Assessment of the protective effect of saffron against Khat induced testicular dysfunction in male rats

ABSTRACT

Catha edulis is a widespread herb used by many people for its euphoric effects, but it has an adverse effect on some vital organs. Saffron is abundant in secondary metabolites which have antioxidant and anti-inflammatory properties. So, this study was established to examine the impact of saffron in minimizing khat-induced testicular toxicity in rats. In this study, 40 male albino rats were used, the rats were divided into four groups (n = 10). Control, Saffron group, Khat group, and Khat + Saffron group. Rats treated with Khat showed marked changes in the testicular histological architecture. Histochemically, the testicular issues displayed low protein and polysaccharide contents. The immunohistochemical results showed low testicular expression for vimentin, E-cadherin proteins, and Bcl-2 while strong positive expression for BAX protein in compared with control. The antioxidant enzymes superoxide dismutase and catalase activity showed a marked decrease while malondialdehyde levels were elevated. However, rats given both khat followed by saffron showed an improvement in testicular histology, histochemistry, immunohistochemistry, and biochemical results; the results were close to the control ones. In conclusion, saffron proved its efficiency in improving testicular toxicity induced by khat in the testis of albino rats due to its richness with secondary metabolites at the histological, and biochemical levels and can be used as a safe daily supplementary.

KEYWORDS:

• Khat
• saffron
• histology
• vimentin
• E-chardin

Introduction

Addiction is a global problem that causes severe damage to physical and mental health, in addition to social problems as stated by Nawi et al. [1]. Narcotic substances are produced directly from natural plants such as hashish, khat, opium, and cannabis plants [2].

Catha edulis (Khat) is a shrub with evergreen leaves found in the Middle East and East Africa [3]. The availability and prevalence of chewing khat have increased and are expanding throughout the world [4]. The most prevalent alkaloid in fresh Catha edulis leaves is cathinone, which is also the main ingredient, accountable for khat stimulant effects [5].

Khat was found to cause esophagitis, psychosis, and cardiovascular issues [6], alterations in the kidneys, liver, and lungs [7], and cytotoxicity in cultured human cells [8]. Also, khat extract has an impact on the development of testes in immature albino rats [9,10]. Chewing khat resulted in producing reactive oxygen species (ROS), which impaired the antioxidants’ effectiveness [11,12].

Natural herbal medicines are attracting increasing attention because they have multi-target, multi-component, few adverse effects, and their cost is low [13,14]. Crocus sativus (saffron) is an autumnal perennial herbaceous flowering plant following the family Iridaceae [15] that is cultivated on a large scale in Iran, Spain, Greece and India [16]. The precious part of saffron is its stigma; these dried stigmas are frequently used as food flavoring and coloring [17].

Saffron is abundant in secondary metabolites like flavonoids, terpenes, anthocyanins, and carotenoids like safranal, carotene, crocin, picrocrocin, crocetin, zeaxanthin and vitamins like riboflavin and thiamine [18,19]. Different saffron extracts have proved their effectiveness in decreasing the severity of morphine withdrawal symptoms in mice [16] and in rats [20]. Saffron extract had cytotoxic and antiproliferative effects on cancer cells by modulating carcinogen metabolism, inhibiting cell proliferation, and regulating cell cycle progression and cell growth [21].

Saffron consumption increases male reproductive potential [16,22]. Also, Abo Edra et al. [23] indicated that crocin has a protective effect on testicular damage because it has an anti-apoptotic and antioxidant effect.

Sertoli cells nourish spermatogenic cells and offer structural support (cell junctions) to them. The attachment between different spermatogenic cells and Sertoli cells is essential for spermatogenesis [24]. The intermediate filament vimentin is an essential protein (type III intermediate filament) that is essential for preserving the mechanical support between the cells as well as the structural integrity of each individual cell. Vimentin facilitates the transport of spermatozoa via the testes ducts as they mature and gain viability [25]. Moreover, it is used to identify Sertoli cell damage and spermatogenic dysfunction [26,27].

Cell-cell adhesion is a crucial function of epithelial cadherin or E-Cadherin, in order to preserve the stability and integrity of tissues [28]. Loss or malfunction in E-cadherin has been correlated with a variety of clinical diseases, including cancer [29]. In all, cadherin adheres to the junctions between Sertoli and germ cells, while vimentin has a vital role in spermatogenesis [24]. This recent study evaluates the impact of khat extract on testicular tissues from the histopathological, histochemical, immunohistochemical, and biochemical terms. As well as evaluating the ameliorating effect of saffron as an antioxidant agent and assessing its ability to reduce damage affecting the testes.

Materials and methods

Material

Khat

Khat was obtained from the Substance Abuse Research Centre in Saudi Arabia.

Saffron

Saffron was purchased from Spain. IBERPRIME IMPORT & EXPORT SL – Lot No. 130416.

Methods

Khat extract preparation

To prepare the khat extract, the green plant leaves (350 grams) were repeatedly rinsed with distilled water, then dried, and finally crushed to a fine powder (dry weight became 56 grams). The ground dried khat powder was soaked (4 h) and then stirred in 500 ml of distilled water for two hours then filtrated and the filtrate was dried by using a rotary evaporator as modulated according to Al-Zubairi et al [30], the final weight of the extract is calculated, and it was 24 grams. To prepare a dose of 200 mg/kg body weight of khat 3 g of khat extract was dissolved in 60 ml distilled water and was prepared freshly before being orally administrated to the animals [31].

Saffron extract preparation

The saffron extract was prepared by immersing one gram of Crocus sativus L. powder in twenty ml of warmed distilled water, stirring for two hours, and then filtrated. A dose of 100 mg/kg body weight of saffron was orally administered to animals. The aqueous solution of the extract was prepared freshly before administrating it to the animals [22].

Animals and experimental design

Albino rats aged ten weeks were reared and maintained at 12 h light/12 h dark cycle under definite conditions of temperature (20 ± 4 °C) and Rh (55 ± 10%). Rats had unlimited access to food and water. The study has been authorized by the Animal Care and Bioethics of the Egyptian Committee, and the animal work was done at the Faculty of Science, Menoufia University, Egypt (Approval No. MNSH 323).

The experimental animals were grouped into four groups, each with ten rats: Control group: Rats received distilled water only. Saffron group: orally administered 100 mg/kg body weight of saffron extract every other day. Khat group: orally administered 200 mg/kg body weight of khat extract every other day. Khat + Saffron group: rats were given khat extract and on the same day after two hours, they took saffron extract with the same doses used in the second and third group every other day for three months.

At the end of the 3 months, animals were fasted for 12 hours before being dissected the following morning. Testis were rapidly immersed in 10% neutral buffered formalin (fixation) for histopathological, histochemical, and immune-histochemical studies.

Histological preparation

Immediately after dissection, the testis was removed, washed in saline, and put in formalin solution (10% neutral buffered) for 24 hours, and 5 micron sections were prepared and stained in hematoxylin and eosin [32].

Morphometric measurements

The diameter and epithelial height of seminiferous tubules were measured. By using linear ocular micrometer lens, the diameter of seminiferous tubules was calculated by taking the average of the two vertical and horizontal diameters of the tubules. The epithelial height was measured from the spermatogenic cells on the inner surface of the basement membrane through the most advanced cell types lining the lumen of the tubules. Measurements were done using 20× per 10 sections (5 rounded tubules/section) [33].

Histochemical investigation

The total polysaccharide was determined by using PAS reaction and total protein is determined by mercury bromophenol blue method as described in [34].

Immunohistochemical investigation

The sections were subjected to incubation with an anti-rabbit monoclonal antibody to assess the expression of B cell lymphoma 2 (Bcl-2) (Abcam, Shanghai Trading Co. Ltd. China, catalog code: ab194583; 1:100), Bcl-2-associated X protein (Bax) (LabVision, Thermo Fisher Scientific; catalog code: MA5–14003; 1:100), vimentin (Abcam, Shanghai Trading Co. Ltd. China, catalog code: EPR3776; 1:100) and Epithelial Cadherin (E- Cadherin) (LabVision, Thermo Fisher Scientific; catalog code: PA5–8508; 1:100) [35].

Image analysis

The immunohistochemical expressions of Bcl-2, Bax, vimentin and E-Cadherin positive cells (brown stained) in testes sections were analyzed by a semi-quantitative scoring system (Fiji-Image J software, Java based application for analyzing images). The percentage of colored stained area (area fraction) per field area was determined by measuring five randomly photographed high-power fields (X400 magnifications).

Biochemical estimations

The other parts of the testis were separated, cleaned as rapidly as possible, weighed then homogenized in ice-cold water and frozen at −20°C for subsequent analysis of catalase (CAT) [36], superoxide dismutase (SOD) [37], and malondialdehyde (MDA) [38].

Statistical analysis

The statistical package for social science (SPSS) program, version 20, was used to analyze and compare the data. The results were expressed as Mean ± standard deviation (Mean ± SD). The data were obtained in the form of average concentration ± SD. Kolmogorov-Smirnoff test (p < 0.05) was used to determine data normality then the Levene test (p < 0.05) was used to assess data homogeneity. Furthermore, data were evaluated using one-way ANOVA (p < 0.05) and the Turkey test to know the significance among cohorts.

Results

Biochemical results

Changes in antioxidants

The control and saffron group showed normal MDA levels and activity of CAT and SOD in testicular homogenate. While the khat extract group resulted in a decrease in CAT enzyme and SOD content while increasing lipid peroxidation product (MDA) concentration. However, the combined group of saffron and khat extract showed prevention of khat effects; by increasing CAT and SOD activity, and on contrast MDA levels decreased in the teste’s homogenate (Table 1).

Table 1. Change in MDA level, CAT and SOD activity in the experimental groups.

Animal groups	MDA (nmol/g)	CAT (U/mg)	SOD (µg /g)
Control	16.94 ± 1.58	4.49 ± 0.32	901.33 ± 3.48
Saffron	17.17 ± 1.35	3.87 ± 0.25	883.69 ± 2.83
Khat	22.86 ± 1.96*	2.49 ± 0.68*	700.43 ± 3.87*
Khat+ saffron	15.70 ± 1.97**	3.18 ± 0.11**	809.34 ± 3.60**

Data were expressed as mean ± standard deviation (n = 10). MDA: malondialdehyde, CAT: catalase, SOD: superoxide dismutase.

*Significant compared to the control group at p < 0.05.

**Significant compared to the khat group at p < 0.05.

Morphometric results

The diameter and epithelial height of the seminiferous tubules in rats treated with saffron and control groups do not differ significantly. Rats given khat extract showed a highly significant decrease in the diameter and epithelial height of their seminiferous tubules when compared to the control group. Rats treated with both khat, and saffron extract showed significantly increased seminiferous tubule diameter and epithelial height when assessed and compared to the khat group, but these values were still lower than those of the control group (Table 2).

Table 2. Change in the epithelial height and diameter of the seminiferous tubules (µm) in the experimental groups.

Animal groups	Epithelial height (µm)	Diameter (µm)
Control	40.52 ± 1.05	210.62 ± 8.55
Saffron	38.58 ± 1.25	208.55 ± 10.23
Khat	20.98 ± 1.66*	140.73 ± 5.66*
Khat+ saffron	27.65 ± 1.16**	165.96 ± 9.11**

Data are represented as (Mean±SD).

*Significant compared to the control group at p < 0.05.

**Significant compared to the khat group at p < 0.05.

Histological observations

The testes from control and saffron rats are encapsulated by dense fibrous connective tissue, the tunica albuginea. The seminiferous tubules are the structural unit of the testes. They are enclosed by basal lamina and are attached by loose connective tissue (interstitial tissue) containing clusters of Leydig cells. Leydig cell contains a large, round nucleus in fine-grained, strongly acidic cytoplasm. Within the seminiferous tubules, the spermatogonia lies in contact with the basal lamina of the tubule. They have a round nucleus with very dense chromatin granules.

The second layer is the primary spermatocytes which have large oval cells and round nuclei with condensed chromatin materials. Their division results in the formation of haploid secondary spermatocytes that rapidly enter and complete the second meiotic division to form spermatids. They are small with very light eccentric nuclei. Differentiation of spermatids leads to spermatozoa. Among spermatogonia, Sertoli cells are present with slightly stained oval nuclei (Figure 1(a–c)).

Figure 1. A photomicrograph of testicular tissue (a,b) control group, (c) saffron group showing: spermatogonia (black arrow), sertoli cell (arrowhead), primary spermatocyte (PS), secondary spermatocytes (SS), spermatids (sp), sperms (S), and interstitial tissue (blue arrow) containing leydig cell (green arrow); (d-g): khat group showing irregular seminiferous tubules (blue arrows) with variable shape and size, edematous lesions (*); thickening in the tunica albuginea (arrowhead), اثةdetachment of the spermatogenic layers (#), degenerated (black arrows) spermatogenic cells, exfoliated dead cells (red arrows); (h): khat and saffron group showing nearly normal seminiferous tubules filled with sperms (S) (H&E).

Marked histological alterations were observed when rats received khat for 3 months. Some seminiferous tubules had irregular membranes and variable shape and size. Edematous lesions were detected between the tubules. Spermatogenic cells are degenerated and the spermatogenic layers appear separated. The degenerated spermatogenic cells had pyknotic nuclei. Besides, dead cells were exfoliated into the tubule lumen and complete inhibition of spermatozoa formation was recognized (Figure 1(d–g)).

When rats were subjected to the dual treatment (khat and saffron extract together) every other day for 3 months, the testes showed fewer notable histopathological alterations. when compared with animals treated with khat only. The seminiferous tubules returned to their distinctive shape (nearly normal) and filled with sperms. The spermatogenic layers seemed to be better organized and healthier than those in the khat-treated group (Figure 1(h)).

Histochemical observations

Polysaccharides

Examination of polysaccharides distribution in the testicular tissue from the control and saffron group showed a magenta color which indicated a PAS-positive reaction. The reaction appeared moderate in the tunica albuginea and the connective tissues between the tubules. While the reaction is high in the membrane of the seminiferous tubule, the head of sperm. While the spermatogenic cells showed weak PAS reaction (Figure 2(a–d), Table 3). In general, the total amount of polysaccharide is high in the control and saffron group. On the other hand, examination of the testicular tissue from rats in the khat group showed a reduction in the amount of polysaccharides particularly in spermatids and the seminiferous tubules membrane (Figure 2(e), Table 3). Examination of PAS sections from testes of rats treated with both khat and saffron revealed a gradual increase in polysaccharide content compared to the khat group (Figure 2(f), Table 3).

Figure 2. A photomicrograph of testicular tissue (a,b): control; (c,d): saffron group showing strong PAS reaction as a magenta color; (e): khat group showing weak reaction of PAS reaction as a magenta color; (f): khat and saffron group showing moderate PAS reaction as a magenta color. Tunica albuginea (black arrow), the membrane of the seminiferous tubule (yellow arrow); spermatogenic cells (blue arrow); sperm (S) and the connective tissues (arrowhead).

Table 3. The area percentage of polysaccharides and protein content (Mean±sd) in the different groups.

Animal groups	The area percentage of polysaccharides and protein content (Mean±SD)
	% polysaccharides	% protein
Control	12.46 ± 1.91	46.61 ± 2.58
Saffron	11.99 ± 1.11	40.25 ± 1.88
Khat	2.61*±0.77	20.64 ± 1.20*
Khat+saffron	9.17**±0.23	30.50 ± 1.14**

Data represented as (Mean±SD), n = 10 animals per group.

*Significant compared to the control group at p < 0.05.

**Significant compared to the khat group at p < 0.05.

Total proteins

Testes from rats in the control and saffron group showed high protein content with an intensive blue color with the mercury bromophenol blue within the membrane of the seminiferous tubules, the cytoplasm and nuclei of the spermatogonial cells and also in the interstitial connective tissue (Figure 3(a,b), Table 3). But when rats were treated with khat every other day for 3 months a marked decrease of the protein materials was observed (Figure 3(c), Table 3). While treating rats with both khat and saffron together every other day for 3 months showed a gradual retrieval in the protein content compared to khat group (Figure 3(d), Table 3).

Figure 3. A photomicrograph of testicular tissue (a): control, (b): saffron group showing high protein content (c): rat treated with khat for 3 months showing a reduction in the protein content (d): rat treated with khat and saffron for 3 months showing a moderate increase in the protein content; membrane of seminiferous tubules (arrowhead), spermatogenic cells (arrow), and sperms (S); (bromophenol blue).

Immunohistochemical observations

Bcl₂ expression

When sections of the testes from control and saffron-treated rats were examined after 3 months, almost spermatogenic cells showed cytoplasmic Bcl2 expression as a brown color (Figure 4(a,b)). The area percentage of Bcl2 expression did not show any significant change between control and saffron group (Table 4). While in the khat-treated group, few spermatogenic cells showed Bcl2 expression in their cytoplasm (Figure 4(c)). Also, when compared to the control group, the area percentage in the khat group increased substantially (Table 4). In khat and saffron group, the Bcl2 expression showed an increase in the spermatogenic cells cytoplasm when compared to khat alone (Figure 4(d)).

Figure 4. A photomicrograph to testicular tissue a-d) Bcl2 expression; (a) control; (b) Saffron group showing cytoplasmic Bcl2 expression in the spermatogenic cells as a brown color; (c) khat group showing Bcl2 expression in few spermatogenic cells; (d) Khat and saffron group showing Bcl2 expression in the moderate number of spermatogenic cells; (e-h) Bax expression (e): control; (f) Saffron group showing negative expression of Bax in the nuclei of spermatogenic cells, sperm and interstitial cells; (g): Khat group showing nuclear Bax expression in a large number of spermatogenic cells; (h): Khat and saffron group showing expression of nuclear Bax in moderate number of the spermatogenic cells (Bcl2 & BAX immunostain).

Table 4. The area percentage of Bcl2, Bax, Vimentin and E-cadherin expression in the different groups.

Animal groups	The area percentage of Bcl2, Bax, Vimentin and E-cadherin (Mean±SD)
	% Bcl2	% Bax	% Vimentin	%E-cadherin
Control	27.806 ± 1.2	Zero	31.25 ± 2.28	33.08 ± 2.56
Saffron	10.483 ± 2.5	Zero	30.69 ± 1.02	29.06 ± 2.36
Khat	10.48*±0.58	25.93 ± 1.59*	3.78 ± 0.96*	10.78 ± 2.87*
Khat+saffron	16.88**±0.96	10.65 ± 1.57**	23.67 ± 1.69**	18.98 ± 1.58**

Data represented as (Mean±SD), n = 10 animals per group.

*Significant compared to the control group at p < 0.05.

**Significant compared to the khat group at p < 0.05.

BAX expression

In control and saffron-treated rats, the nuclei of the spermatogenic cells showed negative Bax expression. Between these two groups, the area percentage did not show any significant difference (Table 4 and Figure 4(e,f)). In the khat group, a strong positive expression of Bax (as a brown color) appeared in a large number of the nuclei of spermatogenic cells. Furthermore, rats given khat showed a highly remarkable increase in the percentage of area of the Bax expression (Table 4 and Figure 4(g)). When rats were treated with both khat and saffron, the number of nuclei of spermatogonia and primary spermatocytes that showed Bax expression was decreased (Table 4 and Figure 4(h)).

Vimentin expression

Immunostaining of vimentin in the testes tissue from the control and saffron group showed strong positive expression as a brown color in Sertoli cells and their adjoining spermatogenic cells and tail of spermatozoa and interstitial tissue (Table 4 and Figure 5(a,b)). On the other hand, vimentin immunoreaction revealed a highly substantial decline in the khat group. It was seen as a faint brown color in the interstitial tissue only (Figure 5(c)). When rats were treated with khat and saffron a highly significant rise in the vimentin expression was observed. It appeared as a moderate brown color in the Sertoli cells, spermatogenic cells, tail of spermatozoa, and interstitial tissue (Figure 5(d)).

Figure 5. A photomicrograph from testicular tissue (a-d) vimentin immunoreaction; (a) control; (b) saffron group: showing strong positive expression of vimentin as a brown color; (c) khat group showing a highly significant reduction in the vimentin expression as faint brown color; (d) khat and saffron group showing vimentin expression as a moderate brown color; sertoli cells (arrowhead), spermatogenic cells (red arrows), tail of spermatozoa (S) and interstitial tissue (black arrows); (e-h) E-Cadherin immunoreaction (e): control; (f) saffron group showing strong expression of Cadherin; (g): khat group showing a marked reduction in cadherin immunoreaction; (h): khat and saffron group showing expression of cadherin as a moderate brown color; (arrowhead) membrane of the seminiferous tubules, (yellow arrow) sertoli cell, (black arrow) spermatocytes, (red arrow) interstitial tissue.

E-cadherin expression

The cadherin immunoreaction in the control and saffron groups was visible as dense brown bands encircling the tubules, in Sertoli cells, the connection between Sertoli cell and the spermatocytes, as well as in the interstitial tissue (Figure 5(e,f)). Rats in the khat-treated group showed a marked diminution in cadherin immunoreaction in the membrane surrounding the seminiferous tubules, Sertoli cell, between Sertoli and the different spermatogenic cells and the interstitial tissue (Figure 5(g)). When rats were treated with khat and saffron a highly significant rise in the cadherin expression was observed. It appeared as a moderate brown color surrounding the tubules, in Sertoli cells, the connection between Sertoli cells and the spermatocytes, and in the interstitial tissue (Table 4, Figure 5(h)).

Discussion

In the present results, rats received khat recorded alterations in the testicular morphometric measurements and histology compared to the control group. These findings could be due to the oxidative stress caused by khat. Abou-Elhamd et al. [10] reported that khat extract and cathinone altered the seminiferous tubules’ morphology and inhibited the production of protein, RNA, and DNA in dividing germ cells, which decreased spermatogenesis. Our results agree with Nyongesa [39] who said that cathinone had a direct effect on impaired spermatogenesis and developing germ cells in the testis of rabbits.

Dosing saffron and khat together reduced the harmful effect of khat by inhibiting the morphometric and histopathological changes in testicular tissues. This may be attributed to the saffron scavenging activity against the free radicals, so shields cells from oxidative damage. Gudarzi et al. [40] and Yari et al [41]. reported that the free radical scavenging and antioxidant capabilities of saffron have modulating effects on oxidative stress. Our results agree with Sakr et al. [42] who demonstrated that saffron showed a significant improvement in germ cell height and in tubular diameter in albino rats treated with sodium valproate.

Histochemical studies on rat testes treated with khat showed a remarkable depletion in the cytoplasmic polysaccharides and protein content. The primary active ingredient in khat cathinone increases serum cortisol levels, stimulates catecholamine release, activates adrenergic receptors, and contributes to the inhibition of insulin release, which lowers the amount of polysaccharides in the cells [43,44].

In accordance with our study, Abd El-Aziz [45] demonstrated that cell protein decreased in the liver, heart, and placenta after khat extract injection during pregnancy in rats.

In the present study, polysaccharides and protein content are greatly recovered in the testicular tissue after rats received saffron and khat together. The improvement in protein and carbohydrate contents by saffron may be attributed to the scavenging ability of saffron components to free radicals. Hosseinzadeh and Talebzadeh [16] reported that saffron has a decrease in damage happened on intracellular DNA and RNA.

In the current study, rats administrated khat showed a decrease in the immunoreaction of Bcl-2, vimentin, and E-cadherin proteins while BAX expression was increased in the different spermatogenic cells. This result could be attributed to khat toxicity which increases ROS generation and activates mitochondrial-mediated cell death pathways (apoptosis). According to Al-Qadhi et al [8], khat impacted the survival and proliferation of several cell types in vitro by raising Bax expression and decreasing the expression of Bcl-2 protein. Also, Karau et al. [46] demonstrated that khat increased apoptosis in the prefrontal cortex.

The disintegration of spermatogenesis is linked to vimentin filament damage. The changes in testicular tissue are caused by the inhibition of the filaments of vimentin in Sertoli cells, which leads to the separation of spermatogenic cells, so the cells undergo apoptosis due to the absence of Sertoli cell support and nurture [24,47].

E-cadherin contributes to cell adhesion and participates in signaling pathways that control the migration, differentiation, and proliferation of cells. The deficiency in E-cadherin expression, reported in the current study, may explain the cause of testicular degeneration and the depletion of free sperms in the seminiferous tubules. In harmony with our results, Abou-Elhamd et al. [10] showed that in the human ovarian cancer SKOV3 cell line, khat extracts lowered the expression of E-cadherin.

Rats given saffron and khat showed a marked rise in the expression of Bcl2 vimentin and E-cadherin protein and a fall in the expression of BAX in their testes. This may be attributed to the anti-apoptotic properties of saffron. Riazi et al. [48] said that saffron is rich in phytochemicals like crocin, crocetin, and carotenoid derivatives which have anti-apoptotic properties. Session [49] mentioned that crocin may be able to prevent testicular damage caused by doxorubicin via vimentin reorganization in Sertoli cells and enhance oxidant and antioxidant systems. Festuccia et al. [50] support our findings, the authors found that saffron treatment enhances cell-cell adhesion and cell mobility, by increasing E-cadherin expression.

Concerning the biochemical results, an increase in the MDA concentration and a decrease in SOD, and CAT enzyme activity were detected in khat-treated rats. These results run in parallel with the histological and immunohistochemical observations. These biochemical alterations induced by khat could be attributed to the oxidative damage of khat extract and testicular alteration. Shewamene and Engidawork [51] & Abdelwahab et al [52]. reported that khat administration triggered tissue toxicity in rats by inducing a condition of oxidative stress, augmentation of prooxidant components, or reduction of antioxidative mechanisms in tissues. Corresponding to the current result, Masoud et al. [53] found that CAT activity was lower in the plasma of khat chewers, which results in an accumulation of H2O2 and higher levels of hydroxyl radicals.

In the current study, the effect of saffron as an antioxidant is demonstrated by the reduction of MDA concentration and increasing the activity of SOD and CAT in rats treated with both khat and saffron. The marked reduction in lipid peroxidation may be due to the saffron active components, which may be very important in managing the antioxidation constituents as free radical scavengers, thus making saffron effective in preventing or alleviating oxidative stress [54]. Moreover, pre-treatment with crocetin decreased MDA production and increased SOD activity, suggesting that crocetin may ameliorate oxidative stress brought on by lipopolysaccharide/D‑galactosamine in rat’s liver [55,56]. The result is in accordance with that of Hashemi et al [57]. who noticed that the CAT activity in the liver and tumor tissues was elevated by saffron carotenoids, such as crocetin and crocin. Kocaman et al. [58] reached the same result that dosing 100 mg/kg/day of crocin in rats resulted in a reduction in MDA and an increase in CAT, GSH, and SOD. Many recent research resulted in the same antioxidant effect of saffron which decreased MDA levels and increased SOD and CAT activity [59,60].

Conclusion

From the present study, we conclude that khat adversely affected the testis of male rats via enhancement of oxidative stress. Treating rats with both saffron and khat extract revealed an improvement in histological, immunohistochemical, and biochemical results when compared to the khat group. This could be linked to the scavenging capacity of saffron to the free radicals and to its antioxidant and anti-inflammatory effects.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate

The study has been authorized by Animal Care and Bioethics Committee, Egypt (Approval No. MNSH 323).

Disclosure statement

No potential conflict of interest was reported by the author(s).