Bee Product Royal Jelly Reduces Oxidative Stress in Healthy MRC-5 Cells and Upregulates GSTP1 Expression ^†

Abstract

Redox homeostasis in the human body is strictly regulated by reducing molecules, such as glutathione, as well as various antioxidant enzymes. Examination of the antioxidant effects of natural products is necessary in order to prevent and treat various pathological conditions considering the correlation of their occurrence with oxidative stress damage. RJ has been identified as a very potent regulator of many metabolic processes and is considered as a medicinal agent that can cope with the oxidative stress. The present study evaluated the RJ’s ability to scavenge superoxide anion radicals O₂^∙− and modulate the expression of GSTP1 marker in healthy lung fibroblasts (MRC-5 cell line) after 24 h. Assessment was performed with the NBT test and quantitative real-time polymerase chain reaction (qPCR). Our results show that RJ successfully reduced the O₂^∙− concentration for ~12% and upregulated GSTP1 gene expression (1.75 fold-change) whose protein product is responsible for catalyzation of glutathione (GSH) binding to oxidative stress metabolites and their further neutralization in cells. We found that RJ has an important protective effect against oxidative damage of healthy human cells and these properties could be used to explore new resources for pharmacological treatments, as well as to improve application of natural medicine for maintaining human health.

1. Introduction

Oxidative stress, as an imbalance between oxidants and antioxidants in favor of the oxidants, causes irreparable cell damage due to oxidation of nucleic acids, proteins, carbohydrates, and lipids, leading ultimately to cell death. In the case of oxidative stress, reactive oxidative species (ROS) are produced in excessive amounts as a result of aerobic breathing and metabolism. In addition to nitrites, the most common ROS are superoxide anion radicals (O₂^∙−) [1]. This deregulation in redox homeostasis deeply impacts human health and causes various pathological conditions, with already shown correlation of oxidative stress damage and majority of diseases. Redox homeostasis in human body is strictly regulated by the “redox-buffering” intracellular thiols, reducing molecules, such as glutathione (GSH), as well as various antioxidant enzymes [1]. Glutathione S-transferase proteins (GSTPs) control drug metabolism and protect the cell from oxidative stress by conjugating GSH with a wide variety of highly electrophilic/lipophilic substrates and reactive species, such as NO [2,3]. Also, it has been reported that ROS, including O₂^∙−, lead to inhibition of soluble GSTs in cells [3].

Considering that the increase in oxidative stress and cell damage is mostly associated with aging, it could be concluded that a decline in antioxidant defense systems is of particular significance for scientific research. Also, the commercial use of many natural antioxidants, dietary supplements, functional foods and nutraceuticals showed to possess the ability to reduce ROS and oxidative stress, resulting in an increase in life span [4]. Therefore, discovery, analysis, and application of natural products with prominent antioxidative properties is mandatory in prevention of various diseases, as well as in their treatment.

As a well-known bee product, royal jelly (RJ) has been traditionally used since ancient times due to its abundance in bioactive activities and numerous biological properties, and it is considered nowadays as an effective dietary supplement. It possesses significant beneficial effects on human wellbeing. RJ is designed as a very potent regulator of many processes like inflammation, where this natural product exerts anti-inflammatory and immuno modulatory activity. Also, it maintains cellular homeostasis, reduces blood pressure by causing vasodilatation, and aids in the recovery from pathological conditions [5]. Its potential to regulate the level of sugar and lipids in the blood, as well as neurotrophic, antitumor, hepatoprotective, antibiotic, anti-allergic, and antiaging potential has been previously reported [6]. Moreover, RJ is considered a medicinal agent that can cope with the oxidative stress that has been demonstrated in various in vitro and in vivo studies [6,7,8].

Therefore, the subject of the current investigation is the evaluation of the RJ’s ability to scavenge superoxide anion radicals O₂^∙− and modulate GSTP1 expression in healthy cells in order to offer a scientific basis for further development and use of this natural bee product in maintaining human wellbeing.

2. Materials and Methods

An RJ sample, produced by Apis mellifera L. bee species, was obtained from beehives located in the central region of Serbia and was kept at 4 °C. The RJ was diluted in PBS (phosphate-buffer solution, Capricorn Scientific, Ebsdorfergrund, Germany) and complete cell culturing medium DMEM (Dulbecco’s Modified Eagle Medium, Lonza, Basel, Switzerland) supplemented with 10% of FBS (Fetal bovine serum, Capricorn Scientific, Ebsdorfergrund, Germany) and penicillin/streptomycin (Gibco, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Healthy lung fibroblasts, MRC-5 cell line, were purchased from American Type Culture Collection (ATCC, Manassas, VI, USA) and propagated at 5% CO₂ and 37 °C in a humified atmosphere.

The obtained working concentration of 100 µg/mL was previously tested on MRC-5 cells for its impact on cell viability and showed no cytotoxic effects [9]; thus, it was furtherly tested for its antioxidant potential.

The NBT test was applied according to already published protocol [10] and the analysis of RJ was performed after 24 h. Resulting absorbances were read at a 550 nm wavelength on a Multiskan SkyHigh Microplate Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The obtained results are presented from two independent experiments performed in triplicates as nmol O₂^∙− per mL and calculated in relation to the number of viable cells [9].

Furthermore, a quantitative real-time polymerase chain reaction (qPCR) technique was used to evaluate the expression of antioxidative marker GSTP1 on gene level, according to the protocol already described in detail [11]. Isolation of mRNA from control and treated MRC-5 cells was performed using TRIzol and chloroform method, while the reverse transcription was performed by applying Fast Gene Scriptase Basic cDNA Reverse Transcription Kit (Nippon Genetics, Tokyo, Japan) and Mastercycler^® PCR (Eppendorf, Hamburg, Germany) aparature. The relative expression of housekeeping gene β-actin and GSTP1 was determined using the EURx qPCR kit (Gdańsk, Poland) according to the manufacturer’s instructions on Mic qPCR Cycler (Biomolecular Systems, Yatala, Australia). The calculation of relative gene expression of GSTP1 in tested MRC-5 cells was performed by using the 2^−ΔΔCt method [11]. The results were acquired from two independent experiments performed in duplicates and represented as relative mRNA expression.

One-way ANOVA and Student’s t test were used within SPSS statistical software package, ver. 20 (IBM, Armonk, NY, USA) to perform statistical analysis, with * p < 0.05 designated as statistical significance.

3. Results

3.1. RJ Reduces O₂^∙− Concentration

The antioxidative potential of RJ was investigated in control and treated MRC-5 cells within 24 h using the NBT test. According to obtained results, this natural bee product significantly (with * p < 0.05 significance) reduced the level of O₂^∙− in tested cells at the applied concentration of 100 µg/mL, when compared to the control values, as can be observed in Figure 1.

3.2. RJ Upregulates Expression of GSTP1

Considering this prominent antioxidative potential of the tested RJ sample, and the role of GSTP1 in the antioxidative protection of cells, we aimed to determine the possible change in GSTP1 gene expression after treatment with this bee product. The GSTP1 marker is responsible for the catalysation of glutathione (GSH) binding to oxidative stress metabolites, such as free radicals, and their further neutralization in cells.

The gene expression of this marker was investigated by the qPCR method, and as can be observed in Figure 2, RJ treatment significantly (* p < 0.05) upregulated GSTP1 mRNA levels (1.75 fold-change) in MRC-5 cells after 24 h, when compared to the control values (Figure 2).

4. Discussion

Many previous studies proved RJ’s antioxidative potential, which is based on the increase in the concentration of enzymatic and non-enzymatic components of the antioxidative system, as well as the inhibition of nitrite production and lipid peroxidation [6,7,8]. As a very effective antioxidant, RJ protects DNA from oxidative damage and possesses the ability to suppress free radicals [6,12,13]. The analysis of RJ’s chemical composition has shown that this natural product consists of many bioactive antioxidative components among which are unsatturated fatty acid 10H2DA and phenolics, which are known to be able to modulate redox status in cells [5,7].

However, the analysis of O₂^∙− concentration in MRC-5 healthy lung fibroblasts has not been reported so far; therefore, results of this in vitro study showed for the first time that this natural agent can reduce oxidative stress in tested cells within 24 h through affecting the concentration of these free radicals. Khoob et al. [14] showed that RJ modifies the activity of superoxide dismutase, the enzyme directly responsible for neutralizing O₂^∙− by increasing it ~3 times [14]. Therefore, the most probable mechanism responsible for reduced level of O₂^∙− presented in our study is the increased activity of SOD.

Moreover, according to our results, RJ elevated the expression of GSTP1 in tested MRC-5 cells, contributing to this antioxidative potential. Glutathione-S-transferases (GST) are a family of phase II enzymes that play critical roles in cell metabolism and detoxification, where these proteins catalyze the inactivation of various substrates, including intracellular reactive oxygen species, by GSH conjugation and their further excretion from the cell by multidrug resistance proteins (MRPs) [15]. Glutathione S-transferase P1 (GSTP1) protein is widely expressed in various types of tissues such as lungs, and plays several roles in healthy cells. This includes phase II xenobiotic metabolism, the neutralization of a variety of endogenous electrophiles produced by oxidative stress, exogenous substances (xenobiotics), carcinogens, and their metabolism. GSTP1 can interact with proteins involved in many celular processess such as apoptosis, cell cycle regulation, proliferation, regulation of inflammation, differentiation, etc. [2,15]. Several recent studies have reported the great therapeutic potential of GSTP1 in lung injury, asthma, and other diseases [16]. All of the above stated indicate that the elevation of GSTP1 levels in healthy cells and tissues can improve the antioxidant protection and mainteinance of redox homeostasis. In addition, GSTP1 also interacts with GPX4 and inhibits the oxidation of polyunsaturated fatty acids on the cell membrane, therefore, inhibiting the occurrence of ferroptosis [17]. Based on our findings, it can be concluded that RJ induced desirable effects in tested MRC-5 by elevating the expression levels of the investigated marker GSTP1.

On the other hand, it has been generalized that the reactive oxygen species (ROS) inhibit cytosolic GSTs and vice versa [3]; therefore, our results indicate the lower concentration of O₂^∙− and, as expected, the consequent elevation of GSTP1, which can lead to increased level of GSTP1 protein in cells.

5. Conclusions

We found that RJ has an important protective effect against oxidative damage of healthy human lung fibroblasts by suppressing the level of superoxide anion radicals O₂^∙− and upregulating GSTP1, thus improving the antioxidative protection in these cells. However, more detailed and extensive studies are necessary regarding this natural product on in vivo models and other molecular targets involving in this metabolic mechanism. These studies can be useful in order to explore new resources for pharmacological treatments, as well as to improve natural medicine. Also, due to its notable antioxidant activity, it becomes an ideal option for being used with other treatments such as chemotherapeutics, or sole supplement for cell and tissue recovery.