|Year : 2021 | Volume
| Issue : 1 | Page : 92-103
Moringa (Moringa oleifera) leaves aqueous extract enhances fibronectin type III domain-containing protein 5 gene expression and serum irisin liberation in an obesity model
Reem K Abdellah1, Nadia M El-Beih1, Fatma A Morsy2, Khaled G Abdel-Wahhab3, Enas A El-Hussieny1
1 Department of Zoology, Faculty of Science, Ain-Shams University, Egypt
2 Department of Pathology, National Research Centre, Cairo, Egypt
3 Department of Medical Physiology, National Research Centre, Cairo, Egypt
|Date of Submission||15-Sep-2020|
|Date of Decision||04-Oct-2020|
|Date of Acceptance||14-Oct-2020|
|Date of Web Publication||26-Mar-2021|
PhD Reem K Abdellah
Department of Zoology, Faculty of Science, Ain-Shams University, Cairo, 11566
Source of Support: None, Conflict of Interest: None
Background Obesity, a risk agent for many chronic diseases, leads to increased mortality and poses one of the major public health problems.
Objective This study aimed to investigate the thermogenic and antiobese efficiency of Moringa aqueous extract (MAE) on obese-modeled rats.
Materials and methods Adult male rats (150–170 g) were randomly divided into four groups, with 10 animals each, as follows: (a) healthy rats served as control, (b) healthy rats administrated with MAE (400 mg/kg/day), (c) obese-modeled rats, and (d) obese-modeled rats treated with MAE.
Results After 30 consecutive days of treatment, the obtained results declared that MAE possessed antiobesity, thermogenic, antilipidemic, and antiinflammatory potential. MAE succeeded significantly in reduction of the BMI and serum leptin level coupled with up-regulation of fibronectin type III domain-containing protein 5 gene mRNA expression and serum irisin level. It clearly increased serum paraoxonase-1 activity and improved lipid profile values. Moreover, it markedly reduced serum tumor necrosis factor α and increased antioxidant activity, which was achieved from the marked improvement in malondialdehyde, nitric oxide, catalase, superoxide dismutase, and glutathione values in cardio-hepatic tissues. These findings were confirmed by the regeneration of the hepatic histopathological structure.
Conclusion MAE, as a food supplement, could play a beneficial role in management of obesity and restoring its complications. This could be exhibited through multiple pathways, mainly via upregulation of fibronectin type III domain-containing protein 5 gene expression and production of the soluble myokine ‘irisin,’ which is responsible for browning of white adipose tissue as well as increment of total body energy expenditure.
Keywords: fibronectin type III domain-containing protein 5 gene, irisin, Moringa, obesity, paraoxonase-1
|How to cite this article:|
Abdellah RK, El-Beih NM, Morsy FA, Abdel-Wahhab KG, El-Hussieny EA. Moringa (Moringa oleifera) leaves aqueous extract enhances fibronectin type III domain-containing protein 5 gene expression and serum irisin liberation in an obesity model. Egypt Pharmaceut J 2021;20:92-103
|How to cite this URL:|
Abdellah RK, El-Beih NM, Morsy FA, Abdel-Wahhab KG, El-Hussieny EA. Moringa (Moringa oleifera) leaves aqueous extract enhances fibronectin type III domain-containing protein 5 gene expression and serum irisin liberation in an obesity model. Egypt Pharmaceut J [serial online] 2021 [cited 2021 May 15];20:92-103. Available from: http://www.epj.eg.net/text.asp?2021/20/1/92/312843
| Introduction|| |
With respect to the estimations of the WHO, more than 50% of the global adult population has obesity or is at least overweight . In several areas worldwide, obesity abundance is still growing rapidly, and if the rate trends continue, it will record globally 18% in men and exceed 21% in women by 2025, inflicting severe burdens upon persons, societies, and health care system . Depending on these statistics, obesity has been justifiably identified as a recent global epidemic health problem . It is a risk agent for many chronic diseases, such as diabetes mellitus (type-2), nonalcoholic fatty liver disease, hypertension, hyperlipidemia, chronic kidney disease, cardiovascular disease, obstructive sleep apnea, osteoarthritis, and malignancies (e.g. breast, colon, and prostate), leading to increased mortality observed in obese individuals . Thus, obesity poses one of the major public health problems nowadays and has a great relevance to both the health care system as well as individual health. Obesity is classically defined based on BMI, which is the anthropometric index that is widely used to identify obesity . Contracting skeletal muscle secretes bioactive molecules called ‘myokines.’ One of the key molecules orchestrating many exercise-induced changes, including mitochondrial biogenesis and/or adipose tissue browning, is a transcription cofactor, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) ,. Interestingly, a link between myokines and PGC1α was recently discovered in the form of the new attractive PGC1α-driven myokine, irisin. Irisin was identified as a proteolytic cleavage product of the fibronectin type III domain-containing protein 5 (FNDC5), a transmembrane protein present mainly in skeletal muscle. In the original report by Boström et al. , FNDC5/irisin was induced by both PGC1α overexpression and physical activity in mice, and circulating irisin was shown to increase in humans in response to exercise. The evidence from in vitro and animal studies suggested the induction of the brown fat-like phenotype by FNDC5/irisin. Furthermore, an overexpression of FNDC5 gene in animal models of obesity resulted in marked up-regulation of uncoupling protein 1 and several mitochondrial genes, increase in oxygen consumption, amelioration of glucose tolerance, and reduction of insulinemia, demonstrating a greatly improved metabolic profile, most likely via elevated energy expenditure .
For many obese individuals, diet and behavioral modification need to be supplemented by pharmacotherapy, or increasingly, bariatric surgery, because attempted adherence to a balanced diet and healthy lifestyle has not addressed this problem . At present, because of dissatisfaction with high costs and potentially hazardous adverse effects, the potential of natural products for treating obesity is under exploration, and this may be an excellent alternative strategy for developing future effective and safe antiobesity drugs . A variety of natural products, including crude extracts and isolated compounds from plants, can induce body weight reduction and prevent diet-induced obesity . One of those natural herbs is Moringa oleifera, which is known as Moringa in the literature, belonging to the family Moringaceae. It has been used for many health benefits . M. oleifera is the most-studied and used species owing to its high phytochemical and pharmacological properties close to human health. It is largely cultivated in tropical and subtropical areas. Moringa is also usually defined as ‘horseradish tree’ or ‘drumstick tree’ . As its leaves are rich in nutrients, especially vitamins and proteins, they can be cooked or eaten fresh to be used for prevention of malnutrition in populations . It is commonly known as miracle tree, as various parts of the plant including roots, leaves, and seeds possess various medicinal as well as nutritional values. Therefore, various preparations of M. oleifera exhibited antibiotic, hypotensive, antiulcer, antiinflammatory, and anticancer properties . The edible leaves of M. oleifera tree have been known as an antidiabetic food for centuries . The aqueous extract of M. oleifera leaves demonstrated potent antioxidant and antidiabetic activity . The pharmacological effects of its leaves have been reasoned to the synergistic actions of the different bioactive constituents included in its leaves that involve trace elements, vitamins , alkaloids (moringinine), carotenoids (β-carotene), and essential amino acids . Moreover, M. oleifera contains three structural classes of phytochemicals with medicinal benefits, that is, glucosinolates (glucomoringin), flavonoids (quercetin and kaempferol), and phenolic acids . It was reported that these phytochemicals possess antioxidant, antihyperglycemic, antihypertensive, lipid-lowering, anticancer, and antiinflammatory characteristics ,. In addition, leaves of this plant are rich source of riboflavin, nicotinic acid, folic acid, and pyridoxine; therapeutically, these leaves are known to have antiatherosclerotic, tumor-suppressive, immune-boosting, and cardiovascular disease-preventive properties . This study aimed to investigate the antioxidant exhibition of the aqueous extract of Moringa leaves in vitro; to explore Moringa aqueous extract (MAE) effect on the expression of FNDC4 gene (responsible for irisin secretion); and to interpret the relation among irisin level and paraoxonase-1 (PON1) activity with the obesity anthropometric and biochemical markers in obese-modeled rats in a trial to evaluate the obesity-modulating role of MAE as well as its lipid-lowering, antiinflammatory, and antioxidant efficiencies.
| Materials and methods|| |
Plant materials and extraction
Moringa (M. oleifera) herb was obtained from a local supplier (Abd El-Rahman Harraz, Bab El-Khalk Zone, Cairo, Egypt). It was identified and authenticated by scientific botanists at Botany Department, National Research Center and found carrying a taxonomic serial number 503874. The aqueous extract of dry Moringa leaves was carried out according to the method of Berkovich et al. . In brief, 50 g of powdered dry leaves was soaked in 500 ml of distilled water and placed in a warm water bath (70°C) for 2 h, and then filtered through sterile filter paper (Whatman number 42, Whatman International Ltd, Maidstone, England). After lyophilization, the dry yield was stored at −20°C till use.
Determination of total phenolic content
Phenolic content of the MAE was performed by dissolving 5 mg of the extract in a 10-ml mixture of acetone and water (6 : 4 v/v). Then, a sample of 0.2 ml was mixed with 1.0 ml of Folin-Ciocalteu reagent (10 fold diluted) and 0.8 ml of sodium carbonate solution (7.5%). After 30 min at room temperature, the absorbance was measured at 765 nm using Cary 100 ultraviolet–visible spectrophotometer. Estimation of phenolic compounds as catechin equivalents was carried out using standard curve of catechin .
DPPH radical scavenging activity
The capacity of antioxidants of MAE to quench DPPH radical was determined as previously described . In this method, a certain amount (0.2 g) of the crude extract was dissolved in methanol to obtain a concentration of 200 ppm. A volume of 0.2 ml of this solution was completed to 4 ml by methanol, and 1 ml DPPH solution (6.09×10−5 mol/l), in the same solvent, was then added. The absorbance of the mixture was measured at 516 nm after 10-min standing. The reference sample (blank) was 1 ml of DPPH solution and 4-ml methanol. Triplicate measurements were made, and the percentage of radical scavenging activity was calculated according to the following equation:
Reducing power assay
The ferric ion (Fe3+)-reducing ability of MAE was assessed according to the method of Sethiya et al. , with slight modification. In this method, 0.5 ml of different fractions (50, 100, 200, 400, and 800 μg/ml) was mixed with 2.5 ml of phosphate buffer (pH 7.4) and 2.5 ml of potassium ferricyanide (0.1 M). This mixture was kept at 50°C in water bath for 20 min. After cooling, 2.5 ml of trichloroacetic acid (10%) was added and centrifuged at 3000 rpm for 10 min. Then, 2.5 ml of upper layer of the solution was mixed with 2.5 ml of distilled water and 0.5 ml of a freshly prepared ferric chloride solution (40% w/v). The absorbance of both sample and control (without samples) was measured at 700 nm. Ascorbic acid at various concentrations was used as standard, and the reducing power was calculated as equivalent to ascorbic acid from the standard curve.
Animals and induction of obesity
A total of 40 adult male Wistar albino rats (Rattus norvegicus) weighing 150–170 g were obtained from Animal Colony, National Research Center, Cairo, Egypt. The animals were housed in suitable plastic cages 1 week before the experimental study for acclimatization. Excess tap water and standard rodent food pellets [20.3% protein (20% casein and 0.3% DL-Methionine), 5% fat, 5% fiber, 3.7% salt mixture, and 1% vitamin mixture], obtained from Meladco Company for animals and rodents food pellets, El-Obour City, Cairo, Egypt, were always available. All animals received humane care in compliance with the standard institutional criteria as cited by animal ethical committee number FWA00014747, National Research Center. After the animals were acclimatized, the rats were fed for 3 months on a high-fat diet (raising standard rodent diet fat to 46% by corn oil, lard, and cholic acid). The weight and nose-anus length of each rat were measured at the end of the 3 months, and BMI was calculated by dividing the weight (g) by the square of nose-anus length (cm2) . Animals with BMI greater than 0.68 g/cm2 were considered obese .
After induction of obesity, both normal and obese rats were randomly divided into four groups (10 rats each) as follows: (a) healthy or control animals, (b) healthy animals orally received a freshly prepared MAE (400 mg/kg/day) dissolved in distilled water for 4 weeks, (c) obesity-modeled animals without any treatment, and (d) obesity-modeled animals orally treated with MAE (400 mg/kg/day) for 4 weeks.
Blood and tissue sampling
At the end of the experimental period, the animals were fasted overnight, and following diethyl ether anesthesia, blood specimens were withdrawn from the retro-orbital plexus into vacutainer and centrifuged. Then the sera were separated, divided into aliquots, and stored at −80°C until biochemical measurements could be carried out as soon as possible. After blood collection, the animals were rapidly killed and parts of the liver and the heart of each animal were dissected, washed with saline, dried, and homogenized for tissue determinations of oxidative stress markers. Another part of the liver was soaked in formalin-saline buffer (10%) for microscopic examination. Vastus lateralis (muscle tissue) biopsies were taken, rolled in aluminum foil, and preserved at −80°C until molecular quantification of FNDC5 gene expression of irisin .
Tissue homogenate preparation
A specific weight (0.3 g), from either liver or heart, was homogenized in 3 ml of ice-cold phosphate buffer (50 mM, pH 7.4) to give 10% homogenate (w/v), and then the homogenates were cool centrifuged at 9000 rpm for 20 min, and supernatants were separated in aliquots and stored at −80°C until determination of oxidative stress markers.
BMI and body weight gain
Body weight gain and BMI of both obese and normal rats groups were recorded at start and end of the experiment and were calculated according to the following equations .
Where W1 is the animals’ weight at start andW2 is the animals’ weight at the end of the experiment.
Biochemical, myokine, and cytokine determinations
Serum glucose level was determined, at the time of blood sampling, using reagent kits purchased from Vitro Scient Co. (Cairo, Egypt). Serum insulin level was performed using ELISA (Dynatech Microplate Reader Model MR 5000, Canada) and reagent kits purchased from Immunospec Co, Ltd, No. 7018 Owensmouth Ave, Suite 103, Canoga Park, United States. Creatinine and urea levels were determined using reagent kits purchased from Vitro Scient Co. (Cairo, Egypt). Serum leptin concentration was measured using reagent kits (EL2001-1) purchased from Assaypro (USA). Irisin and tumor necrosis factor α (TNFα) levels were measured using reagent kits (SG-10179 and SG-10127, respectively) purchased from SinoGeneClon Biotech Co. (Hang Zhou, China). Serum alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) activities were determined using reagent kits purchased from Vitro Scient (Cairo, Egypt). Serum gamma-glutamyl transferase (GGT) activity was measured using reagent kits purchased from BioSystems (Spain). Alkaline phosphatase (ALP) activity was assayed using reagent kits purchased from Diagnostic Systems GmbH (IVD Alte Strasse 9 65558 Holzheim, Germany). Total proteins and albumin, total cholesterol, triglycerides, and high-density lipoprotein (HDL) cholesterol levels were determined with reagent kits obtained from Spectrum Diagnostics (Cairo, Egypt). Low-density lipoprotein (LDL) cholesterol level was calculated according to equation of McNamara et al. . Very Low-density lipoprotein (VLDL) cholesterol concentration was calculated according to the equation of Friedewald et al. . Atherogenic indices (AIP, CRI-I, and CRI-II) were calculated according to Aboulgasem and Azab . Creatine kinase and lactate dehydrogenase activities were estimated according to the kinetic methods using reagent kits purchased from Diagnostic Systems GmbH. Serum PON1 activity was determined according to the kinetic spectrophotometric chemical method described by Eckerson et al.  using a substrate buffered mixture [Paraoxon (1.0 mol/l), CaCl2 (1.0 mmol/l), glycin buffer (50 mmol/l)]. Under the aforementioned system, PONase can hydrolyze paraoxon (sigma) to ρ-nitrophenol and diethylphosphate. The rate of paraoxon hydrolysis can be measured spectrophotometrically at 405 nm and 37°C by monitoring the increase of absorbance at zero time and each 2-min interval for 10 min. All samples were run in duplicate. The average value was used for activity calculation using a molar extinction coefficient of 18, 300 M/cm for p-nitrophenol. Results are expressed as U/l for PON1 activity (nmole paraoxon hydrolyzed per minute).
Oxidative stress markers of liver and heart tissues
Tissue reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and nitric oxide (NO) levels were determined using reagent kits obtained from Biodiagnostic Co. (Giza, Egypt). Lipid peroxidation level was estimated chemically according to the method described by Ruiz-Larrea et al.  on the basis of malondialdehyde (MDA) reaction with thiobarbituric acid, which forms a pink complex that can be measured photometrically. In this method, 0.5 ml of liver homogenate supernatant (1 g Liver or heart tissue was homogenized in 10 ml phosphate buffer pH 7.4 and cool centrifuged at 5000 rpm for 10 min) was added to 4.5 ml of working reagent (0.8 g thiobarbituric acid was dissolved in 100 ml percloric acid 10% and mixed with 20% trichloroacetic acid in volume ratio 1–3, respectively). In a boiling and shaking water bath, the sample-reagent mixture was left for 20 min, and then carried out to cool at room temperature, and centrifuged for 5 min at 3000 rpm. The absorbance of the clear pink supernatant was measured photometrically at 535 nm against reagent blank (0.5 ml of distilled water +4.5 ml of working reagent).
RNA extraction and reverse transcription PCR
Total RNA was isolated from liver samples using BIOZOL reagent purchased from Bioer Technology Co. Ltd. (Hang zhou, China) following manufacturer’s instructions. Reverse transcription (RT) of total RNA to cDNA was performed by mixing 1 μg of total RNA, 1.5 µl from 10 μΜ oligo (dT) (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA), 2 µl of 10 mM dNTP mix (Promega Corporation, Madison, Wisconsin, USA), 4 µl of 25 mM MgCl2 (Promega Corporation), 2 µl of ×10 RT buffer (SibEnzyme Ltd, Novosibirsk, Russia), 2 µl RNase-inhibitor (Promega Corporation), and 7.5 µl RT-enzyme (M-Mul V) (SibEnzyme Ltd.). The volume of this reaction mixture was completed to 25 μl of DEPC-treated water and incubated at 70°C for 10 min, then at 37°C for 10 min, and 42°C for 1 h, followed by final extension stage at 72°C for 10 min . cDNA product was kept at −20°C. RT was carried out in Biometra thermocycler (Analytik Jena Company, Göttingen, Germany).
Quantitative real-time PCR analysis
PCR was performed with specific primers for FNDC5 (F:
3′CTTCATT CTCCGCTGGTCCCTG 5′; R: 5′ CAGATGCAAGCAGCCGTCTCAG 3′) and GAPDH (F: 3′TCAAGAAGGTGGTGAAGCAG-5′; R: 5′AGGTGGAAGAAT GGGAGTTG-3′) as a house-keeping gene. The primers were obtained from Sigma Co. (Sigma Aldrich, Egyptian International Center for Import, Cairo, Egypt). All PCR reactions were performed using Maxima SYBR Green qPCR Master Mix (Bioline, London, UK) and were carried out using Agilent Mx3005P QPCR System (Agilent Technologies Co., Santa Clara, California, USA). Mixtures were prepared in a total volume of 20 μl containing 1 μg of cDNA sample, 0.8 μl of forward primer, 0.8 μl of reverse primer, 10 μl of Sybr Green mastermix, and the volume was completed to 20 μl with RNase/DNase-free sterile water. The PCR reaction consisted of one cycle involved initial denaturation at 95°C for 10 min, followed by 40 cycles of 15 s at 95°C, 30 s at 62°C, and 30 s at 72°C. Each sample was analyzed in triplicate. Differences in gene expression between groups were calculated using the △△Ct (cycle threshold, Ct) method , which were normalized against GAPDH and expressed as relative mRNA levels compared with controls. Ct indicates the fractional cycle number at which the amount of amplified target reaches a fixed threshold.
Portions from the liver of different groups were sectioned into 5-μm-thick paraffin sections, stained with hematoxylin and eosin , and investigated by a light microscope.
Comparisons between means were carried out using one-way analysis of variance followed by post-hoc (Tukey) multiple comparisons test at significance level of P value more than or equal to 0.05 according to Steel and Torrie . This was carried out using statistical analysis system (SAS) program software; copyright (c) 1998 by SAS Institute Inc. (Cary, North Carolina, USA).
| Results|| |
In vitro estimation results’ yield, total phenolic compounds, radical scavenging activity, and reducing power of MAE are shown in [Figure 1]a, b.
|Figure 1 (a) Yield, TPC, and RSA of MAE (mean of three replicates±SEM). (b) Reducing power of MAE (mean of three replicates±SEM). MAE, Moringa aqueous extract; RSA, radical scavenging activity; TPC, total phenolic compounds.|
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BMI was not disturbed in MAE-treated healthy animals, whereas obese-modeled rats recorded a significant elevation compared with those of healthy control. Moreover, treatment of obese rats with MAE led to a significant reduction in BMI compared with obese rat group ([Figure 2]).
|Figure 2 BMI of control, obese, and MAE-treated rats. The data represented as mean±SEM. *Significant from healthy group, #Significant from obese group. MAE, Moringa aqueous extract.|
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Treatment of healthy animals with MAE neither deteriorated the activities of serum ALAT, ASAT, GGT, and ALP nor the levels of total proteins, albumin, globulin, A/G ratio, creatinine and urea; in contrast, obese animal group recorded a significant increase in the activity of ALAT, ASAT, GGT, and ALP, but did not disturb the other measurements when MAE-treated group was compared with the healthy group. Fortunately, treatment of the obese rat group with MAE resulted in improvements in the values of ALAT, ASAT, GGT, ALP, albumin, and total proteins level compared with obesity rat group ([Table 1]).
|Table 1 Liver and kidney functions of control, obese, and Moringa aqueous extract-treated animals groups|
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Comparing with the control group, administration of rats with MAE did not disturb serum glucose, insulin, lipid profile, atherogenic indices, creatine kinase, and lactate dehydrogenase values, whereas obese rat group revealed a marked deterioration in these measurements. Promisingly, treatment of obese rats with MAE resulted in a significant improvement in the mentioned parameters ([Table 2]).
|Table 2 Serum glucose, insulin, lipid profile, atherogenic indices, creatine kinase, and lactate dehydrogenase of control, obese, and Moringa aqueous extract-treated animal groups|
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Similarly, neither serum irisin, TNFα, leptin, nor PON1 values were disturbed after MAE treatment of healthy animals, whereas obese-modeled ones showed a significant reduction in serum irisin and PON1 coupled with a significant rise in both TNFα and leptin levels compared with control group. Interestingly, treatment of obese animals with MAE significantly downregulated the serum TNFα and leptin levels and upregulated serum irisin and PON1 values ([Figure 3],[Figure 4],[Figure 5],[Figure 6]).
|Figure 3 Serum TNFα value of nonobese and obese adult male albino rats treated with MAE. The data are represented as mean±SEM. *Significant from healthy group, #significant from obese group. MAE, Moringa aqueous extract; TNFα, tumor necrosis factor α.|
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|Figure 4 Serum PON1 activity of nonobese and obese adult male albino rats treated with MAE. The data are represented as mean±SEM. *Significant from healthy group, #significant from obese group. MAE, Moringa aqueous extract; PON1, paraoxonase-1.|
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|Figure 5 Serum irisin of nonobese and obese adult male albino rats treated with MAE. The data are represented as mean±SEM. *Significant from healthy group, #significant from obese group. MAE, Moringa aqueous extract.|
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|Figure 6 Serum leptin of nonobese and obese adult male albino rats treated with MAE. The data are represented as mean±SEM. *Significant from healthy group, #significant from obese group. MAE, Moringa aqueous extract.|
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Compared with normal rats, MAE-administrated group showed neither adverse oxidative stress voltage (NO and MDA) nor the antioxidant battery (GSH, SOD, and CAT) of liver and heart, whereas obese rats recorded a significant reduction in GSH, SOD, and CAT values coupled with a significant increase in NO and MDA levels. Interestingly, treatment of obese rats with MAE resulted in a marked decrease in MDA and NO levels, matched with a marked rise in the GSH, SOD, and CAT values compared with the corresponding values of obese rat group ([Table 3]).
|Table 3 Hepatic and cardiac oxidative markers (glutathione, superoxide dismutase and catalase, malondialdehyde, and nitric oxide) of control, obese, and Moringa aqueous extract-treated animal groups.|
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The obtained result showed insignificant reduction in skeletal muscle FNDC5 mRNA expression in obese rats as compared with the control group, although a marked upregulation was observed in obese rat treated with Moringa in comparison with obese rats. Additionally, oral administration of healthy rats with MAE recorded insignificant change in the expression of FNDC5 when compared with the control group. Favorably, oral treatment of obese group with MAE upregulated the expression of FNDC5 as compared with obese group ([Figure 7],[Figure 8],[Figure 9],[Figure 10],[Figure 11]).
|Figure 7 Fibronectin type III domain-containing protein 5 (FNDC5) expressed in muscle of nonobese and obese adult male albino rats treated with MAE. The data represented as mean±SEM. *Significant from healthy control group, #significant from obese group. MAE, Moringa aqueous extract.|
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|Figure 8 Section of the liver of healthy control rats showing normal histological structure of hepatic lobules and central vein (cv) (hematoxylin and eosin, ×400).|
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|Figure 9 Section of the liver of healthy rats treated with MAE (400 mg/kg body weight) showing normal histological structure of hepatic lobules and central vein (cv) (hematoxylin and eosin, ×400). MAE, Moringa aqueous extract.|
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|Figure 10 (a) Section of liver of obese-rats showing micro vesicular (red arrow) steatosis and sings of degeneration in the form of pyknosis (P) (hematoxylin and eosin, ×200). (b) Another filed of liver of obese-rats showing dilated, congested portal vein (orange arrow), fibrosis in portal area, dilated bile duct (red arrow) and cellular infiltration around (black arrow) (hematoxylin and eosin, ×100).|
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|Figure 11 (a) Section of liver of obese-rats treated with MAE (400 mg/kg body weight) showing that the liver still suffering from pathological changes in the form of dilated and congested portal (red arrow). Some singes of degeneration are seen in the form of minute vacuolar degeneration (green arrow) and pyknotic hepatocyte (black arrow) (hematoxylin and eosin, ×200). (b) Another filed of liver of obese rats treated with MAE (400 mg/kg body weight) showing red blood cells in dilated blood sinusoids could be seen (black arrow) (hematoxylin and eosin, ×200). MAE, Moringa aqueous extract.|
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| Discussion|| |
This study declared that MAE neither disturbed liver, kidney, or heart physiological functions ; oxidative status ; BMI; glucose; insulin; lipid profile; nor atherogenic indices , reflecting a physiological safe effect of MAE. Obese animals showed a marked increase in ALAT, ASAT, GGT, and ALP levels without any effect on serum urea, creatinine, and proteins. Liver histological structure was also deteriorated, as was suggested before ,. Fat accumulation and lipid peroxidation in both membranes and cytosol of hepatocytes caused escaping of liver enzymes from the hepatocyte-cytosol into the circulation , indicating disruption in hepatocytes integrity and function . In addition, partly infiltrated inflammatory cells, macrosteatosis/microsteatosis, and inflammation features were observed in obese livers, confirming that effect .
FNDC5 gene was slightly expressed in healthy MAE-treated rats and markedly expressed in MAE-treated obese ones, reflecting the FNDC5-expressing and irisin-releasing potential of MAE. Both in humans and rodents, the positive correlation of brown fat tissue formation with antiobesity effects suggests a central role of beige adipocyte thermogenesis in whole-body energy metabolism . It was suggested that irisin ameliorates obesity and glucose disorders  and that circulating irisin is negatively associated with BMI and percentage of fat mass ; in harmony with that, the current study showed that the skeletal muscle FNDC5 mRNA expression level was decreased in obese rats, combined with a significant reduction in circulating irisin level. The current study highlights, for the first time, that MAE improved the obesity status by increasing FNDC5 gene mRNA expression and serum irisin level, as a consequence. One or more mechanisms could be proposed; it could be postulated that FNDC5 gene was cleaved under the effect of MAE constituents, resulting in production of the soluble protein ‘irisin,’ which is responsible for browning of white adipose tissue as well as increment of total body energy expenditure, and secondly, the observed downregulation and upregulation of FNDC5 in obese and Moringa-treated rats, respectively, may be associated with the observed leptin level change in our study because leptin has a regulatory role on FNDC5/irisin as mentioned by Rodriguez et al. . FNDC5 mRNA expression may also be induced by elevation of NO level, a second messenger molecule, contractility, and blood flow in the skeletal muscles, as previously reported by Chatterjee et al. . Generally, MAE therapy leads to marked improvement in liver and kidney functions, as well as liver histological structures of MAE-treated obese animals, as stated by Yousef et al. .
Obesity model showed a marked increase in BMI and glucose levels as previously reported ,; this may be owing to intake of excessive calories and the fat accumulation, as there is a direct correlation between the intake of fat and BMI as well as deposition of fat in the liver , which is evidenced herein by the raised cholesterol, triglycerides, LDL, and VLDL and atherogenic indexes ,. It was reported that adipose tissue lipids are greatly derived from triglycerides that circulate in blood, especially during feeding on high-fat diet . The disturbed lipid profile in this study also could be owing to the reversed transport of cholesterol from the circulation to the liver ; high-fat diet leads to internal disorders of lipid metabolism manifested as hypercholesterolemia, hypertriglyceridemia, and high LDL . Glycolipid metabolism disorders are associated with major risk factors leading to cardiovascular disease such as atherosclerosis, thrombosis, and blood supply disorders ; that is why, the atherogenic indexes were markedly elevated herein our study.
Oxidative stress results from an imbalance between pro-oxidants and antioxidants . The significant reduction in hepatic, renal, and cardiac GSH, SOD, and CAT accompanied with a marked elevation of MDA and NO in obese rats is against the findings of Della Vedova et al.  and Yang et al. . Indeed, elevated production of reactive species is suggested to be resulted by activation of inflammatory cells at the sites of adipose tissue inflammation and hypertrophy . As a positive correlation between BMI and lipid peroxidation was evidenced , the increased NO level could be a consequence of the expression of endothelial NO synthase in subcutaneous adipose tissues . Similarly, the reduction of SOD, CAT, and GSH values could be attributed to the increased oxidative stress after lipid metabolism disorder and excessive ROS generation ,. The marked decrease of irisin and PON1 values and increase of leptin and TNFα levels of obese-rats serum were also confirmed ,.
Under oxidative stress, PON1 activity could be inactivated as a result of altered formation and/or release of HDL, or by S-glutathionylation via a redox regulatory mechanism characterized by the formation of mixed disulfide between a protein thiol and oxidized GSH . Leptin is secreted by the adipocytes and over-released during obesity; this could be owing to many factors: the excessive ROS generation, reduced hypothalamic leptin receptor expression , or and the extensive progression of the adipocytes; therefore enlargement of adipose tissue. The impaired blood flow results in hypoxia, which leads to necrosis and infiltration of macrophages in adipose tissue  and overproduction of adipokines, which include pro-inflammatory TNFα . Moreno-Navarrete et al.  illustrated that the amount of brownadipose tissue significantly decreased in obesity and was negatively related with BMI. They attributed the decreased irisin level in obese patients to the lower amounts of brown adipocytes in adipose tissue. In agreement with this hypothesis, circulating irisin level was significantly decreased in obese animal group in the current study.Selvakumar and Natarajan  attributed the hepatoprotective property of MAE to presence of many bioactive components, mainly, quercetin and kaempferol. MAE-treated obese rats showed a significant improvement in insulin, HDL cholesterol, glucose, BMI, cholesterol, triglycerides, LDL, and VLDL, and atherogenic indices near the control values; this result is against the reports of Tuorkey  and Elabd et al.  The hypolipidemic effect of different medicinal plants has been related to their bioactive components; cholesterol downregulation property of MAE may be owing to flavonoids, which may augment the activity of lecithin acyltransferase, which regulates blood lipids through incorporation of free cholesterol into HDL and transferring it back to VLDL and LDL, which are taken back later in liver cells .
The significant decrease in serum leptin coupled with marked elevation of PON1 in MAE-treated obese animals comes in line with Daba et al.  and Sierra-Campos et al. . Leptin antagonizes the effect of insulin and increases inflammation  which is confirmed by the rise in both serum TNFα and hepatocardiac oxidative parameters in obese animals. The marked improvement in GSH, SOD, CAT, MDA, and NO of both hepatic and cardiac tissues concomitant with significant decrease in TNFα posttreatment of obese rats with MAE, comes in line with many previous findings , who attributed that ameliorating role to the presence of large quantities of phenolics and flavonoids, such as quercetin and kaempferol and ascorbic acid in MAE. The large number of bioactive compounds, with a high reducing power and radical scavenging activity, might explain the pharmacological properties of MAE; thus, its bioactive ingredients were found capable of binding to PON1, which leads to increase in maximal velocity of the reaction. Atrahimovich et al  showed that the polyphenols of MAE bind to an allosteric site on recombinant PON1 and improve the enzyme function and biology. The results of this study indicated the antiobesity potential of MAE may be mediated by improved plasma irisin and skeletal muscle FNDC5, suggesting a scenario in which irisin enhances white adipocyte transdifferentiation (by increasing UCP1) into beige adipocytes, and as a consequence, the capacity of adipose tissue to uptake glucose or to synthesize fatty acids increases, promoting lipid mobilization or burning these fuels (by increasing PGC1α). FNDC5 gene expression was also negatively associated with TNFα, which is a negative regulator of white transdifferentiation to brown adipose tissue .
| Conclusion|| |
This study demonstrated that circulating MAE possesses antiobesity efficacy that is evidenced through the upregulation of FNDC5 gene expression and increase of the circulating irisin as well as antidyslipidemic battery that protects the body from the complications that are associated with obesity induced by high-fat diet. Furthermore, the current study demonstrated that the extract, to a high extent, reverses the formation of hepatic steatosis and nonalcoholic hepatic disorder.
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Conflicts of interest
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11]
[Table 1], [Table 2], [Table 3]