Protection of apigenin against acrylonitrile-induced sperm and testis injury in rats: involvement of activation of ASK1-JNK/p38 signaling pathway

Abstract

This study aims to clarify if apigenin (AP) could play a pivotal role in attenuating acrylonitrile (ACN)-induced sperm and testis injury by inhibiting ASK1-JNK/p38 signaling pathway. Male Sprague–Dawley rats were randomly divided into five groups: a control group (corn oil), an ACN group (ACN 46 mg kg⁻¹), an ACN + AP1 group (ACN + AP 117 mg kg⁻¹), an ACN + AP2 group (ACN + AP 234 mg kg⁻¹) and an ACN + AP3 group (ACN + AP 351 mg kg⁻¹). The ACN + AP groups were given ACN by gavage after a pretreatment with different dosages of AP for 30 min, whereas the rats in the control group received an equivalent volume of corn oil. The gavage was conducted for 6 days per week in 4 weeks. The results showed that AP reduced sperm deformity rate and DNA fragment index and attenuated the testicular injury induced by ACN. AP could also alleviate oxidative stress, downregulate ASK1-JNK/p38 signaling pathway and eventually inhibit mitochondria-mediated testicular apoptosis. In brief, AP could dampen oxidative stress thereby inhibiting testicular apoptosis mediated by ASK1-JNK/p38 signaling pathway, alleviating ACN-induced sperm and testis injury and exerting a protective effect on male reproductive system.

Introduction

Acrylonitrile (ACN), a crucial organic chemical synthetic monomer, is widely used in the manufacture of synthetic resins, rubbers, plastics, fibers and acrylamide. ACN is highly toxic and carcinogenic, which is classified as category 2B by IARC [1]. Exposure of workers occurs at every step from synthesis of ACN to its incorporation into commercial polymeric materials [2]. In addition, ACN is also detected in kitchen utensils [3] and cigarette smoke [4], inducing a low-dose exposure to human, which should not be ignored. Absorbed ACN is able to get into body through oral intake, vapor inhalation and skin contact [2]. The metabolism of ACN is mainly carried out in two different ways, i.e. the conjugation with glutathione (GSH) directly, and the epoxidation catalyzed by cytochrome P-450 (Cyt P-450) to cyanoethylene oxide (CEO). The metabolism consumes GSH with a release of cyanide (CN⁻) [5], which causes a generation of reactive oxygen species [6] and initiates a free radical reaction cascade, making the body undergo a lipid peroxidation. The depletion of GSH and free radicals generated from metabolism of ACN leads to oxidative damage [7]. The treatment of ACN resulted in a lipid peroxidation and a decreased antioxidant capacity in the brain and liver of rats [8]. The administration of ACN also decreased the levels of testosterone and androsterone, which may be related to the increase of oxidative stress [9]. ACN would contribute to a testicular apoptosis for triggering oxidative stress and activating NF-κB signaling pathway [10]. In view of available articles, oxidative stress, activation of intracellular signaling pathways and an imbalance of apoptosis were involved in the male reproductive injury caused by the introduction of ACN. Therefore, the supplementation with antioxidants might be an effective treatment against the toxicity of ACN.

Apigenin (AP), 4′, 5, 7-trihydroxy flavonoid, is rich in a variety of vegetables, fruits and some Chinese herbs, among which the content of celery is the highest [11]. In recent years, AP has grabbed great attention as a health-promoting agent due to its low intrinsic toxicity and selective response to normal versus cancer cells, compared with other flavonoids [12]. Numerous studies have proved the beneficial biological properties of AP, including antioxidant, anti-inflammatory and anticancer [13]. Moreover, AP has a significant effect on the reproductive and endocrine systems of rats [14–17]. Primarily acting on the antioxidant system in the body, the specific effect of AP is not always favorable, i.e. to militate against the antioxidant system at a high dose [18, 19]. In addition, AP suppressed the phosphorylation of IκBα and downregulated the expression of caspase 3 [20], suggesting that AP alleviated the inflammatory response mediated by NF-κB signaling pathway and inhibited the ACN-induced apoptosis of germ cells. Therefore, a suitable dosage range of AP is highly desired for its appropriate applications.

It was demonstrated that ASK1-JNK/p38 signaling pathway is crucial to apoptosis and can be activated by oxidative stress [21]. Our previous studies [22] revealed that ACN induced an oxidative stress to cause apoptosis in rat testis, which involved the activation of ASK1-JNK/p38 signaling pathway, while AP inhibited the activation of JNK and p38 MAPK in keratinocytes [23]. Thus, the purpose of this current study is to evaluate whether AP plays a protective effect against the ACN-induced reproductive injury by inhibiting the ASK1-JNK/p38 signaling pathway in male rats, to protect from the reproductive toxicity of ACN by utilizing of AP.

Materials and methods

Animal preparation and administration

Sixty adult male Sprague–Dawley (SD) rats weighing 180–220 g were obtained from the Medical Animal Center (Gansu University of Chinese Medicine, China) and bred in the experimental center of the School of Public Health (Lanzhou University, China). The rats were housed in a temperature-controlled room with cycles of 12:12 light/dark and provided with ad libitum water and standard rat chow. Animal care and experimental protocol coincided with the guidelines set by the Laboratory Animal Committee of Lanzhou University. All experiments were approved by the Animal Ethics Committee of the Animal Care Center (School of Public Health of Lanzhou University).

Sixty male SD rats were randomly divided into five groups (12 rats for each group), i.e. control group (corn oil), ACN group (ACN 46 mg^.kg⁻¹), ACN + AP1 group (ACN + AP 117 mg^.kg⁻¹), ACN + AP2 group (ACN + AP 234 mg^.kg⁻¹) and ACN + AP3 group (ACN + AP 351 mg^.kg⁻¹). ACN (Tianjin Kaixin Chemical Co., Ltd. China, 99% purity) and AP (Shaanxi Ciyuan Biotechnology Co., Ltd. China, 98% purity) were reconstituted in corn oil, respectively, to achieve the required dosages, which were based on our previous experiments [24]. AP was administered intragastrically 30 min before the treatment of ACN, and the animals were treated 6 days per week in 28 days. The control group was given the same volume of corn oil. On the day after the last administration, all rats were weighed and euthanized under the ether inhalation anesthesia for 1–2 min.

Measurement of body weight and organ coefficient

After being removed and suctioned out the blood immediately, the testes and epididymides of rats were weighed. The organ coefficient was calculated according to the following formula: organ coefficient (%) = organ wet weight/body weight × 100%.

Detection of sperm morphology and DNA fragmentation in rats

Six rats in each group were selected for detecting sperm morphology. The epididymis of rats incised with three transverse cuts were bathed in an ampoule containing 0.9% of saline at 37°C for 10 min, allowing sperms to swim out. Smears made using 15 μL of sperm suspension were air-dried, fixed in methanol and stained in 2% of solution of eosin for 1 h. Sperms displaying morphological abnormalities (e.g. head defects, neck and middle segment defects and main segment defects) were identified according to the laboratory manual of the WHO for the examination of human semen and sperm–cervical mucus interaction [25]. At least 1000 sperms were counted per smear to calculate the sperm deformity rate via dividing the number of sperms with abnormal morphology by the total number of sperms assessed.

The as-made smears were fixed in Carnoy’s solution for 12 h, stained with 0.01% of acridine orange (AO) for 5 min and then analyzed using a fluorescence microscope at the wavelength of 450–490 nm. At least 300 sperms were counted per smear, and the DFI was calculated by the division of the orange fluorescence to the total of orange and green fluorescence.

Pathological examination of testis

After being polyoxymethylene fixed and paraffin wax embedded, three testis samples from each group were sectioned at 5 μm and stained with hematoxylin–eosin (H & E) staining. Then, the pathological changes of rat testes were analyzed by a light microscope. The histological diagnoses were confirmed by two independent pathologists.

Measurement of activity of the marker enzyme in rat testis

The activities of alkaline phosphatase (AKP), acid phosphatase (ACP), succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH) were measured with a commercial enzyme linked immunosorbent assay kit following the manufacturer’s instructions (NanJing JianCheng Bioengineering Institute Co., Ltd, China).

Western blot assay

Testicular tissue of rats was lysed on ice with an RIPA lysis buffer and centrifuged at 12 000 g for 15 min at 4°C. Protein concentrations were measured using the BCA Protein Assay Kit (Thermo, USA). Equal amount of protein (60 μg) in each lane was separated by the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene-difluoride (PVDF) membrane. The membrane was blocked in a prepared Tris-buffered saline with Tween 20 (TBST) containing 5% of skim milk for 3 h at room temperature and incubated overnight at 4°C with primary antibodies against ASK1, p-ASK1, MKK7, p-MKK7, p38, p-p38, JNK, Cyt c, Caspase 3 (Cell Signaling Technology, USA); p- JNK, Caspase 9 (Abcam, USA); Bax, Bcl-2 and GAPDH (Signal Antibody, USA) in TBST. The PVDF membrane was rinsed five times with TBST for 5 min, and then processed with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. The protein bands were visualized with an ECL western blotting substrate (Beyotime Biotechnology, China), and images were acquired with a gel imaging system. Image J software (National Institutes of Health, USA) was used to analyze the densities of protein bands, and the relative expression of protein was calculated. GAPDH was used as a reference protein.

Quantitative real-time polymerase chain reaction

Total RNA of testis tissue was extracted with TRIzol reagent (Takara Bio Inc., Japan), and reverse transcription was performed using the Takara PrimeScript RT Master Mix Reagent Kit (Takara Bio Inc., Japan). Rat-specific premiers of ASK1, MKK7, p38, JNK, Cyt c, Caspase 9, Caspase 3, Bax, Bcl-2 were designed by Takara Bio Inc, as showed in Table 1. Quantitative RT-PCR was conducted using the SYBR Green PCR Kit and StepOnePlus real-time PCR system (Takara Bio Inc., Japan). β-actin was used as an internal reference.

Table 1.

Primer sequences of genes

Genes	Primers (5′-3′)	Sequences (5′-3′)
ASK1	Forward	5’-GCTCCTGGTACAGCCATTGAAGA-3’
	Reverse	5’-AGGACATCCACCAGCGTGTAATC-3’
MKK7	Forward	5’-CGCCTGGTCACATGGGCTTCT-3’
	Reverse	5’-CGACATCCACTTCGAGTGTCTCATA-3’
p38	Forward	5’-ATGGGTGCATGTGTGCATGA-3’
	Reverse	5’-CTACTGATGGCAGGAGCCTGTG-3’
JNK	Forward	5’-TCCCAGCTGACTCAGAACATAACAA-3’
	Reverse	5’-TGGACGCATCTATCACCAGCA-3’
Cyt c	Forward	5’-TGATCCTTTGTGGTGTTGACCAG-3’
	Reverse	5’-GACCATGGAGGTTTGGTCCAGT-3’
Caspase 9	Forward	5’-CTGAGCCAGATGCTGTCCCATA-3’
	Reverse	5’-GACACCATCCAAGGTCTCGATGTA-3’
Caspase 3	Forward	5’-GAGACAGACAGTGGAACTGACGATG-3’
	Reverse	5’-GGCGCAAAGTGACTGGATGA-3’
Bax	Forward	5’-CATTGACACCAAAGAGTACGC-3’
	Reverse	5’-TGTTGATGAATCTCAGCAGGA-3’
Bcl-2	Forward	5’-GACTGAGTACCTGAACCGGCATC-3’
	Reverse	5’-CTGAGCAGCGTCTTCAGAGACA-3’
β-actin	Forward	5’-GGAGATTACTGCCCTGGCTCCTA-3’
	Reverse	5’-GACTCATCGTACTCCTGCTTGCTG-3’

Detecting the apoptosis of rat testes

Three of rat testes were detected to evaluate the level of apoptosis using terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay (Wuhan Boster Biological Technology Co., Ltd. China). The number of brown particles (TUNEL-positive cells) in five seminiferous tubules was counted with a light microscope and averaged per slice.

Statistical analysis

Statistical analysis was performed using the SPSS 22.0 (IBM, USA). Data were presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was applied for the multiple comparison. P < 0.05 was considered statistically significant.

Results

Effect of AP on the general situation and organ coefficient in rats

During the experiment, only the rats in the ACN group had symptoms of salivation, restlessness and irritability in the second week, which were not observed in other groups. There was no significant difference in the wet weight and organ coefficient of testis among the five groups (P > 0.05) (Fig. 1A). The wet weight and organ coefficient of epididymis in the ACN group were significantly lower in comparison with the control group (P < 0.05), and the organ coefficient of epididymis in the ACN + AP1 and ACN + AP2 group were significantly higher than that in the ACN group (P < 0.05) (Fig. 1B).

Figure 1.

Effect of AP on wet weight and organ coefficient of testis and epididymis in rats treated with ACN. ^*P < 0.05 vs. control group; ^#P < 0.05 vs. ACN group.

Effect of AP on sperm morphology and fragmentation in rats

Effect of AP on sperm morphology

The head deformity rate and tail deformity rate of sperm in the ACN group increased (P < 0.05), while those deformity rates of sperm were obviously decreased in the ACN + AP1 and ACN + AP2 group (P < 0.05), as shown in Fig. 2 and Table 2.

Figure 2.

Effect of AP on sperm morphology after the treatment of ACN (n = 6). Arrows indicated teratospermia. A. control group, B. ACN group, C. ACN + AP1 group, D. ACN + AP2 group, E. ACN + AP3 group. Magnification × 100.

Table 2.

Effect of AP on sperm deformity rate in rats exposed to ACN

Groups	Sperm count	Number of sperm	Head deformity	Tail deformity
		deformity n (%)	n (%)	n (%)
Control	6022	272 (4.52%)	221 (3.67%)	51 (0.85%)
ACN	5022	384 (7.65%) *	312 (6.21%) *	72 (1.43%) *
ACN + AP1	5017	301 (6.00%) #	235 (4.68%) #	66 (1.32%)
ACN + AP2	6014	309 (5.14%) #	213 (3.54%) #	96 (1.60%)
ACN + AP3	6012	432 (7.19%)	314 (5.22%)	118 (1.96%)
χ2		69.933	62.233	28.18
P		0.000	0.000	0.000

^* P < 0.05 vs. control group.

^# P < 0.05 vs. ACN group.

Effect of AP on sperm fragmentation induced by ACN

The DFI of spermatozoa in the ACN group was higher than that in the control group (P < 0.05), which could be improved by the addition of AP, as shown in Fig. 3 and Table 3.

Figure 3.

Effect of AP on sperm fragmentation after the treatment of ACN (n = 6). Green and red fluorescence represented the normal and DNA-damaged sperm respectively. A. control group, B. ACN group, C. ACN + AP1 group, D. ACN + AP2 group, E. ACN + AP3 group. Magnification × 100.

Table 3.

Effect of AP on the sperm DFI in rats exposed to ACN

Groups	Sperm count	DFI (%)	χ2	P
Control	1800	45 (2.50%)	784.452	0.000
ACN	1500	589 (39.27%) *
ACN + AP1	1800	371 (20.60%) #
ACN + AP2	1800	235 (13.06%) #
ACN + AP3	1800	429 (23.83%) #

^* P < 0.05 vs. control group.

^# P < 0.05 vs. ACN group.

Effect of AP on testicular injury induced by ACN

Morphological changes of testicular tissue

The pathological sections of testis were observed using an optical microscope. The spermatogenic tubules in the control group were intact and arranged closely. Spermatogenic cells in different stages were arranged neatly and clearly, and displayed as spermatogonia, primary spermatocytes, secondary spermatocytes and spermatocytes from the basement membrane to lumen sequentially. In the lumen, mature spermatozoa were visible, and Leydig cells were distributed in groups between seminiferous tubules (Fig. 4A and a). In the ACN group, the seminiferous tubules atrophied and deformed, and the diameter of these tubules diminished. The layers of spermatogenic cells decreased significantly with a decline of the numbers of primary spermatocytes, secondary spermatocytes, mature spermatozoa and Leydig cells (Fig. 4B and b). In the ACN + AP1 group, spermatogenic tubules were partially damaged with a decline of the layers of spermatogenic cells and the number of mature spermatozoa (Fig. 4C and c). In the ACN + AP2 group, the walls of spermatogenic tubules were relatively intact and arranged in a regular manner. Only a few layers of spermatogenic cell reduced, and both spermatogenic cells and mature sperm were found in the lumen (Fig. 4D and d). In the ACN + AP3 group, the seminiferous tubules atrophied with narrowing diameters. The layers and number of spermatogenic cells, and the number of mature spermatozoa and Leydig cells all decreased (Fig. 4E and e).

Figure 4.

Effect of AP on the pathology of rat testes detected using H & E staining (n = 3). A, a. control group, B, b. ACN group, C, c. ACN + AP1 group, D, d. ACN + AP2 group, E, e. ACN + AP3 group. A ~ E. Magnification × 100. a ~ e. Magnification × 400.

Marker enzymes in rat testes

The activity of AKP and SDH in rat testes decreased after the treatment of ACN (P < 0.05) (Fig. 5A and C), while AP increased the activity of AKP (P < 0.05). The slight increase of LDH was weakened by AP (Fig. 5D). However, there was no statistical difference in the activity of ACP for these groups (Fig. 5B).

Figure 5.

Effect of AP on the marker enzymes in rat testes after the treatment of ACN. ^*P < 0.05 vs. control group; ^#P < 0.05 vs. ACN group.

Effect of AP on oxidative stress indicators in testes

The activities of SOD and CAT in testes that were suppressed by the addition of ACN, AP could restore the activity mildly. However, the change did not show statistical significance (Fig. 6A and B). Compared to the control group, the activity of GSH-Px and the level of T-AOC in testes of the ACN group decreased significantly (P < 0.05) (Fig. 6C and D). Compared to the ACN group, the level of T-AOC and content of GSH of testes in the ACN + AP1 group significantly increased (Fig. 6D and E), while the activity of GSH-Px in the ACN + AP3 group significantly decreased (P < 0.05) (Fig. 6C). No statistical difference was found in the content of MDA (Fig. 6F), as shown in Fig. 6.

Figure 6.

Effect of AP on oxidative stress indicators in the rat testis (n = 6) ^*P < 0.05 vs. control group; ^#P < 0.05 vs. ACN group.

Effect of AP on the ASK1-JNK/p38 signaling pathway

The protein expressions of ASK1, p-ASK1, MKK7, p-MKK7, JNK, p-JNK, p38 and p-p38 were detected to figure out if AP was involved in the ASK1-JNK/p38 signaling pathway. Results showed that the protein expressions of ASK1, p-ASK1, MKK7, p-MKK7, JNK, p-JNK, p38 and p-p38 protein in rat testes increased after the treatment of ACN, implying the activation of ASK1-JNK/p38 signaling pathway. In the ACN + AP1 and ACN + AP2 groups, the protein expressions of ASK1, p-ASK1, MKK7, p-MKK7, JNK, p-JNK, p38 and p-p38 significantly decreased (Fig. 7B and D). The mRNA expression of these factors was consistent with the protein (Fig. 7E and F). The ratios of p-JNK/JNK, p-p38/p38, p-ASK1/ASK1 increased, which revealed that the ASK1-JNK/p38 signaling pathway was activated by the addition of ACN, while suppressed by the addition of AP (Fig. 8A and B).

Figure 7.

Effect of AP on the protein and mRNA expression of the ASK1-JNK/ p38 pathway in rat testes after the treatment of ACN (n = 6). ^*P < 0.05 vs. control group; ^#P < 0.05 vs. ACN group.

Figure 8.

Effect of AP on the p-ASK1/ASK1, p-MKK7/MKK7, p-JNK/JNK and p-p38/p38 in the protein expression in rat testes after the treatment of ACN (n = 6). ^*P < 0.05 vs. control group; ^#P < 0.05 vs. ACN group.

Effect of AP on testicular apoptosis

Testicular apoptosis was displayed in the Fig. 9. Compared to the control group, the number of TUNEL-positive cells in the ACN group multiplied, which could be notably reduced by the pretreatment with 117 and 234 mg^.kg⁻¹ of AP (P < 0.05) (Fig. 9F).

Figure 9.

Effect of AP on the testicular apoptosis after the treatment of ACN (n = 3). A. control group, B. ACN group, C. ACN + AP1 group, D. ACN + AP2 group, E. ACN + AP3 group, and F. comparison of TUNEL-positive cells in each seminiferous tubule. Arrows pointed at TUNEL-positive cells. Magnification × 400. ^*P < 0.05 vs. control group; ^#P < 0.05 vs. ACN group.

Effect of AP on the protein and mRNA expression of apoptosis-related factors

Eventually, the examined expression of some apoptosis-related factors (Fig. 10A) showed that Cyt c, Caspase 9, Caspase 3 and Bax increased, while Bcl-2 and Bcl-2/Bax decreased after the treatment of ACN, which could be restored by the addition of AP dramatically (Fig. 10B and C). The mRNA expression was consistent with proteins partially (Fig. 10D and E).

Figure 10.

Effect of AP on the protein and mRNA expression of apoptosis-related factors in rat testes after the treatment of ACN (n = 6). ^*P < 0.05 vs. control group; ^#P < 0.05 vs. ACN group.

Discussion

In the animal experiment, ACN caused injury to several organs in rats, i.e. liver, brain and testis [26], which is also a multisite carcinogen to mice [27]. We focused on its toxic effect on the male reproduction system and intended to look for a protective agent against its toxicity. It was shown that wet weight and organ coefficient of epididymis reduced after the treatment of ACN in comparison to the control group. The pretreatment of AP of 234 mg kg⁻¹ played a protective role. Commonly, changes of organ wet weight and organ coefficient indicate a pathological injury. The current results indicated that AP conferred a protection on epididymis, which contributed to the sperm maturation in male rats.

Among ACN-exposed workers, ACN impaired the sperm quality by inducing the DNA strand breakage and sex chromosome aneuploidy in spermatozoa [28]. Animal studies revealed that ACN significantly increased the sperm deformity rate in male mice, which mainly were the head deformity [29, 30]. Pathological studies showed that ACN induced the degenerative lesions of spermatogenic tubules and lowered the number of sperm and spermatogenic cells [31]. Our results suggested that sperm deformity rate (mainly as a head deformity) and sperm DFI both increased after the treatment of ACN but decreased by the pretreatment of 117 and 234 mg^.kg⁻¹ of AP, which were in agreement with those reports in literatures, i.e. AP improved the sperm damage induced by various chemicals in rodent [32, 33]. The treatment with the addition of AP exerted an antioxidant effect by the oral perfusion at 234 and 468 mg kg⁻¹ of AP for 35 days, while 936 mg kg⁻¹ of AP promoted the oxidation in rat testes [34]. It is noteworthy that 351 mg^.kg⁻¹ of AP exerted a lower protection on the sperm deformity in this experiment. The difference may be ascribed to the combined effect of ACN and AP to increase the sensibility of rats. And a pro-oxidant effect of AP may account for the sperm changes in rats receiving the highest dose [35], since the same sperm changes in the ACN + AP3 group were observed as in the ACN group. Namely, a certain dose of AP protected against the sperm deformity and DNA damage induced by the addition of ACN.

AKP, ACP, SDH and LDH, as testicular marker enzymes, are identified as functional indicators of spermatogenesis [36]. AKP mainly involves in the division of spermatogenic cells and nutrients transport to spermatogenic cells [37]. ACP, termed as an indicator of dyszoospermia, is related to the phagocytosis of Sertoli cells, which plays a vital role in maintaining the normal metabolism and physiological function of spermatogenic cells [38]. SDH involves in the energy metabolism of sperm by converting sorbitol to fructose [39]. LDH is associated with the glucose metabolism and maturation of spermatogenic cells and spermatozoa [38, 40]. The present data suggested that the addition of ACN suppressed the activities of AKP and SDH, indicating that ACN interfered with the testicular nutrient transport and energy metabolism, inducing serious damage to testes. Nevertheless, the pretreatment of AP improved the activity of AKP and reduced the activity of LDH, which revealed that AP was beneficial for the testicular energy metabolism and maturation of spermatogenic epithelium. The activity of ACP showed no statistical difference in each group, which meant that the addition of ACN did not cause spermatogenic disorders yet. In brief, AP improved the testicular marker enzymes observed in the ACN-induced testis damage to a certain extent, and this effect was verified by histopathological changes.

The exposure to ACN and its metabolism can both initiate the oxidative stress [6]. The male reproduction system is extremely sensitive to the oxidative damage because it is rich in unsaturated fatty acids. Therefore, it is generally believed that the treatment of ACN causes the testis injury in rats. AP, a potent antioxidant and a free radical scavenger [41], can reinforce the cellular antioxidant defense system [42]. As previously reported [43], AP obviously reduced the MDA content, increased the SOD activity and the T-AOC level in hyperuricemia rats. In the present study, we detected the activities of SOD, CAT and GSH-Px, levels of T-AOC, GSH and MDA, respectively. The results showed that the addition of ACN inhibited the activity of GSH-Px and reduced the level of T-AOC notably, which indirectly verified that the antioxidant ability in testes was impaired and the oxidative stress occurred. Whereas AP restored the T-AOC level, increased the GSH content to an even higher level than the normal, which was in line with the study that AP has a beneficial effect against the ACN-induced oxidative stress in brain [44]. The inconspicuous reduction of CAT, SOD and MDA by the addition of ACN may be associated with the treatment time. The results denoted that AP is beneficial in alleviating the oxidative stress caused by ACN, mainly by acting on a series of reactions referring to the antioxidant ability.

Oxidative stress can activate numerous intracellular signaling pathways, such as mitogen-activated protein kinases (MAPKs) [45], which have crossovers and feedbacks with other pathways [46]. Apoptosis signal-regulating kinase 1 (ASK1), as a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, is activated in response to the oxidative stress and transmits signals to MAPKs such as JNK and p38 [47]. ASK1 at Thr⁸⁴⁵ phosphorylated in response to the oxidative stress activates and recruits MKK4/7 and MKK3/6, which are necessary for the activation of JNK and p38 [48–50]. Previous reports confirmed that the JNK/p38 pathway was actively involved in the apoptosis of testicular germ cells induced by various stimuli [51, 52], which was also activated in the testicular tissue of testicular torsion and blood–testis barrier dysfunction [53, 54]. These evidences prove that the JNK/p38 pathway appears closely related to the testis damage. In this experiment, the expression levels of ASK1, p-ASK1, MKK7, p-MKK7, JNK, p-JNK, p38 and p-p38 increased markedly after the treatment of ACN, so did ratios of p-JNK/JNK and p-p38/p38. Whereas the pretreatment with AP inhibited the activation of the ASK1-JNK/p38 signaling pathway induced by the addition of ACN.

Both p38 MAPK and JNK, belonging to members of the MAPK family, involves in the regulation of cell survival and apoptosis [55]. JNK regulates apoptosis through two mechanisms [56]: on the one hand, it phosphorylates c-Jun, activates transcription factor-2 (ATF-2), leading to the activation of AP-1, and increases the expression of Fas/FasL signaling pathway-related proteins, which contribute to the apoptosis [57, 58]. On the other hand, JNK activates Bax through Bid-Bax-dependent mechanism by translocating cytochrome c (Cyt c) from the inner mitochondrial membrane into the cytosol. Then Cyt c combines with the intracellular apoptotic protease activating factor-1 (Apaf-1) and Caspase 9 precursor to become an active complex, namely apoptosome. In this process, Caspase 9 self-activates, which in turn triggers the apoptosis cascade mediated by the apoptosis-executing factor Caspase 3 [59, 60]. Besides, JNK also phosphorylates mitochondrial proteins especially Bcl-2 and Bcl-xl, inhibits their anti-apoptotic activity and induces apoptosis [60–62]. Upon the activation, p38 phosphorylates a variety of substrates and affects the activity of transcription factors such as c-Jun, p53 and ATF-2, thereby inducing the germ cell apoptosis by causing abnormal changes in the cell cycle, cell proliferation and differentiation or the reduction of cytokine synthesis [63, 64]. ACN induced the spermatogenic cell apoptosis in male mice, and the incidence of apoptosis increased with the increasing dosage and time [65]. The relative mRNA expression of Bax and Caspase 3 in rat testes increased after the exposure to 50 mg kg⁻¹ of ACN for 13 weeks, indicating that the addition of ACN affected the expression of Bcl-2 family and promoted the apoptosis [10]. In another research, AP restored the ischemic heart by inhibiting p38 MAPK, upregulating the Bcl-2 and down-regulating Bax, suppressing the activity of Caspase 3 and apoptosis of cardiomyocytes [66]. Similarly, we found that the addition of ACN up-regulated the expression of Cyt c, Bax, Caspase 9 and Caspase 3, decreased Bcl-2 and the ratio of Bcl-2/Bax significantly. Nevertheless, the expression of Bax, Caspase 9 and Caspase 3 reduced, Bcl-2 and the ratio of Bcl-2/Bax increased markedly in all groups pretreated with AP. Thus, AP inhibited the ACN-induced apoptosis in rat testis, which was also confirmed by TUNEL assay that number of TUNEL-positive cells in the ACN group multiplied significantly but reduced notably in the ACN + AP1 and ACN + AP2 groups.

Conclusion

In the present study, we found that AP exhibits the potential to protect against the ACN-induced sperm injury and pathological damage of testes by alleviating the oxidative stress, ASK1 activation, JNK and p38 phosphorylation and subsequently the mitochondria-mediated apoptosis, showing characteristics of antioxidant and anti-apoptosis.

Conflict of interest

None declared.