Abstract

Objectives

The aim of this study is to examine the anti-aging effect of Hibiscus sabdariffa (HS) against oxidative stress induced by hydrogen peroxide (H₂O₂) on immortalized human ovarian epithelial cells (IHOECs).

Materials and Methods

The obtained IHOECs were combined with 12.5 mg/mL HS extracted by adding 1mL of distilled water (dH₂O) and different concentrations of H₂O₂ for oxidative effect. IHOECs cell viability was increased with HS extract and determined by MTT test assay. Real-time polymerase chain reaction was conducted to amplify and detect apoptosis-related BAX and BCL₂, stem cell markers SOX₂ and OCT₄, and antioxidative nuclear factor erythroid 2-related factor 2 (NRF2) gene expressions. For the statistical analysis Kruskal-Wallis analysis and Mann-Whitney U test were used in SPSS program.

Results

The increase in HS in cell proliferation was 14%. While there is no significant increase of cell viability in the combined group with HS and H₂O₂, there were only statistically significant groups are 200, 800, 900 and, 1,000 µl. It was indicated that HS has apoptotic, stem cell promotion and antioxidative effects on high H₂O₂ concentrations of these groups (p<0.05).

Conclusion

In combined groups with both HS and H₂O₂ induced oxidative stress, HS increased the expression of proapoptotic gene BAX and decreased antiapoptotic gene BCL₂. Although, the remaining cells which are abundantly stem cells expressed genes OCT₄ and SOX₂ remarkably decreased, at high H₂O₂ concentration opposite effects were observed. NRF₂ gene expression also increased in this high oxidative stress.

Introduction

A wild tropical plant in the Malvaceae family Hibiscus sabdariffa (HS) is also known as roselle. There are various historical uses for HS in many cultures and established benefits of HS that are as follows: antihypertensive, anti-inflammatory, anti-obesity qualities; nephron-, hepato- and cardio- protective effects etc (1). Although further investigations are needed on its anti-aging properties, studies of this medicinal plant are still ongoing.

Immortalized human ovarian epithelial cells (IHOECs) were derived from human ovarian surface epithelium, which contains stem cells and expresses SOX₂, OCT₄ and nuclear factor erythroid 2-related factor 2 (NRF₂) genes (2, 3). We aim to investigate if and how we can use HS to slow down aging in IHOECs and improve ovarian health.

A number of the body’s primary processes gradually deteriorate with age, making aging an extremely complicated process. Hereditary factors, lifestyle choices, and environmental factors including xenobiotic pollutants, infectious agents, UV radiation, diet-borne chemicals, and so forth all have an impact on this unavoidable process. Aging and senescence are accompanied by a number of internal and exterior signs and symptoms, such as wrinkles and dry skin, diabetes, atherosclerosis, cancer, and neurological diseases. The most prevalent and deadly kind of ovarian cancer is epithelial and was linked to aging in many studies. In animal models, the removal of aging cells slows or stops the aging process. It has been suggested that a reduction in mitochondrial activity causes aging because it increases reactive oxygen species (ROS) generation and macromolecule damage (4, 5).

The imbalance between oxidants and antioxidants leads to oxidative stress, which is one of the primary initiating elements of aging-related damages that culminate in apoptosis. Free radicals have the ability to harm proteins, DNA, and fatty tissues in human body when their numbers exceed the antioxidants’ capacity to keep them in balance. Antioxidants can prevent these processes lowering the level of oxidative stress (6).

The previous works support the scientific hypothesis that HS plants enriched with bioactive constituents play an imperative role in the management of degenerative and chronic diseases, lipid metabolism (anti-cholesterol), anti-diabetic and anti-hypertensive effects that are associated with oxidative stress (7).

Within the overall antioxidant defense strategy, the function and efficacy of the first line defensive antioxidants, such as catalase (CAT), glutathione peroxidase (GPX), and superoxide dismutase (SOD), are critical and vital. Our body’s defense system against aging and oxidation is antioxidants like SOD, CAT and GPX can be enhanced by HS (8).

In this study we aimed to define the antiaging effect of HS on the IHOECs through some mechanisms and genes like BAX, BCL₂, SOX₂, OCT₄ and NRF₂.

In determining the survival of a cell there are many factors. There are two main routes that regulate apoptosis: the intrinsic (mitochondrial pathway) and extrinsic (death receptor pathway). The B-cell lymphoma 2 (BCL-₂) protein family orchestrates and regulates the intrinsic pathway. This protein family can be categorized based on their functions as either anti-apoptotic, including BCL-₂, BCL-x, BCL-w, MCL-₁, and A₁/BFL-₁, or pro-apoptotic, such as BAX, BAK. In addition, recent studies show BAX and BCL₂ contribute also to the cell cycle and apoptosis regulation (9).

SOX₂ and OCT₄ genes are embryonic stem cell markers that maintain pluripotency and regulate differentiation of stem cells. The upregulation of these genes under oxidative stress conditions with the addition of HS aqueous extract will indicate that the stem cell population is preserved. When cells transition away from a pluripotent state, SOX₂ signaling becomes vital for the development of various endodermal and ectodermal tissues during fetal development (10).

OCT₄ belongs to the Oct family of POU transcription factors. It holds a crucial position in controlling both the pluripotency and differentiation of stem cells. Considering the stem cell’s role in tissues we can relate the expression of these genes to a tissue aging, the ability to regenerate and preservation (11).

NRF₂’s protective function extends to averting chemical- and radiation-induced carcinogenesis by facilitating the enzymatic modification and elimination of carcinogens, as well as using gene transcription to repair oxidative damage and suppress ROS. Consequently, NRF₂ is widely regarded as a cytoprotective transcription factor, pivotal for cellular defense mechanisms and survival. Its deficiency renders organisms more susceptible to carcinogenesis and metastasis, highlighting its role as a tumor suppressor NRF’s significance in maintaining cellular integrity and adaptation to environmental stressors (12, 13).

Exposing HS, an antioxidant that uses the cascade mechanism, to the cell at an optimal level will delay the aging of the IHOECs and will extend healthy longevity. As a main result, it will prolong the reproductive age.

Materials and Methods

The cells used in the study are commercially available so ethics committee approval is not required. The study protocol was appropriate in accordance with the Declaration of Helsinki.

1. Cell Culture 

IHOECs were obtained from American Type Culture Collection. IHOECs were maintained in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F12) medium supplemented with 2.5mM L-glutamine (Bio-Ind, USA), 15mM HEPES, 100 U/mL penicillin and 100 mg/mL streptomycin (Sigma, St Louis, MO, USA) and 10% fetal bovine serum and incubated at 37 °C in humidified 5% CO₂ incubator. Every 48 hours, the growth medium was changed, and when the cells achieved 75-85% confluence, 0.25% trypsin was used to separate them.

2. HS Extract Preparation and Applying   

The flower part of HS were collected and identified by the Department of Biochemistry at Ankara University Faculty of Medicine. The 12.5 mg/mL of HS were dried, mashed and extracted by adding 1 mL of dH₂O. Before applying the 12.5 mg/mL extract underwent filtration a mesh with apertures measuring 0.22 mm in diameter. Subsequently, an extract of HS was added to the cell plates and incubated for 24 hours.   

3. MTT Assay   

To determine the viability of IHOECs, 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed. Cells were inoculated onto 96-well plates as 2x10⁴ cells per well and expected to adhere overnight. The cell groups cells were incubated with 12.5 mg/mL HS extract for 24 hours. Then they were treated with solutions of H₂O₂ within a concentration range of 200 to 800 µM and waited for 3 hours. Following this the 10 µM (5 mg/mL) of MTT reagent was introduced to each cell group for 2 hours at 37 °C.  Later, 100 μL detergent reagent was added. The color change was detected by using a spectrophotometric plate reader (Biotek, USA), each sample’s optical density was measured at 570 nm, using 690 nm as a reference.    

4. Real-time Polymerase Chain Reaction

IHOECs cell lines being already treated were cultured in 6-well plates with seeding density of 5×10⁵ cells/well. The total RNA was isolated by Trizol reagent (Invitrogen, USA). Complementary DNA (cDNA) synthesis was carried out using reverse transcriptase (iScript cDNA synthesis kit; Biorad). To amplify and detect apoptosis-related BAX and BCL₂, stem cell marker SOX₂, OCT₄ and antioxidative NRF₂ genes Real-time polymerase chain reaction (RT-PCR) was conducted using the CFX Connect RT-PCR Detection System (Biorad, CA, USA). To normalise the expression level of mRNAs, the expression of GAPDH mRNA was used as an endogenous control. This system uses the SYBR Green PCR Master Mix (iTaq Universal SYBR Green Supermix, Biorad, CA). The amplification parameters were heating to 95 °C for 10 min, followed by 50 cycles of each denaturation to 95 °C for 30 s, annealing to 60 °C for 30 s, and extension to 72 °C for 30 s.

Statistical Analysis

The results were expressed as the mean ± standard deviation (mean ± SD) and were analysed at SPSS Statistics using Kruskal-Wallis analysis and Mann-Whitney U test. Differences with values of p<0.05 were considered statistically significant.

Results 

1. Optimization of HS Concentration and Cell Viability Relation 

By applying HS at different concentrations without exposing IHOECs to any oxidative effect, the most appropriate concentration that provided 14% maximum cell proliferation for our experiment was found to be 12.5 mg/mL HS extract, and this concentration was used in all experiments. Cell viability of IHOECs in DMEM F12 medium for 24 hours was analysed by MTT test assay. The quantitative data are presented as mean ± SD in 3 repetitive experiments (p<0.05) (Figure 1).

According to Shapiro-Wilk test results, only 200, 800, 900 and 1,000 µL combined experiment results provide a test of normality (p=0.013). IHOECs were fed with HS were under oxidative influence, it was expected that a high rate of cell proliferation would be maintained due to the anti-oxidative effect of HS. Contrary to the expected results, the predicted increase in cell viability in MTT assay was not observed. MTT results indicated that cell viability in H₂O₂ and HS combined group had no significant effect on cell proliferation to change HS’s anti-aging effect (p≥0.05) (Figure 2).

It has been determined that H₂O₂ at concentrations of 200 µL, 800 µL, 900 µL, 1,000 µL with HS may have a negligible antioxidant effect (p=0.05). H₂O₂ concentrations of 200 µL and 800 µL with HS showed a minor antioxidant effect and were linked to decreased cell viability, suggesting HS may alter this (Figure 2). These concentrations were further studied for their slight contribution to proliferation and potential apoptotic effects in IHOECs. They are key to demonstrating HS’s impact on the expression of SOX₂, OCT4, BAX, BCL₂, and NRF2 genes.

2. HS Treatment Protects Stem Cell Marker Gene Expressions

In the control HS group, OCT₄ and SOX₂, which are stem cell markers of survived cells, are extremely abundant. Cell lines contain a high amount of stem cell gene expressions also has been demonstrated with this experiment even without any treatment. 

HS does not have an increasing effect on stem cell marker gene expression in 200 µL H₂O₂ concentrations but an increase in stem cell marker gene expressions was observed at high 800 µL concentration of H₂O₂ (Figure 3).

Although the expected anti-oxidative effect was not observed, it was observed that HS increased the SOX₂ and OCT₄ gene expression at a concentration of 800 µL, where the cells started to receive the most oxidative damage.

In the HS + 800 µL H₂O₂ group, a 4-fold increase was observed for the OCT₄ gene expression and a 5-fold increase for the SOX₂ genes under the excessive oxidative stress.

3. HS Treatment Increase the Apoptosis of Damaged Cells 

Apoptosis is referred to as programmed cell death because it occurs due to biochemical instructions in the cell’s DNA, is a type of protection mechanism of cells under stress (14). Apoptosis signaling is controlled by certain genes within the cell, and BAX (Figure 4a) is one of these apoptotic genes whereas BCL₂ is one of the anti-apoptotic genes (Figure 4b).

According to the RT-PCR reults while BAX gene expression increased, BCL₂ gene expression decreased. There was a strong positive correlation between HS and BAX/BCL₂ ratio. Between HS + 200 µL H₂O₂ and HS + 800 µL H₂O_{2 ,}180-fold difference in the ratio of BAX/BCL₂ genes (Figures 4a and 4b).

This fold increase in BAX/BCL₂ proved to us that the cell was using the apoptotic pathway. The death type of cells leads that HS might protect IHOECs which under the high concentration of oxidative stress via apoptosis. In the high oxidative conditions, induction of apoptosis by the HS in the damaged cells causes clearence of vulnerable IHOECs.

4. HS Treatment Increase Cellular Defence and NRF₂ Expressions in High Concentrations

The transcription factor NRF₂ controls the production of genes involved in drug detoxification and the oxidative stress response, which in turn protects cells against toxic and oxidative assaults. Cells become resistant to inflammatory stimuli and chemical carcinogens when NRF₂ is activated (15).

In this four repetitive NRF₂ gene expression determination in different concentration of H₂O₂ (200 µL H₂O₂ and 800 µL H₂O₂) experiment, there is any effect of HS in terms of cell proliferation in the control group or cellular defence against 200 µL H₂O₂ applied group (Figure 5).

Also, there is a higher expression rate in HS + 800 µL H₂O₂ than HS + 200 µL H₂O₂. These results indicate that HS may be more effective in terms of anti-oxidative and cellular defence effects in high concentration compared to low concentrations. P<0.001 indicates there was strong significance difference (Figure 5).

Discussion 

HS is a rich source of bioactive compounds which makes it a significantly rewarding topic of research. In recent literature, it is found to have many effects including antiseptic, diuretic, antioxidant and antimutagenic properties (16).

The anti-aging and antitumoral effects of HS were investigated on different types of cells including fibroblasts of skin (17), breast cells (18) and to name few.

However, IHOECs cell lines were not commonly studied until our study, although we see many examples of similar cell lines such as immortalized human ovarian surface epithelial cells. 

The expected anti-oxidative effect of HS was not observed when the cells were under oxidative influence. Despite the anticipation that HS would maintain a high rate of cell proliferation due to its anti-oxidative effect, an increase in cell viability was not observed. This suggests that while HS may have some antioxidant properties, its effectiveness may be limited to concentrations of oxidative stress. 

None of the previous studies in the literature examined stem cell properties in IHOECs. With this study, we examined the basal levels of SOX₂ and OCT₄ genes which have embryonic stem cell pluripotency in IHOECs for the first time and showed with our experiments that these cells contain stem cells.

It is known that ovarian epithelial cells are an important source of stem cells. Before being used therapeutically, it must have the proliferative potency and the capacity to maintain stem cells and progenitor cells without undergoing genetic modification during ex vivo culture (19). 

In the context of IHOECs, in addition to showing high expression in the control group, it was observed that SOX₂ and OCT₄ gene expressions decreased dose-dependently only under the influence of H₂O₂. However, when the oxidative stress in the cells was increased following HS treatment application, an increase in the expression of stem cell biomarkers was observed. Cell death was observed in the group in which 200 µL of H₂O₂ was applied together with HS solution and it was stated that stem cell markers decreased. In the group where 800 µL H₂0₂was applied together with HS solution, although cell death was observed, the expression of stem cell markers increased.

The increase in the expression of SOX₂ and OCT₄ in the presence of high oxidative stress following HS treatment, may provide evidence that HS solution has a protective effect on stem cells in IHOEC.

An increase in stem cell markers could suggest a pre-cancerous effect due to potential DNA damage under high oxidative stress. To verify this, DNA damage should be assessed in these cells under varying oxidative stress levels.

We studied the cell death mechanism in IHOEC due to oxidative stress, a topic not previously explored. We found a significant increase in BAX, a protein that promotes cell death, and a notable decrease in BCL₂, a protein that prevents it. This suggests a high BAX/BCL₂ ratio and indicates apoptosis in the IHOECs.

NRF₂, a cellular oxidative stress sensor, increases the cell’s antioxidant capacity by upregulating genes to eliminate ROS (20, 21). HS enhances antioxidant defense by reducing ROS and increasing SOD and CAT (22). Our results show NRF₂ expression decreased at low stress but increased at high stress, indicating HS’s antioxidant effect at high oxidative stress. Contrary to studies showing NRF₂ upregulates BCL₂ and downregulates BAX (23), our study found an exponential increase in BAX/BCL₂. A study found boric acid’s antioxidant and antineoplastic effects on damaged cells by increasing NRF₂ and BAX/BCL₂ (24). Similarly, HS may activate NRF₂ pathway and stimulate apoptosis in stressed IHOEC cells. Parallel to our study, HS may have an antioxidant effect on IHOEC cells by the activation of the NRF₂ antioxidant pathway and stimulation of apoptotic death of the stressed cells. 

Nevertheless, in other studies, NRF₂ was found to play an unexpected role in cancer development (25, 26). Related to that, an increase in the expression of NRF₂ may be related to the initiation of cancer in the epithelial ovarian cells. 

It was found significantly in our study that H₂O₂ application reduced the number of cells. Results can conclude that in the presence of high oxidative stress, HS has the potential to preserve the viability of stem cells while driving normal cells to apoptosis. Since maintaining the viability of stem cells is critical for tissue regeneration and repair, we can say that HS has a positive effect on the general condition of IHOEC. 

The effect of HS impact on gene expression and its potential geno-protective effects make it a promising area for further research. Future studies could explore these effects in more detail, potentially uncovering new therapeutic applications for HS. These findings contribute to our understanding of the complex interactions between plant extracts, oxidative-stress, cell viability, stem cell protection and open new avenues for research in this area. 

From an alternative perspective, adult-onset ovarian cancer and neo-oogenesis may be caused by stem cells found in IHOECs. This situation may indicate that it controls neo-oogenesis in addition to the pathophysiology of epithelial ovarian cancer. Thus, ovarian cancer and epithelial stem cells are closely related. As a result, a model can be developed to investigate the initial stages of ovarian cancer and produce novel insights.

Conclusion

In conclusion, this study suggests that adding HS to IHOEC during culture has the potential to cause clearance of damaged cells and induce stemness of IHOECs for the first time. It was reported that HS stimulated apoptosis in highly oxidative damaged cells and cause an increase in stem cell markers.