Protective effects of Azilsartan against cyclophosphamide-induced ovarian toxicity in rats model

Abstract

Background

Cyclophosphamide (CP) is an effective alkylating anticancer agent that is widely used in cancer chemotherapy, and it can cause ototoxicity and infertility in women.

Objectives

So, this study aimed to evaluate the protective effects of Azilsartan (AZ) as an antioxidant and anti-inflammatory agent in a rat model of CP-induced ovarian toxicity.

Materials and Methods

After receiving the 28 female Wister rats, they were acclimatized in proper environmental conditions for a week and then randomly divided into four groups based on the study protocol. After 15 days of the experiment, they were sacrificed, and organs were collected for biomarker detection (Using the ELISA technique) and histopathological analysis.

Results

The level of IL-10 was significantly (P < 0.05) decreased in all treated groups compared to control hostile groups, while the TNF-α level was significantly (P < 0.05) increased in AZ (220.67 ± 7.88 ng/mL) and AZ + CP groups (221.78 ± 9.11 ng/mL) compared to control negative/CP groups. Regarding the oxidative biomarker level, a significant increase was only found in the AZ + CP group (176.02 ± 6.71 nmol/mL) compared to the control negative group. On the other hand, histopathological findings revealed that ovarian sections in animals that received a single dose of CP had severe ovarian atrophy with significant follicular regression and deterioration, as well as depletion of stromal supportive tissues.

Conclusions

Azilsartan drastically reduced CP-induced ovarian toxicity in vivo by enhancing oxidative stress and inhibiting inflammatory effects in ovarian cells.

Introduction

A local renin-angiotensin system (RAS) has been found in the ovary. This ovarian RAS may regulate ovarian steroidogenesis and is therefore considered an essential system for regulating physiologic pathways.¹ The role of the ovarian renin-angiotensin system (OVRAS) in ovarian physiology and disease, angiotensin and angiotensin receptors are widely distributed in the ovarian follicle, pre-ovulatory theca and granulosa cells, and postovulatory mural granulosa-lutein cells and regulate steroidogenesis.²

Cancer chemotherapy has side effects, especially in young female patients, such as reducing the number of primordial and other growing follicles, leading to early menopause, amenorrhea, and infertility that might negatively impact the quality of life of young cancer survivors.³ Chemotherapeutic agents with alkylating properties have demonstrated high gonadal toxicity, especially in females. In response to the needs of a young cancer population undergoing highly gonadotoxic treatments, medical strategies to overcome infertility have been developed, and those are offered in clinical practice.⁴

Cyclophosphamide (CP) is an economical and effective therapy; it is widely used in various diseases, including cancer, hematologic diseases, primary angiitis of the central nervous system (CNS), chronic inflammatory demyelinating polyneuropathy (CIDP), rheumatoid arthritis, and nephrotic syndrome.⁵ Therapeutic and toxic effects of CP are related to its active metabolites that produce DNA and tissue damage via reactive oxygen species (ROS)-induced toxicity and dysregulation of the antioxidant defence mechanisms.⁶ Significant toxicity has been noticed, particularly in the reproductive system. CP promotes the maturation of ovarian follicles, decreases follicular reserve, and ultimately leads to ovarian failure or even premature ovarian failure (POF).⁷

Azilsartan (AZ) is a unique angiotensin receptor blocker (ARB) with a peroxisome proliferator-activated receptor-gamma (PPAR-γ) agonistic activity.⁸ PPAR-γ activation exerts anti-inflammatory effects and reduces ROS production.^9,10 AZ may further reduce inflammatory chemokine expression and suppress apoptotic cell death, especially in rheumatic arthritis.¹⁰ AZ suppresses osteoclastogenesis and ameliorates ovariectomy-induced osteoporosis by inhibiting ROS production and activating Nrf2 signaling.¹¹

Various therapeutic strategies have been developed to overcome ovarian toxicity. Hence, the mechanisms involved in CP undesired iatrogenic ovarian damage have not been intensively studied and remain unclear. Thus, we aimed to study the effect of AZ in reducing CP-induced ovarian toxicity in rat models.

Materials and methods

Materials

Azilsartan (AZ) medixomil powder was obtained from Apollo Healthcare Resources, Singapore, while carboxymetheylcallouse (CMC; 0.5%) was obtained from Hebei Tanpeng, China and was used for AZ preparation. Ketamine was purchased from Bioveta Company, Czech Republic and Xylazine (2%) was obtained from Interchem company, Netherlands. Bioassay Technology Company, UK, obtained the enzyme-linked immunosorbent assay (ELISA) kits for biomarker estimation. Cyclophosphamide was obtained from Sandoz, Switzerland.

Animals

Female Wister rats, aged 6–8 weeks, weighing 180–220 gm, were provided by the College of Education, University of Tikrit, Tikrit, Iraq. The rats were acclimatized on distilled water and a standard rat chow for seven days and kept in a well-ventilated room with a 12-h’ dark/light cycle at 25 ± 3.0 ° C before the study.

Experimental protocol

Twenty-eight animals were allocated into four groups (7 rats each). Group I was control negative rats drenched daily with normal saline for 15 consecutive days. Group II rats were treated orally with AZ (1.0 mg/kg) daily for 15 consecutive days.¹² Group III rats were injected with CP intraperitoneally (100 mg/kg) on day 9 of the experiment and treated daily with 0.5 mL of CMC in normal saline until sacrifice day. Group IV rats were treated orally with AZ (1.0 mg/kg) for 15 consecutive days, and on day 9 of the experiment, they were given a single dose of CP intraperitoneally (100 mg/kg). All animals were sacrificed on day 16 of the experiment under deep general anaesthesia using a mixture of xylazine and ketamine. Initially, rats were fasted for at least 10 h before scarification date and then euthanized in a humane practice. All the experiments were performed according to institutional guidelines for the ethical care of animals. The drenching processes were done using force-feeding needles.

Sample collection

After animal sacrifice, ovaries were harvested and weighed, and one of them was fixed in a 10% buffered formalin solution at room temperature for histopathological evaluation. On the other hand, another ovary was rinsed with 5 mL of 1X phosphate buffer saline (PBS) to remove excess blood, chopped into 1–2 mm pieces on ice in ice-cold buffer, and then stored at −80 °C for determination of the levels of interleukin-10 (IL-10), tumour necrosis factor-alpha (TNF-α), and malonaldehyde (MDA) using Enzyme Linked Immuno-Sorbent Assay (ELISA) technique. For every 0.1 mg of ovarian tissue, 500 μL of complete extraction buffer was added to the tube and homogenized. Then, tissue homogenates were centrifuged for 5 min at 5,000 × g. The supernatant was removed immediately and assayed directly, following the instructions of the manufacturing company with minor modifications.

Histopathological technique protocol

Ovaries were immobilized and secured into tissue cassettes, then fixed with 10% neutral buffered formaldehyde solution for about 48 h. After that, sections were dehydrated by passing through ascending ethanol alcohol (50, 60, 70, 90, and 100%), followed by a couple of steps of xylene clearance. Next, the processed tissues were infiltrated and embedded in melted paraffin blocks using an automated wax embedder at 60–70 °C. Tissue blocks were sectioned to 5 μm using a rotary microtome. Afterwards, tissue sections were fixed on glass slides and dried using a hot plate tissue dryer. Later, glass slides with their mounted tissue sections were deparaffinized and cleaned with xylene solution for 30 min, then dried in a hot oven at 50 C for 5 min. Finally, tissue sections were stained with Harris’s hematoxylin and eosin solution, and the cover was slipped.

Semi-quantitative lesion scoring

Basically, during necropsy detection, ovaries were scored for the presence of severe ovarian atrophy. Then, during microscopic examination, ovarian sections were evaluated for follicular regression, area of severe vascular congestion and stromal depletion. Regarding the ovarian sections, the mean percentage of atrophic, atretic, deteriorated and intact ovarian follicles was measured and calculated in 10 randomly selected fields within the section. Furthermore, the areas of vascular congestion and stromal depletions were evaluated as a total mean percentage. On the other hand, within the liver sections, vacuolar degenerations were estimated and measured in percentage of calculated cell numbers from randomly selected fields. In contrast, vascular congestion was assessed in μm and statistically evaluated as mean percentage.

Meanwhile, in kidney sections, vacuolar degenerations and cellular swelling within the renal tubular epithelia were measured in the same manner as for liver tissue. Lesion scoring was estimated semi-quantitatively via image analyzer software (Am Scope, 3.7) using a microscope eye-piece camera (MD500, 2019), and tissue samples were analyzed under the light microscope (NOVEL XSZ-N107T, China). In conclusion, the mean percentage of all calculated values were expressed as following lesion scoring system (score 0%–10% as no lesions; score 10%–25% as mild; score 25%–50% as moderate; score 50%–75% as severe; and score 75–100 as critical lesions).

Statistical analysis

Statistical analyses were performed using GraphPad Prism 8 (Inc., San Diego, CA, USA). Results are reported as means and standard deviations. The studied groups were compared using an independent sample t-test and Mann-Whitney U test. A P-value of <0.05 was considered statistically significant.

Results

The level of IL-10 was significantly (P < 0.05) decreased in all treated groups compared to control hostile groups. In respect of TNF-α, it was significantly (P < 0.05) increased in group AZ (220.67 ± 7.88 ng/mL) and AZ + CP (221.78 ± 9.11 ng/mL) treatments compared to control negative (180.03 ± 19.77 ng/mL) and CP groups (190.25 ± 25.58). Regarding the oxidative biomarker level, a significant increase was only found in the AZ + CP group (176.02 ± 6.71 nmol/mL) compared to the control negative group (159.35 ± 6.71 nmol/mL) (Table 1 and Fig. 1).

Table 1.

Shows the levels of anti-inflammatory and antioxidant biomarkers among treated rats with various agents.

Study Group	IL-10 (pg/mL)	TNF-α (ng/mL)	MDA (nmol/mL)
Group I (CN)	1.22 ± 0.06b	180.03 ± 19.77	159.35 ± 6.71
Group II (AZ)	1.05 ± 0.16a	220.67 ± 7.88a,b	160.98 ± 12.86
Group III (CP)	1.05 ± 0.10a	190.25 ± 25.58	171.32 ± 9.66
Group IV (AZ with CP)	1.01 ± 0.08a	221.78 ± 9.11a,b	176.02 ± 6.71a

Values are expressed as the mean and standard deviation; AZ: Azilsartan; CN: Control negative; IL: Interleukin; TNF: Tumor necrosis factor; MDA: Malonaldehyde; CP: Cyclophosphamide. a, b = P < 0.05 when compared with the CN; and CP group, respectively.

Fig. 1.

Shows interleukin (IL-6), tumour necrotizing factor (TNF-α), and malonaldehyde (MDA) levels expression among treated animals; ^*Significant difference (P < 0.05).

Histopathological findings

Microscopically examined ovarian sections in G1 showed the typical histological architecture of a standard control ovary, which exhibited a normal arrangement of ovarian follicles with a regular amount of stromal tissue. In addition, animals treated with AZ in G2 did not show any significant morphological changes except for moderate vascular congestion compared to ovaries in G1. In contrast to G1 and G2, ovarian sections in animals that received a single dose of CP (G3) reveal the presence of severe ovarian atrophy, significant follicular regression and deterioration, and the depletion of stromal supportive tissue.

On the other hand, animals in G4 treated with AZ, followed by a single dose of CP, still displayed significant atrophy to the ovarian tissue. However, substantial and apparent follicular regenerative changes are notable, evident by an increase in the primary follicles within a proliferated stromal connective tissue in the ovarian medulla (Fig. 2).

Fig. 2.

Photomicrograph of ovary from groups; G1: Control negative group received normal saline. The ovarian section shows no obvious abnormal morphological changes, represented by typically arranged ovarian follicles (OF) filled with proteinaceous fluid antrum (A) embedded within a connective tissue stroma (CS), the section also reveals large degraded corpus luteum (CL). G2: Treated with Azilsartan (1.0 mg/kg/day), illustrated multifocal distribution of many ovarian follicles (OF), together with the presence of large recently ovulated graffian follicle (GF) and small scraggy atretic follicle (yellow arrow), all fixed in the CS. The section also shows large congested ovarian vein (VC). G3: Injected with a single dose of Cyclophosphamide (CP) (100 mg/kg) elucidate significant and severe ovarian atrophy and degeneration with the regression of many follicles (RF). The section also expresses the presence of many atrophic follicles (AF) together with smaller CL and OF. G4: Treated with Azilsartan (1.0 mg/kg/day) and then injected with CP (100 mg/kg), show significant ovarian atrophy with the presence of many smaller OF and other degenerative AF. Moreover, the section reveals other regenerative grown follicles with extended antrum (A) together with smaller areas of CS. Scale bar: 4 mm.

Regarding the micro-morphological quantitative assay of ovarian sections, the mean of ovarian atrophy (74.32%), follicular atrophy (72.51%), and follicular deterioration (73.46%) in the CP group were non-significantly decreased (61.44, 59.72, and 56.18%, respectively) in AZ + CP group (Table 2).

Table 2.

Micromorphological quantitative assay of ovarian sections.

Experimental Group (No = 7)	Ovarian Atrophy (Mean %)	Follicular Atrophy (Mean %)	Follicular Deterioration (Mean %)	Lesion Scoring (0–100%)	Lesion Grading
GI (CN)	2.76a#	3.45a	4.19a	0–10	No lesion
GII (AZ)	6.38a	5.29a	5.42a	0–10	No lesion
GIII (CP)	74.32d	72.51d	73.46d	50–75	Severe
GIV (AZ+ CP)	61.44d	59.72d	56.18d	50–75	Severe

Follicular atrophy and follicular degeneration were estimated in % of cell numbers. Ovarian atrophy was calculated as a mean percentage of regressed ovaries per treatment group. Area of vascular congestion estimated in μm. Each value represents mean percentage (n = 7). #Statistical comparison among groups. Mean values with different capital letters have significant differences at P < 0.05. CN: Control negative; AZ: Azilsartan; CP: Cyclophosphamide.

Discussion

Although little information is available about the pathogenic mechanism of CP-induced ovarian damage, its toxicity is attributed to oxidative stress, inflammation, and apoptosis. Using compounds with antioxidant and cytoprotective properties to protect ovarian function from deleterious effects during chemotherapy would be a significant advantage.¹³ To the best of our knowledge, this is the first research to be done on AZ’s protective activity against CP cytotoxicity in animal models worldwide.

The mechanism by which CP exerts its toxic effects on the different cellular components of the ovary should be thoroughly investigated using microarray analysis, bioinformatic analysis, serological investigation, and microscopical findings. Thus, in this study, AZ as a cytoprotective compound was used to facilitate CP-induced ovarian injury and its possible mechanisms of action were investigated through inflammatory and oxidative biomarkers. Understanding these mechanisms is essential for developing efficient and targeted pharmacological complementary therapies that could protect and prolong female fertility.

In the current study, a significant increase in TNF-α and IL-6 ovarian levels indicates the prominent inflammatory role in CP-induced ovarian toxicity, reflecting that the cytokines production may result from either cellular DNA damage with inhibition in the cell cycle progression and eventually cell death or biochemical changes in the cellular environment and metabolism induced by the interaction of chemotherapy with the target cell.¹⁴ Consequently, in comparison to CP treated group, the level of IL-10 was significantly (P < 0.05) decreased in the AZ group. Similarly, the level of MDA was reduced in the AZ group compared to the CP group but without significant correlation (P > 0.05), while the level of TNF-α was significantly (P < 0.05) increased in the AZ group compared to the CP group. These outcomes indicated that AZ decreased the inflammatory effects caused by CP injection and also enhanced the antioxidant potential in injured ovarian tissues. These results align with Abdel-Aziz et al.¹⁴ which found that Cilostazol (selective phosphodiesterase-3 inhibitor) with antioxidant, anti-inflammatory, and antiapoptotic activities exhibits protective effects against CP-induced ovarian damage in female rats.¹⁴ Also, genistein inhibited the alleviation of oxidative stress and inflammation significantly compared to the CP-treated group.¹⁵ Moreover, Zhang et al.¹⁶ found a significant reduction in body weight (P < 0.01), spleen coefficient (P < 0.01), leukocyte density (P < 0.01) and alanine transaminase (ALT) (P < 0.01); however, superoxide dismutase (SOD), malondialdehyde (MDA), and creatinine significantly (P < 0.05) increased in mice after five days treatment with CP.¹⁶

With the growing numbers of young female cancer survivors and the aim of fertility preservation, the maintenance of reproductive potential after cancer therapy has become a paramount concern. Though the alkylating agent, CP, is considered to be an effective chemotherapeutics for cancer treatment, it has many side effects, including drastic histopathological changes to ovarian tissues.¹⁴

It is well known that oxidative stress in granulosa cells, after CP treatment, causes apoptosis of these cells, leading to ovarian follicle atresia mostly by granulosa cell apoptosis or proliferation inhibition,¹⁷ and also causes a reduction in the follicular number.¹⁸ In this study, CP elucidated significant and severe ovarian atrophy and degeneration with the regression of many follicles. Also, it causes atrophy of the follicles, inhibiting the growth of the corpus luteum and ovarian follicle. Similar results were seen by other agents such as Tamoxifen¹⁷ and Atorvastatin¹⁹ through mitigation of acute inflammation, degeneration of stroma and follicles, stromal oedema, vacuolization, atresia of the follicles and congestion of blood vessels in the CP-treated animals.

On the other hand, this study showed that AZ treatments displayed multifocal distribution of many ovarian follicles, with large recently ovulated graffian follicles and small scraggy atretic follicles that fixed in the connective tissue stroma. Simultaneously, AZ + CP showed potential ovarian atrophy with many smaller degenerative-atrophic follicles. The results suggested that the combination of AZ and CP mitigated the cytotoxic effect of CP. Similar results were found by other compounds in ovarian tissues.^14,15,19 In the same manner, N-acetylcysteine and vitamin E coadministration significantly decreased the side effects of CP in ovarian tissue.²⁰

Conclusions

Azilsartan played a significant role in reducing CP-induced ovarian toxicity in vivo by restoring the oxidative stress and inflammatory biomarkers to their normal levels, with marked improvement in the histopathological picture of ovarian damage.

Author contributions

NMAM: Conceptualization, study registration, methodology, data collection, data analysis, prepared tables and figures, and writing the original manuscript.

Funding

The study is self-funded; no grant or fund was obtained from national/international agencies, organizations, or Universities.

Conflict of interest statement. There is no conflict of interest related to this study.

Data availability

The datasets used and analyzed during the current study are provided in the manuscript, and some of them are with the author, who can provide them upon request.

Ethical approval

This study was based on the ARRIVE Guidelines for Animal Care and Use. The experimental protocol was approved by the scientific and ethical committees of the College of Pharmacy, University of Sulaimani, Iraq (No. PH20-21 on 23 August 2021-UoS) after intensive revision. Thus, procedures are used to diminish animal pain/discomfort.