Effect of oleoylethanolamide on systemic inflammation in the development of alimentary-induced obesity in mice

doi:10.21203/rs.3.rs-3094720/v1

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Effect of oleoylethanolamide on systemic inflammation in the development of alimentary-induced obesity in mice

https://doi.org/10.21203/rs.3.rs-3094720/v1

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The anorexigenic effect of oleoylethanolamide (OEA, C₂₀H₃₉NO₂) was studied in a model of diet-induced obesity in mice. Physiological, biochemical and immunohistochemical methods were used to reveal differences in the changes in the weight of experimental animals, morphological changes in the spleen tissues, as well as changes in the cytokine expression profile in the spleen, blood plasma and in macrophage cell culture. First, it has been shown that a hypercaloric diet high in carbohydrates and cholesterol leads to the development of systemic inflammation accompanied by organ morphological changes and increased production of proinflammatory cytokines. In parallel, the use of OEA reduces the intensity of cellular inflammatory reactions accompanied by a decrease in markers of cellular inflammation and proliferation, such as CD68, Iba-1 and Ki67 in the spleen tissue and stabilizes the level of proinflammatory cytokines (IL-1β, IL6, TNFα) both in animals and in cell culture. In addition, in macrophage cell culture (RAW264.7) it was shown that OEA also suppresses the production of reactive oxygen species and nitrites in LPS-induced inflammation. The results of this study indicate the complex action of OEA in obesity, which includes the reduction of systemic inflammation.

OEA

oleoylethanolamide

obesity

inflammation

spleen

cytokines

The high incidence of obesity and its associated metabolic and somatic disorders (diabetes mellitus, atherosclerosis, cardiovascular pathology) dictate the need to identify new molecular mechanisms involved in the complex and systemic regulation of these pathophysiological processes (Hideki et al., 2007). Despite the high prevalence of obesity, methods of pharmacological therapy are presently limited (Hofbauer et al., 2007; Padwal & Majumdar, 2007; Brethauer et al., 2006; Chaput et al., 2007; Bray & Ryan, 2007). Currently, there are only three available prescription drugs studied in large randomized placebo-controlled clinical trials and approved for long-term treatment of obesity. These include phentermine, appetite suppressant that increases catecholamine levels and induces a feeling of satiety; orlistat, a gastrointestinal lipase inhibitor that reduces fat absorption; and sibutramine, noradrenaline-serotonin-dopamine reuptake inhibitor that suppresses appetite and may also increase energy demand (Hofbauer et al., 2007). The usage of these drugs is complicated by serious gastrointestinal (orlistat) or cardiovascular (sibutramine) side effects (Padwal & Majumdar, 2007), which often ultimately leads to polypragmasy. Moreover, the pharmacotherapy results exceed the effects achieved by simple dietary restrictions and physical activity by only a few percent.

In this regard, the development of fundamentally new treatments for obesity is a priority for both patients and the health care system. The most promising pharmacological targets for the development of drugs with metabolic and anorexigenic effects can be elements of the human and animal body's own endocannabinoid system, which is represented by a group of lipid nature compounds - N-acylethanolamides (NAE) of saturated and polyunsaturated fatty acids (Uberto et al., 2006). In the presented work, the essential fatty acids of olive oil were used as a source for the OAE synthesis. In general, extra virgin olive oil (EVOO) consists predominantly of monounsaturated fatty acids (MUFAs), with oleic acid (C18: 1) is 55–83%, followed by polyunsaturated fatty acids (PUFAs) of 4–20%, mainly linoleic acid (C18:2), and saturated fatty acids (SFAs) of 8–14%, such as palmitic acid (C16:0) (Covas et al., 2015; Montserrat-de la Paz et al., 2016; Foscolou et al., 2018). Accumulated data suggests that OEA may be involved in various pathophysiological aspects of appetite regulation and lipid and carbohydrate metabolism. This endogenous metabolite is synthesized in the upper small intestine during lipid absorption, and it modulates the expression of genes involved in fat absorption and fatty acid metabolism (Drover & Abumrad, 2005; Chung et al., 2015). Due to its wide range of biological effects, OEA has great potential in the pharmacotherapy of obesity and associated lipid disorders (Cota et al., 2003; Bermudez-Silva et al., 2010; Dipasquale et al., 2010). OEA administration appears to reduce appetite and inhibit weight gain in both lean and obese rodents (Hildebrandt et al., 2003; Piomelli, 2013; Okamoto et al., 2004). These effects of OEA are mediated primarily by activation of the peroxisome proliferator-activated receptor-α (PPAR-α) (Fu et al., 2003). Other close structural analogs of OEA, such as linoleylethanolamide (LEA) and palmitoylethanolamide (PEA) also have high affinity for PPAR-a and show similar anti-inflammatory properties (Ishida et al., 2013; Lambert et al., 2002), However, their anorexic effects are much inferior to those of OEA. In terms of reducing food intake, PEA is significantly less effective, LEA is similar in potency to OEA, and oleic acid itself has no effect (Rodriguez de Fonseca et al., 2001; Diep et al., 2011). Nevertheless, endogenous levels of OEA, PEA, and LEA are regulated by the nutritional status of the animals. Fasting generally reduces NAE levels in the initial small intestine, while food intake stimulates duodenal and jejunal mucosal cells to produce endogenous OEA (Fu et al., 2007; Fu et al., 2011), PEA and LEA (Diep et al., 2011; Igarashi et al., 2015). Dietary fat content can also influence endogenous intestine levels of OEA, LEA and PEA in a time- and dose-dependent manner, which suggests their potential role as so-called fat sensors in the intestine (Diep et al., 2011). Moreover, a diet high in sucrose and low in fat may reduce the mobilization of OEA and LEA from the small intestine during intraduodenal lipid administration (Igarashi et al., 2015). These observations suggest that changes in the NAE data pathways also play a role in lipid metabolism. It is now known that obesity is accompanied by a state of chronic systemic inflammation associated with elevated levels of circulating cytokines and acute phase proteins (Schmidt-Arras & Rose-John, 2016; Vicennati et al., 2002; Visser et al., 1999). Serum IL6 is increased in diabetics and non-diabetic obese people compared with non-obese controls (Bastard et al., 2002; Goyal et al., 2012; Yang et al., 2006). Levels of other inflammatory cytokines such as TNFα, MCP-1, IL-1 and IL-8 also tend to raise in obesity (Kim et al., 2006). With the development of systemic inflammation, an immune response, accompanied by corresponding molecular and cellular changes in the organs of the immune system, usually occurs. The thymus and spleen are the main reservoir for T lymphocytes, which can regulate the innate immune response and provide protection against pathogens and tissue damage. Oxidative stress and inflammation that develop in obesity, among others, can lead to dysfunction of these organs (Ding et al., 2021). The spleen size has been shown to increase in obese individuals (Altunkaynak et al., 2007; Benter et al., 2011). Recently, an increase in spleen volume, a marker of chronic inflammation and immune system activation, has been shown to correlate with the progression of fatty liver dystrophy (Tarantino et al., 2009a; Tarantino et al., 2009b). Finally, elevated levels of proinflammatory cytokines detected in the spleen, particularly IL6, were found to be directly proportional to BMI and waist circumference (Engeli et al., 2003). Given the available data, we can assume that endogenous NEAs, including OEAs that interact with the PPAR-α receptor, help maintain homeostasis by preventing the triggering of systemic inflammation. It can be supposed that OEA, having a multifactorial complex effect on both metabolic and immune processes, may be a promising candidate for the pharmacological adjustment of alimentary disorders and concomitant pathologies of carbohydrate and lipid metabolism.

In this study, the OEA-based dietary supplement (OEA-DS) was shown to have anorexic effects in a model of alimentary-induced obesity in mice, and its effects were accompanied by a decrease in systemic inflammation.

2.1. Production of a dietary supplement

A dietary supplement based on oleoylethanolamide (OEA-DS) was prepared from olive oil (containing 72.3% oleic acid). Methyl esters were isolated from olive oil by methanolysis in the presence of a sodium methoxide as an alkaline catalyst. Further, fatty acid ethanolamides were obtained from the methyl esters according to the Farris method (Farris, 1979). The esters and ethanolamine were taken in the ratio 1:1.1 (mol/mol), sodium methoxide was used as a catalyst, the reaction proceeded at 90^oC for 4 hours under reduced pressure.

Fatty acid esters were analyzed by gas chromatography on a Shimadzu GC-17A chromatograph (Japan) with a flame ionization detector, a 30 m × 0.25 mm i.d. capillary column Suppelkovax 10 (USA). The analysis was performed under the following conditions: the column temperature was 205°C, the temperature of the injector and detector was 250°C. Helium was used as a carrier gas. The peaks of the fatty acid methyl esters were determined from the retention times of the individual FA esters by comparing their equivalent carbon length numbers. The results of the analysis are shown on the chromatogram (Fig. 1). The percentage of fatty acids in the dietary supplement is presented in the table.

2.2. Experimental model

The experiment was performed on three-month-old male C57BL/6 mice. The animals were divided into 4 groups, 10 animals in each group: "CTL", a group receiving standard diet; "CTL + OEA-DS", a group receiving standard diet with the OEA-DS added at a dosage of 200 mg/kg per day orally along with food; "DIO", a group of animals with diet-induced obesity; and "DIO + OEA-DS", a group of animals with diet-induced obesity receiving the OEA-DS at a dosage of 200 mg/kg per day.

The "CTL" and "CTL + OEA-DS" groups received a ready-made feed containing: grains, high-protein components (vegetable and animal proteins), vegetable oil, amino acids, organic acids, vitamin-mineral complex (Delta Feds - C-19, Russia). Animals of groups with induced obesity received modified feed containing: 60% standard feed + 20% sucrose + 20% sunflower oil + 10% cholesterol. Weight measurements were taken once a week. Daily caloric intake was assessed once every 4 days by weighing the remaining mass of feed with further recalculation to its caloric value (the caloric value of standard feed was 250 kcal/100 g, the modified feed was 400 kcal/100 g). The period of the experiment was 2 months. At the end of the experiment, such parameters as the total weight gain over the entire period and the average daily caloric intake per animal were evaluated.

Excision of material for subsequent immunohistochemical study was performed at the end of the experiment, i.e., after 2 months from the beginning of the diet. For this purpose, mice were anesthetized (3% isoflurane in 100% oxygen solution) using a vaporizer for rodent anesthesia (VetFloTM, Kent Scientific Corporation, Torrington, Connecticut, USA), then the chest cavity was opened and blood was taken from the left ventricle using a syringe, then the biomaterial was sampled. To collect the material for histological and immunohistochemical studies, some animals underwent transcardially perfusion with cold 0.1 M phosphate buffer (PBS), pH 7.2, and then with fixative solution (10% formalin in 0.1 M PBS, pH 7.2). The spleen was then immediately excised and placed into freshly buffered 10% formalin for 24 h at 4°C. After 5-6-fold washing with PBS, the biomaterial samples were dehydrated and transferred into paraffin according to the standard protocol. Blood plasma and spleen for Western blotting were extracted without preperfusion and immediately frozen in liquid nitrogen, with further storage at -80°C.

2.3. Immunohistochemical Staining

Paraffin sections of the spleen (7 µm) were dewaxed, then incubated in 3% hydrogen peroxide for 15 minutes to block endogenous peroxidase. This was followed by 3 washes with 0.1 M phosphate buffer (pH 7.2), each for 10 minutes. The sections were subsequently incubated for 1 hour in blocking buffer (PBS, 2% BSA (Santa Cruz, SC-2323, USA), 0.1% Tween20, 0.25% Triton X-100 (Gerbu, USA)). Next, without washing, primary mono- and polyclonal rabbit antibodies dissolved in blocking buffer were added to the next markers: Iba-1 (1:2000, ab108539, Abcam, UK) CD68 (1:1000, ab125212, Abcam, UK), PPAR-α (1:1000, ab245119, Abcam, UK), CD163 (1:1000, ab182422, Abcam, UK), Ki67 (1:1000, PA5-19462, Thermo Fisher, USA). Sections with primary antibodies were incubated in a wet chamber in a refrigerator at 4°C overnight. A negative control (without primary antibodies) was also incubated in parallel. The sections were subsequently washed with phosphate buffer (0.1 M, pH 7.2) 3 times for 10 min, after which the sections were incubated in a solution of secondary antibodies (Biotinylated Goat Anti-Rabbit IgG, ab178846, Abcam, UK) for 15 min. After 3 washes with buffer (0.1 M, pH 7.2), the sections were incubated for 10 minutes with streptavidin (ab64269, Abcam, UK) and then washed again three times with phosphate buffer (0.1 M, pH 7.2). Then the sections were processed with chromogen (Nova Red, Vector Laboratories, USA) for 5 min. The sections were washed with distilled water, then dehydrated and mounted under glass slides using mounting medium. Immunohistochemical staining area was assessed using ImageJ 1.41 software package (NIH, USA).

Every fifth serial section was used for immunohistochemical staining. The area of the immunohistopositive zone was assessed using ImageJ 1.41 software package (NIH, USA). In the case of Iba-1, CD68, CD163 and PPAR-α markers, the ratio of the area of interest to the total area of immunopositive staining was assessed, which was expressed as a percentage. For the Ki67 marker, the number of immunopositive cells/mm³ was measured, which was calculated according to the formula: d=(10⁹×n)/(S×7), where d is the cell density; 10⁹ is the conversion factor of µm² to mm²; n is the number of immunopositive cells; S is the area of the area under study (µm²); 7 is the slice thickness (µm).

2.4. Histological Staining

For histological staining, the deparaffinized sections were placed for 5 min in Mayer's hematoxylin solution (BioVitrum, Russia), then washed for 15 min under running water, followed by eosin staining (BioVitrum, Russia). Sections were placed in concentrated eosin solution for 5 seconds, then washed three times in 96% ethanol. After that, the sections were transferred into xylene for 10 minutes and then covered with slides using mounting medium.

2.5. Cell Culture

The mouse macrophage cell line RAW264.7 was used for in vitro studies. The cells were cultured in standard DMEM medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA), and 0.5% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C in a humidified atmosphere with 5% CO2. Trypsin-EDTA (0.05%) (Thermo Fisher Scientific, Waltham, MA, USA) was applied for cell passaging. For cytotoxicity studies, cells were seeded in 96-well microplates (1 × 10³ cells/well); for Western blotting, cells were placed in 6-well plates (1 × 10⁵ cells/well). Each experiment was performed independently three times.

2.6. MTS-test

The cytotoxic activity of OEA-DS was assessed using the MTS test. Cells were plated in transparent 96-well plates (1 × 10³ cells/well) and incubated in standard medium for 1 hour. After cell adhesion, the medium was replaced with OEA-DS solution at concentrations of 0.01; 0.1; 1 and 10 µg/mL and incubated overnight. Cells cultured in standard medium were used as a negative control. The next day, MTS reagent (ab197010, Abcam, UK) was added to each well and incubated for 2 h at 37°C. The optical density was then measured on a microplate reader (Biorad) at 490 nm. The results were presented as percentages relative to the negative control.

2.7. Analysis of Reactive Oxygen Species (ROS) and Nitric Oxide (NO)

The antioxidant activity of OEA-DS was studied in a culture of macrophage cells activated by bacterial lipopolysaccharide (LPS). The experiments were carried out in 96-well plates, where cells were plated (1 × 10³ cells/well); 1 hour after cell adhesion, the culture medium was replaced with a medium supplemented by OEA-DS at concentrations of 0.001, 0.01, 0.1, 1, and 10 µg/mL. Then, LPS (1mg/ml) was added to some wells to activate the cells. Cells activated only by LPS served as a positive control, and cells in a standard culture medium served as a negative control. ROS activity was studied after 24-hour incubation by adding 10 µg/mL. 20 µL of 2,7-dichlorodihydrofluorescein diacetate solution (D399, Invitrogen) according to the manufacturer's recommendations. A 10 µm solution of DAF-FM diacetate (D23844, Thermo Fisher Scientific, USA) was used for NO level analysis according to the manufacturer's recommendations. Fluorescence intensity was measured using a SPARK TECAN 10M tablet reader for ROS at λex/λem = 485/518 nm and for NO at λex/λem = 460/524 nm. The results obtained were presented as a percentage relative to the negative control.

2.8. Western Blotting

Macrophage culture was placed into 6-well plates at 1×10⁹ cells per well and divided into 4 groups: intact cells, cells treated with LPS at 1 µg/ml, and groups of cells treated with OEA-DS at concentrations of 1 and 10 µg/ml. After 24 hours incubation cells were sonicated homogenized in phosphate buffer (7.2 pH) with the addition of 150 mM serine protease inhibitor (PMSF, Helicon GC207002). Spleen tissues were homogenized manually by pestle grinding. Plasma was not homogenized. The protein concentration in the solution was then measured, followed by equalization to a concentration of 2 mg/ml for spleen tissue and cell culture and and 1 mg/ml for plasma. Then the samples were diluted 1:1 with 1x Sample buffer (Biorad) containing 5% 2-mercapnoethanol (Sigma-Aldrich M6250), followed by incubation for 5 minutes in a water bath at 94 ^oC. Electrophoresis was performed using ready-to-use gel cartridges (Protean mini gel Any kDa, Biorad) and a molecular ladder (Spectra Multicolor Broad Range Protein Ladder, Thermo Fisher) in a Biorad chamber. Each well was filled with 20 µl of sample, and the amperage per gel was set to 15 mA. After electrophoresis, proteins were transferred to the PVDF membrane. All transfer materials were used from the Transblot Turbo Transfer kit (Biorad). After transfer was completed, the membranes were placed in blocking buffer (PBS containing 2% BSA) for one hour.

Next, the blocking buffer was washed three times with PBS + 0.1% Tween 20 (PBS-T) solution, 5 min each time, then left overnight with primary antibodies to the markers: ASAHL (1:1000, Santa Cruz, sc-100470), IL-1β (1:1500, Abcam ab9722), IL6 (1:1500, Abcam ab208113), TNFα (1:1000, Thermo Fisher 701135), α-Tubulin (1:500, Abcam ab7291) dissolved in PBS buffer with 0.02% BSA (Sigma-Aldrich A2153) and 0.1% Tween20. After incubation with primary antibodies, PBS-T was washed again 3 times for 5 min each, then incubated for 1 hour with secondary rabbit (Vector laboratories, PI-1000) and mouse antibodies (Vector laboratories, PI-2000) dissolved in PBS-T, and then washed again 3 times with phosphate buffer. Chemiluminescence Western Blot ECL Substrate (Biorad) was used for detection in the amount of 1 ml of substrate per membrane, incubation time was 5 min. Detection was performed using the ChemiDoc gel documentation system (Biorad). The obtained images were analyzed using ImageJ 1.41 software package (NIH, USA).

2.9. Statistical Analysis

Further statistical analysis and plotting was performed using GraphPad Prism 8.00 software (GraphPad Software, USA). Data were statistically processed using single-factor analysis of variance followed by Tukey's posterior test. Using the Shapiro-Wilk test, all data were tested for normal distribution. Data obtained from the weight distribution and macrophage culture were compared using Student's t-test. Data were presented as mean ± SEM (standard error of the mean), and p < 0.05, p < 0.01, p < 0.001 were considered statistically significant.

3.1. OEA has an anorexic effect

The results of the body weight measurements, in addition to the increase in the weight of all animals due to their growth and development, show a tendency for inhibition of weight gain in the "CTL + OEA-DS" group compared to the control (22 ± 0 grams at the beginning and 24.2 ± 0.5 grams at the end in the "CTL" group; 24.6 ± 0.4 grams at the beginning and 26.4 ± 0.7 grams at the end in the "CTL + OEA-DS" group ), and a significant decrease in the "DIO + OEA-DS" group compared to the "DIO" group (23.6 ± 0.4 grams at the beginning and 30.4 ± 0.2 grams at the end in the "DIO" group; 23.8 ± 0.7 grams at the beginning and 28.2 ± 0.9 grams at the end in the "DIO + OEA-DS" group) (Fig. 2, a).

The caloric intake of the "DIO" group animals is doubled compared to the control (10.5 ± 0.3 kcal in the "CTL" group and 21.6 ± 0.9 kcal in the "DIO" group). Use of OEA-DS in obesity reduced the amount of calories consumed by 14% (18.9 ± 0.5 kcal in the "DIO + OEA-DS" group). Also, use of OEA-DS slightly reduces caloric intake in the "CTL + OEA-DS" group (9.9 ± 0.3 kcal, 5% less than in the control group). (Fig. 2, b).

3.2. OEA-DS reduces the intensity of morphological changes and cell proliferation in the spleen

In the "CTL" and "CTL + OEA-DS" groups the main structural elements of the organ are clearly distinguishable: white pulp and red pulp. There is a slight accumulation of hemosiderin in the red pulp (Fig. 3a, c). The diet-induced obesity significantly expands the hemosiderin inclusion zone, yellow and brown grains are noted in the white pulp, indicating a more intense process of erythrocyte damage (Fig. 3. b). Also, the red pulp of animals from the "DIO" group shows a mild diffuse accumulation of leukocytes, somewhere there is swelling of trabeculae. These phenomena are accompanied by the growth of white pulp follicles, which is expressed by a significant increase in the W/R index (1.3 ± 0.1). In the "DIO + OEA-DS" group the lymphoid infiltration is less pronounced, the accumulation of blood pigment is less (Fig. 3, d). W/R with OEA-DS almost returns to the control level (0.9 ± 0.03) (Fig. 3, i).

Quantification of Ki67-positive cellular elements show a significant increase in the number of proliferating cells in lymphoid follicles in obesity (5389 ± 281.8 cells/mm³) − 73% higher than in the control group (3115 ± 446.4 cells/mm³), and 135% higher than in the "CTL + OEA-DS" group (2292 ± 494.5 cells/mm³). Administration of OEA-DS to obese animals contributes to a significant decrease in the number of Ki67+ -cells in the white pulp, 68% lower than the control level (1845 ± 440.1 cells/mm³) (Fig. 3, j).

3.3. OEA-DS administration is accompanied by an increase of PPAR-α receptor expression in the spleen

OEA-DS induces a significant increase in the staining area of PPAR-α receptor-expressing cells in the white pulp of standard diet animals (1.1 ± 0.1% in the CTL group; 1.8 ± 0.2% in the CTL + OEA-DS group) and the animals with diet-induced obesity (1.0 ± 0.03% in the DIO group; 1.6 ± 0.2% in the DIO + OEA-DS group) (Fig. 4, i).

At the same time, in the red pulp no significant differences between the groups were observed, but there was a similar tendency for the PPAR-α positive staining area to increase in the groups treated with OEA-DS (Fig. 4, j).

3.4. The action of OEA-DS is accompanied by a decrease in inflammatory reactions at both cellular and molecular levels

In the "DIO" group, the development of alimentary-induced obesity is characterized by the formation of foci of accumulation of Iba-1-positive macrophages in the entire area of lymphoid follicles. Taken together, the areas occupied by macrophage accumulations of tissue take up an average of 7.9 ± 0.2% of the observed area, which is almost 5 times higher than those in the control group (1.7 ± 0.1%) and in the control group treated with OEA-DS (1.6 ± 0.1%). Administration of OEA-DS to obese animals reduces white pulp cellular infiltration to control levels (1.3 ± 0.1%). (Fig. 5, i)

In the red pulp the density of Iba-1-positive cells in the "DIO" group (9.5 ± 0.4%) is 7 times higher than in the "CTL" (1.5 ± 0.1%) and the "CTL + OEA-DS" group (1.7 ± 0.1%), the values in the "DIO + OEA-DS" group (1.6 ± 0.2%) are almost equal to those of control (Fig. 6, m).

Quantification of the proinflammatory marker CD68 in the "DIO" group in the white (1.4 ± 0.1%) and red (5.9 ± 0.3%) pulp shows a significant increase - more than 2-fold compared to "CTL" (in the white pulp − 0.4 ± 0.4%; 2.4 ± 0.2% in the red pulp) and "CTL + OEA-DS" (0.3 ± 0.03% in the white pulp; 1.8 ± 0.2% in the red pulp). Activation of CD68-positive cell elements is also observed in the white and red pulp of the "DIO + OEA-DS" group, but of a lower intensity (in the white pulp − 0.6 ± 0.5%; in the red pulp − 3.5 ± 0.2%) (Fig. 5j; Fig. 6n).

The "DIO" group demonstrates an increase in CD163-positive area in the red pulp by 40% (2.8 ± 0.1%) compared to the "CTL" group (2.0 ± 0.1%). Administration of OEA-DS to obese animals almost doubled the staining area compared to the control (3.6 ± 0.1%) (Fig. 6o).

Alimentary-induced obesity enhances the release of pro-inflammatory cytokines into the blood. In the plasma of animals from the "DIO" group the concentration of IL6 doubles, and IL1β triples compared with animals from the "CTL" group. Administration of OEA-DS to animals with a standard diet does not affect the plasma concentrations of cytokines, but leads to a significant decrease in IL1β and IL6 in animals with obesity. With the development of obesity, the release of TNFα into plasma also increases 5-fold, but the usage the OEA-DS has no influence at this marker (Fig. 7, c).

The results of western blotting of spleen tissues also demonstrate increased expression of all studied pro-inflammatory cytokines in the "DIO" group animals, several times higher than in the control group. OEA-DS therapy in obese animals prevents the development of inflammation, thereby inhibiting the synthesis of pro-inflammatory cytokines IL6 and IL1β in the spleen. Similarly to plasma, administration of OEA-DS has no effect on changes in the TNFα expression (Fig. 7, d).

3.5. OEA-DS induces metabolic changes in macrophage cell culture

On the macrophage cell line RAW264.7, the MTS test reveals no cytotoxic effect of OEA-DS at concentrations of 0.01, 0.1, 1, and 10 µg/mL (Fig. 8, a). Macrophage activation by LPS (1 µg/mL) is accompanied by a significant increase in reactive oxygen species (ROS) and nitric oxide (NO). Addition of OEA-DS to LPS-activated macrophages at concentrations (0.001-10 µg/mL) leads to a significant decrease in both ROS (p < 0.05) production (Fig. 8, c). and nitric oxide (Fig. 8, e). At the same time, treatment of intact cells with OEA-DS also doesn’t lead to changes in the levels of both ROS (Fig. 8, d) and nitric oxide (Fig. 8, b).

Western blotting data demonstrate that LPS activation makes cytokine production (TNFα, IL1β, and IL6) considerably higher. Addition of OEA-DS at concentrations of 1 and 10 µg/mL reduces the production of proinflammatory markers to almost control levels (Fig. 9, d). Western blotting also reveals that that macrophages preferentially use the ASAHL enzyme to metabolize OEA-DS. OEA-DS treatment activates cells to increasing the ASAHL synthesis in a dose-dependent manner (Fig. 9, c).

A significant reduction in caloric intake during OEA-DS administration in obese animals, in addition to the activation of lipolysis processes, may indicate the involvement of central mechanisms in the hunger and satiety state regulation. This hypothesis is also confirmed by studies (Pelchat & Schaefer, 2000; Waters et al., 2001), which showed by using physiological and biochemical tests on animals, that a decline in caloric intake was not associated with a high stress in animals, impaired motor activity, cognitive processes, malaise, pain or body temperature changes. Also, the study by Romano et al. (Romano et al., 2020) found no effect of OEA-DS on eating behavior modification of animals which did not eat any tasty food during the experimental period. The findings suggest that the effects of OEA are probably directed specifically against overeating by inducing faster satiety, but the exact mechanisms require further study.

The lower body weight gain in both obese and non-obese animals receiving OEA-DS suggests that the anorexigenic effects of OEA-DS extend not only to excess fat mass and not only to the consumption of tasty hypercaloric food. And the presence of significant differences in the morphological profile of the spleen confirms the presence peripheral effects of the dietary supplement, manifested in the reduction of general inflammation, enhancement of lipolysis and normalization of metabolic processes by modulating the activity of the immune system cells.

The ability of macrophages to exhibit tissue-specific diversity as well as to change their phenotype to produce pro- and anti-inflammatory cytokines is well known (Krzyszczyk et al., 2018). The division of macrophage into pro-inflammatory (M1) and alternatively activated anti-inflammatory (M2) phenotypes was introduced in 2000 (Mills et al., 2000). In turn, one or another pathway of macrophage activation is determined mainly by the local tissue microenvironment, which ensures their constant adaptation to environmental conditions (Lavin et al., 2014; Gosselin et al., 2014). Macrophages, due to their primary involvement in the uptake of pathogen particles, apoptotic cells or cell debris, have a set of scavenger receptors (SRS), which are able to recognize a wide range of ligands (Penberthy et al., 2016).

The transmembrane receptor CD163 belongs to the scavenger receptors and is highly expressed in macrophages (Kristiansen et al., 2001). As a rule, CD163 is expressed on the cell surface of alternatively activated M2 macrophages (Barros et al., 2013). The presence of anti-inflammatory cytokines such as IL-4 and IL-10 in the medium is known to induce CD163 expression, whereas the proinflammatory cytokines IL6, TNFα, IFNγ and LPS suppress its expression (Etzerodt et al., 2013). In the spleen tissue of obese animals treated with OEA-DS, the expression of CD163 is enhanced, which was proved by both immunohistochemical and Western blotting methods. In vitro data also show a reduction in the synthesis of pro-inflammatory factors, that occurs simultaneously with an increase in CD163 expression, and also confirms the direct effect of OEA-DS on activation of cells with an anti-inflammatory phenotype. The heightened activity of CD163-positive cells in the DIO group compared to the "CTL" and "CTL + OEA-DS" groups can be explained by the fact that the reparation processes will also take place during the development of inflammation in any case.

CD68 is also considered as a member of the scavenger receptor type D (SCARD) family. CD68, is predominantly expressed in the late endosomes and lysosomes of macrophages (Jiang. Et al., 1998), and can be significantly increased in macrophages responding to inflammatory factors (Rabinowitz & Gordon, 1991; O'Reilly et al., 2003) and are capable of binding apoptotic cells (Sambrano & Steinberg, 1995; Penberthy & Ravichandran, 2016). The highest number of CD68-positive elements in the "DIO" group indicates the presence of inflammation and active apoptosis processes in both white and red pulps, which agrees with the histological data. OEA-DS regulates the ratio of pro- and anti-inflammatory factors in the tissue environment, which, according to Western blotting of spleen tissue and cell culture, leads to a reduction the number of CD68-immunopositive cells.

Ionized calcium-binding adaptor molecule 1 is selectively expressed by microglia/macrophages (Imai et al., 1996). Normally, Iba1 expression is elevated in activated macrophages during inflammation and takes a key part in phagocytosis processes (Ohsawa et al., 2000). The abundant activation of macrophages observed in the spleen of animals with alimentary-induced obesity indicates, firstly, the presence of inflammatory process in the organ and, secondly, the intensive processes of phagocytosis of destroyed apoptotic cells in both white and red pulp, which is supported by both immunoperoxidase reaction results for other inflammatory markers, and histological staining results.

The proliferation marker Ki67 is expressed in all active phases of cell cycle (G1, S and G2), but is absent in resting cells (G0 phase), which makes it an indicator of cell proliferation (Scholzen & Gerdes, 2000). Significant increase in the number of Ki67-positive cell elements directly in the lymphoid follicles of the spleen on the background of high levels of proinflammatory cytokines both in the spleen and in the blood plasma indicates the initiation of proliferation processes of T-lymphocytes, induced by inflammation. Proliferating T cells, in turn, also contribute to a secondary increase in proinflammatory cytokine production, which ultimately causes the characteristic cellular pattern of inflammation. The in vivo results are consistent with the in vitro data obtained on the macrophage cell line RAW264.7. Treatment of macrophages with OEA-DS neutralizes LPS-induced activation of the synthesis of pro-inflammatory factors: TNF, IL1β and IL6, and also inhibits the production of ROS and nitric oxide. Other studies also confirm the anti-inflammatory activity of both OEA itself and the other main components of this dietary supplement (PEA, LEA), expressed, in particular, in the reduction of LPS-induced inflammation (D’Aloia et al., 2021; Ishida et al., 2013; Yang et al., 2016).

N-acylethanolamide acid amidase (NAAA/ASAHL) is the enzyme responsible for the inactivation of NAE to ethanolamine and the corresponding fatty acid (Tsuboi et al., 2005; Tsuboi et al., 2007; Ueda et al., 2010). ASAHL is most highly expressed in macrophages and other cells of the immune system (Ueda et al., 2001). The dose-dependent increase in the expression of this enzyme in macrophages upon treatment with OEA-DS confirms the cellular uptake of the dietary supplement in the form used.

Elevated numbers of PPAR-positive cells in the spleen of animals treated with OEA-DS without visible differences between the obese and non-obese groups indicates that the increased expression of this receptor is associated specifically with the intake of the substance and in this case is not mediated by pathology. A lot of studies confirm the anti-inflammatory effect of PPAR-α agonists, which in most cases is mediated by a dysregulation of the transcription factor NFkB (Delerive et al., 1999; Madej et al., 1998), which leads to the inhibition in the production of proinflammatory factors such as TNFα, IFN-γ, IL6 and IL-1β (Staels et al., 1998; Chung et al., 2015). Probably the observed effect of OEA-DS on PPAR-α expression underlies the suppression of inflammatory responses at both the molecular and cellular levels.

In this way, OEA-DS therapy suppresses the synthesis of key pro-inflammatory cytokines, especially in plasma, and modulates the activity of other immune cells. This is observed in the spleen tissue, where the activity of cells with a pro-inflammatory phenotype reduces and, conversely, the mechanisms of cell activation towards the anti-inflammatory phenotype are triggered. The spleen in this case is an illustrative example of how a hypercaloric diet high in cholesterol can not only contribute to weight gain, but also lead to systemic inflammation affecting other internal organs. Based on this, the suppression of systemic inflammation in obesity is a significant part of comprehensive therapy, and dietary supplements such as OEA combining both anorexigenic and anti-inflammatory effects are promising candidates for this medical strategy.

Author Contributions: Conceptualization, D.I., I.M., I.D.; methodology, D.I., A.P., R.S.; validation, D.I., I.M.; formal analysis, D.I., I.M., I.D; investigation, D.I., I.M., A.P., R.S., I.D.; resources, A.P., I.M., R.S.; data curation, I.M., I.D.; writing-original draft preparation, D.I.; writing-review & editing, I.M., I.D.; visualization, D.I..; supervision, I.M., I.D.; project administration, D.I., I.M.; funding acquisition, I.M. All authors have read and agreed to the published version of the manuscript.

Funding: No funding.

Institutional Review Board Statement: All experimental procedures were approved by the Animal Ethics Committee at the A.V. Zhirmunsky National Scientific Center of Marine Biology, Far Eastern Branch, Russian Academy of Sciences (№16150-208-01/122 of January 31, 2023) according to the Laboratory Animal Welfare guidelines and the European Communities Council Directive 2010/63/EU.

Informed Consent Statement: Not applicable.

Data Availability Statement: The datasets generated during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest: The authors declare no conflict of interest.

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Effect of oleoylethanolamide on systemic inflammation in the development of alimentary-induced obesity in mice

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Production of a dietary supplement

2.2. Experimental model

2.3. Immunohistochemical Staining

2.4. Histological Staining

2.5. Cell Culture

2.6. MTS-test

2.7. Analysis of Reactive Oxygen Species (ROS) and Nitric Oxide (NO)

2.8. Western Blotting

2.9. Statistical Analysis

3. Results

3.1. OEA has an anorexic effect

3.2. OEA-DS reduces the intensity of morphological changes and cell proliferation in the spleen

3.3. OEA-DS administration is accompanied by an increase of PPAR-α receptor expression in the spleen

3.5. OEA-DS induces metabolic changes in macrophage cell culture

4. Discussion

5. Conclusion

Declarations

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