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Original Article

Anat Cell Biol 2023; 56(4): 526-537

Published online December 31, 2023


Copyright © Korean Association of ANATOMISTS.

Effect of Sofosbuvir on rats’ ovaries and the possible protective role of vitamin E: biochemical and immunohistochemical study

Neven A. Ebrahim1 , Hussein Abdelaziz Abdalla2,3 , Neimat Abd Elhakam Yassin4 , Aya Elsayed Maghrabia5 , Amira Ibrahim Morsy1

1Department of Anatomy and Embryology, Faculty of Medicine, Mansoura University, Mansoura, 2Department of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Mansoura University, Mansoura, Egypt, 3Department of Medical Biochemistry, Faculty of Medicine, Taibah University, Medina, Saudi Arabia, 4Department of Clinical Pharmacology, Faculty of Medicine, Mansoura University, Mansoura, 5Veterinary in Medical Experimental Research Center, Faculty of Medicine, Mansoura University, Mansoura, Egypt

Correspondence to:Neven A. Ebrahim
Department of Anatomy and Embryology, Faculty of Medicine, Mansoura University, Mansoura 35516, Egypt
E-mail: nevenebrahim@mans.edu.eg

Received: March 21, 2023; Revised: June 14, 2023; Accepted: June 22, 2023

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Hepatitis C virus (HCV) infection is a major health problem worldwide and its eradication is mandatory. Direct acting HCV polymerase inhibitors, such as Sofosbuvir (SOF), is an effective regimen. However, it has some side effects like mutagenesis, carcinogenesis, and the impairment of testicular function. It is important to evaluate the safety of SOF on the ovary, as there are no studies yet. Increasing the production of Reactive Oxygen Species (ROS), causes oxidative stress, which affects ovulation process, female reproduction, and fertility. Accumulation of SOF in the cells was demonstrated to promote ROS generation. Vitamin E (Vit E) is an antioxidant agent that has an essential role in the female reproductive system, its deficiency can cause infertility. We explored the effect of SOF treatment alone and co-treated with Vit E on ovarian ROS level and ovarian morphology experimentally using biochemical and immunohistochemical studies. Significant changes in oxidative stress markers; nitric oxide and malondialdehyde lipid peroxidation, antioxidant enzymes; catalase, super oxide dismutase, and reduced glutathione, proliferating markers; proliferation cell nuclear antigen and Ki-67 antigen and caspase 3 apoptotic marker were demonstrated. It was shown that where SOF induced oxidative stress, it also aggravated ovarian dysfunction. The essential role of Vit E as an antioxidant agent in protecting the ovarian tissue from the effect of oxidative stress markers and preserving its function was also displayed. This could be guidance to add Vit E supplements to SOF regimens to limit its injurious effect on ovarian function.

Keywords: Ovary, Sofosbuvir, Oxidative stress, Apoptosis, Vitamin E

Hepatitis C virus (HCV) infection is a major health burden causing advanced liver diseases worldwide. Egypt has the highest ratio of HCV globally, especially genotype four which represents more than 90% of the HCV infections [1]. The treatment with direct acting antiviral drugs for HCV (HCV polymerase inhibitors) has evolved recently, such as Sofosbuvir (SOF), which is effective in suppressing HCV replication and is recommended in its treatment regimens [2]. However, SOF has some side effects like mutagenesis, carcinogenesis, and impairment of fertility [3]. It was demonstrated to reduce the level of testosterone hormone and caused marked degeneration of testicular seminiferous tubules in rats [4]. It is mandatory to evaluate the safety of certain drugs on ovarian toxicity, as any morphological abnormities induced in the ovaries cause impairment of the female reproductive cycle [5]. The old HCV treatment regimen: interferon and ribavirin, were shown to affect the ovarian reserve in treated women [6]. There are no studies on the effect of SOF on ovarian tissue or function yet, and it is the aim of this study.

Proliferation cell nuclear antigen (PCNA) is a nonhistone protein that assists DNA polymerase which is essential for cellular replication. Its expression affects mitotic activity, so it’s usually used as an indicator for cellular proliferation [7-9]. The Ki-67 antigen is also a cell proliferation marker [10]. It is expressed within the nucleus, while in mitosis it is relocated to the chromosomes’ surface, it’s present through cell division at the S, G2, and M stages of the cell cycle, and after mitosis at G1 stage. Its expression is only absent in quiescent cells at the G0 stage [11]. Those markers are used to follow up the effect of drugs on the ovarian follicle’s proliferation and cytotoxicity [12-14].

Caspase 3 is one of the most significant apoptotic caspases, related to intracellular proteins proteolysis and to the apoptotic morphological changes [15]. Its activity measurement helps the assessment of the cytotoxicity of certain drug or chemicals on ovarian tissue or cell [16, 17].

Increasing the production of Reactive Oxygen Species (ROS), can cause oxidative stress, which is involved in many diseases, ROS are generated from leakage of electrons during production of adenosine triphosphate and oxidative phosphorylation from the inner mitochondrial membrane. ROS affects reproduction and fertility, several studies demonstrated that accumulation of toxic metabolites from oxidative stress can cause follicular atresia [18-20]. It could cause mitochondrial damage [21], steroidogenic enzymes inhibition [22], cellular membrane and DNA damage which may end up with apoptosis or necrosis [23].

Lipid peroxidation, which is unsaturated fatty acids’ oxidation, is one of the most harmful effects of free radical attack. Malondialdehyde (MDA) is one of the end products of lipid peroxidation [24]. Nitric oxide (NO) is a short-lived free radical gas that is synthesized by NO synthases from L-arginine. It is involved in many physiological functions like vasodilation, and neurotransmission. It is a highly diffusible molecule and forms stable oxidized metabolites, such as nitrates and nitrites [25]. NO was shown to modulate granulosa cell function that was associated with follicular maturation and ovulation in women [26-28].

The antioxidant enzymes, such as Super Oxide Dismutase (SOD), Catalase (CAT), and reduced glutathione (GSH) and the nonenzymatic antioxidants, such as vitamin E (Vit E), and vitamin C, located in the ovarian follicles, help elude oxidative damage [29, 30].

Antioxidants detoxify ROS and maintain body oxidant/antioxidant balance. Vit E (α-tocopherol) is a fat-soluble vitamin, acts as antioxidant that reserves lipoproteins and cell membranes from peroxidation, it inhibits the formation of free radical and reduces lipid peroxidation [31]. Vit E has an essential role in the female reproductive system, its deficiency can cause infertility [32]. It has a protective effect on ovarian follicles against certain drugs [12, 33].

HCV is certainly a major problem causing marked morbidity and mortality worldwide, its eradication is mandatory and SOF treatment showed promising responses [34], however as most of the drugs, it has multiple side effects including its reproductive dysfunction effects. Avoiding or reducing these side effects of the drug would help in safely using this medication and thus continuing eradication of the infection with minimum drawbacks.

Aim of the work

The purpose of this study is to investigate the effect of SOF on rats’ ovarian tissue through exploring the morphological changes in ovarian cortex and medulla and the role of ROS in pathogenesis. In addition, to examine whether Vit E could be a protective agent against the cytotoxic effect of SOF. In a trial for better understanding of the drawbacks of SOF on ovarian function and try to protect against it.

Ethical approval

The animal experiments were conducted according to the guidelines for the Principles of Laboratory Animal Care. All rats’ experimental procedures and protocols were approved by the institutional review board (IRB), Faculty of Medicine, University of Mansoura (Ref: R.22.06.1730).

Animal model preparation

Twenty-four virgin female Sprague-Dawley rats, average age 6–8 weeks (each 150–250 g weight) were purchased from Mansoura Medical Experimental Research Center to be used in this study. Animal experiments were performed in accordance to the guidelines for the Principles of Laboratory Animal Care. The rats had been fed standard pellet diet and water ad libitum. Rats had been divided randomly into four groups: control, drug treated, sham, and drug treated+Vit E co-treated. The rats were given the treatment via oral gavage, the therapeutic dose of SOF (Mpiviropack 400 Mg/tablet; Gardenia Pharmacy) used in concentration 40 mg/kg body weight, dissolved in 0.9% NaCl, and had been given daily for 1 month [35, 36]. Vit E (Vitamin E 450 Mg 1,000 IU/capsule; Saha Pharmacy), used in concentration 100 mg/kg and had been administrated also daily by gavage [33]. Rats were individually housed in pathogen-free conditions at (20°C–22°C) and (45%–55%) humidity in a 12 hours light-dark cycle. Rats had been sacrificed 24 hours after day 30.

Experimental groups

Twenty four rats had been divided into 4 groups: Negative control group: (6 rats) had been fed on a basal diet, then sacrificed (to be compared with experimental groups). Experimental group I (Sofosbuvir treated): (6 rats) had been given Sofosbuvir for 30 days, then sacrificed (to confirm its effect on the ovary). Experimental group II (SOF and Vit E-treated): (6 rats) had been given SOF together with Vit E for 30 days then sacrificed (to confirm its protective effect on the ovary).

Specimen collection

After 30 days, rats were sacrificed, and ovarian samples were collected, weighted, and prepared for chemical, histological and immune-histochemical studies to explore the effect of the drug on female rats’ ovaries and the potential protective role of Vit E.

Chemical assessment

The ovary was weighted and homogenized with Tris-HCl (5 mmol/l containing 2 mmol/l EDTA, pH 7.4), homogenate was centrifuged at 1,000 ×g for 10 minutes at 40°C. Supernatants were used for measuring NO, CAT, SOD, MDA and GSH activity after the calculation of the exact wet weight per ml [37].

The level of NO in the homogenates was measured by colorimetric determination of nitrite levels using Griess reagents [38], by nitric oxide assay kits (2533; Bio Diagnostic Co.).

MDA assay Kit (118970; Abcam), SOD activity assay kit (65354; Abcam), CAT activity assay kit (83464; Abcam) and GSH assay kits (TA2511; Bio Diagnostic Co.) were used for colorimetric determination of MDA, SOD activity, Catalase activity, and GSH according to the manufacturers’ instructions.

Tissue processing, histology, and immunohistochemistry

At the end of the experiment, ovarian tissues had been harvested, cut, and fixed in buffered formalin. Tissue samples were embedded in paraffin and sections were 5-μm thickness cut and prepared for hematoxylin and eosin (H&E) staining to study the ovarian morphology and whether there is any effect on ovarian follicles.

Immunohistochemical studies were carried out following the same protocol discussed in a previous publication [39]. Normal goat serum was used to block nonspecific receptors, PCNA (ab265585; Abcam), Ki-67 (ab279653; Abcam) and caspase 3 (PA5-86276; ThermoFisher) primary antibodies were used with the recommended dilution (PCNA anti-mouse monoclonal antibody, 1:200 dilution; Ki-67 monoclonal mouse, 1:500 dilution; caspase 3 anti-mouse polyclonal, 1:50 dilution), then specimens were incubated with corresponding secondary antibodies. Diaminobenzidine (DAB) and Streptavidin peroxidase were used to obtain the brown color, counterstained with Mayer’s hematoxylin for 30 seconds, then washed with PBS and with distilled water. Finally, it was mounted and examined with an Olympus microscope (CX41) to evaluate ovarian follicles proliferation and apoptosis, to investigate the effect of SOF treatment and the protective role of Vit E on ovarian functions.

Morphometric studies

H&E-stained slides, five from each group were used for manually counting the follicles and corpora lutea. For each slide, follicles were blindly counted in five non-overlapping fields (×200).

The area percentage of brown stained PCNA, caspase 3 and Ki 76 immunostaining sections were measured also from non-overlapping fields of immuno-stained slides of each group (×200), using the NIH Image J program (National Institute of Health). Image J program was used to separate the brown-colored immuno-histochemically stained pixels from the background. To define color threshold range for the brown-colored immuno-histochemically stained regions, the brightness and saturation for the randomly selected image samples were adjusted. Then, the selected threshold was applied to separate the background (H&E-stained pixels) from the positively stained color pixels (DAB) in the chosen images.

Statistical analysis

Statistical analysis was performed using unpaired t-test for two groups’ comparison and One-way ANOVA for more than two groups’ comparison (GraphPad Prism 9; GraphPad Software Inc.). Numerical data were presented as mean±standered error of the mean and differences among groups were compared using P value ≤ 0.05 as significant.

Biochemical assays

Lipid peroxidase studies demonstrated statistically significant increases in the concentration of NO and MDA (P<0.00001) after SOF treatment compared to the control (Fig. 1A, B), then it was reduced significantly after SOF+Vit E co-treatment (NO, P<0.0001 & MDA P<0.00001) (Fig. 1A, B). However, it showed decreases in the activity of CAT (P<0.0001) and SOD (P<0.00001) and decreasing concentration of GSH (P<0.00001) in SOF treated ovaries (Fig. 1C–E) compared to control group. Then, it was significantly increased after SOF+Vit E co-treatment (P<0.0001 for SOD, GSH and P<0.01 for CAT) (Fig. 1C–E).

Figure 1. Histograms showing the mean ovarian tissue oxidative stress markers (NO, MDA, SOD, GSH, and CAT). (A, B) Levels of NO and MDA in different experimental groups were demonstrated. The SOF-treated group shows a significant upregulation in the level of NO and MDA compared to the control group, ****P<0.00001. The oxidative stress markers’ levels are reversed considerably in the SOF+Vit E-treated group compared to the SOF group, ###P<0.0001, ####P<0.00001. (C–E) The ovarian tissue CAT, SOD, and GSH levels in different experimental groups. The SOF-treated group demonstrates a significant downregulation in CAT, SOD, and GSH levels compared to the control group, ***P<0.0001, ****P<0.00001. The oxidative stress markers are considerably altered in the SOF+Vit E co-treated group compared to SOF group, #P<0.01 and ###P<0.0001. One-way ANOVA was used to compare between all the groups, unpaired t-test was used to compare each group with the control, and the SOF+Vit E co-treated with SOF group. NO, nitric oxide; MDA, malondialdehyde; SOD, super oxide dismutase; GSH, reduced glutathione; CAT, catalase; SOF, Sofosbuvir; Vit E, vitamin E. *P<0.01 and **P<0.001.

Histopathological studies

Microscopic examination

H&E-stained sections of the controlled rats’ ovaries demonstrated a normal preserved ovarian histological structure with an ovarian cortex filled with multiple follicles at different developmental stages, and a central medulla with vascularized stroma (Fig. 2A). The ovarian sections of SOF-treated rats showed cystic atretic follicles with degenerated ovum and multiple vacuolation. Moreover, there were dilated congested blood vessels in the ovarian medulla (Fig. 2B, C). Ovarian sections from Vit E-treated rats revealed presence of growing follicles and an increase in the number of corpora lutea (Fig. 2D). In SOF+Vit E treated group, healthy graafian follicles reappear with multiple corpora lutea and healthy mildly vascularized medulla (Fig. 2E).

Figure 2. Micrographs of hematoxylin and eosin-stained rats’ ovarian sections (A–E). (A) Ovarian sections in the control group illustrate multiple growing GF, CL, vascularized M, PR with observable ovum. (B, C) The SOF-treated ovary demonstrating atretic follicile (T), congested B and multiple V. (D) The Vit E-treated ovary illustrates healthy follicles with multiple corpora lutea. (E) The SOF+Vit E co-treated ovary demonstrates health primary follicles with multiple corpora lutea and mildly vascularized medulla. (F–H) Histograms illustrate the morphometric study of the effect of SOF and Vit E on treated rats’ ovaries: (F) healthy follicles. (G) Atretic follicile and (H) corpora lutea. The SOF-treated group illustrates a significant reduction in the number of atretic follicles and corpora lutea compared to the control. While the number of atretic follicles was significantly increased in the SOF group compared to the control, *P<0.01, **P<0.001, ****P<0.00001. In the SOF+Vit E co-treated group, the number of healthy follicles and corpora lutea are significantly upregulated, while the atretic follicles are significantly downregulated, ####P<0.00001. One-way ANOVA was used to compare between multiple groups, unpaired t-test was used to compare between two groups. Scale bar: 50 μm. GF, graafian follicles; CL, corpus luteum; M, medulla; PR, primary follicle; SOF, Sofosbuvir; B, congested blood vessles; V, vacuolations; Vit E, vitamin E.

Morphometric studies

The morphometric studies revealed a significant reduction (P<0.00001) of healthy follicles’ numbers and a significant increase (P<0.00001) in the number of atretic follicles in the SOF treated rats group compared to the control and Vit E groups (Fig. 2F, G). The number of corpora lutea demonstrated a significant reduction in SOF treated rats (P<0.001) compared to the control and increased significantly in SOF+Vit E-treated (P<0.00001) compared to SOF treated group (Fig. 2H).

Immunohistochemistry of PCNA, Ki-67 and caspase 3

The number of proliferating cells (PCNA positive cells) was observed to be the least in SOF-treated group compared to control, Vit E and SOF+Vit E co-treated groups (Fig. 3A–D). There was a significant reduction in the intensity of PCNA stained area % in SOF-treated group compared to control and Vit E-treated groups. There is also a significant increase in SOF+Vit E co-treated group compared to SOF-treated group (P<0.0001) (Fig. 3E).

Figure 3. Micrographs of rat’s ovaries immuno-histochemicaly stained with PCNA. PCNA-positive nuclei are localized in brown (A–D). (A) Control group, (B) SOF-treated group, SOF illustrates significant reduction in the PCNA-positive cells in comparison to the control group, (C) Vit E-treated group, and (D) the SOF+Vit E treated group, where PCNA-positive cells increased compared to the SOF group. (E) Histogram illustrates the quantitative analysis of the intensity of PCNA stained areas in different experimental groups. The SOF-treated group demonstrates a statistically significant reduction compared to the control group, ****P<0.00001. The SOF+Vit E co-treatment significantly reduces the PCNA immuno-staining in comparison with the SOF treated group, ####P<0.00001 (Bar=20 μm). PCNA, proliferation cell nuclear antigen; SOF, Sofosbuvir; Vit E, vitamin E.

The proliferating marker Ki-67 in the control group’s ovarian sections demonstrated intense nuclei staining of the most growing Graafian follicles granulosa and theca cells (Fig. 4A). Ki-67 immuno-stained SOF-treated rats’ sections showed negatively stained cystic atretic follicles (Fig. 4B). Vit E-treated rats showed positively stained graafian follicle granulosa and theca cells (Fig. 4C). SOF+Vit E co-treated group demonstrated return Ki-67 positive granulosa and thecal cells in growing follicles’ wall (Fig. 4D). The area percentage of the Ki-67-immuno-stained sections revealed a significant (P<0.00001) reduction in SOF-treated rats, in comparison with the control group, which was significantly increased (P<0.00001) after co-treatment with Vit E compared to the SOF treated group (Fig. 4E).

Figure 4. Micrographs of rat’s ovaries after Ki-67 immunohistochemical technique. Ki-67-strongly stains the cell nuclei (A–D). (A) The control group, granulosa cells and theca cells in the graafin folliciles (GF) are intensely stained. (B) The SOF-treated group demonstrates Ki-67-negativily stained cystic atretic follicles (CF), (C) The Vit E-treated group, and (D) the SOF+Vit E-treated group, where the number of Ki-67-positive cells localized in granulosa and theca cells of GF. (E) Histogram illustrates the quantitative analysis of the intensity of Ki-67 stain area percentage in different experimental groups. The SOF-treated group demonstrates a statistically significant reduction compared to the control group, *P<0.01, ****P<0.00001. The SOF+Vit E Co-treatment reduces Ki-67 immuno-staining significantly in comparison with the SOF treated group, ####P<0.00001 (Bar=20 μm). SOF, Sofosbuvir; Vit E, vitamin E.

Apoptosis marker (caspase 3) was detected in the luteal and granulosa cells layer of ovarian follicles and distinctly in the SOF-treated group, and less intensely in the control, Vit E treated and SOF+Vit E-treated groups (Fig. 5A–D). In the SOF-treated group, the intensity of caspase-3 staining was significantly increased (P<0.00001) compared to the control group and the SOF+Vit E co-treated group showed significant reduction in caspase-3 intensity (P<0.00001) compared to the SOF treated group (Fig. 5E).

Figure 5. Micrographs immunolocalize caspase 3 in rat’s ovaries (A–D). (A) The control group demonstrates limited stain. (B) The SOF-treated group in which SOF significantly upregulates the caspase 3 positive cells, which are predominantly located in the cytoplasm of the cells, compared to the control group. The brown-stained cells concentrate at granulosa and luteal cells. (C) The Vit E-treated group, and (D) the SOF+Vit E treated group, where the number of caspase 3 positive cells are reduced as compared to the SOF group. (E) Histogram illustrates the quantitative analysis of the intensity of caspase 3 stain in different experimental groups. The SOF-treated group demonstrates a statically significant upregulation compared to the control group, ****P<0.00001. The SOF+Vit E co-treatment significantly reduces the caspase 3 immuno-staining in comparison with the SOF treated group, ####P<0.00001 (Bar=20 μm). SOF, Sofosbuvir; Vit E, vitamin E.

In this study, we explored the effect of SOF on the rat’s ovarian oxidative stress markers, and its possible impact on ovarian dysfunction. The underlying ovarian morphological changes were examined to investigate its association with the observed biochemical changes induced by the drug. The data revealed that SOF induced oxidative stress markers (NO and MDA) significantly, which was reduced by the co-treatment with Vit E. However, the reverse took place regarding the antioxidant enzymes; CAT, SOD, and GSH markers, they were significantly decreased in SOF treated rats and increased after co-treatment with SOF+Vit E. The histopathological data demonstrated evident ovarian damage.

Accumulation of SOF in the cells promotes the ROS generation in recent studies [40, 41]. Oxidative stress and ROS have a major role in ovulation process and female reproduction [33]. Oxidative stress takes place when the cells fail to protect themselves against the exaggerated raise in ROS with sufficient levels of antioxidant enzymes [42]. Our study showed significant induction in NO and MDA lipid peroxidation, which was associated with significant reduction in SOD, CAT, and GSH antioxidant enzymes in SOF treated group compared to control and Vit E groups. All data supported the hypothesis that SOF-induced oxidative stress and reduced the antioxidant activities in the ovarian tissue which might explain the observed ovarian morphological dysfunction in the drug treated group. However, co-treatment with SOF+Vit E corrected the increase in NO and MDA. It also upregulated the antioxidant enzymes; CAT, SOD, GSH, and improved the morphological appearance in immunohistochemical studies.

Multiple vacuolations were noticed in SOF-treated rats, this was also observed in colazapine treated rats’ ovaries [43]. This could be related to steroidogenesis dysfunction, which leads to ovarian cholesterol level elevation that causes vacuolation in nicotine-treated rats, as reported by Patil et al. [44]. SOF had been reported to induce massive tubular vacuolation in association with congested glomerular capillaries in previous studies [35, 45]. However, it partially improves renal architecture damage induced by thioacetamide when it combined with declatasvir [46]. Multiple atretic follicles and dilated congested blood vessels were also demonstrated in the SOF treated group and the number of those atretic follicles were significantly higher in the drug treated group compared to the control. This was reversed by adding Vit E to the SOF treatment. The opposite occurred regarding the healthy growing graafian follicles and corpora lutea, they were reduced significantly in the SOF treated rats compared to the control. However, in the SOF+Vit E co-treated group, they were significantly higher compared to the SOF treated group.

Vit E is an antioxidant that protect the cell membrane against oxidative stress induced by free radicals produced in the lipid peroxidation chain reaction and limit its propagation [47]. Vit E administration with Acrylamide drug ameliorated ovarian biochemical and histological parameters [33]. Vit E seems to protect ovarian tissue against SOF induced oxidative stress, via its antioxidant effect, this was also reflected on the cellular and morphological appearance of ovarian tissue in the immunohistochemical studies, where the numbers of atretic follicles were reduced, the healthy graafian follicles and corpora lutea increased significantly. Vit E is known to be an anti-infertility vitamin due to its powerful antioxidant effect, that protect the cells and tissues against oxidative stress, which induces damage by increasing the antioxidant defense abilities against the free radicals [48].

PCNA was chosen in addition to Ki-67 to assess cellular proliferation, the former protein starts accumulating in cell cycle G1 phase and extends to its highest level in the S phase and reduces in the G2/M phase. Thus, it’s a good marker to study cell proliferation [49]. PCNA stain was localized in granulosa, theca cells in all graafian follicles and luteal cells in both the control and the Vit E treated group, which demonstrated intense PCNA stain. SOF-treatment reduced the number of PCNA-positive cells, the percentage of PCNA-immuno-positive cells (proliferation index, area %) was significantly attenuated compared to the control and Vit E treated groups. While this proliferation index upregulated significantly after co-treatment with Vit E.

Ki-67 antigen is a nuclear non-histone protein, essential for the synthesis of ribosomal RNA, cellular proliferation, and absent in quiescent cells [50]. In the current study, Ki-67 exhibited a similar pattern to PCNA immuno-stained. It intensely stained the cell nuclei of growing graafian follicles’ granulosa and theca cells in the control, Vit E, and SOF+Vit E co-treatment groups. SOF-treated rats’ ovaries showed a significant reduction in the intensity of Ki-67 immunostaining, suggesting inhibition of cell proliferation. Moreover, oxidative stress can cause inhibition of cell proliferation [51]. This supports the hypothesis that oxidative stress is linked to the attenuation of cellular proliferation and the morphological dysfunction observed in the ovarian tissue that is induced by the SOF treatment.

Caspase 3 is a marker of apoptosis; it’s activated by other caspase enzymes and initiates the apoptotic cascade [52]. ROS produced by the mitochondria causes membrane damage, and cytochrome c release, which links to apoptotic protease activation factor-1 and stimulates caspase-9. That by turn activates caspase 3 and initiates the apoptosis [53]. SOF unregulated the expression of caspase 3 significantly compared to the control and Vit E groups. While co-treatment with SOF+Vit E significantly downregulated this apoptotic marker. This was concomitant with the significant upregulation in the oxidative stress markers and the atretic follicles numbers in the SOF treated group, which was reversed by adding Vit E to the SOF treatment. This is supportive to our hypothesis that SOF induced ovarian morphological dysfunction is related to ROS induction, and Vit E could be a potential protective factor to be used with SOF treatment, to protect against its oxidative stress effect and its subsequent ovarian dysfunction.

The potential protective role of Vit E against ovarian injury, biochemical and histological changes induced by SOF in rats was explored in the present study, rats that received the drug showed ovarian damage in the form of significant upregulation of the number of atretic follicles and significant decrease in ovarian follicular, luteal counts with congested and dilated blood vessels. The downregulation in the number of growing follicles after SOF treatment was evident in the current study by Ki-67, PCNA, and caspase-3 immuno-histochemical study, which is concomitant with the altered lipid peroxidation and antioxidant enzyme activities, indicating an oxidative stress induced follicular atresia. Vit E is a major chain-breaking antioxidant that protects against harmful effects of ROS, it also helps in cell to cell signaling, or across various structures inside the cell [54]. It demonstrated a protective role in the present study against the oxidative stress and its subsequent ovarian dysfunction induced by the SOF treatment.

Our data provides in vivo proof of SOF induced oxidative stress as a persuader of ovarian cytotoxicity with its subsequent reproductive dysfunctions. The potential role of Vit E antioxidant in reversing the ovarian cytotoxicity through attenuation of oxidative stress induced by SOF is also an important finding. Additional future studies will focus on validation of the results with additional techniques including gene expression and western blotting. Blocking of Ki-67, PCNA or caspase-3 receptors will also confirm the involvement of those markers in the pathogenesis of ovarian dysfunction induced by SOF drug. In situ localization of ROS in rats’ ovaries by immunohistochemical techniques will be helpful to detect which cell or structure is induced by SOF to produce ROS.

In conclusion, this in vivo experimental study anticipates a biochemical and immuno-histochemical evident of the SOF role in oxidative stress induction that results in ovarian dysfunction. Moreover, it demonstrates the essential role of Vit E as an antioxidant agent in protecting the ovarian tissue from the effect of oxidative stress markers and preserve its function. This could be guidance to add Vit E supplements to the SOF regimen to prevent its harmful reproductive adverse effects.

The authors are thankful to the Medical Experimental Research Center (MERC) Staff, Mansoura University, for their advice and support. They also extend gratitude to Jana Hassanein for her help with language editing.

Conceptualization: NAE, HAA, AIM. Data acquisition: NAE, AIM, AEM, NAEY, HAA. Data analysis or interpretation: NAE. Drafting of the manuscript: NAE. Critical revision of the manuscript: AIM, HAA. Approval of the final version of the manuscript: all authors.

No potential conflict of interest relevant to this article was reported.

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