NOS 2, and NRF-2 in the ventral prostate. Like other exogenous stimuli, chia oil can promote the NRF-2 activation pathway, to control the pro-oxidative response 31 , once activated, participates in the regulation of programmatic functions stimulated by oxidants, including autophagy, reticulum stress, and mitochondrial biogenesis.
Omega-3 fatty acids exhibit known inflammatory properties that suppress prostate carcinogenesis, we investigate the potential role of fish oil and chia in reducing the inflammatory effects on HFD-induced prostate epithelial cells. Statements have been published in the literature that omega-3 PUFAS are important in the quantity and quality of immune responses 32 . Fish oil intake, containing a mixture of omega-3 PUFAS, reduces the expression of IL-6, TNF-α, and NF-κB in the ventral prostate and was more efficient when associated with aerobic physical exercise (Fig. 8). NF-κB is a pro-survival nuclear transcription factor activated by a variety of stimuli, including oxidative stress. Evidence suggests that fish oil components such as DHA can attenuate the transcriptional activity of NF-κB by inhibiting translocation to the nucleus in obesity-induced prostate cells 33 . Omega-3 PUFAS -activated PPAR α can also directly interfere with the NF-κB (p50-p65 dimer) and consequently inhibit expression of the gene encoding pro-inflammatory cytokineIL-6. Possibly the higher concentration of omega-3 PUFAS in fish oil inhibited AR/NF-κB promoted down-regulation in TNF-α and COX-2, and this modification reduced PIN. Physical exercise associated with fish oil supplementation (concentration of EPA and DHA) significantly reduced prostate inflammation for increased IL-10 in the prostate. Another described effect of EPA and DHA is COX inhibition that reduces inflammation and ROS production 34 .

Conclusions
Physical exercise and encouraging PUFA consumption are of utmost importance in the treatment of obesity and related diseases, which are characterized by variations in adipose tissue deposition, lipoprotein profiles. Employing both strategies concurrently provides additional benefits for reducing the negative effects of obesity and prostatic diseases. When we incorporate fish and chia oil supplementation into a high-fat diet, we verify the ability to prevent prostatic damage by reducing the circulating lipid profile, increasing antioxidant activity and its anti-inflammatory capabilities. This protection was more effective when associated with aerobic exercise, suggesting regulation of antioxidant activity such as higher expression of CAT and SOD-1, lower expression of www.nature.com/scientificreports/ IL-6, TNF-α and NF-κB as well as an increase in anti-apoptotic proteins of BAX and FAS/CD95 and reduction of BCL-2.

Materials and methods
Ethics statement. Experiments, all animal procedures, were conducted in accordance with the ethical principles in animal research adopted by the Brazilian College of Animal Experimentation (COBEA) and the study protocol was approved by the Ethics Committee on Animal Use (CEUA) of the Universidade do Oeste Paulista-Unoeste, Presidente Prudente (protocol number 3962).
Animals and experimental procedures. Forty-nine adult Wistar rats (60 days old) were individually housed and maintained at 22 ± 1 °C, 60-70% humidity, and kept on a 12-h light/dark cycle for the duration of the experiment. Animals were randomized into seven treatment groups (n = 7): Adaptation phase (1st to 6th week) of the high-fat diet (HFD) was realized with all groups and control group (CT) treatment that received ad libitum standard diet and water; HFD (HF) treatment that received ad libitum high-fat diet and water; HFD with fish oil supplement (HF + FO) treatment; HFD with physical exercise (HF + Ex) treatment; HFD with fish oil supplement and physical exercise (HF + FO + Ex) treatment; HFD with chia oil supplement (HF + CO) treatment; and HFD with chia oil supplement and physical exercise (HF + CO + Ex) treatment. Beginning at 60 days old, all animals in the HFD groups underwent an HFD induction period in which they had ad libitum access to HFD, standard ration and water. At seven weeks post-adaptation (102 days old), rats began the experimental phase in which the fish oil, chia oil, and physical exercise groups began oil supplement intake and the physical exercise protocol (Fig. 9). All procedures with the animals were carried out from 1 to 6 pm.
Dietary composition. At 60 days of age, rats were maintained on standard rat chow (commercial Supralab) or began the HFD induction period. The HFD used in this research was previously described by Estadella et al. 35 and consisted of a hypercaloric mixture (normoproteic and HFD) containing ground and mixed commercial Supralab ration, roasted peanuts, and milk chocolate and cornstarch in a 3:2:2:2. The high-fat diet was composed by lipids (59%), carbohydrates (28%) and proteins (13%). The proximate composition of the experimental diets was evaluated according to the analytical methods recommended by the Association of Official Analytical Chemists. The commercial diet was composed of 24.11% of proteins, 4.27% of lipids and 52.20% of carbohydrates,  (Table 3).
Aerobic physical exercise protocol. Rats in the aerobic physical exercise groups were subjected to 30 min of swimming while wearing a weighted vest in a tank divided into sections using plastic dividers. The swimming tank was filled with water maintained at 29 °C. During the 1-week adaption phase, the rat was habituated to the swimming protocol and the vest without weights attached (Fig. 9). The training protocol was conducted three times per week. Following the adaptation phase, a weight corresponding to 3.5% of the animal's total mass was attached to the vest at the posterior region of the thorax that corresponds to a 70% exercise intensity moderate 37 . This intensity was adjusted to 70% exercise intensity at the end of the first four weeks of training in order to avoid adaptation to the protocol.
Samples collection. At 172 days of age, 48 h after the last physical training session, after sacrifice, the animals remained overnight fasting for 12 h, had their blood collected under intraperitoneal anesthesia of ketamine (60 mg/kg) and xylazine hydrochloride (1 mg/kg), abdominal-pelvic laparotomy was performed, the ventral prostate and epididymal, retroperitoneal and mesenteric adipose tissues were removed, weighed, and processed for future analysis. The slides were analyzed and photographed in a light microscope, model AxioCam ECR5s Zeiss.
Blood sample. Serum levels of total cholesterol (TC), high-density lipoprotein (HDL), and TAG were analyzed by blinded experimenters using a colorimetric method with Cobas C111 equipment (Roche Diagnostics- Figure 9. Timeline of intervention protocol for 14 weeks of a high-fat diet, fish oil supplementation, chia oil, and aerobic physical exercise. www.nature.com/scientificreports/ Brazil) and the ROCHE commercial kit according to the manufacturer's instructions. The TC/HDL levels were calculated based on total cholesterol (mg/dl) divided by HDL (mg/dl), the VLDL levels were calculated by TAG divided per five, the LDL was calculated using total cholesterol values minus HDL mg/dl minus VLDL ratios.
Body weight and nutritional analyses. Body weight was also measured, to evaluate weight gain, we calculated body mass gain (Δ = final weight-starting weight). Relative prostate weight, used to evaluate the growth of prostate in different interventions, was determined as the ratio between absolute prostate weight and total animal bodyweight (g). During the experimental period, weekly consumption of water and food and changes in rat body mass were monitored. Food intake value and caloric value of ration for rodents (3 kcal/g for standard ration and 9 kcal/g for HFD) were used to obtain total energy consumption (TEI, kcal/day = average food consumption per day [g] × 3) and (ii) feed efficiency (FE, g/kcal = mean bodyweight gain/total TEI mean) 38 .
Histologic analysis of prostate. The  Immunofluorescence analysis. To block the endogenous peroxidase, the sections were subjected to a solution of hydrogen peroxide + methanol and peroxidase block. Protein blockade was performed by incubation in a blocking solution with bovine serum albumin for 1 h diluted with PBS buffer. The sections were subjected to reaction with specific primary antibody IL-10 (NYRm, sc-73309) and incubated in a humid chamber overnight. After wash, all sections were incubated at room temperature with FITIC (goat anti-mouse (626511), Invitrogen NOVEX), and the DAPI was applied (DAPI:DI306). The sections were mounted with Vectashield (H-1000, CA94010 Burlingame, Vector Laboratories) and examined using an inverted confocal microscope. The intensity of immunoreactivity of IL-10, antigens was examined in 10 fields per animal using Image-J software version 1.50i (National Institutes of Health, Bethesda, MD, USA), and the percentage of tissue marking was quantified for each image and used for percentage for the area.
Quantitative RT-PCR. The prostate samples were stored in the freezer at -80ºC, immersed in trizol, crushed in the tissue homogenizer, and submitted to the Trizol extraction protocol, following the protocol of the manufacturer. The concentration of the total RNA recovered was measured by spectrophotometry, all samples of total RNA were treated with DNAse before being submitted to RT-qPCR, according to the instructions of the DNAse I-Amplification Grade. Reverse transcription was performed according to the high capacity protocol using random primers as a primer oligonucleotide. The expression of NOS2, GSS, NFR2, SOD1, and CAT genes was evaluated by real-time PCR (Table 4), and for the normalization of the relative expression of the target genes, mean expression values of the GAPDH gene were used 40 . The initial standardization of real-time PCR amplification occurred on an Applied Biosystems 7500 Real-Time PCR Systems thermocycler. The calibration curve for each gene under study was made with serial dilutions of a pool of cDNA synthesized from 20 μg of prostate mRNA.
Statistical analysis. Analysis of variance was performed for repeated measures with a 95% confidence interval adjusted by the control variables group and time. Mann-Whitney and Chi-Square statistical analysis were performed to compare the histopathological data of the ventral prostate, the analysis of variance of bidirectional estimated marginal means (ANOVA) was used to compare the mobility of the seven groups analyzed, followed by the post-test Tukey's. For nutritional analyzes up to 6 weeks samples was used between groups a Student's t-test for independent. All analyzes were performed using the SPSS version 25 statistical program, the p-value < 0.05 was considered statistically significant.  Table 4. Sequences of forward and reverse primers used for RT-PCR. Determined by the formula 2(-ΔCq) (Pfaffl, 2001). GSS Glutathione synthetase, NRF-2 Nuclear factor erythroid 2-related factor-2, NOS-2 Nitric Oxide Synthase-2, SOD-1 Superoxide Dismutase, CAT catalase, GAPDH Glyceraldehyde 3-phosphate dehydrogenase gene. GSS  F-TAT CTC TGC CAG CTT TGG GG  95  R-TCT TGG AAG CTT CGT TGG TCT   NFR2  F-TCC ATT CCC GAG TTA CAG TGTC  91  R-TCT CTG TCA GTG TGG CCT CT   NOS2  F-AAA CAA CAG GAA CCT ACC AGCT  100  R-GAC CAC TGA ATC CTG CCG AT   SOD1  F-GCG TCA TTC ACT TCG AGC AG  191  R-CCT CTC TTC ATC CGC TGG AC   CAT  F-GCG GAT TCC TGA GAG AGT GG  188  R-GAG GGT CAC GAA CTG TGT CA   GAPDH  F-CCA TCA CCA TCT TCC AGG AG  102  R-TCT CCA TGG TGG TGA AGA CA