Efficacy of Carotenoid-Loaded Gelatin Nanoparticles in Reducing Plasma Cytokines and Adipocyte Hypertrophy in Wistar Rats

Abstract

The present study investigated the effect of gelatin-based nanoparticles (EPG) loaded with a carotenoid-rich crude extract (CE) on systemic and adipose tissue inflammatory response in a model with inflammation induced by a high glycemic index and high glycemic load diet (HGLI). Nanoparticles synthesized were characterized by different physical and chemical methods. The in vivo investigation evaluated Wistar rats (n = 20, 11 days, adult male with 21 weeks) subdivided into untreated (HGLI diet), conventional treatment (nutritionally adequate diet), treatment 1 (HGLI + crude extract (12.5 mg/kg)), and treatment 2 (HGLI + EPG (50 mg/kg)) groups. Dietary intake, caloric intake and efficiency, weight, inflammatory cytokines tissue concentration, visceral adipose tissue (VAT) weight, histopathological analysis, and antioxidant activity in plasma and VAT were investigated. EPG showed the same physical and chemical characteristics as previous batches (95.2 nm, smooth surface, and chemical interactions between materials). The EPG-treated group was the only group promoting negative ∆dietary intake, ∆caloric efficiency, and ∆weight. In addition, it presented a significant reduction (p < 0.05) in IL-6 and leptin levels and a greater presence of multilocular adipocytes. The results suggest that EPG can act as a nutraceutical in adjuvant therapy for treating inflammatory diseases associated with adipose tissue accumulation.

Keywords: Cucumis melo L.; adiposity; anti-inflammatory agents; nanoparticles.

Figure 1

Characterization of gelatin-based nanoparticles containing carotenoid-rich extract of Cantaloupe melons obtained by the O/W emulsification technique. (A) Representative Scanning Electron Microscopy image. (B) Laser diffraction. (C) Fourier Transform Spectroscopy: a—EPG: crude extract rich in carotenoids from Cantaloupe melons nanoencapsulated in porcine gelatin; b—porcine gelatin; c—Tween 20; d—CE: crude extract rich in carotenoids from Cantaloupe melons. O/W: oil in water.

Figure 2

Plasma concentrations of IL-6, TNF-α, and leptin in adult male Wistar rats (31 weeks) with inflammation induced by the HGLI diet submitted to different treatments and evaluated after ten days of the experiment (11th day). (A) IL-6. (B) TNF-α. (C) Leptin. No treatment: HGLI diet + 1 mL of water by gavage; conventional treatment: nutritionally adequate diet (Labina^® feed) + 1 mL of water per gavage; test treatment 1: HGLI diet + 1 mL of CE at a concentration of 12.5 mg/kg by gavage; test treatment 2: HGLI diet + 1 mL of EPG at a concentration of 50 mg/kg by gavage. HGLI diet: mixture composed of Labina^®, condensed milk, and sugar (1:1:0.21 w/w/w); HGLI: high glycemic index and high glycemic load diet; CE: crude extract rich in carotenoids from Cantaloupe melons; EPG: crude extract rich in carotenoids from Cantaloupe melons nanoencapsulated in porcine gelatin. Values are expressed as mean (standard deviation). For the evaluation of data normality, the Kolmogorov–Smirnov test was used. Plasma concentrations of IL-6, TNF-α, and leptin showed a parametric distribution; therefore, the ANOVA test with Tukey’s post-test was used to determine the significant differences between the evaluated groups. Values of p ≤ 0.05 (*** p < 0.0001) were considered statistically significant.

Figure 3

Weight of different types of visceral adipose tissue of adult male Wistar rats (31 weeks), with inflammation induced by HGLI diet, submitted to different treatments and evaluated after ten days of the experiment (11th day). (A) Retroperitoneal adipose tissue. (B) Epididymal adipose tissue. (C) Perirenal adipose tissue. No treatment: HGLI diet + 1 mL of water by gavage; conventional treatment: nutritionally adequate diet (Labina^® feed) + 1 mL of water per gavage; test treatment 1: HGLI diet + 1 mL of CE at a concentration of 12.5 mg/kg by gavage; test treatment 2: HGLI diet + 1 mL of EPG at a concentration of 50 mg/kg by gavage; HGLI diet: mixture composed of Labina^®, condensed milk and sugar (1:1:0.21 w/w/w); HGLI: high glycemic index and high glycemic load diet; CE: crude extract rich in carotenoids from Cantaloupe melons; EPG: crude extract rich in carotenoids from Cantaloupe melons nanoencapsulated in porcine gelatin. Values are expressed as mean (standard deviation). The weights of the retroperitoneal, epididymal, and perirenal adipose tissues showed a parametric distribution, and therefore, the ANOVA test with Tukey’s post-test was used to determine the significant differences. Values of p ≤ 0.05 were considered statistically significant.

Figure 4

Representative images of histological sections of adipose tissue from adult male Wistar rats (31 weeks), with inflammation induced by the HGLI diet, submitted to different treatments and evaluated after ten days of the experiment (11th day), stained with hematoxylin-eosin (HE) belonging to the following: (A) Untreated group. (B) Conventional treatment. (C) Test treatment 1. (D) Test treatment 2. Total magnification 100× (Objective Lens 10×), scale bar: 200 μm. Panoramic evidence of the presence of white adipose tissue consisting of numerous unilocular adipocytes () primarily intact, showing focal areas of membrane destruction (), extensive regions of lipolysis channels (), vast areas of fibrosis consisting of dense non-patterned connective tissue in areas of adipocyte destruction (), and the presence of the focal regions of multilocular adipocytes (). No treatment: HGLI diet + 1 mL of water by gavage; conventional treatment: nutritionally adequate diet (Labina^® feed) + 1 mL of water per gavage; test treatment 1: HGLI diet + 1 mL of CE at a concentration of 12.5 mg/kg by gavage; test treatment 2: HGLI diet + 1 mL of EPG at a concentration of 50 mg/kg by gavage; HGLI diet: mixture composed of Labina^®, condensed milk and sugar (1:1:0.21 w/w/w); HGLI: high glycemic index and high glycemic load diet; CE: crude extract rich in carotenoids from Cantaloupe melons; EPG: crude extract rich in carotenoids from Cantaloupe melons nanoencapsulated in porcine gelatin.

Figure 5

TNF-α concentration in visceral adipose tissue of adult male Wistar rats (31 weeks) with inflammation induced by HGLI diet, submitted to different treatments and evaluated after ten days of the experiment (11th day). (A) Retroperitoneal adipose tissue. (B) Epididymal adipose tissue. (C) Perirenal adipose tissue. No treatment: HGLI diet + 1 mL of water by gavage; conventional treatment: nutritionally adequate diet (Labina^® feed) + 1 mL of water per gavage; test treatment 1: HGLI diet + 1 mL of CE at a concentration of 12.5 mg/kg by gavage; test treatment 2: HGLI diet + 1 mL of EPG at a concentration of 50 mg/kg by gavage; HGLI diet: mixture composed of Labina^®, condensed milk and sugar (1:1:0.21 w/w/w); HGLI: high glycemic index and high glycemic load diet; CE: crude extract rich in carotenoids from Cantaloupe melons; EPG: crude extract rich in carotenoids from Cantaloupe melons nanoencapsulated in porcine gelatin. Values are expressed as mean (standard deviation). The TNF-α concentrations in the epididymal and perirenal adipose tissues showed a parametric distribution; therefore, the ANOVA test with Tukey’s post-test was used to determine the significant differences. The TNF-α concentration of the retroperitoneal adipose tissue showed a non-parametric distribution; therefore, the Kruskal–Wallis test with Dunn’s post-test was used to verify differences between the assessed groups. Values of p ≤ 0.05 were considered statistically significant.

Figure 6

IL-6 concentration in visceral adipose tissue of adult male Wistar rats (31 weeks) with inflammation induced by HGLI diet, submitted to different treatments and evaluated after ten days of the experiment (11th day). (A) Retroperitoneal adipose tissue. (B) Epididymal adipose tissue. (C) Perirenal adipose tissue. No treatment: HGLI diet + 1 mL of water by gavage; conventional treatment: nutritionally adequate diet (Labina^® feed) + 1 mL of water per gavage; test treatment 1: HGLI diet + 1 mL of CE at a concentration of 12.5 mg/kg by gavage; test treatment 2: HGLI diet + 1 mL of EPG at a concentration of 50 mg/kg by gavage; HGLI diet: mixture composed of Labina^®, condensed milk and sugar (1:1:0.21 w/w/w); HGLI: high glycemic index and high glycemic load diet; CE: crude extract rich in carotenoids from Cantaloupe melons; EPG: crude extract rich in carotenoids from Cantaloupe melons nanoencapsulated in porcine gelatin. Values are expressed as mean (standard deviation). The IL-6 concentrations in adipose tissues showed a parametric distribution, and therefore, the ANOVA test with Tukey’s post-test was used to determine the significant differences between the evaluated groups. Values of p ≤ 0.05 were considered statistically significant.

Figure 7

Malondialdehyde in plasma and visceral adipose tissue of adult male Wistar rats (31 weeks) with inflammation induced by HGLI diet, submitted to different treatments and evaluated after ten days of the experiment (11th day). (A) Plasma. (B) Retroperitoneal adipose tissue. (C) Epididymal adipose tissue. No treatment: HGLI diet + 1 mL of water by gavage; conventional treatment: nutritionally adequate diet (Labina^® feed) + 1 mL of water per gavage; test treatment 1: HGLI diet + 1 mL of CE at a concentration of 12.5 mg/kg by gavage; test treatment 2: HGLI diet + 1 mL of EPG at a concentration of 50 mg/kg by gavage; HGLI diet: mixture composed of Labina^®, condensed milk and sugar (1:1:0.21 w/w/w); HGLI: high glycemic index and high glycemic load diet; TBARS: thiobarbituric acid reactive substances; CE: crude extract rich in carotenoids from Cantaloupe melons; EPG: crude extract rich in carotenoids from Cantaloupe melons nanoencapsulated in porcine gelatin. Values are expressed as mean (standard deviation). Plasma and retroperitoneal adipose tissue concentrations showed non-parametric distribution, so the Kruskal–Wallis test with Dunn’s post-test was used to verify differences between the evaluated groups. The concentration in the epididymal adipose tissue showed a parametric distribution; therefore, the ANOVA test with Tukey’s post-test was used to determine the significant differences between the evaluated groups. Values of p ≤ 0.05 (** p = 0.01) were considered statistically significant.

Figure 8

Sulfhydryls in plasma and visceral adipose tissue of adult male Wistar rats (31 weeks) with inflammation induced by HGLI diet, submitted to different treatments and evaluated after ten days of the experiment (11th day). (A) Plasma. (B) Retroperitoneal adipose tissue. (C) Epididymal adipose tissue. No treatment: HGLI diet + 1 mL of water by gavage; conventional treatment: nutritionally adequate diet (Labina^® feed) + 1 mL of water per gavage; test treatment 1: HGLI diet + 1 mL of CE at a concentration of 12.5 mg/kg by gavage; test treatment 2: HGLI diet + 1 mL of EPG at a concentration of 50 mg/kg by gavage; HGLI diet: mixture composed of Labina^®, condensed milk and sugar (1:1:0.21 w/w/w); HGLI: high glycemic index and high glycemic load diet; CE: crude extract rich in carotenoids from Cantaloupe melons; EPG: crude extract rich in carotenoids from Cantaloupe melons nanoencapsulated in porcine gelatin. Values are expressed as mean (standard deviation). Plasma concentrations showed non-parametric distribution, so the Kruskal–Wallis test with Dunn’s post-test was used to verify the difference between the evaluated groups. The concentration in adipose tissues showed a parametric distribution; therefore, the ANOVA test with Tukey’s post-test was used to determine the significant differences between the evaluated groups. Values of p ≤ 0.05 were considered statistically significant.

Figure 9

Superoxide dismutase (SOD) activity in the plasma of adult male Wistar rats (31 weeks) with inflammation induced by the HGLI diet, submitted to different treatments and evaluated after ten days of the experiment (11th day). No treatment: HGLI diet + 1 mL of water by gavage; conventional treatment: nutritionally adequate diet (Labina^® feed) + 1 mL of water per gavage; test treatment 1: HGLI diet + 1 mL of CE at a concentration of 12.5 mg/kg by gavage; test treatment 2: HGLI diet + 1 mL of EPG at a concentration of 50 mg/kg by gavage; HGLI diet: mixture composed of Labina^®, condensed milk and sugar (1:1:0.21 w/w/w); HGLI: high glycemic index and high glycemic load diet; CE: crude extract rich in carotenoids from Cantaloupe melons; EPG: crude extract rich in carotenoids from Cantaloupe melons nanoencapsulated in porcine gelatin. Values are expressed as mean (standard deviation). Plasma concentrations showed non-parametric distribution, so the Kruskal–Wallis test with Dunn’s post-test was used to verify the difference between the evaluated groups. Values of p ≤ 0.05 were considered statistically significant.