Docosahexaenoic acid-flurbiprofen combination ameliorates metaflammation in rats fed on high-carbohydrate high-fat diet

Biomedicine & Pharmacotherapy 109 (2019) 233–241

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Docosahexaenoic acid-flurbiprofen combination ameliorates metaflammation in rats fed on high-carbohydrate high-fat diet Nahla E. El-Ashmawy1, Ghada M. Al-Ashmawy1, Asmaa A. Kamel1,

T

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Department of Biochemistry, Faculty of Pharmacy, Tanta University, Postal Code: 31527, Egypt

A R T I C LE I N FO

A B S T R A C T

Keywords: Metaflammation Obesity DHA Flurbiprofen AMPK Resolvin D1

Objective: Potential benefits of combining docosahexaenoic acid (DHA), an omega-3 fatty acid with flurbiprofen (Flu), a non-steroidal anti-inflammatory drug in ameliorating obesity remain to be elucidated. This study aimed to investigate the possible protective effects of DHA and Flu, either alone or in combination, against obesityinduced metaflammation and to clarify the underlying molecular mechanisms. Methods: Seventy-five male Wistar rats were divided into five groups: normal diet (ND) group, high-carbohydrate high-fat diet (HCHFD) control group, DHA group (HCHFD + 200 mg/kg DHA), Flu group (HCHFD + 10 mg/kg Flu), and DHA + Flu group (HCHFD + DHA + Flu). Treatments were administered orally daily for 8 consecutive weeks, parallel with the start of diets. Results: Plasma levels of glucose, insulin, and TGs were significantly reduced in DHA, Flu, and DHA + Flu treated groups, while HDL-C concentrations were significantly elevated in the same groups, compared to HCHFD control group. Only Flu and DHA + Flu groups showed a significant decrease in plasma levels of leptin, TC, and LDL-C, relative to HCHFD control group. Concentrations of phosphorylated adenosine monophosphate-activated protein kinase (pAMPK) and resolvin D1 (RvD1) in epididymal adipose tissue (EAT) were significantly increased in the three treated groups, compared with HCHFD control group. Expression of AMPK-α1 subunit in EAT was significantly increased, whereas expression of nuclear factor kappa B (NF-κB) was significantly decreased in EAT of the three treated groups, relative to HCHFD control group. Conclusions: Docosahexaenoic acid-flurbiprofen combination showed an ameliorative effect on obesity-associated metaflammation and its consequences in rats.

1. Introduction Obesity is a major health hazard marked by excessive body fat accumulation and elevated body mass index (BMI) that exceeds 30 kg/m2. It is prevalently escalating with a present count of more than 500 million having a high risk of morbidity [1] and it is likely to double by 2040 [2]. The most common cause of obesity was found to be a longterm imbalance between energy intake and expenditure [3], which occurs primarily due to sedentary lifestyles and unhealthy dietary habits, specifically diets rich in saturated fats and carbohydrates [4]. White adipose tissue (WAT) plays a crucial endocrine role in balancing metabolic homeostasis and inflammation-modulatory activities beyond the paradigm of fuel storage [5]. During conditions of prolonged over-nutrition, there is an expansion of adipose tissue mass through hypertrophy and hyperplasia, causing a reduction in tissue

vascularization which leads to areas with lower oxygen availability [6]. Furthermore, adipocytes undergo a profound change in their secretome profile by an increased expression of pro-inflammatory cytokines and alterations in the level of adipokines [7]. These changes result in an undesirable inflammatory cells infiltration [8] associated with an increased necrosis of adipocytes [9]. This chronic inflammation linked to metabolic cells (metaflammation) is deleterious and leads to local WAT dysfunction and systemic metabolic complications including insulin resistance, type 2 diabetes, dyslipidemia, etc. [10]. Monotherapies often offer small to moderate weight loss benefits, while polytherapies seem to show more promising results in the management of obesity and related comorbidities [11]. Therefore, development of new therapeutic strategies is of vital importance [12]. Flurbiprofen (Flu), a nonsteroidal anti-inflammatory drug (NSAID),

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Corresponding author at: Department of Biochemistry, Faculty of Pharmacy, Tanta University, Egypt. E-mail addresses: [email protected] (N.E. El-Ashmawy), [email protected] (G.M. Al-Ashmawy), [email protected] (A.A. Kamel). 1 Department of Biochemistry, Faculty of Pharmacy, Tanta University, Egypt. https://doi.org/10.1016/j.biopha.2018.10.049 Received 4 August 2018; Received in revised form 7 October 2018; Accepted 9 October 2018 0753-3322/ © 2018 Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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was revealed to have, in addition to cyclooxygenase (COX) inhibition, pleiotropic actions including decreasing endoplasmic reticulum stress and lowering circulating leptin levels [13,14]. However, the effects of flurbiprofen on the regulation of blood glucose and lipid profile in obesity along with related molecular pathways need to be clarified. Docosahexaenoic acid (DHA), an omega-3 fatty acid, was demonstrated to have potent anti-inflammatory, hypolipidemic, and insulinsensitizing properties [15]. Herein, it was hypothesized that these protective properties of DHA can be enhanced when provided in conjunction with Flu. Accordingly, the objective of the current study was to investigate the possible protective role of DHA and Flu against obesityassociated inflammation in rats.

Table 2 The composition of the powdered rat food provided in normal chow and high-carbohydrate high-fat diets.

2. Material and methods 2.1. Animals

Ingredients

Quantity (g/kg)

Yellow corn Casein Sun flower oil Cellulose Dicalcium phosphate Calcium carbonate Sodium chloride Sodium bicarbonate Antibiotic and antifungal Choline chloride L-lysine HCl DL-methionine

560 295 45 50 10.5 28 3 1 1 1 3 2.5

The powdered rat food was composed of 29.5% protein, 4.5% fat, 56% carbohydrate, 5% fiber and moisture.

Seventy-five male Wistar rats weighing 95–110 g at approximately five weeks of age, were purchased from the National Research Center, Giza, Egypt. Rats were housed in wire cages for one week under constant environmental conditions for adaptation and allowed free access to normal chow diet and water.

4 °C and 3000 rpm for 20 min using a cooling centrifuge (Sigma®, Germany). The obtained plasma samples were then separated and stored at −20 °C till biochemical assays. After blood collection, rats were sacrificed, then the liver and epididymal adipose tissue (EAT) samples were carefully dissected, washed with saline, dried, and weighed. The EAT samples were divided; one portion was used for histopathological examination, and the other portions were kept frozen at −80 °C for subsequent biochemical analysis.

2.2. Experimental design This study was conducted in accordance with the institutional guidelines for care and use of laboratory animals approved by the Research Ethical Committee (Faculty of Pharmacy, Tanta University, Egypt). After seven days of acclimatization, rats were randomly assigned into five groups (n = 15/group). The first group, the normal diet (ND) group, was maintained on a normal chow diet and water ad libitum. The other four groups were allowed free access to high-carbohydrate high-fat diet (HCHFD) in addition to 25% fructose provided in the drinking water for 8 weeks [16]. The composition of the normal chow and HCHF diets is shown in Tables 1 and 2. According to the given treatment, the four groups of HCHFD maintained rats were assigned as follows: HCHFD control group, DHA group, Flu group, and DHA + Flu group. Non-esterified DHA powder (eBioChem. Co., China) with high purity of > 99% was administered orally daily at a dose of 200 mg/kg [17]. Flu (Abbott Logistics BV Co., United Kingdom) was administered orally daily at a dose of 10 mg/kg [13]. DHA and Flu were prepared as suspension in 1% carboxymethylcellulose (CMC) [18]. ND group was given 1% CMC, the same vehicle given to the other groups. The treatment continued for eight weeks, and the body weights of rats were recorded weekly.

2.4. Histopathological examination EAT samples were fixed in 10% formalin solution, dehydrated using serial dilutions of alcohol, then embedded in paraffin. Tissue blocks were sectioned at 4 μm thickness using microtome (Leica®, Germany). The obtained tissue sections were stained with hematoxylin and eosin (H&E), examined using light microscope (Olympus®, Japan), and then photographed at ×200 magnification. Five fields per sample and three samples from each group were analyzed using Image J, an image analysis software, to determine adipocyte area which was calculated from the average value of the cell area in all of the measured fields [19]. Crown-like structures (CLS), indicative of inflammatory macrophages surrounding dead adipocytes, were also identified based on aggregates of nucleated cells surrounding individual adipocytes [20]. 2.5. Calculation of Lee index, change in body weight and body weight gain Lee index for each rat was calculated at the end of the experiment to verify obesity as follows: Lee index =

2.3. Specimen collection

Table 1 The composition of normal chow diet and high-carbohydrate high-fat diet (HCHFD).

*

Powdered rat food Fructose Beef tallow Sweetened condensed milk Vitamins and trace minerals Fiber Energy (Kcal/Kg)

Normal chow diet (g/kg)

HCHFD (g/kg)

945 – – – 25 30 3615

200 400 200 145 25 30 4518

Body weight (g ) Naso − anal length (cm)

. If the Lee

index values were ≤ 0.3, rats were considered to be of normal weight, whereas for values > 0.3, rats were classified to be obese [21]. Change in body weight for ND, DHA, Flu, and DHA + Flu groups was measured weekly according to Choi et al. [22] as follows: change in body weight (g/week) = mean body weight of HCHFD control group (g) – mean body weight of each group (g). Body weight gain for each of the five groups was measured according to Choi et al. [22] as follows: body weight gain (g) = final body weight – initial body weight.

At the end of the experiment, rats were fasted overnight and blood was withdrawn by cardiac puncture under diethyl ether anesthesia and transferred into EDTA-lined tubes. Blood samples were centrifuged at

Diet composition

3

2.6. Determination of plasma glucose and lipid profile Fasting plasma level of glucose was determined by the enzymatic colorimetric method described by Trinder [23], using kits obtained from Biodiagnostic Co., Egypt. Plasma triglycerides (TGs), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) levels were measured according to enzymatic colorimetric methods described by Bucolo & David [24], Allain et al. [25], and Friedewald et al. [26], respectively, using kits obtained from Biodiagnostic Co., Egypt. Plasma low-density lipoprotein cholesterol (LDL-C) level was then calculated

* The detailed composition of powdered rat food (El-Gibali®, Tanta, Egypt) is shown in Table 2. 234

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Fig. 1. (A) Body weight during the experimental period. (B) Change in body weight measured weekly. (C) Body weight gain. Data are represented as a mean ± SD (n = 15/group), significance was set at p < 0.05; a: significant vs ND group, b: significant vs HCHFD control group, c: significant vs DHA group, d: significant vs Flu group. ND group: normal chow diet-fed rats for 8 weeks; HCHFD control group: high carbohydrate-high fat diet-fed rats for 8 weeks; DHA group: (HCHFD + 200 mg/ kg of docosahexaenoic acid); Flu group: (HCHFD + 10 mg/kg of flurbiprofen); DHA + Flu group: (HCHFD + DHA + Flu). Treatments were administered orally daily for 8 consecutive weeks, parallel with the start of diets.

follows; AMPK α1-subunit gene forward primer: (5`-GGTCCTGGTGGT TTCTGTTG-3`), reverse primer: (5`-ATGATGTCAGATGGTGAATT-3`), NF-κB gene forward primer: (5`-GAACTTGTGGGGAAGGACTG-3`), reverse primer: (5`-GGGGTTATTGTTGGTCTGGA-3`), Glyceraldehyde 3phosphate dehydrogenase (GAPDH) gene forward primer: (5`-TGGAC CACCCAGCCCAGCAAG-3`), reverse primer: (5`-GGCCCCTCCTGTTGT TATGGGGT-3`). PCR program was adjusted to start with initial activation for 2 min at 94 °C, followed by 45 cycles (94 °C for 5 s, 62 °C for 10 s, and 72 °C for 20 s) using RT- qPCR system Pikoreal 5100 (Thermo Fisher Scientific Co., Finland). Each sample was analyzed and normalized to the level of reference gene (GAPDH) and expressed as relative copy number (RCN). Threshold cycle (Ct) values of the samples were calculated, and transcript levels were analyzed by the 2−ΔCt method [28].

according to Friedewald et al. [26] as follows: LDL-C (mg/dL) = TC – TGs/5 – HDL-C. 2.7. Determination of fasting plasma insulin and leptin Plasma insulin level was assayed using rat insulin enzyme-linked immunosorbent assay (ELISA) kit, purchased from Calbiotech Inc Co., USA. Plasma leptin was assayed using rat leptin ELISA kit, obtained from Shanghai Sunred Biological Technology Co., China. 2.8. Calculation of HOMA-IR Insulin resistance has been estimated according to the homeostasis model assessment-estimated insulin resistance (HOMA-IR) index described by Matthews et al. [27], which was calculated as follows: [fasting insulin (μIU/mL) × fasting plasma glucose (mg/dL)] / 405.

2.11. Statistical analysis Analysis of data was performed using statistical package for social science (SPSS) software version 22 [29]. Data are presented as mean ± SD and % change. Statistical comparison among groups was performed by one-way analysis of variance (ANOVA) using Fisher’s leastsignificant differences (LSD) method for comparison between two groups. Statistical significance was set at p < 0.05.

2.9. Measurement of resolvin D1 (RvD1) and phosphorylated adenosine monophosphate-activated protein kinase (pAMPK) concentrations in the EAT In addition to anthropometric measurements and biochemical analysis of the previous metabolic parameters in plasma, the local WAT inflammation needed to be investigated by measuring the concentrations of both RvD1, a pro-resolution signal, and pAMPK, a key regulator of inflammatory defense. EAT homogenate was prepared by weighing 0.1 g of EAT, followed by homogenization in 1 ml of 0.1 M phosphate buffered saline (pH 7.4) using Polytron homogenizer (Kinematica®, Switzerland) to prepare 10% w/v homogenate. The homogenates were centrifuged at 3000 rpm for 20 min at 4 °C, and the isolated supernatants were used to measure RvD1 and pAMPK concentrations, using rat ELISA kits obtained from Shanghai Sunred Biological Technology Co., China.

3. Results 3.1. Effect on body weight, Lee index, and weight of liver and EAT Body weight of rats of all groups over the 8 weeks of the experiment is shown in Fig. 1A. Final body weight of HCHFD control group was significantly increased by 68% (p < 0.05), compared with ND group. In contrast, both DHA and Flu groups showed significant reductions in final body weight by 9% and 26% (p < 0.05), respectively, compared with HCHFD control group. Interestingly, DHA + Flu group decreased body weight to a level near to that observed in ND group throughout the experimental period (Fig. 1B). Body weight gain in DHA, Flu, and DHA + Flu groups was significantly lower by 13% (143.93 ± 11.24 g, p < 0.05), 42% (95.87 ± 9.37 g, p < 0.05), and 53% (69.47 ± 7.17 g, p < 0.05), respectively, than that of HCHFD control group (166 ± 12.90 g), as shown in Fig. 1C. EAT weights were 90% (p < 0.05) higher in HCHFD control group than in ND group. DHA, Flu and DHA + Flu groups showed a significant reduction in EAT weight by 7%, 31%, and 41% (p < 0.05), respectively, compared with HCHFD control group. Liver weight was significantly higher by 60% (p < 0.05) in HCHFD control group than that observed in ND group. Flu and DHA + Flu groups showed a significant reduction in liver weight by 22% and 27% (p < 0.05), respectively, while there was no significant change in DHA group,

2.10. Gene expression of AMPK α1-subunit and nuclear factor kappa B (NF-κB) by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) Total RNA was extracted from frozen EAT using total RNA extraction kit of Bioer Technology (China) according to the manufacturer’s instructions. Concentration and purity of the obtained RNA were detected spectrophotometrically at 260/280 nm using a ScanDrop Nanovolume spectrophotometer (Analytik Jena®, Italy). The extracted RNA was then reversely transcribed into complementary DNA (cDNA) using HiSenScript RH (-) cDNA synthesis kit (iNtRON Biotechnology, Korea). The obtained cDNA was amplified using SensiFAST ™ SYBR No-ROX kit (Bioline, USA) as described by the manufacturer. Sequence of primers (Invitrogen Co., USA) used in RT-qPCR were as 235

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Table 3 Initial and final body weight, naso-anal length, Lee index, epididymal adipose tissue (EAT) weight, and liver weight of rats in all experimental groups. Parameters

ND

HCHFD control

Initial body weight (g) Final body weight (g) Naso-anal length (cm) Lee index EAT weight (g) Liver weight (g)

113.47 ± 4.86 166.67 ± 10.71 19.20 ± 0.71 0.29 ± 0.01 1.78 ± 0.29 5.0 ± 0.56

115.27 ± 8.17 279.53 ± 21.8 19.69 ± 0.84 0.33 ± 0.01 a 3.38 ± 0.32 a 7.97 ± 0.7 a

DHA 111.13 ± 5.21 254.47 ± 18.73 19.40 ± 0.76 0.33 ± 0.01 a 3.15 ± 0.34 a,b 7.56 ± 0.66 a

a

a,b

Flu

DHA + Flu

108.00 ± 5.29 208.20 ± 18.78 a,b,c 19.45 ± 0.8 0.30 ± 0.01 a,b,c 2.32 ± 0.3 a,b,c 6.21 ± 0.49 a,b,c

111.40 ± 6.57 180.60 ± 19.81 a,b,c,d 19.42 ± 0.74 0.29 ± 0.01 b,c,d 2.0 ± 0.24 b,c,d 5.80 ± 0.3 a,b,c

All values are expressed as mean ± SD (n = 15/group), significance was set at p < 0.05; a: significant vs ND group, b: significant vs HCHFD control group, c: significant vs DHA group, d: significant vs Flu group. ND group: normal chow diet-fed rats for 8 weeks; HCHFD control group: high carbohydrate-high fat diet-fed rats for 8 weeks; DHA group: (HCHFD + 200 mg/kg of docosahexaenoic acid); Flu group: (HCHFD + 10 mg/kg of flurbiprofen); DHA + Flu group: (HCHFD + DHA + Flu). Treatments were administered orally daily for 8 consecutive weeks, parallel with the start of diets.

Fig. 2. (A) Fasting plasma glucose level in rat groups. (B) Plasma insulin level in rat groups. (c) HOMA-IR in rat groups. Data are represented as a mean ± SD (n = 15/group), significance was set at p < 0.05; a: significant vs ND group, b: significant vs HCHFD control group, c: significant vs DHA group, d: significant vs Flu group. ND group: normal chow diet-fed rats for 8 weeks; HCHFD control group: high carbohydrate-high fat diet-fed rats for 8 weeks; DHA group: (HCHFD + 200 mg/ kg of docosahexaenoic acid); Flu group: (HCHFD + 10 mg/kg of flurbiprofen); DHA + Flu group: (HCHFD + DHA + Flu). Treatments were administered orally daily for 8 consecutive weeks, parallel with the start of diets.

compared with HCHFD control group. Initial and final body weight, naso-anal length, Lee index, as well as liver and EAT weights are presented in Table 3.

of HCHFD control group. Interestingly, DHA + Flu group showed a reduction in the plasma insulin to a level comparable to that of ND group. HOMA-IR score was significantly increased by 189% (19.40 ± 1.94, p < 0.05) in HCHFD control group compared to ND group (6.72 ± 0.73). On the other hand, the HOMA-IR score in DHA, Flu and DHA + Flu groups was significantly reduced by 36% (12.44 ± 1.08, p < 0.05), 49% (9.83 ± 1.05, p < 0.05), and 60% (7.84 ± 1.17, p < 0.05), respectively, relative to HCHFD control group. Changes in HOMA-IR score in all of the studied groups are represented in Fig. 2C.

3.2. Effect on fasting plasma glucose, insulin level, and HOMA-IR Plasma glucose and insulin levels in our studied groups are shown in Fig. 2(A & B). In this study, untreated animals fed on HCHFD exhibited significant increases in plasma glucose and insulin levels by 69% (165.07 ± 11.23 mg/dL, p < 0.05) and 70% (47.56 ± 3.08 μIU/mL, p < 0.05), respectively, compared to ND group (97.56 ± 7.67 mg/dL and 27.93 ± 2.15 μIU/mL, respectively). On the contrary, DHA, Flu and DHA + Flu groups showed a significant reduction in fasting plasma glucose by 20% (132.30 ± 7.00 mg/dL, p < 0.05), 25% (124.30 ± 11.75 mg/dL, p < 0.05), and 33% (110.04 ± 10.07 mg/dL, p < 0.05), respectively, compared with HCHFD control group. Increased levels of insulin evoked by HCHFD diet were significantly attenuated by DHA and Flu to be 20% (38.11 ± 2.31 μIU/mL, p < 0.05) and 32% (32.12 ± 2.72 μIU/mL, p < 0.05) lower than that

3.3. Effect on plasma leptin Plasma leptin concentrations were increased in HCHFD control group by 79% (239.84 ± 14.61 pg/mL, p < 0.05), compared with ND group. Rats of Flu and DHA + Flu groups showed significant reduction in plasma leptin by 23% (184.24 ± 8.68 pg/mL, p < 0.05) and 33% (159.79 ± 13.39 pg/mL, p < 0.05), respectively, while there was no significant change in DHA group (231.09 ± 11.66 pg/mL) in Fig. 3. (A) Plasma leptin level in rat groups. (B) Plasma lipid profile in rat groups. Data are represented as a mean ± SD (n = 15/group), significance was set at p < 0.05; a: significant vs ND group, b: significant vs HCHFD control group, c: significant vs DHA group, d: significant vs Flu group. ND group: normal chow diet-fed rats for 8 weeks; HCHFD control group: high carbohydrate-high fat diet-fed rats for 8 weeks; DHA group: (HCHFD + 200 mg/ kg of docosahexaenoic acid); Flu group: (HCHFD + 10 mg/kg of flurbiprofen); DHA + Flu group: (HCHFD + DHA + Flu). Treatments were administered orally daily for

8 consecutive weeks, parallel with the start of diets. 236

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AMPK- α1 subunit by 81% (1.34 ± 0.064 RCN, p < 0.05), 111% (1.56 ± 0.08 RCN, p < 0.05), and 136% (1.75 ± 0.02 RCN, p < 0.05), respectively, compared to untreated rats fed on HCHFD. On the other hand, EAT from HCHFD control group showed a significant increase in the mRNA levels of NF-κB by 104% (1.06 ± 0.016 RCN, p < 0.05), compared to ND group (0.52 ± 0.014 RCN) (Fig. 5B). DHA, Flu, and DHA + Flu treatments significantly decreased the expression of NF-κB by 20% (0.85 ± 0.006 RCN, p < 0.05), 29% (0.75 ± 0.013 RCN, p < 0.05), and 39% (0.65 ± 0.008 RCN, p < 0.05), respectively, relative to untreated HCHFD-fed rats.

comparison with HCHFD control group (Fig. 3A). 3.4. Effect on plasma lipid profile HCHFD feeding without receiving any treatment resulted in significant increases in plasma TC, TGs, and LDL-C by 41% (96.27 ± 6.52 mg/dL, p < 0.05), 65% (118.45 ± 9.76 mg/dL, p < 0.05), and 100.1% (56.74 ± 7.40 mg/dL, p < 0.05), respectively, compared to ND group (68.33 ± 5.41 mg/dL, 71.84 ± 6.23 mg/dL, and 28.21 ± 3.60 mg/dL, respectively). HCHFD control group showed significant lower HDL-C levels by 41% (15.83 ± 1.09 mg/dL, p < 0.05), compared to ND group (27.04 ± 1.74 mg/dL). DHA, Flu and DHA+Flu groups showed significant reduction in plasma TGs by 21% (93.49 ± 8.87 mg/dL, p < 0.05), 30% (82.67 ± 7.68 mg/dL, p < 0.05), and 39% (72.05 ± 6.44 mg/dL, p < 0.05), respectively, relative to HCHFD control group. Both Flu and DHA + Flu groups showed 13% (84.12 ± 6.77 mg/ dL, p < 0.05) and 24% (72.74 ± 5.10 mg/dL, p < 0.05), respectively, lower levels of plasma TC than HCHFD control group, while there was no significant change in DHA group (94.88 ± 7.47 mg/dL) relative to HCHFD control group. Similarly, Flu and DHA + Flu groups significantly decreased LDL-C concentrations by 29% (40.06 ± 6.45 mg/ dL, p < 0.05) and 42% (32.82 ± 4.60 mg/dL, p < 0.05), respectively, compared to HCHFD control group. DHA, Flu, and DHA + Flu treatments increased plasma HDL-C levels significantly by 31% (20.73 ± 1.52 mg/dL, p < 0.05), 39% (21.96 ± 1.72 mg/dL, p < 0.05), and 63% (25.75 ± 2.18 mg/dL, p < 0.05), respectively, relative to HCHFD control group. The effects of different treatments on plasma lipids are summarized in Fig. 3B.

3.7. Effect on EAT histology As shown in Fig. 6(A & B), adipocyte area of EAT was significantly increased in the HCHFD control group by 152% (1810.52 ± 353.06 μm2, p < 0.05) compared to ND group (715.85 ± 100.35 μm2, p < 0.05). Crown-like structures (CLS) were observed in HCHFD control group indicating infiltrated macrophages which surround dead adipocytes (Fig. 6B). On the contrary, rats fed on HCHFD and treated with DHA displayed a significant reduction in adipocyte area by about 33% (1220.24 ± 223.32 μm2, p < 0.05), compared to that of HCHFD control rats (Fig. 7A). Flu-treated group showed an EAT with significantly smaller adipocyte size by about 55% (809.80 ± 166.20 μm2, p < 0.05) than that of HCHFD control rats (Fig. 7B). Similarly, treatment with DHA + Flu tended to decrease the adipocyte area significantly to 795.25 ± 118.71 μm2, making it almost similar to that of ND group (Fig. 7C). Importantly, no CLS were observed in the three treated groups. 4. Discussion

3.5. Effect on RvD1 and pAMPK concentrations in EAT In the present study, we have demonstrated that rats fed on HCHFD diet for 8 weeks developed signs of low-grade inflammation concomitant with some metabolic abnormalities including body weight gain, fat deposition in liver and epididymal region, hyperglycemia, hyperinsulinemia, insulin resistance, and dyslipidemia. Weight gain observed in this study was driven primarily by increased energy intake from fat and reduced metabolic rate as a consequence of high fructose intake [30]. These findings were supported by the histopathological changes in EAT of HCHFD control group, which showed significant hypertrophy and inflammatory cell infiltration with appearance of CLS. These observations were in agreement with previous reports [3,16]. Our results indicated that treatment of rats with DHA or Flu significantly decreased the epididymal adipocyte area relative to HCHFD control group, an effect which was more observable in Flu group, whereas DHA + Flu group was found to have adipocyte area near to that of ND group. Furthermore, CLS were less detected in the EAT of the three treated groups, indicating the ameliorative effect of DHA and Flu against EAT inflammation. These findings were consistent with those of Bargut et al. [31], who showed that the treatment of mice fed a highfructose diet supplemented with DHA significantly decreased epididymal adipocyte area. The present work indicated that DHA-treated rats showed a

As shown in Fig. 4(A & B), EAT concentrations of RvD1 and pAMPK were significantly decreased in the HCHFD control group by 41% (0.77 ± 0.08 ng/g tissue, p < 0.05) and 42% (369.64 ± 59.09 ng/g tissue, p < 0.05), respectively, compared to ND group (1.29 ± 0.13 ng/g tissue and 640.43 ± 71.35 ng/g tissue, respectively). Rats of DHA, Flu, and DHA + Flu groups exhibited significant increases in the EAT concentrations of RvD1 by 42% (1.09 ± 0.13 ng/ g tissue, p < 0.05), 43% (1.10 ± 0.09 ng/g tissue, p < 0.05), and 56% (1.19 ± 0.12 ng/g tissue, p < 0.05), respectively, relative to HCHFD control rats. Similarly, DHA, Flu, and DHA + Flu treatments significantly increased EAT concentrations of pAMPK by 26% (465.31 ± 45.41 ng/g tissue, p < 0.05), 38% (508.32 ± 48.58 ng/g tissue, p < 0.05), and 63% (601.01 ± 60.66 ng/g tissue, p < 0.05), respectively, compared to HCHFD control rats. 3.6. Effect on expression of AMPK α1-subunit and NF-κB in EAT Fig. 5A shows that the expression of the AMPK-α1 subunit was significantly decreased in HCHFD-control group by 61% (0.74 ± 0.048 RCN, p < 0.05), relative to ND group (1.91 ± 0.051 RCN). Rats treated with DHA, Flu, and DHA + Flu showed increases in the expression of

Fig. 4. (A) Epididymal adipose tissue concentration of resolvin D1 in rat groups. (B) Epididymal adipose tissue concentration of pAMPK in rat groups. Data are represented as a mean ± SD (n = 15/group), significance was set at p < 0.05; a: significant vs ND group, b: significant vs HCHFD control group, c: significant vs DHA group, d: significant vs Flu group. ND group: normal chow diet-fed rats for 8 weeks; HCHFD control group: high carbohydrate-high fat diet-fed rats for 8 weeks; DHA group: (HCHFD + 200 mg/kg of docosahexaenoic acid); Flu group: (HCHFD + 10 mg/kg of flurbiprofen); DHA + Flu group: (HCHFD + DHA + Flu). Treatments were administered orally daily for 8 consecutive weeks, parallel with the start of diets. 237

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Fig. 5. (A) AMPK-α1 subunit gene expression (relative copy number “RCN”) in epididymal adipose tissue of rat groups. (B) NF-kB gene expression (RCN) in epididymal adipose tissue of rat groups. Data are represented as a mean ± SD (n = 15/ group), significance was set at p < 0.05; a: significant vs ND group, b: significant vs HCHFD control group, c: significant vs DHA group, d: significant vs Flu group. ND group: normal chow diet-fed rats for 8 weeks; HCHFD control group: high carbohydrate-high fat diet-fed rats for 8 weeks; DHA group: (HCHFD + 200 mg/kg of docosahexaenoic acid); Flu group: (HCHFD + 10 mg/kg of flurbiprofen); DHA + Flu group: (HCHFD + DHA + Flu). Treatments were administered orally daily for 8 consecutive weeks, parallel with the start of diets. Fig. 6. Representative photomicrographs of epididymal adipose tissue (EAT) sections stained with (H&E) (×200). (A): ND group showing normal EAT structure with normal adipocyte area (715.85 ± 100.35 μm2). The fat inside adipocytes was dissolved due to use of xylol in histological process, so the inclusions appear as its negative image (arrowhead). (B): HCHFD control group showing EAT with adipocyte area of 1810.52 ± 353.06 μm2 indicating significant hypertrophy by about 152% (p < 0.05) compared to ND group. Adipocyte death with inflammatory cell infiltration was detected with appearance of CLS (star).

normalizing body and organ weights compared to either of the treatments solely. This may be attributed to the cumulative effects of the two drugs exerted via different mechanisms. Circulating leptin levels are positively associated with fat mass, adipocyte size, and BMI suggesting a possible leptin resistance in obese individuals as they do not show the expected anorexic response [30]. This was consistently observed in our study where plasma leptin levels were clearly increased in HCHFD control group compared with ND group. In accordance with the previous experiment [36], our study demonstrated that the slight decrease in body and EAT weights observed in the DHA group was found to be parallel with the insignificant reduction of plasma leptin levels observed in this group. On the contrary, the significant decrease in adipose tissue mass observed in DHA + Flu group resulted in a significant reduction in circulating leptin levels, an effect which was more observable than that obtained by Flu alone. These observations were confirmed by the previous report of Hosoi et al. [13]. One of the critical regulators of the whole body energy homeostasis and inflammatory defense when treating obesity is AMPK [37].

significant decrease in body weight gain and EAT weight, compared to HCHFD control group. These findings were consistent with previous reports [32,33], which documented that DHA could decrease body fat primarily by reducing preadipocyte differentiation, decreasing the number of adipocytes, promoting lipolysis, and suppression of lipogenesis. DHA administration to HCHFD-fed rats didn’t alter liver weights compared to HCHFD control rats, which could be attributed to accumulated TGs within the liver. Our results can be explained by the work of Gaiva et al. [34], who reported that diet rich in fish oil produced impaired TGs transportation out of rats hepatocytes due to the declined synthesis of apolipoprotein B-48 of very low-density lipoprotein. Furthermore, our study demonstrated that Flu-treated rats showed a significant decrease in body weight gain, EAT weight, and liver weight. This effect was consistent with the results obtained by Hosoi et al. [14] and Hosoi et al. [35], who demonstrated that Flu could decrease body weight by attenuating leptin resistance induced by endoplasmic reticulum stress. Interestingly, our work demonstrated that combination between DHA and Flu showed the most significant effect on

Fig. 7. Representative photomicrographs of epididymal adipose tissue (EAT) sections stained with (H&E) (×200). (A): DHA group showing EAT with a significant decrease in adipocyte area by about 33% (1220.24 ± 223.32 μm2, p < 0.05) relative to HCHFD control group. (B): Flu group showing EAT with significantly smaller adipocyte area by about 55% (809.80 ± 166.20 μm2, p < 0.05) than that of HCHFD control rats. (C): DHA + Flu group showing EAT with adipocyte area of 795.25 ± 118.71 μm2, that is nearly similar to that of ND group. No CLS were observed in these treated groups. 238

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individually. The RvD1-increasing effect exerted by Flu may be secondary to the up-regulation of 15-lipoxygenase-1 which contributes to resolution of inflammation through formation of resolvins [53,54]. Induced gene expression of NF-κB found in adipose tissue of obese individuals supports the role of adipose-derived inflammation as a factor contributing to alteration of insulin signaling and glucose uptake, favoring the development of insulin resistance [55]. In our study, the increased blood glucose together with increased serum insulin level suggest that insulin action on glucose regulation was impaired in HCHFD control group, which is supported by the previous study of Boonloh et al. [16]. The degree of insulin resistance was distinctly high as indicated by elevated HOMA-IR scores in HCHFD control group. It was also suggested that one of the consequences of the hypertrophic response is the decrease in insulin-dependent glucose uptake caused by the impairment of insulin-dependent glucose transporter 4 (GLUT4) translocation to the plasma membrane [6]. Results of the present work showed that supplementation of HCHFD-fed rats with DHA resulted in decreased plasma glucose and insulin as well as HOMA-IR scores. Accumulating evidence showed that DHA might improve insulin signal transduction in adipocytes through induction of the gene expression and translocation of GLUT4 [56,57]. Interestingly, the inhibition of NF-κB and activation of AMPK by Flu may improve the insulin resistance and explain the observed decrease in glucose and insulin levels in plasma of rats treated with Flu in our study. Consequently, co-treatment with DHA and Flu resulted in ameliorating the insulin resistance and normalizing plasma glucose and insulin levels.

Activated AMPK phosphorylates key proteins concerned with suppression of lipogenesis, promotion of fatty acid oxidation, and enhancement of glucose transport [22]. To further focus of attention on AMPK, the correlation of active pAMPK and AMPK gene expression was studied. In the present investigation, treatment with DHA or Flu individually induced AMPK gene expression and increased the pAMPK in EAT, while co-treatment with both drugs significantly increased both pAMPK and AMPK gene expression, relative to either treatment alone. These results were confirmed by findings obtained by King et al. [38], who demonstrated that AMPK could be activated by some NSAIDs including Flu. Our results are also in agreement with previous reports by Kim et al. [39] and Kim et al. [40], who found that AMPK mRNA expression was elevated in EAT of mice fed DHA diet. The results described in our study illustrated that the regulation of carbohydrate and lipid metabolism is enhanced by transcriptional induction of AMPK in rat EAT following either individual or combined treatment with DHA and Flu. Concerning the lipid profile measured in the current study, DHAtreated animals showed lower levels of TGs and higher levels of HDL-C associated with no significant effect on either TC or LDL-C, compared to HCHFD control group. These results are supported by previous reports [41,42], which demonstrated that the decrease in TGs associated with the insignificant change in LDL-C obtained by DHA may result from massive lipoprotein lipase activation which promotes the clearance of TGs and conversion of very low-density lipoprotein to LDL. Furthermore, a greater reduction in cholesteryl ester transfer protein (CETP) activity by DHA may explain the exhibited increase in HDL-C [41,43]. Our results also demonstrated that Flu-treated group could alleviate HCHFD diet-evoked dyslipidemia. This may be attributed to the activation of AMPK in rodent adipocytes that decreased lipid synthesis through inactivation of lipogenic enzymes such as acetyl CoA carboxylase (ACC) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) [22]. Interestingly, the combination of DHA and Flu could normalize the lipid profile to be near to that observed in ND group. Beyond the effects already described, decreasing the AMPK activity in obesity may lead to activation of NF-κB signaling [44,45]. NF-κB is an oxidative stress-sensitive transcription factor, which activates transcription of several pro-inflammatory genes including COX-2, interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α) [46,47]. Interestingly, either DHA or Flu monotreatment decreased HCHFDevoked up-regulation of NF-κB, however, a further decrease was achieved with the combined treatment. The inhibitory effect of DHA on NF-κB gene expression in many cell types was previously reported by Polus et al. [48] and Bargut et al. [31]. Furthermore, it was reported by Birch & Cheung [49] that flurbiprofen could potently inhibit NF-κB activation and its target genes. In addition to obesity-induced production of pro-inflammatory mediators, inflamed obese adipose tissue shows an intrinsic inability of generating specialized pro-resolving mediators, such as resolvins, which are a family of lipid mediators generated enzymatically from n-3 polyunsaturated fatty acids [50,51]. RvD1 has been reported to blunt the production of proinflammatory cytokines, decrease adipose tissue macrophages accumulation, and improve insulin sensitivity in obese animals [52]. This was consistently observed in our study where the EAT concentration of RvD1, derived from endogenous DHA, was found to be decreased in HCHFD control group compared to ND group. Our results indicated that treatment with DHA could reverse the obesity associated-decrease in RvD1 which may explain the enhancement of the anti-inflammatory environment and the absence of CLS in the EAT of DHA group despite having hypertrophic adipocytes. Therefore, DHA does not only modulate the production of n-6 polyunsaturated fatty acids’ metabolites, but also produces lipid mediators such as resolvins and protectins that have highly potent anti-inflammatory and pro-resolution properties. Furthermore, the present work showed that treatment with DHA + Flu increased RvD1 to a greater extent compared to either treatment with DHA or Flu

5. Conclusions In the present study, the combination of DHA with Flu exerted an anti-inflammatory effect and protected against obesity-associated metaflammation in HCHFD-fed rats. On the molecular level, both drugs upregulated AMPK and downregulated NF-κB gene expression, while elevated the level of the endogenous anti-inflammatory RvD1. Because co-treatment with DHA and Flu could correct the deterioration of adipose tissue morphology and function, obesity-associated dyslipidemia, hyperglycemia, hyperinsulinemia, and insulin resistance, it could be considered a promising strategy against obesity-associated complications. Further prospective studies are needed to speculate whether the observed effects of DHA + Flu are likely to be achieved when therapy is given to animals after 8 weeks of HCHFD, i.e. therapeutic regimen. Future studies are also warranted to investigate the therapeutic efficacy of DHA and Flu combination against obesity and metabolic syndrome in clinical settings. Authors contributions El-Ashmawy NE: analyzed the data, critically reviewed the manuscript and approved its final version for publication. Al-Ashmawy GM: designed the study, reviewed the manuscript and approved it for publication. Kamel AA: designed the study, conducted the research, drafted the manuscript and approved it for final approval. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Acknowledgments The authors gratefully acknowledge Dr. Marwa S. Gad Allah (Lecturer of Pathology, Faculty of Medicine, Menofia University) for her help in conducting and interpreting the histopathological investigations. 239

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