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 Table of Contents  
Year : 2020  |  Volume : 4  |  Issue : 4  |  Page : 219-227

Alteration of apoptotic gene expression and reproductive parameters through Thymus vulgaris administration following paclitaxel-induced testopathy in micea

1 Department of Anatomical Sciences, Medical School, Kermanshah University of Medical Sciences, Kermanshah, Iran
2 Department of Anatomical Sciences, Medical School, Tehran University of Medical Sciences, Tehran, Iran

Date of Submission08-Jan-2020
Date of Decision03-Feb-2020
Date of Acceptance03-Mar-2020
Date of Web Publication31-Dec-2020

Correspondence Address:
Mohammad Reza Salahshoor
Department of Anatomical Sciences, Medical School, Kermanshah University of Medical Sciences, Kermanshah
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2096-2924.305929

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Objective: Paclitaxel (PAC) is a chemotherapy drug and has an active role in the treatment of lung, breast, and ovarian cancers; however, its use causes increased levels of free radicals, leading to severe and irreversible organ damage. Thymus vulgaris is a species of flowering plant that contains various antioxidant chemical compounds. This study aimed to investigate the effect of T. vulgaris on PAC-induced oxidative damage in mice testis and sperm parameters.
Methods: In this study, 40 BALB/c mice were divided into five groups: normal control (NC); positive control (20 mg/kg of PAC); and three treatment groups (4.5, 9, and 18 mg/kg doses of T. vulgaris extract + 20 mg/kg PAC). Treatments were administered intraperitoneally daily for 50 days. Finally, the levels of serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone were measured using immunoassay method. Testicular stereological features and sperm parameters were also calculated. Antioxidant parameters were measured using nitrite oxide (NO) and perioxidation levels and ferric-reducing ability of plasma assay. The expressions of p53, caspase-3, Bax, and Bcl-2 were measured through real-time quantitative polymerase chain reaction.
Results: T. vulgaris + PAC treatments at all doses significantly increased all parameters in NC and three treatment groups compared with the positive control group (20 mg/kg of PAC) (P < 0.05), except the LH, FSH, and NO levels, which were decreased. Further, significantly downregulated levels of p53, caspase-3, and Bax genes and? upregulated levels of Bcl-2 gene expression were noted in NC and three treatment groups in comparison with the positive control group (P < 0.05).
Conclusions: T. vulgaris administration attenuates the toxic effects of PAC on male reproductive parameters.

Keywords: Apoptotic; Paclitaxel; Reproductive; Testopathy; Thymus vulgaris

How to cite this article:
Roshankhah S, Hassanzadeh G, Faramarzi A, Salahshoor MR. Alteration of apoptotic gene expression and reproductive parameters through Thymus vulgaris administration following paclitaxel-induced testopathy in micea. Reprod Dev Med 2020;4:219-27

How to cite this URL:
Roshankhah S, Hassanzadeh G, Faramarzi A, Salahshoor MR. Alteration of apoptotic gene expression and reproductive parameters through Thymus vulgaris administration following paclitaxel-induced testopathy in micea. Reprod Dev Med [serial online] 2020 [cited 2021 May 11];4:219-27. Available from: https://www.repdevmed.org/text.asp?2020/4/4/219/305929

  Introduction Top

Male infertility is known to be a clinical disease with physical, familial, social, and psychological complications. Various studies have shown that 15% of couples in the US suffer from infertility, while 25% of couples have sexual dysfunction related to male infertility.[1] One of the major infertility factors, especially in athletes, is reactive oxygen species (ROS) damage following the administration of anticancer drugs.[2] ROS has a crucial role in intracellular signaling pathways, sperm capacity, acrosome reaction, and sperm-oocyte fusion. DNA and unsaturated fatty acids in the membrane of spermatogonia and sperm cells are strongly sensitive to free radicals, resulting in oxidative-reducing reactions, elimination of cellular integrity, and necrosis. These phenomena alter the morphology, motility, and viability of the sperm, which may reduce the number of active sperm with fertilization ability.[3] One of the most well-known side effects of anticancer drugs is disruption of spermatogenesis, leading to infertility in many cases.[4] Anticancer drugs such as paclitaxel (PAC) have substantial side effects, including amenorrhea, and sexual impotence.[5] PAC is a DNA-destructive chemotherapy agent that is used for long-term treatment of chronic myelogenous leukemia and ovarian cancers in low doses and also for bone marrow suppression in patients undergoing bone marrow transplantation in high concentrations.[6] Pectasides et al. showed that the long-term administration of nontherapeutic doses of PAC could increase the atrophy rate, reduce the number of germinal testicular cells, and also change the serum levels of luteinizing hormone and follicle-stimulating hormone (LH and FSH) by induction of free radical generation as well as serum levels of testosterone.[7] PAC inhibits cell division especially in spermatogonial cells that have high division rates.[8] According to the various published studies, the incremental process in the concentration of antioxidant compounds available in semen can prevent free radicals from attacking the sperm membrane and DNA. Thus, sperm accumulation, decreased testosterone level, reduced motility rate (due to low mitochondrial ATP production), reduced sperm capacity, and acrosome reaction will be inhibited.[9] Some plants contain antioxidant and even aphrodisiac compounds that are effective in the sexual process and levels of male and female hormones.[10] Thymus vulgaris, a member of the mint family, is native to the northern and northwestern parts of Iran; it is a thornless shrub with heart-shaped leaves that have toothed edges.[11] Other species of this family (garden thyme) grow in Russia, Georgia, and some other Eastern European countries. The fruitful plant grows up to 1–1.5 m and has 20–40 flowers.[12] It contains various flavonoid antioxidant compounds, including gallic acid, protocatechuic acid, gentisic acid, chlorogenic acid, ferulic acid, p-coumaric acid, salicylic acid, and also anthocyanins, including delphinidin, petunidin and malvidin.[13] The total phenolic content in the flowers of T. vulgaris is calculated as being 164.9 mg GAC/g, which is more than the content of the same chemical compound in the leaves (60.1 mg QE/g).[14] The high phenolic content of the flowers has made them one of the most important sources of natural antioxidants. Studies have shown that this plant, with its high antioxidant content, can protect sperm against toxic chemicals.[15] The aim of this study was to evaluate the protective effects of hydroalcoholic extracts of the T. vulgaris plant on testicular tissue using stereological principles, sperm parameters, and antioxidant properties, following tissue damage induced by PAC.

  Methods Top

Preparation of herbal extract

T. vulgaris flowers were collected from the highlands of Gilan (Asalem) in late summer. They were dried in a dark medium at 60°C. The dried plant was ground (300 g) and dissolved in 70% ethanol. Next, 72 h later, the solution was filtered using filter paper (Whatman No. 2, UK); it was then condensed at 50°C using a vacuum distillation unit (Heidolph Collegiate, LABOROTA 4000, Germany). The final dried extract (20 g) was stored at 4°C.[9]

Experimental design and treatments

Forty BALB/c male mice (30 ± 5 g) were housed according to the 12-h light/12-h dark photocycle at 24°C–30°C and 50%–55% relative humidity. They were kept in appropriate conditions with regard to food (standard pellets) and water under the rules of laboratory animal manipulation. All investigations conformed to the ethical principles of animal research and were approved by the Ethics Committee of Kermanshah University of Medical Sciences (No. 1397.497). The animals were divided into five groups (eight mice in each). Group 1, the normal control (NC) group, received 0.5 mL distilled water (DW). Group 2, the PAC-positive control group, received 20 mL/kg PAC dissolved in 0.5 mL DW (ratio of 1:1). Groups 3–5, the treatment groups, received 4.5, 9, and 18 mg/kg, respectively, of T. vulgaris dissolved in 0.5 mL DW + 20 mg/kg PAC dissolved in 0.5 mL DW (T. vulgaris extracts and PAC were administered at 2-h intervals at specified times for 50 days). The extract and PAC were administered for 50 consecutive days through intraperitoneal injection.[5]

Hormones assay

At the end of the study (day 51), the mice were humanely destroyed by cervical dislocation using the standard laboratory animal protocols. Blood samples were aspirated from the heart, and serum was collected by centrifugation (1,000 ×g, 15 min). The serum levels of testosterone (Elisa Kit – Pishtazteb, Iran), LH (Beckman Coulter Kit, Czechoslovakia), and FSH (Beckman Coulter Kit, Czechoslovakia) were measured using immunoassay method.[16]

Sperm count

The animal epididymis was washed with normal saline solution. To conduct a sperm count, the epididymis tail was crushed in a Petri dish containing 2 mL of Ham's F10 medium. Five hundred microliters of the sperm suspension was diluted (1:10) with formaldehyde (10%). Finally, the settled spermatozoa were counted at × 400 (Nikon Microscope ECLIPSE E600W, Tokyo, Japan) in accordance with the previous study protocol. The mean number of spermatozoa in the four small corners (0.2 mm2) with large center squares (1 mm2) was counted; then, the final number of sperms was counted in the specified volume: Counted number of spermatozoa × dilution volume/number calculated in mm2 × depth of the slide chamber.[15]

Sperm motility

To evaluate the sperm motility according to the previous study and WHO protocols, a drop of the suspension was placed onto a glass slide and covered with a coverslip. Thirty fields were calculated using a light microscope at ×400 (Nikon microscope ECLIPSE E600W, Tokyo, Japan). Sperm classification was carried out according to protocol as follows: (1) Immotile sperm (motility I); (2) nonprogressive sperm (in situ sperm) (motility II); (3) forward sperm (motility III).[17]

Sperm viability

Eosin Y was used for the differentiation of living cells from dead ones. For this purpose, 40 μL of sperm suspension was mixed with 10 μL of eosin Y (0.5% in saline) and was then assessed at ×400. No eosin staining occurs in the head and neck of a living sperm, while dead sperm shows eosin color due to defects in their cell membranes; thus, the heads and necks of dead cells turn red.[5]

Stereological parameters

The samples were placed in 10% formalin for 72 h. Then, eight slabs were obtained through the orientator method. Paraffin-embedded blocks were prepared from each slab, then cut into 5-μm sections (LEICA SM2010RV1.2 Microtome Machine, Germany), and stained using hematoxylin and eosin to analyze the volume and length parameters of each slab. To estimate the stereological parameters, such as volume (parenchyma, interstitial tissues and seminiferous tubules), length (seminiferous tubules), and the lumen diameter (LD) and height of the germinal epithelium in the testicular tissue, the slides were analyzed according to the previous study under a Nikon light microscope (Nikon microscope ECLIPSE E600W, Tokyo, Japan) equipped with KEcam (KEcam Technologies, Lekki Lagos, Nigeria) with stereological probes and using Adobe Photoshop CC (Adobe system, San Jose, CA, USA) [Figure 1].[18]
Figure 1: (a) Point probe (35 points) for calculation of volumetric density in testis structures (to estimate the volume density of the seminiferous tubules and interstitial tissue). The total number of points located on each structure is counted in each field of view and is compared with the total number of points in the volume measurement formula: Vv = SP structure/SP reference (H and E, ×200). (b) Framework probe, for estimating the length density of seminiferous tubules (H and E, ×200). The probe consists of banned lines (black lines) and counting lines (yellow lines). Tubules are inserted into the frame probe or are contacted by counting lines. Length density formula: Lv = 2(SQ/a (frame) × Sframe), where a is frame area, SQ is sum of the structures counted, and Sframe is the total number of the random counted frames (conventionally, 200 field of view) (H and E, ×200).

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Determination of nitric oxide

The Griess colorimetric method was used to measure the NO levels. Briefly, following deproteinization of 500 μL of the serum specimens by adding zinc sulfate (5 mg), the specimens were centrifuged (16,000 ×g for 12 min), and the supernatants were mixed with equal amounts of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine in 2.5% phosphoric acid) in the wells of 96-well ELISA plates. After 10 min incubation at 37°C, the absorbance was measured at 540 nm with the use of a microplate reader. Nitrite concentrations were calculated using a sodium nitrite standard curve as μmol/L.[15]

Lipid peroxidation levels

The reaction of thiobarbituric acid (TBA) with malondialdehyde (MDA) was applied to measure the lipid peroxidation levels. For this procedure, the homogenate of testicular tissue from a part of the frozen sample was used. The testes were washed with the phosphate-buffered saline at a pH of 7.4. An ultrasonic homogenizer in a cold phosphate buffer containing ethylenediaminetetraacetic acid (EDTA) was used to homogenize the sample groups. Finally, the resultant supernatant was stored at -70°C. Based on the chlorogenic process, the dye generated by the reaction of TBA with MDA in the commercial kit was read at 532 nm. In this order, 50 μL of tissue supernatants was added to the test tubes containing 2 μL of butylated hydroxytoluene and then 50 μL of phosphoric acid (1 mol/L) and 50 μL of TBA solution. The tubes were incubated for 60 min at 60°C and were centrifuged at 11,000 ×g for 4 min. 75 μL of the supernatant was poured into the spectrophotometer tubes to read at 532 nm, and finally, the MDA level was reported in nmol/mg protein.[19]

Determination of ferric reducing ability of plasma

The total antioxidant potential of the testis homogenate was determined using a modification of ferric reducing ability of plasma (FRAP). The FRAP reagent was prepared as a mixture of 5 mL of 10 mmol/L 2, 4, 6-tripyridyl-s-triazine in 50 mL of 0.1 mol/L acetate buffer (pH 3.6), 5 mL of 20 mmol/L ferric chloride, and 40 mmol/L of hydrochloric acid. All solutions were mixed according to the volume ratio of 25:2.5:2.5. The FRAP assay was performed using a reagent preheated to 37°C for 15 min, 200 μL of a testis homogenate sample was mixed with 90 μL of reagent, and the reaction was incubated at 37°C for 4 min. The mixture was centrifuged at 16,000 ×g for 15 min. After centrifugation of the resulting mixture (25,000 ×g/10 min), the absorbance of the supernatant was read at 593 nm, compared with a standard curve of ferrous sulfate (0–1,000 μmol/L).[9]

RNA extraction and real-time quantitative polymerase chain reaction

The total RNA was extracted using a QIAGEN RNA purification mini kit according to the manufacturer's protocol. In this procedure, 30 mg of testicular tissue was placed in RLT buffer; ethanol (96%) was added to the lysate, and the resulting mixture was recentrifuged. The supernatant was added to the RNeasy Mini Spin Column. Then, the total RNA was bound to the column membrane, the contaminants were efficiently washed away, and the high-quality RNA was eluted in RNase-free water. The quality of the extracted RNA was checked by a spectrophotometer (UV1240, Shimadzu, Kyoto, Japan) at a 260/280 nm wavelength absorbance ratio. The DNA was synthesized using a commercial BioFact kit (BioFact RT Series, Korea). According to the kit instructions, 1 μg of total RNA, 10 μL of mastermix, 0.5 μL of oligo-d (T) primer, and 0.5 μL of Random Hexamer primers were added. Then, the final volume with RNase-free water was increased to 20 μL. The RT reaction was carried out at 70°C (45 min) followed by heat inactivation at 95°C (3 min). The expressions of p53, Bax, and Bcl-2 were evaluated using High ROX BioFact™ 2X Real-Time PCR Smart mix SYBR Green PCR master mix. The real-time PCR light cycler device (StepOne™ Real-Time PCR System, USA) was based on the manufacturer's protocol. The polymerase chain reaction (PCR) primers were designed by? Oligo software (La Jolla, CA, USA) and the sequences were blasted in the NCBI database. The sequences of all the genes are listed in [Supplementary Table 1]. The PCR reactions for mRNAs expression consisted of 95°C for 5 min (denaturing cycle) followed by variable amplification cycles (38–42 cycles) at 90°C for 30 s (annealing cycle) and 72°C for 1 min (extension cycle). All the quantitative real-time PCR reactions were carried out in duplicate, and β-actin was used as a housekeeping gene. Gene expression levels were measured using the? Ct (2-?? CT) method (fold changes).[20],[21]

Statistical analysis

Data analysis was applied using the? GraphPad Prism statistical software package (version 8, Inc, San Diego, USA,). Normality and homogeneity of data were determined by Kolmogorov–Smirnov test (P > 0.05). Data were presented as mean ± standard deviation. Differences between the experimental groups were assessed by one-way ANOVA followed by Tukey's test (using a significance level of P < 0.05 in the tests).

  Results Top

Hormones assay

PAC increased the serum levels of LH (P = 0.004) and FSH (P = 0.015) and decreased testosterone level (P = 0.015) significantly compared with the NC group (negative control). The extract of T. vulgaris changed the levels of sex hormones; thus, all doses of the extract led to a significant increase of the testosterone serum level (P = 0.015) and decrease of the LH (P = 0.006) and FSH (P = 0.0025) levels in treatment groups compared with the positive control group [Figure 2].
Figure 2: Comparison of serum levels of testosterone, FSH, and LH (μIU/mL) in NC, PC, and the extract (TV) groups (mean ± SD). *Statistically significant (P < 0.05) between the PC and NC group andstatistically significant (P < 0.05) between extract and PC group. FSH: Follicle-stimulating hormone; LH: Luteinizing hormone; NC: Normal control; PC: Positive control; TV: Thymus vulgaris; SD: Standard deviaton.

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Stereological parameters

In stereological studies, it was found that PAC changed the size and length of the testicles. PAC caused a significant increase in the LD (P = 0.023) and the total intertubular compartment volume (TICV, P = 0.0021) and a significant reduction in the seminiferous epithelium height (SEH, P = 0.011), total tubular diameter (TTD, P = 0.022) and testis parenchyma volume (TPV) in the positive control group compared with the negative control group (healthy). However, in all doses of T. vulgaris extract, significant increases in SEH (P = 0.022), TTD (P = 0.012), and TPV (P = 0.021) and significant reductions in LD (P = 0.021) and TICV (P = 0.007) in the extract groups were observed compared with the positive control group [Figure 3].
Figure 3: SEH, LD, TTD, TICV, and TPV in NC, PC, and treatment extract groups (TV 4.5, 9, and 18 mg/kg) (mean ± SD). *Statistically significant (P < 0.05) between the PC and NC groups.Statistically significant (P < 0.05) between treatment and positive groups. NC: Normal control; PC: Positive control; TV: Thymus vulgaris; SD: Standard deviaton; SHE: Seminiferous epithelium height; LD: Lumen diameter; TTD: Total tubular diameter; TICV: Total intertubular compartment volume; TPV: Testis parenchyma volume.

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Sperm parameters

PAC caused a significant reduction in motility (forward [P = 0.011], in situ [P = 0.021]) and in immotile (P = 0.039), viability (P = 0.029), and sperm count (P = 0.016) in the NC group compared with the positive control group (healthy). In all groups, at all doses of extract treatment, the sperm count (P = 0.02), motility (forward, P = 0.042), and viability (P = 0.039) were increased significantly compared with the positive control group [Table 1].
Table 1: Number (×106/mL), motility (%) and viability (%) of sperm in normal control group (N), positive control group (PAC), and T. vulgaris treatment extract groups (TV 4.5, 9 and 18 mg/kg) (mean±SD)

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Testis ferric reducing ability of plasma, lipid peroxidation, and serum level of nitric oxide

The FRAP levels in testis tissue of the PAC group were significantly (P = 0.008) lower than the NC group. In all extract groups, the doses of T. vulgaris extract increased the FRAP considerably compared with the positive control group. PAC significantly (P = 0.021) increased the NO level in the positive control group compared with the NC group. All doses of T. vulgaris extract significantly (P = 0.008) decreased the mean level of serum NO in the extract groups compared with the positive control group. Lipid peroxidation levels in the testis tissue of the PAC group were significantly (P = 0.008) lower than the NC group. All doses of T. vulgaris extract significantly (P = 0.014) increased the lipid peroxidation in the extract groups compared with the positive control group [Figure 4].
Figure 4: The effect of TV extract on serum NO, FRAP, and LP levels of testis tissue (n = 8 per group) in NC, PC, and treatment extract groups (TV 4.5, 9 and 18 mg/kg) (mean ± SD). *Statistically significant (P < 0.05) between the PC and NC groups andstatistically significant (P < 0.05) between treatment and positive groups. NC: Normal control; PC: Positive control; TV: Thymus vulgaris; SD: Standard deviaton; NO: Nitric oxide; FRAP: Ferric reducing ability of plasma; LP: Lipid peroxide.

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P53, Bax, Bcl-2, and caspase-3 expression levels

PAC significantly upregulated the apoptosis-regulating genes of caspase-3 (P = 0.022), p53 (P = 0.011), and Bax (P = 0.002) and also significantly downregulated the Bcl-2 (P = 0.008) mRNA in the positive control group compared with the NC group. In all doses of T. vulgaris extract, significant downregulation of apoptosis-regulating genes, including caspase-3 (P = 0.022), p53 (P = 0.015), and Bax (P = 0.011), was detected. Further, upregulation of Bcl-2 (P = 0.013) mRNA in the extract groups was seen at all doses of T. vulgaris compared with the positive control group. However, all doses of T. vulgaris extract significantly upregulated the Bcl-2 (P = 0.033) mRNA in the extract groups compared with the positive control group [Figure 5].
Figure 5: The effect of TV on C3, p53, Bax, and Bcl-2 gene expression of testis (n = 6) in the NC, PC, and treatment groups (TV, 100, 200, and 400 mg/kg) (mean ± SD). *Statistically significant (P < 0.05) between the PC and NC groups andStatistically significant (P < 0.05) between treatment and PC groups. NC: Normal control; PC: Positive control; TV: Thymus vulgaris; SD: Standard deviaton; C3: Caspase 3.

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Histopathological findings

In the NC group, the germ cells were located on the basement membrane of the seminiferous tubules and had enlarged euchromatin nuclei and eosinophil cytoplasm. These cells were located in an entirely distinct and integrated layer throughout the germinal epithelium. The tubular lumen space was filled with normal spermatozoa (with a distinct tail), and the walls of the spermatozoa were unified. Spermatogenic lineage cells, including spermatogonia, primary spermatocytes, early spermatids, late spermatids, and Sertoli cells, were seen to be healthy (integrated cell membrane and intracellular junctions, eosinophilic cytoplasm, no degeneration and vacuolization); while in the positive control group, the walls of the tubules were fragmented, with no integration, and there were degenerated spermatogenic and Sertoli cells, and lost vacuolated cytoplasm. Their heterochromatin nuclei represented that the cells were inactivated. Degenerated cells filled the seminiferous lumen, vacuolization of the seminiferous epithelium was seen, and partial-to-complete absences of germ cells were also noticed. The interstitial space of the tubules was increased and filled with inflammatory and necrotic Leydig cells. The volume of vessels in the same areas was increased, resulting in hyperemia, and intestinal edema with congestion. In the treatment groups, the number of necrotic and vacuolated cells was decreased and the germ cell density was increased. The morphologies of the nucleus and the cytoplasm of the cells were similar to the same samples of the NC group, and only the spermatid cells were filled in the lumen of the tubules [Figure 6].
Figure 6: Histopathological changes in testicular tissue (H and E, ×400) in (1) normal control group, (2 and 3) positive control group, (4) Thymus vulgaris 4.5 mg/kg, (5) Thymus vulgaris 9 mg/kg, (6) Thymus vulgaris 18 mg/kg. The black arrows in 2: Lumen space in abnormal conditions filled with degenerated cells. The square box in 1, 4, 5, and 6: Germinal epithelium containing spermatogenic cells. The circle box in 2 and 3: Degeneration of spermatogonial cells and release of cell debris into the lumen space. The stars in 2 and 3: Destruction of the wall of seminiferous tubules. The red arrows in 3: Identifies vacuolization.

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  Discussion Top

In the present study, PAC increased LH and FSH and decreased testosterone levels. Generally, PACl damage the spermatogenic function of the testis, and then affect the function of Sertoli cells, leading to elevated FSH and LH.[22] Akbari Bazm et al. also showed that this stanozolol, testosterone, and nandrolone compound changes the sexual hormone levels in male mice.[23] As a result, division and differentiation of Sertoli, Leydig, and spermatogenic cells are impaired. FSH is essential as a stimulant of Sertoli cells and is involved in the differentiation of spermatogonia cells.[24] A recent systematic review and meta-analysis revealed that long-term AAS administration increases the serum levels of LH and FSH and reduces testicular volume and weight; hypogonadism is eventually seen.[25] In this study, the T. vulgaris extracts increase the serum testosterone levels and decreased the LH and FSH levels. Chromatographic, chemical, and spectroscopic methods showed that extracts of this plant contain important anthocyanins such as delphinidin, petunidin, and malvidin, which are crucial to the therapeutic properties of this plant.[26] Rojas-GarcÍa et al. showed that testosterone increases sperm count and decreases motility.[27] A study by Omotuyi et al. showed that consumption of anthocyanin-containing extracts increase testosterone levels and decreases serum FSH in male rabbits.[28] A study by Arabi et al. also showed that extract consumption of anthocyanin-containing plants might improve serum male hormones (increased testosterone and decreased FSH and LH levels) as well as improve sperm parameters (increased sperm count and motility) in mice.[29] As well as Vitamin C and E, the T. vulgaris plant contains important flavonoid compounds, such as quercetin, apigenin, catechin, and epicatechin.[14] These compounds have high levels of antioxidant activity. Further, apigenin, as a flavonoid compound present in T. vulgaris, regulates the secretion of LH by Leydig cells and also increases the serum testosterone level.[30] Sertoli cells, which are affected by FSH, increase the secretion of water into the seminiferous tubules to maintain the size and structure of the tubules in normal conditions and regulate the nutrition and differentiation of spermatogenic cells.[31] Borovskaya et al. showed that administration of a dose of 20 mg PAC for 28 days reduced the seminiferous tubule diameter and increased the interstitial tissue thickness in mice.[32] In the present study, it was found that the PAC increased the LD and the TICV and decreased the SEH, TPV, and TTD following 50 days of administration. PAC causes the atrophy of seminiferous tubules and SEH, which leads to a reduction in the number of spermatogonia, Sertoli cells, and spermatogenic cells.[32] Our study also showed that the T. vulgaris extract containing anthocyanin and flavonoid compounds improved the structure of the seminiferous tubules in comparison with the PAC group (positive control group) within 50 consecutive days, as well as reduce the interstitial connective tissue and, furthermore, the function and structure of the germinal epithelium were preserved against ROS. By reduction in the number of germ cells and the spermatogenesis process, the SEH and the diameter of the tubular lumen are decreased and increased, respectively. As a result of the atrophy of the seminiferous tubules, their volume was decreased; thus, the interstitial tissue of the tubules was increased, while the volume of parenchymal tissue was decreased.[15] PAC reduces the sperm motility of the flagella by deactivation of the ion pumps (Na/K ATPase enzymatic activity), increases the amount of ROS, and reduces the level of mitochondrial energy in the sperm neck.[7] ROS could also induce the destruction of sperm DNA or alter the intracellular concentration of Ca2+, consequently initiating apoptosis.[33] Studies show that the dolphinidine available in this plant improves spermatogenesis and reduces damage to testicular tissue in the varicocele model of rats. This compound improves the sperm parameters (number and motility) and reduces the apoptosis of spermatogonial cells.[34] Studies also show that PAC, by increasing the number of free cellular radicals, could decrease cellular antioxidant capacity (due to the reduction in catalase and MDA levels), leading to damage of the membrane, sperm, and spermatogonia DNA.[6] The result of our previous study on PAC is in line with the results of the present study, suggesting an increase in NO level and a decrease in the FRAP following administration of this anticancer drug.[5] In this study, a dose of 20 mg/kg reduced the tissue level of lipid peroxidation and FRAP and increased the serum NO level. Since the sperm membrane contains a large amount of unsaturated fatty acids, it is highly vulnerable to free radical attack, by which the membrane structure disappears.[15] The phenolic structure of anthocyanin contributes to the antioxidant action of ROS elimination, including superoxide, singlet oxygen, peroxide, hydrogen peroxide (H2O2), and the hydroxyl radical.[35] The present study showed that the accumulated ROS levels due to PAC administration were reduced following usage of the T. vulgaris extracts. It was also shown that the serum NO level decreased after this extract administration and that the testis tissue FRAP and also lipid peroxidation increased. In this study, it was shown that the administration of PAC increased the expression of preapoptotic factors (such as p-53, Bax, and caspase-3), whereas the expression of antiapoptotic (Bcl-2) factors was decreased. P-53 regulates the activities of preapoptotic factors such as Bax, caspases, and endonucleases by releasing cytochrome C from the mitochondria, while Bcl-2 prevents the release of cytochrome C complex.[36] Studies showed that PAC leads to upregulation of preapoptotic factors in tissues with androgen receptors, such as the prostate and breast.[37] These compounds induced the apoptosis process in the ovarian granulosa cells and testicular tissue by increasing serum testosterone levels.[38] This drug binds to the related receptor to be translocated into the nucleus and may up- or down-regulate the genes with appropriate hormone response elements.[39] Cells resist environmental damage and oxidative stress attack by cellular enzymes, while PAC decreases the activity of these enzymes and facilitates the process of apoptosis and cellular damage.[40] ROS regulates the apoptosis-associated transcription factors, such as p53, caspase3, and Bax/Bcl-2 ratio. The p53 gene is responsible for cell cycle control at G1/S and G2/M checkpoints and overexpression during cellular damage to induce apoptosis in the cells arrested at these stages.[41] In the process of spermatogenesis, the germ cells are damaged or overexpressed by Sertoli phagocytes through the Fas/FasL apoptotic system-dependent manner to maintain equilibrium, which is essential to normal spermatogenesis. In damage to testicular tissue, the expression of caspase-3, along with the activation of cytochrome C, could increase the rate of the apoptosis process.[42] Zhang et al. showed that the testicular anabolic–androgenic steroid (AAS) in a monkey model led to upregulation of Bax and p53, downregulation of Bcl-2 genes, and increased apoptosis in spermatogonial cells over a period of 60 days of administration.[43] El-Hanbuli et al. showed that the AAS in a rat's testes increased the caspase-3, p-53 and apoptosis in spermatogonial cells.[44] In this study, T. vulgaris extract reduced the caspase-3, p53, and Bax and increased the Bcl-2 expression and ultimately protected the cells from ROS-induced apoptosis. Studies have shown that PAC reduced the expression of Bax by reduction in the amount of ROS and increase of Bcl-2 genes by decreasing the p53 expression.[45]

In conclusion, T. vulgaris significantly reversed the effect of PAC-induced male fertility parameters injury through reduced the ROS (decreased the serum NO and increased the testis FRAP and lipid peroxidation) and improved the levels of testosterone hormones, which resulted in spermatogenic cells being protected from apoptosis (maintenance of volume and SEH), and then to improvement of sperm parameters (motility, number, and viability). The extent of male fertility protection showed by T. vulgaris against PAC-induced male fertility parameters damage in rats proves that T. vulgaris could be commercialized as a pharmaceutical drug for male reproductive ailments. Further research is needed to validate the antioxidant strength of T. vulgaris on other diseases caused by manifestation of oxidative stress.


Kermanshah University of Medical Sciences' Research and Technology Deputy is gratefully acknowledged for the financial support for the research project.

Financial support and sponsorship

This research was funded by the Research and Technology Deputy of Kermanshah University of Medical Sciences with grant number 97497.

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

  [Table 1]


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