Eicosapentaenoic acid/docosahexaenoic acid 1:1 ratio improves histological alterations in obese rats with metabolic syndrome

Abstract

Background: Marine polyunsaturated fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been associated with improvement in the Metabolic Syndrome (MS). The aim of this study is to evaluate how three fish-oil diets with different eicosapentaenoic acid/docosahexaenoic acid ratios (EPA/DHA ratio) affect the histology of liver, kidney, adipose tissue and aorta in a preliminary morphological study. This work uses an animal model of metabolic syndrome in comparison with healthy animals in order to provide information about the best EPA:DHA ratio to prevent or to improve metabolic syndrome symptoms.

Methods: 35 Wistar rats, as a control, and 35 spontaneously hypertensive obese rats (SHROB) were fed for 13 weeks with 3 different supplementation of fish oil containing EPA and DHA ratios (1:1, 2:1 and 1:2, respectively). All samples were stained with haematoxylin/eosin stain, except aorta samples, which were stained also with Verhoeff and van Gieson's stain. A histological study was carried out to evaluate changes. These changes were statistically analyzed using SPSS IBM 19 software. The quantitative data were expressed by mean ± SD and were compared among groups and treatments using ANOVA with post-hoc tests for parametric data and the U-Mann-Whitney for non-parametric data. Qualitative data were expressed in frequencies, and compared with contingency tables using χ² statistics.

Results: EPA:DHA 1:1 treatment tended to improve the density and the wrinkling of elastic layers in SHROB rats. Only Wistar rats fed with EPA:DHA 1:1 treatment did not show mast cells in adipose tissue and has less kidney atrophy. In both strains EPA:DHA 1:1 treatment improved inflammation related parameters in liver and kidney.

Conclusions: EPA:DHA 1:1 treatment was the most beneficial treatment since improved many histological parameters in both groups of rats.

Figure 1

Aortic wall thickness and wrinkling of elastic layers. Aortic wall thickness and wrinkling of elastic layers. Venhoeff & Van Gienson stain. (400x). Images on the left: Wistar rats. Images on the right: SHROB rats. (A and B) EPA:DHA 1:1 treatment. (C and D) EPA:DHA 2:1 treatment. (E and F) EPA:DHA 1:2 treatment. As we can see, images on the right, are thicker and less dense, and have less wrinkling, therefore are less functional.

Figure 2

Aorta results. At the top Histological cuts of abdominal aorta, Hematoxilin-eosin stain (400x) (A and B) EPA:DHA 1:1 in Wistar rat and SHROB rat respectively. We can observe the wall of aorta with the muscular cells nucleus and intuit elastic bands. As we can see SHROB rats presents a nuclei hypertrophy of aortic muscle cells than Wistar rats aortic muscle cells. When there was a nuclear hypertrophy of aortic muscle cells, the size of the cells increased and, consequently, the density decreased and the elastic layers were less wrinkled. This indicates a decreased health status. At the bottom four graphics (C) The different treatments did not affect the thickness of the aortic wall in either Wistar or SHROB rats. The same letters in different strains mean significant statistical differences. p = 0.023^a, p < 0.001^b. (D) In aortic lumen area there were no differences between Wistar and SHROB for any treatment or among treatments in each strain. This indicates that the portion of aorta was taken from the same zone, which enables results to be compared. (E) Wrinkling was significantly higher in Wistar than SHROB for the EPA:DHA 2:1, EPA:DHA 1:2 treatments (p = 0.002^g and p = 0.002^h respectively). The same letters mean significant statistical differences in different strains. (F) SHROB rats showed less density than Wistar without significant differences. In SHROB rats group the best value was given by EPA:DHA 1:1 treatment.

Figure 3

Adipose tissue results. At the top Histological cuts of adipose tissue, Hematoxilin-eosin stain (A) EPA:DHA 1:1 in SHROB rat (100x), we can observe the variable size of adipocytes and intuit the presence of inflammatory cells. (B) EPA:DHA 1:2 in SHROB rat (300x) mast cells marked up by black arrows. At the bottom four graphics in which: (W) refers to Wistar strain, (S) refers to SHROB strain. (A) EPA:DHA 1:1, (B) EPA:DHA 2:1, (C) EPA:DHA 1:2. Equal letters, a, b, c, d, e means statistical differences among strains. (C) In both strain of rats there weren’t statistically significant differences in the proportion of different adipocyte sizes among the treatment groups. Comparing the two strains, it was higher in SHROB rats than in WISTAR rats for the EPA:DHA 1:1 diet (p = 0.031^a). (D) As far as the presence of white cells, and therefore inflammation, is concerned; there was a higher effect of PUFA n-3 in Wistar rats than in SHROB rats. In these strain only EPA:DHA 1:2 treatment doesn’t show a significant improvement (p = 0.001^c) (E) Mast cells % in Wistar rats showed differences with EPA:DHA 1:1 (p < 0.001^*). And in SHROB rats (p = 0.022^{^}, p = 0.018^{^} respectively) Comparing the two strains, in the EPA:DHA 1:1 diet, SHROB showed a greater presence of mast cells than WISTAR (p = 0.001^b). (F) The proportion of macrophages in SHROB rats was lower than in Wistar rats but no significant differences were founded.

Figure 4

Liver results. Graphics of liver results in which: (W) refers to WISTAR stain, (S) refers to SHROB stain. (A) EPA:DHA 1:1, (B) EPA:DHA 2:1,(C) EPA:DHA 1:2. Equal letters, a, b, c, d, e means statistical differences among strains. (A) Liver samples of WISTAR rats no showed steatosis. Consequently, between WISTAR and SHROB statistically significant differences were observed in all treatment groups (EPA:DHA 1:1 p = 0.001, EPA:DHA 2:1 p = 0.001, EPA:DHA 1:2 p = 0.031). The graphics showns the grade of stetosi in SHROB rats, its location is shown in graphic (B) we can see how in SHROB rats steatosis tended to be periportal, but no statistically significant differences were found between treatment groups. (C) SHROB rats also showed greater lobular inflammation than Wistar. There were significant differences between strains in the EPA:DHA 2:1 and EPA:DHA 1:2 treatments (p = 0.025* and p = 0.02^{^}, respectively). (D) The presence of microgranulomas was higher in SHROB rats than in Wistar, and the differences were significant in three tretament groups: EPA:DHA 2:1 (p = 0.005a). In SHROB fewer microgranulomas were present in the EPA:DHA 1:1 and EPA:DHA 1:2 treatments, but there were no significant differences with other groups. (E) We found lipogranulomas in the Wistar EPA:DHA 2:1 group and in all the groups of SHROB rats, with significant differences between strains for EPA:DHA 1:1 (p < 0.001), EPA:DHA 2:1 (p = 0.001), EPA:DHA 1:2 (p = 0.002). (F) Portal inflammation was present in both Wistar and SHROB rats but differences were only significant between strains in the EPA: DHA 1:1 treatment (p < 0.001). No differences between treatments were found in the same strain.

Figure 5

Histological cuts of liver. Histological cuts of liver, Hematoxilin-eosin stain (A) EPA:DHA 2:1 treatment in SHROB rat (40x), shown steatosis with periportal location (B) EPA:DHA 2:1 treatment in SHROB rat (400x), lobular inflammation marked up with a black arrow. The picture also allows us to observe steatosi which is marked up, in part, with red arrows (C) EPA:DHA 2:1 in SHROB rat (600x), on the right some macrophages surrounding a lipidic drop, forming a lipogrnaulome and on the left a lipogranulome also formed by macrophages. The picture also allows us to observe steatosi at higher magnification (D) EPA:DHA 2:1 in Wistar rat (200x), portal inflammation, as we can see the inflammatory cells are surrounding portal space.

Figure 6

Kidney results. At the top Histological cuts of kidney, Hematoxilin-eosin stain. (A) EPA:DHA 1:1 in SHROB rat (40x) thyroidization zones are marked by black arrows. Thyroidization it’s a kind of atrophy which gives them the appearance of the follicles in the thyroid gland as we can see in the picture (hence the name). (B) EPA:DHA 1:2 in SHROB rat (200x), inflammation in kidney can be observed by the presence of inflammatory cells, also we can observe a normal glomerulus on the top and a glomerulus with glomerulosclerosis marked by a black arrow. At the bottom three graphics in which: (W) refers to Wistar strain, (S) refers to SHROB strain. (A) EPA:DHA 1:1, (B) EPA:DHA 2:1, (C) EPA:DHA 1:2. Equal letters, a, b, c, d, e means statistical differences among strains. (C) We found greater presence of glomerulosclerosis in SHROB rats than in Wistar rats, but significant differences were found only in the EPA:DHA 1:1 treatment (p = 0.005^a). (D) SHROB rats had greater thyroidization than Wistar rats in all treatment groups p = 0.003^a, p = 0.032^b, p = 0.005^c. (E) Atrophy was also higher in SHROB rats. EPA:1:1 doesn’t shown atrophy in either both treatments, but no significant differences were founded. (F) Inflammation were better also in EPA:DHA 1:1 treatments in both strains, but without significant differences.