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Seven-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-chromen-4- one reduces atherogenic index and Nrf2 and GPx gene expressions in hyperlipidemic rats

Purpose: To investigate the effect of 7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-chromen-4-one isolated from mahogany (Swietenia macrophylla King) seeds on atherogenic index, expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and expression of the glutathione peroxidase (GPx) genes in hyperlipidemic rats. Methods: A total of 25 rats male aged 8 weeks and weighing an average of 200 g were used. They were divided into five groups as follows: (I) normal (N), (II) hyperlipidemic (HL), (III) hyperlipidemic rats treated with simvastatin (HL+SV), (IV and V) hyperlipidemic rats treated with 30 or 90mg, respectively, of 7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-chromen-4-one per 200 g body weight per day for 4 weeks. Atherogenic index (AI) was calculated from the levels of triglyceride (TG) and high-density lipoprotein (HDL) while Nrf2 and GPx gene expressions were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Results: Two different doses of 7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-chromen-4-one in hyperlipidemic rats significantly reduced their atherogenic index (p < 0.05). Nrf2 and GPx expression levels were lower than (p > 0.05) those of hyperlipidemic group. Conclusion: Seven-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-chromen-4-one reduces the atherogenic index and expression levels of Nrf2 and GPx genes in hyperlipidemic rats. Thus, this compound has potential as an antihyperlipidemic agent


INTRODUCTION
Hyperlipidemia, a form of dyslipidemia, is a lipid metabolism disorder characterized by increased levels of cholesterol and/or triglycerides. Hyperlipidemia is caused by many factors, including an unhealthy lifestyle, low physical activity, and a high-fat diet [1].
Oxidative stress causes translocation of the nuclear factor erythroid 2-related factor 2 (Nrf2) from the cytoplasm to the nucleus, where it combines with the antioxidant-responsive element (ARE) and induces the expression of antioxidant genes such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) [2]. Nrf2 is a regulator of oxidative stress in the cytoplasm and is removed by Kelch-like ECH-associated protein 1 (Keap1) for proteosomal degradation [3,4]. Keap1 regulates the expression of Nrf2 and enzymes involved in the Nrf2-Keap1 downstream signaling pathway, such as GPx. In hyperoxic conditions, the conformation of the Keap1 protein is changed and its binding to Nrf2 is prevented. Then, Nrf2 expression is increased, which also increases the expression of downstream antioxidants and resistance to oxidative damage [5].
Oxidative stress can be prevented by endogenous and exogenous antioxidants. Endogenous antioxidants protect cells from damage caused by oxidative stress. In hyperlipidemia, endogenous antioxidants are not sufficient to prevent cell damage. Therefore, exogenous antioxidants are needed to protect cells from oxidative stress-related diseases.
Many studies have reported that flavonoid compounds reduce oxidative stress. A previous study found that 7-hydroxy-2-(4-hydroxy-3methoxyphenyl)-chromen-4-one isolated from mahogany seeds (Swietenia macrophylla King) influences the expression of some genes involved in carbohydrate metabolism in a rat Type 2 diabetes mellitus (T2DM) model [6]. This study aimed to evaluate the effects of 7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-chromen-4-one flavonoid groups on the atherogenic index and expression of Nrf2 and GPx genes in hyperlipidemic rats.

EXPERIMENTAL Animals
Twenty-five male rats (Rattus norvegicus), aged 8 weeks old, with an average weight of 200g, were obtained from the Center for Food and Nutrition Studies, Universitas Gadjah Mada, Yogyakarta, Indonesia. The rats were housed in individual cages and acclimatized to laboratory conditions (22-25C) and a 12-h daylight cycle for 7 days with free access to food and water.

Gene expression analysis using quantitative polymerase chain reaction (qPCR)
The cDNAs were synthesized using the iScript cDNA Synthesis kit (Bio-Rad) according to the manufacturer's protocol. The SsoFast™ Evagreen® Supermix (Bio-Rad) was used for qPCR on an iCycler Model CFX 96 Real-Time System (Bio-Rad). The qPCR reaction was conducted for each gene (Nrf2 and GPx) using the same internal control Beta actin gene ( Table  1). The program for cDNA amplification was 5 min at 95C, followed by 40 cycles at 95C for 60 sec, and 57C for 60sec.

Statistical analysis
The results are expressed as mean ± standard deviation (SD). Differences in atherogenic indices among the groups before and after treatment with 30 or 90mg 7-hydroxy-2-(4hydroxy-3-methoxyphenyl)-chromen-4-one per 200g body weight per day (HL+30 or HL+90, respectively) were analyzed by one-way ANOVA followed by the Games-Howell test. The expression levels of liver Nrf2 and GPx genes after treatment were compared by one-way ANOVA followed by the Games-Howell tests. Paired t-tests were used to analyze the atherogenic index before and after treatment. Differences were considered significant at p<0.05.
Mallick and Khan [9] reported that sweet oranges (Citrus sinensis) and grapefruit (Citrus paradisi) produce antioxidants that have hypolipidemic effects in rats fed with a cholesterol-rich diet. Another study showed that chrysin flavonoid from honey, propolis, and plant extracts exerted     [11] reported that black mulberry (Morus nigra) leaf extracts contained abundant polyphenols, particularly chlorogenic acid. Chlorogenic acid had beneficial effects by reducing cholesterol and controlling fatty accumulations in the liver by increasing peroxisome proliferator-activated receptor alpha (PPAR-α) [12].
In the present study, rats with dyslipidemia had higher Nrf2 expression levels than normal rats, and treatment with 7-hydroxy-2-(4-hydroxy-3methoxyphenyl)-chromen-4-one isolated from mahogany (Swietenia macrophylla King) seeds reduced the Nrf2 expression levels. These results suggest that 7-hydroxy-2-(4-hydroxy-3methoxyphenyl)-chromen-4-one has antioxidant properties that reduce dyslipidemia-induced oxidative stress. Polyphenolic compounds and antioxidant activity also were detected in extracts of white (Morus alba) and black (Morus nigra) mulberry leaf [13][14][15]. GPx is localized in the cytoplasm, in the mitochondrial matrix, and in insoluble forms associated with membranes involved in the neutralization of lipid hydroperoxides [16]. The GPx function is responsible for lowering hydrogen peroxide (H 2 O 2 ) levels and converting lipoperoxides and organic hydroperoxides into suitable hydroxylation compounds, which are less reactive. Quercetin has in vivo antioxidant properties, and quercetin treatment increases hepatic GPx expression in older rats [17].
Research by Martin et al [18] showed that cocoa polyphenolic extract was an effective inducer of GPx. These reports are consistent with the study of Phachonpai et al [19], which reported that quercetin added to rat diet significantly increased the superoxide dismutase (SOD), catalase (CAT), and GPx activities [19,20].

CONCLUSION
The results of this study indicate that 7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-chromen-4-one lowers the atherogenic index and expression levels of Nrf2 and GPx genes in hyperlipidemic rats, and there may be suitable for management of hyperlipidemia but further investigations are required to ascertain this.

DECLARATIONS
terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/ 4.0) and the Budapest Open Access Initiative (http://www.budapestopenaccessinitiative.org/rea d), which permit unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.