1. Introduction
Non-alcoholic fatty liver disease (NAFLD) is characterized by a variety of liver conditions ranging from hepatic steatosis, to non-alcoholic steatohepatitis (NASH) [
1] which, if untreated, can lead to further complications such as hepatic fibrosis, cirrhosis or cancer, and even death [
2,
3]. There are several dietary interventions that can be used to ameliorate hepatic lipid accumulation, oxidative stress and inflammation associated with NAFLD and NASH [
4,
5] including the use of foods that are rich in antioxidants, among which polyphenols and carotenoids play a predominate role [
6,
7].
Moringa oleifera Lam is a softwood tree original from the Himalayas and Northern India, which has been investigated for its uses in human health [
8]. Many parts of this plant (leaves, immature pods, flowers and fruits) are edible and are used for their nutritional content in many countries [
9]. The leaves of the Moringa, the most utilized part of the plant, are characterized by having a great number of bioactive compounds including vitamins, carotenoids, polyphenols (phenolic acids and flavonoids) [
10]. For this reason, Moringa leaves (ML) have been used to treat a number of conditions including insulin resistance, cardiovascular disease, hepatic steatosis, cancer and others [
11].
ML have also been shown to have hepatoprotective activity and decrease the plasma lipids of rats fed a high-fat diet [
12,
13]. The aqueous extract of Moringa leaves has been reported to regulate glucose metabolism in diabetic rats [
14,
15]. In addition, ML have been found to have a cardioprotective role in isoproterenol-induced myocardial infarction by affecting the activities of several enzymes associated with oxidation [
16].
Flavonoids and saponins present in ML are reported to increase HDL (high density lipoprotein) cholesterol (HDL-C) and to lower LDL (low density lipoprotein) cholesterol (LDL-C) and very low-density lipoprotein (VLDL) cholesterol in hypercholesterolemic rats [
17]. It appears that flavonoids and saponins lower cholesterol absorption by the inhibition of cholesterol micellar solubility [
18]. Thus, both of these bioactive compounds found in the aqueous extract have been reported to have hypolipidemic effects [
9,
10].
Several bioactive compounds including nitrile, mustard oil glycosides and thiocarbamate present in ML have been shown to stabilize blood pressure [
19]. In addition, aqueous extracts of leaves, fruit and seeds of Moringa have antioxidant capacities [
20], possibly due to the presence of quercetin and kaempferol in Moringa, as they have been reported to have strong antioxidant activity on hepatocyte growth factor-induced oxidation [
21]. The liver protection of Moringa leaves has been found in other reports. Ethanolic extracts of ML have shown a protective effect against antitubercular drug-induced liver damage in rats [
22]. Further, histological examinations confirmed a decrease in hepatic damages induced by drugs [
23]. ML also showed the ability to reduce carbon tetrachloride-induced liver fibrosis and control the rise of serum aminotransferase activities and globulin level [
24] and to protect liver exposed to ionizing radiation by increasing antioxidant enzymes and inhibiting nuclear factor κB (NF-κB) translocation to the nucleus [
25].
Phenolic acids, including chlorogenic acid (CGA), from ML have been shown to have antioxidant, anti-inflammatory and anti-hyperglycemic properties [
26]. However, the specific mechanisms associated with reduction of hepatic lipids have not been fully elucidated.
The present study was conducted to investigate the effect of ML in a guinea pig model of hepatic steatosis. Our hypothesis was that ML would prevent hepatic steatosis induced by a high cholesterol diet by modulating the gene expression of regulators of hepatic cholesterol and triglyceride homeostasis through the action of bioactive compounds present in ML.
4. Materials and Methods
4.1. Experimental Design
Male Hartley guinea pigs (
n = 8 per group) were allocated into three groups: Control, Low Moringa (LM) (10 g/100 g), and High Moringa (HM) (15 g/100 g) for 6 weeks. This amount of Moringa was chosen based on 20 g of food consumed by guinea pigs per day to have 2 or 3.5 g or Moringa per day similar to human studies (Vergara-Jimenez, personal communication). At the end of the 6 weeks, guinea pigs were killed by exsanguination following heart puncture and blood was centrifuged immediately (2000×
g) to separate plasma. Liver tissue was also collected, snap-frozen and stored at −80 °C until further analysis. All animal protocols were approved by the Institutional Animal Care and Use Committee (Assurance A3124-01) at the University of Connecticut (Protocol #A16-003) on 1 March 2016. Moringa leaves were a gift from Scientech from Health International (Mexico City, Mexico). Macronutrient concentration of the diet was adjusted to account for the inclusion of Moringa in the diet. Moringa composition is presented in (
Table 5). A detailed description of the diets is presented in (
Table 6).
4.2. Plasma Lipids, Glucose and Liver Enzymes
The concentrations of glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and plasma lipids, including total cholesterol (TC), LDL-C, HDL-C and triglycerides (TG) were measured by using Cobas C 111 Analyzer (Roche Diagnostics, Indianapolis, IN, USA).
4.3. Lipoprotein Subfraction and Size
The concentrations of large, medium and small VLDL, LDL and HDL particles were measured by nuclear magnetic resonance (NMR). NMR spectroscopy was performed on a 400-MHz NMR analyzer (Bruker Biospin, LipoScience Inc., Raleigh, NC, USA), as described previously [
27].
4.4. Lecithin Cholesterol Acyl Transferase (LCAT) and Choleterol Ester Protein (CETP) Acitivities
The activity of LCAT was evaluated by use of a commercially available fluorometric assay kit (Cell Biolabs Inc., San Diego, CA, USA). The relative LCAT activity was assessed by the strength of the fluorescence signal following an incubation period. CETP activity was measured by use of a commercially available assay kit (BioVision Inc., Milpitas, CA, USA). The decrease in fluorescence intensity over time was used to calculate CETP activity after incubation of plasma with a self-quenching donor molecule and an acceptor molecule.
4.5. Hepatic Lipids
Hepatic tissue (100 mg) from each sample was homogenized with 200 μL of chloroform/isopropanol/MP40 (7/11/1). The mixtures were centrifuged and the supernatant dried at 50 °C overnight. The quantification for TC and free cholesterol (FC) was done with enzymatic assays kits (Wako Diagnostics, Mountain View, CA, USA) [
28]. Esterified cholesterol (CE) was calculated manually by subtracting FC from TC. Liver TG were extracted (100 mg of tissue) with chloroform:methanol (2:1), dried under nitrogen at 60 °C and solubilized in 1% Triton X-100. The solubilized lipid isolates were analyzed by enzymatic methods (Wako Diagnostics, Mountain View, CA, USA), as previously described [
29].
4.6. Histologic Evaluation
Small pieces taken from the same liver section were immersed in 10% buffered formalin. Formalin-fixed livers were paraffin embedded and sections measuring 3–5 μm were stained with hematoxylin. The stained tissue sections were viewed under bright field microscopy at 200× magnification. An AxioCam ICc3 camera (Zeiss, Thornwood, NY, USA) was used to take the pictures.
4.7. Inflammatory Cytokine Concentration in Liver
Tissue total protein was extracted and protein concentration of the lysates was evaluated by use of the bicinchoninicc acid assay rotein Assay Kit (Cell Signaling Technologies, Inc., Beverly, MA, USA). Using the same concentration of protein for all samples, the following cytokines were measured using Luminex technology (Luminex 200 System, Austin, TX, USA) with the MILLIPLEX MAP Rat Cytokine Immunoassay kit (Millipore corporation, Charles, MO, USA): interleukin (IL)-1β, IL-6, IL-10, interferon (IFN)-γ, monocyte chemotactic protein 1 (MCP-1), and tumor necrosis factor (TNF)-α, as previously described [
30].
4.8. Gene Expression
Primer design: The National Center for Biotechnology Information website was used to design the specific primers of Cavia porcellus. The following genes involved in lipid metabolism and inflammation were analyzed: LDL receptor (LDL-R), cluster of differentiation-68 (CD68); HMG-CoA reductase (HMG-CoA-R); peroxisome proliferator-activated receptor (PPARγ), cluster of differentiation-36 (CD36); diacyl glycerol acyltransferase-2 (DGAT-2), Primer sequences are available upon request.
4.9. RNA Isolation, cDNA Synthesis and Real-Time qRT-PCR
RNA was extracted with TRIzol (Thermo Fisher Scientific, Pittsburgh, PA, USA) after liver tissues were homogenized. RNA was reverse transcribed to cDNA and then amplified and quantified by real-time quantitative polymerase chain reaction using a Bio-Rad CFX96 Real-Time system (BioRad, Hercules, CA, USA), as described [
31]. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was used in normalization using the 2
−ΔΔCt method.
4.10. Statistical Analysis
Differences among groups were analyzed by one-way ANOVA and Tukey post hoc analysis. p < 0.05 was considered to be significant. All analyses were conducted on SPSS for Windows, Version 20 (IBM Corp., Armonk, NY, USA). All data are presented as mean ± Standard Deviation (SD).