The effect of leaf hydroalcoholic extract of Ephedra pachyclada infertility in male rats treated with cyclophosphamide: An experimental study

Abstract Background Cyclophosphamide (CP) has clinical applications in treating diverse malignancies and autoimmune disorders; at the same time, it also has harmful effects on the body tissues, particularly the genitals. The most significant side effects of CP are changing the reproductive system's function and infertility. Objective This study determines the Ephedra hydroalcoholic extract (EP) role on testicular tissue and the pituitary-gonadal axis in CP-treated male rats. Materials and Methods In this experimental study, 48 adult Wistar rats were separated into 6 groups (n = 8/each): control, sham, CP recipients, and CP recipients with gavage-fed EP (250, 500, and 1000 mg/kg). On the 29 th day, the blood of the weighed animals' was drawn from their heart, and serum concentrations of follicle-stimulating hormone, luteinizing hormone, and testosterone were measured. After preparing testicular tissue segments, cells were counted. Results While CP decreased follicle-stimulating hormone, luteinizing hormone, and testosterone levels (p < 0.05), the use of EP changed them and even reached the control. Serum gonadotropin-releasing hormone increased significantly in all EP groups compared to the control and CP groups. Compared to the control, a significant decrease in total antioxidant capacity and plasma glutathione peroxidase was observed in the CP groups. EP (all doses) significantly increased their concentration compared to the CP group (p < 0.05); significant reduction in serum total oxidant status and malondialdehyde in CP groups changed by EP (p < 0.05). Although CP's role on spermatogonia counts (57.5 ± 5.2 in CP, 67.1 ± 6.0 in control), higher doses of EP had no significant effect on this but did affect spermatocyte and spermatid cells count. Conclusion Due to its antioxidant characteristics, EP mitigated the effects of CP on the investigated parameters in rats.


Introduction
Cancer is known to be the second cause of mortality in over 100 countries, affecting life expectancy and patient's quality of life.
From the past decades till now, rational use of effective anti-cancer agents for treatment was felted (1); among all discovered drugs, cyclophosphamide (CP), a nitrogen mustard anti-cancer agent, has been consumed as a chemotherapeutic drug for more than 50 yr after its discovery (2)(3). Despite CP's clinical benefits, some significant adverse effects of CP, such as nephrotoxicity (4), cardiotoxicity (3), severe bladder bleeding, and gonadotoxicity had limited its clinical indication (5)(6)(7). In the presence of cytochrome P450, CP metabolizes into 2 toxic compounds, acrolein, and phosphoramide.
Acrolein is an unsaturated and electrophilic reactive aldehyde that generates noxious reactive oxygen species (ROS) and next, affects the neighboring tissue (8,9). Studies have defined the acrolein-induced excessive ROS effects on testicular microvascular, endocrine signaling, and germ-cell apoptosis which causes testicular infertility (10). Besides them, CP increases the incidence of early menopause and infertility in males and females; this type of infertility is often transitory, but it can potentially be permanent (11,12). In a study by Watcho et al., CP-treated Wistar rats had significant reduction in seminal vesicle weight, testosterone level, sperm count, motility, and viability; furthermore, sperm morphological abnormality was also detected (13). In addition to morphological and qualitative changes in sperm caused by CP, there are also studies that have shown DNA damage and nuclear maturations (14).
In the last few years, considerable interest in the use of medicinal plants with the aim of cancer treatment or reducing anticancer agents' side effects has been increased.
CP applies its effect primarily by persuading oxidative stress and changing gene expression in spermatocytes vastly unlike stages of development (15); therefore, these studies mostly rely on the antioxidant role of plants and consider antioxidants as affective factors (13,14). For example, in a study by Can et

Determination of extract purification
At first, 1 ml of phenol solution was combined with 200 µl EP pachyclada extract and "stored in dark for 6 min, then 2 ml of NaCo 3 (7%) was added and kept for 120 min. The solution absorbance was measured at a wavelength of 765 ɲm" and finally, the total phenolic amount of extract was calculated using the standard curve of gallic acid and reported 9.28 mg/(GAE)4/g (25).

Experiment design
In this experimental study, 48 Wistar male rats

Biochemical analysis
On the 29 th day, the blood of the weighed animals' was drawn from their heart (under anesthesia using Xylazine (10 mg/kg of body weight) and Ketamine (50 mg/kg of body weight)).
Their serums were collected via centrifugation

Histological examination
For histological examination, testis tissue (left and right) was rapidly removed and placed in a 10% formalin container. After 48 hr, the testis tissue was removed from the solution and after routine tissue processing; paraffin sections of 5 μm thickness were prepared as serial sections.

Ethical considerations
This study was in agreement with ethical issues of animal laboratory Ethics of Jahrom University of Medical Science, Jahrom, Iran

Statistical analysis
SPSS software (version 24) was used for data analysis. At first, the normality of the data was measured using Shapiro-Wilk normality test.
Data were analyzed by Independent-samples t test using SPSS software version 16 and the significance level was considered 0.05.

Changes in serum LH, FSH, testosterone, and GnRH
Data analysis showed that CP had affected serum FSH and LH and using EP had altered these; using higher doses of EP (500 and 1000 mg/kg) were not significantly different with the control. All experimental groups showed a significant decrease in serum testosterone compared to the control group at p < 0.05.
The mean testosterone concentration in CP and EP 500 mg/kg did not significantly differ from the CP group; however, the higher doses were significantly different from CP group and the best effect belonged to the highest dose.
Serum GnRH concentration showed a significant increase in all EP groups compared to the control and CP groups. The mean serum concentration of GnRH in experimental groups containing 500 and 1000 mg/kg EP was significantly higher than the group treated with 250 mg/kg EP (p < 0.05).
On comparing CP groups with EP extract ones, it was found that 1000 mg/kg EP had more significant effects on boosting serum-assessed parameters (Table I).

Changes in serum antioxidants, MDA, and TOS
Data analysis showed a significant reduction of serum TAC and GSH-PX in groups that used CP compared to the control. EP (all and EP did not affect them (Figure 1).

Testicular tissue histological results
The histological examination of the testicular tissue showed that no tissue changes were observed in the control and sham groups. In the CP group, cell disorganization and disorders inside the seminiferous tubule, and wrinkling of the germinal epithelium were observed. In groups receiving EP with different doses, tissue disorders showed less severity than the CP group receiving CP. By increasing the concentration of EP, the intensity of these changes decreased so the lowest amount of tissue disorders was observed in the EP 1000 mg/kg group (Figure 2).   Figure 1. The effect of cyclophosphamide (CP) and Ephedra (EP) on male Wistar rats spermatogonia, spermatocytes, spermatid, Leydig, and Sertoli cell. Each group means with at least one shared letter has no significant difference. P < 0.05 is considered statistically significant, Independent-samples t test.

Discussion
Gonadotoxicity has become one of the most notable and commonly encountered side effects of extended CP use, so there is an imperative and crucial need to identify therapeutic strategies to decrease or regulate the reproductive potential of cancer patients subjected to CP therapy (28)  that further studies should be conducted on rats that have been exposed to chemotherapy.

Conclusion
As a result, leaf hydroalcoholic extract of EP pachyclada can be utilized to mitigate the adverse effects of CP, especially in higher doses.
The EP extracts' therapeutic effects are based on its antioxidative function and resilience against the ROS status and oxidative stress in CP toxicity that diminishes steroidogenesis, testosterone production, and sperm functional parameters.