Sulforaphane attenuates irradiation induced testis injury in mice

ABSTRACT

Objectives

The testis is vulnerable to ionizing radiation, sexual dysfunction and male infertility are common problems after local radiation or whole-body exposure. Currently, there are no approved drugs for the prevention or treatment of radiation testicular injury. Sulforaphane (SFN) is an indirect antioxidant that induces phase II detoxification enzymes and antioxidant genes. Herein, we investigated the radiation protective effect of SFN on testicular injury in mice and its potential mechanism.

Methods

In this study, we investigated the protective effect of SFN on radiation-induced testicular injury in mice and its potential mechanism by screen the testicular histoarchitecture, oxidative stress, cell apoptosis and SFN related Nrf2 and its downstream antioxidant genes.

Results

The results show that the harmful effects of ionizing radiation on testes manifest as damage to histoarchitecture, increased oxidative stress and cell apoptosis, thereby impairs male fertility. SFN can resist reproductive toxicity caused by ionizing radiation in vivo by reducing the level of oxidative stress by activating Nrf2 and its downstream antioxidant genes. Besides, SFN also can inhibit the NFκB activity and effectively attenuated the testicular microstructure damaged by ionizing radiation.

Conclusions

Our study reveals SFN can be considered an outstanding candidate for potential clinical applications in the prevention of irradiation-associated testicular injury.

KEYWORDS:

• Sulforaphane
• Ionizing radiation
• Testis injury
• Nrf2
• NFκB
• spermatogenesis
• SSCs
• ROS

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Correction

1. Introduction

With the wide application of ionizing radiation in industry and clinical treatment, radiation injury has become a more common injury. Testis is the primary sexual organ of male and is very sensitive to ionizing radiation [1]. Radiation exposure can cause testis injury, a reduction in sperm count and quality may lead to temporary or even permanent infertility [2]. In addition, it has been reported that male patients often suffer from sexual dysfunction, oligozoospermia, spermatozoosis and infertility during ionizing radiation treatment [3].

Testicular radiation injury is the major factor in reducing fertility in cancer patients [4]. The molecular pathways that control spermatogenesis under radiation and the mechanisms of DNA damage response and homeostasis in spermatogonal stem cells remain unclear [5]. Spermatogonial stem cells support the differentiation of normal germ cell and must be preserved throughout life [6]. However, the recovery of spermatogenesis may be impaired due to the reduction of spermatogonial stem cells after radiation [7]. Radiation dose begin to adversely affect spermatogenesis at 0.1-1.2 Gy, and irreversible damage occur at 4 Gy [8].

Recent studies have shown that ionizing radiation stimulates oxidative stress and generates reactive oxygen species (ROS), such as hydroxyl radicals, hydrogen peroxide, and superoxide anions which can cause a direct damage [9]. Indirectly, Radiation splits water molecules and cause greater damage to biological molecules because of the products of radiation decomposition are highly reactive [10]. Oxidative stress occurs when the production of ROS exceeds the capacity of the cell to defend itself against oxidation. The use of chemical compounds in radiation therapy is an obvious strategy for improving therapeutic outcomes [11]. However, due to their inherent toxicity and high cost, most studied synthetic radioactive protective compounds are underutilized clinically [12]. Therefore, for radiation testicular injury, there is an urgent need for highly effective radiation protective agents with fewer side effects.

In the current study, a naturally-occurring isothiocyanate compound, sulforaphane (SFN), which found in cruciferous vegetables [13] was used to investigate whether it could protect mice from ionizing radiation-induced testicular damage. Our results demonstrate the radioprotective effect of SFN on radioactive testicular injury. Specifically, SFN reduces DNA damage accumulation and germ cells apoptosis, facilitating spermatogenesis recovery postiradiation by preserving the SSCs pool.

2. Materials and methods

2.1. Animals and experimental design

C57BL/6J male mice, 7–8 weeks old, were purchased and raised at Soochow University Laboratory Animal Center. An ambient temperature of 18–24°C and a continuous 12 hours light/dark cycle were maintained. Each mouse was given free access to a standard laboratory diet and water. 48 mice aged 7–8 weeks were randomly divided into six groups: blank control (Ctrl, n = 12), 5mg/kg SFN pretreatment (S1, n =12), 10mg/kg SFN pretreatment (S2, n =12), radiation + no pretreatment (IR, n = 12), radiation +5 mg/kg SFN pretreatment (IRS1, n = 12), radiation +10 mg/kg SFN pretreatment (IRS2, n = 12). In all SFN groups, the mice were intraperitoneally injected with a SFN solution (MedChemExpress, USA) once a day starting 72 hours before radiation until they were sacrificed. Mice in the control group and the radiation + no pretreatment group were given intraperitoneal injections of an equal volume of solvent for dissolving SFN (PBS with a final concentration of 0.1% DMSO) at the same time until they were sacrificed. Mice in the radiation group and the radiation + SFN groups were subjected to 6 Mev-ray radiation to the lower abdominal testis area (total dose 2Gy). Twenty-four hours after radiation, six mice were randomly sacrificed in each group. Seventy-two hours after radiation, the remaining mice were sacrificed. All animal experiments were approved by the Ethics Committee of Soochow University and conducted in accordance with its guidelines.

2.2. Computer-assisted sperm analysis (CASA) and testis testosterone level detection

The cauda epididymidis on one side of the mouse was carefully removed, dissected and incubated in 1 mL buffer containing 75 mM NaCl, 24 mM EDTA, and 0.4% FBS for 5 min at 37 °C in a water bath to allow sperm release. Gently blow and mix well, rinse 3 μL sperm suspension into the matching counting plate, place it in computer-assisted sperm analysis (Beijing Weili New Century Technology, China), and complete the automatic detection. Testosterone was measured by electro chemiluminescence immunoassay (Roche Science, Germany) from testis samples of mice.

2.3. Oxidative stress biomarker assay

MDA assay kit obtained from ABclonal Technology (China) was selected to determine testes MDA content. Assay was conducted according to the manufacturer’s instructions.

2.4. Histomorphological evaluation of the testis

Six testes from each group were removed and fixed in 4% paraformaldehyde, and then embedded in paraffin, sectioned at a thickness of 4μm prior to being stained with hematoxylin and eosin (H&E). The stained sections were studied by light microscopy to evaluate spermatogenesis and histopathological studies. Spermatogenesis was investigated with Johnsen's scoring system [14]. The germinal epithelium of at least 25–30 tubules for each testis was assessed and the mean Johnsen's score per mouse was calculated [15,16]. For quantitative evaluation, five random seminiferous tubules from each testis were selected and the thickness of the epithelium (from the basement membrane to lumen) was measured by Image-Pro Plus software [17]. Apoptosis was analyzed by TUNEL assay kit (BOSTER Biological Technology, China) according to the manufacturer’s instructions.

2.5. RNA extraction and reverse transcription-polymerase chain reaction (RT–PCR) analysis

Total testicle RNA was extracted (Vazyme, China), and then reverse-transcribed to cDNA with HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, China). Real-time PCR was performed on a Sequence Detection System (ABI StepOnePlus, Thermo Fisher Scientific, USA) using the Taq Pro Universal SYBR qPCR Master Mix (Vazyme, China), according to the manufacturer’s instructions. The primer sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and were as follows:

PLZF forward 5′-CGTTGGGGGTCAGCTAGAA-3′,

reverse 5′-GCATGGGGCTCTCTTTCCTT-3′;

CCNA1 forward 5′-GTGGGTTCCCAAGCAGCA-3′,

reverse 5′-CCCTAGCACGGTTCTCTGTG-3′;

Nrf2 forward 5′-GCCCTCAGCATGATGGACTT-3′,

reverse5′-AACTTGTACCGCCTCGTCTG-3′;

DMRT1 forward 5′- CGAGAGCTCGAAGGGAGTTC-3′,

reverse 5′-CACCCTGTGACCAGAGTAGC-3;

HO-1 forward 5′-GTCAAGCACAGGGTGACAG-3′,

reverse5′-CTAGCAGGCCTCTGACGAAG-3′;

SOD1 forward 5′-GGGAAGCATGGCGATGAAAG-3′,

reverse5′-CCCCATACTGATGGACGTGG-3′;

OVOL2 forward 5′-CTGCGAGTAACCTGGAGTGA-3′,

reverse5′-GGCATGGTGGGATCCTCTC-3′;

NQO1 forward 5′-CATTGCAGTGGTTTGGGGT-3′,

reverse5′-TCTGGAAAGGACCGTTGTC-3′;

SYCP2 forward 5′-AAGGAGCTGACTTTCCTTGG-3′,

reverse5′-GCAGAGCCTTTTCCTCTTTCA-3;

Stra8 forward 5′-TTATAATGGCCACCCCTGGAGA-3′,

reverse5′-CCTATTCAGTACCTGCCACAG-3′;

SPO11 forward 5′-GCAACCAAGAGGAGCAATG-3′,

reverse5′-TCCTGGGCACTTTCAGCATA-3′;

Prm1 forward 5′-AGCATCTCGCCACATCTTGA-3′,

reverse5′-TGACAGGTGGCATTGTTCCT-3′.

Actin forward 5′-TGAGCTGCGTTTTACACCCT-3′,

reverse5′-GCCTTCACCGTTCCAGTTT-3′.

Actin served as an internal control for every sample.

2.6. Immunohistochemical analysis

After deparaffinization, the sections were retrieved by antigen retrieval solution for 60 min at 70°C. All sections were blocked with 10% bovine serum albumin (BSA) and then incubated with anti-p-Nrf2 antibody (Abclonal, 1:100) at 4°C overnight. Slides were washed in PBS and incubated with secondary antibodies for 30 min, followed by an additional wash. They were then treated with DAB reagent (Gene Tech, China). After washing the samples with PBS, counterstaining was performed using hematoxylin, and further washing was performed using tap water. Sections were dehydrated in graded alcohol and covered with a cover glass. The number of stained cells was determined in four fields per slide under a microscope.

2.7. Cell culture

The mouse spermatogonia cell line (GC-1spg) was purchased from ATCC (USA) and cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco, China) supplemented with 10% fetal bovine serum (Cegrogen Biotech, Germany) and 1% penicillin-streptomycin (Gibco, China) and incubated in a humidified incubator at 37°C under 5% CO₂.

2.8. Measurement of ROS generation

According to the manufacturer’s instructions, the ROS generation was examined by a ROS Assay Kit (Beyotime, China) which can be detected with a fluorescent enzyme reader. Sulforaphane was dissolved in dimethyl sulphoxide (DMSO) to stock solutions of 100 mM and stored at −80 °C. Working concentrations were freshly prepared in culture medium. The final DMSO concentration was kept at 0.1% for all samples, including controls. After four hours of incubation, the sulforaphane containing medium was removed and replaced by fresh growth medium. the treatment procedure was repeated for three consecutive days. Then, Cells were exposed to ionizing radiation using RS2000 Pro (Rad Source, USA) at a dose rate of 1.225 Gy/min (total 2Gy). After 3 hours, GC-1 cells were incubated with 10 mM DCFH-DA at 37°C for 20 min. Fluorescence was determined with a 48 point well scan on a Synergy2 (BioTek, USA).

2.9. Western blotting

Total testicular protein was extracted and quantified，The proteins (40μg) were separated by sodium dodecyl sulfate (SDS)-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane, which was then blocked in 5% non-fat milk for 1 h. TBST diluted primary antibodies (1:1000) against Nrf2, SOD1, Histone H3, and HO-1 (Proteintech, China); c-Kit, Stra8, 4-hydroxynonenal (4-HNE), NQO1 (1:1000 - 1:3000, Abcam, China); Cleaved-Caspase3, β-actin and γH2AX (1:1000, Cell Signaling Technology, USA); 3-nitrotyrosine (3-NT), PLZF, Phospho-Nrf2-S40 (1:1000, Abclonal, China), were incubated with the membrane at 4^◦C overnight and the membrane was washed three times with TBST. Subsequently treated with appropriated second antibody (1:1000, Beyotime, China) for 1 h at room temperature, and the membrane was washed three times with TBST. Then visualized using an enhanced chemiluminescence detection kit. The results were analyzed by FluorChem M (Alpha, United States).

2.10. Statistical analysis

GraphPad Prism 8.0 (San Diego, United States) was used for statistical analysis. Data were presented as the means ± standard deviation (SD). Comparisons between groups were performed using an unpaired two-tailed Student’s t-test. Asterisks represent the p values as follows:∗ p < 0:05, ∗∗ p < 0:01, ∗∗∗ p < 0:001, p < 0:05 was considered statistically significant.

3. Results

3.1 SFN relieved ionizing radiation -induced seminiferous epithelium injury

As shown in Figure 1A, no significant histopathological changes were observed in Ctrl group. Compared with the control group, spermatogenic tubules atrophied, spermatogenic epithelium thinned with a great reduction of spermatogenic cells in IR group, epithelial thickness and Johnsen’s score were also decreased significantly (p < 0.001), as shown in Figure 1B,C. However, the spermatogenic tubules were slightly degenerated and spermatogenic cells were slightly absent in IRS1 and IRS2 groups. Meanwhile, the Johnsen’s score and epithelial thickness of IRS1 and IRS2 groups were higher than those of IR group (p < 0.001). More precisely, 5 mg/kg SFN exhibited a better ability to cope with irradiation-induced testicular injury than 10 mg/kg SFN. As shown in Figure 1D, 24 hours after radiation, there is no statistically significant difference was found in the testis testosterone level of the mice in each group (p > 0.05). 72 hours after radiation, there is also no statistically significant difference was found in the testis testosterone level of the mice in each group (p > 0.05).

Figure 1. SFN relieved ionizing radiation -induced seminiferous epithelium injury. (A) Hematoxylin and eosin staining of testis samples on the indicated day after ionizing radiation (bar = 50μm). Red star shows the atrophy of seminiferous tubules. Yellow star shows the thickness of epithelium ([). blank control (Ctrl), 5mg/kg SFN pretreatment (S1), 10mg/kg SFN pretreatment (S2), radiation + no pretreatment (IR), radiation+5mg/kg SFN pretreatment (IRS1) radiation+10mg/kg SFN pretreatment (IRS2). (B) Bar diagram showing the epithelial thickness of the seminiferous tubules. (C) Bar diagram showing the Johnsen's score of the testis. (D) Bar diagram showing the level of testis testosterone. n = 6 at least in each group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs Ctrl; # p < 0.05, ## p < 0.01, ### p < 0.001 vs IR.

3.2 Effects of SFN on apoptosis and DNA damage of testicular germ cells after ionizing radiation.

Since ionizing radiation can induce testicular cell apoptosis, we used TUNEL apoptosis detection kit to detect the apoptosis level of mouse testicular cells. As shown in Figure 2A, compared with the IR group, the number of TUNEL-positive cells in the IRS1 and IRS2 groups decreased (p < 0.01). Furthermore, we measured the levels of apoptosis-related proteins. We observed that the expression of pro-apoptotic protein cleaved-caspase3 and DNA double strand breaks marker γH2AX was markedly upregulated in the IR group, while these effects were reversed by SFN treatment (Figure 2C). Overall, these results indicated that SFN affords protection against irradiation-induced apoptosis.

Figure 2. Effects of SFN on apoptosis and DNA damage of testicular germ cells after ionizing radiation. (A) The apoptosis cells were determined by TUNEL staining. Bar =50μm. →Indicates TUNEL-positive cells. (B) The TUNEL-positive cells frequency in mice testes. (C) Relative protein levels of γH2AX and Cleaved-caspase3 detected by Western blot, n = 3 for each group. * p < 0.05, ** p < 0.01, *** p < 0.001vs Ctrl; # p < 0.05, ## p < 0.01, ### p < 0.001vs IR.

3.3 Effects of SFN on spermatogenic function after ionizing radiation

To evaluate the effect of SFN on improving spermatogenic function, we performed computer-assisted sperm analysis. The results showed that SFN significantly improved sperm concentration and total sperm motility in IRS1 and IRS2 mice (Figure 3A,B). However, 10mg/kg SFN treatments seems obviously toxic to sperm. Next, we sought to determine the expression patterns of candidate genes that SFN administration might protect. IRS1 and IRS2 were included in the gene expression analysis to determine the mechanism. We found that 5 mg/kg SFN improved some gene expression better than 10 mg/kg SFN after ionizing radiation.

Our results showed that SFN significantly increased levels of SSC self-renewal genes (PlZF, DMRT1) and SSC differentiation genes (including Stra8, SYCP2, SPO11, OVOL2, and CCNA1) compared with IR group (P < 0.05) (Figure 3). Expressions of PLZF, DMRT1, SYCP2, SPO11, and CCNA1 more increased with the 5 mg/kg SFN treatment compared to the 10 mg/kg SFN treatment after ionizing radiation . The results of PLZF and Stra8 were further verified by western blot. The levels of spermatogenic cell marker was significantly increased in the IRS1 and IRS2 groups compared with IR group. We also evaluated Prm1 expression following SFN administration (Figure 3J). The result showed that Prm1 expression remarkably increased with SFN treatment in IRS1 and IRS2.

Figure 3. Effects of SFN on spermatogenic function after ionizing radiation. (A) Sperm concentration. (B) Total sperm motility (PR+NP). (C) Expression of PLZF mRNA and protein in testis. (D) Expression of Stra8 mRNA and protein in testis. (E) Expression of DMRT1 mRNA in testis tissue. (F) Expression of SPO11 mRNA in testis. (G) Expression of CCNA1 mRNA in testis. (H) Expression of SYCP2 mRNA in testis. (I) Expression of OVOL2 mRNA in testis. (J) Expression of Prm1 mRNA in testis. (K) Relative protein level of c-kit detected by Western blot, n = 3 in each group. * p < 0.05, ** p < 0.01, *** p < 0.001vs Ctrl; # p < 0.05, ## p < 0.01, ### p < 0.001vs IR.

3.4 SFN inhibits ionizing radiation-induced testicular oxidative stress

To investigate the generation of reactive oxygen species in cells, we evaluated the production of ROS. Compared with the control group, ROS production was significantly increased after ionizing radiation (P < 0.05). SFN intervention reduced irradiation-induced ROS levels compared to levels in the IR group (P < 0.05) (Figure 4A). MDA is the main product of lipid peroxidation and is produced by unsaturated fatty acids, free amino acids and DNA exposed to ionizing radiation [18]. Ionizing radiation rapidly increased MDA levels in the testes, but SFN administration showed a decrease in MDA levels (Figure 4B). Western blotting was performed to detect the accumulation of oxidative stress markers 3-nitrotyrosine (3-NT) [19] and 4-hydroxy-2-nonenal (4-HNE) [20] in the testis. The results showed that ionizing radiation significantly increased the expression of 3-NT and 4-HNE in testis compared with Ctrl group, while irradiation-induced expression of 3-NT and 4-HNE was significantly decreased with SFN treatment (Figure 4C,D).

Figure 4. SFN inhibits ionizing radiation-induced testicular oxidative stress. (A) DCFH-DA fluorescence probe was used to detect ROS in GC-1 cells. (B) MDA level in the testis. (C), (D) Western blotting was used to detect the expression of oxidative stress indicator, 3-NT and 4-HNE. n = 3 in each group. * p < 0.05, ** p < 0.01, *** p < 0.001vs Ctrl; # p < 0.05, ## p < 0.01, ### p < 0.001vs IR

3.5. SFN upregulated Nrf2 expression and restricted NFκB activation in the testis after ionizing radiation

These above results suggest that SFN can prevent irradiation-induced testicular injury. Therefore, we first examined whether SFN prevents this irradiation-induced effect by activating Nrf2 by measuring Nrf2 expression and its transcription in testis. Compared with the Ctrl group,ionizing radiation treatment significantly decreased the expression of Nrf2 at the mRNA level (Figure 5A) in the testis at both 1d and 3d. The expression of Nrf2 at the protein level (Figure 5B) decreased at 1d with ionizing radiation treatment but increased at 3d. However, Nrf2 expression in the testis was significantly increased in both 1d and 3d in the IRS1 and IRS2 groups compared to the IR group.

Figure 5. SFN upregulated Nrf2 expression and restricted NFκB activation in the testis after ionizing radiation. (A) Nrf2 expression at mRNA level was detected by real-time PCR. (B) Nrf2 expression at protein level was detected by Western blot. (C) The activation of Nrf2 was examined by immunohistochemical staining (bar = 50μm). →Indicates p-Nrf2 positive cells. (D) Western blot for Nrf2 phosphorylation at Ser40 (p-Nrf2), Keap1, p65, p-p65. (E), (F) Nrf2 function was measured by determining the expression of Nrf2 downstream genes, SOD1, heme oxygenase 1 (HO-1), and NAD(P)H: quinone oxidoreductase (NQO1) at mRNA and protein levels. n = 3 in each group. * p < 0.05, ** p < 0.01, *** p < 0.001vs Ctrl; # p < 0.05, ## p < 0.01, ### p < 0.001vs IR.

Previous studies reported that SFN modified the cysteine residue of Keap1 and directly promoted the uncoupling of Nrf2 with Keap1, thereby translocating Nrf2 to the nucleus [21]. Immunohistochemical staining of p-Nrf2 showed that the expression of p-Nrf2 was mainly located in the nucleus of spermatogenic cells in SFN-treated groups (Figure 5C), Quantitative analysis of p-Nrf2 expressions by western blotting confirmed that the expression of p-Nrf2 increased significantly at 1d and 3d in the IRS1 and IRS2 groups, Keap1 decreased after ionizing radiation (Figure 5D).

Nrf2 transcriptional function was confirmed by increased expression of its downstream antioxidant genes, SOD1, HO-1 and NQO1 at the mRNA (Figure 5E) and protein (Figure 5F) levels. Ionizing radiation decreased the expression of these genes at both 1d and 3d. Compared with that in IR group, testicular expression of these genes was significantly increased in IRS1 and IRS2 groups at both 1d and 3d. Considering the important role in the cell damage caused by ionizing radiation, the influence of SFN on redox-regulated transcription factor nuclear factor-kappa B (NF-κB) was investigated. Ionizing radiation treatment significantly elevated the phosphorylation level NF-κB p65 compared to the control group (Figure 5D), which indicated that the NF-κB activity was activated by ionizing radiation in testis. Treatment of SFN greatly inhibited the phosphorylation of NF-κB. These results suggest that SFN plays an antioxidative role by modulating Nrf2/NFκB signaling pathway.

3. Discussion

This study shows that SFN can resist reproductive toxicity caused by ionizing radiation in vivo. SFN reduced the level of oxidative stress and effectively restored the testicular microstructure damaged by ionizing radiation. The mechanism may involve resistance to oxidative stress-induced apoptosis. SFN can be considered an outstanding candidate for potential clinical applications in the treatment of irradiation-associated testicular injury.

The radiosensitivity of tissue cells is proportional to their proliferative capacity. The testis is where spermatogonia in the epithelium of seminiferous tubules develop into sperm after successive mitosis and meiosis, and is very active in proliferation and differentiation [22]. In this study, spermatogenic tubule atrophy was observed in mice after ionizing radiation, accompanied by spermatogenic cell reduction and thinning. However, the level of testosterone had no significant difference in each group mice, the possible reason is that the Leydig cells of the testis are remarkably more radioresistant than germinal epithelium and are only injured by high therapeutic radiation doses [23] Studies have shown that part of the toxicity of ionizing radiation is caused by massive apoptosis in radiation-sensitive organs. Apoptosis is an important pathological manifestation of irradiation-induced testicular injury. Radiation can lead to increased apoptosis of testicular cells, decrease the total number of germ cells and decrease the epithelial thickness of germinal cells [24], and inhibiting germ cell apoptosis can reduce testicular injury. We believe that the testicular injury caused by ionizing radiation in this study was partly due to apoptosis caused by a large amount of irradiation, which directly leads to a large decrease in the number of spermatogenic cells.

The results of our study showed that increased levels of self-renewal and differentiation genes in SFN-administered testes, which are key determinants in spermatogenesis [25]. In one study, the results showed the importance of PLZF for maintaining SSC self-renewal [26]. In addition, Dmrt1 is a highly conserved gonadal regulator expressed in all mitotic germ cells of mice, including spermatogonial stem cells (SSC), and promotes SSC maintenance at least in part by activating PLZF transcription to regulate self-renewal [27]. It seems that SFN could promote self-renewal in these cells by protecting the expression of SSC self-renewing genes PLZF and DMRT1.

Our results also showed that the genes involved in SSC differentiation (c-Kit, Stra8, SPO11, SYCP2, OVOL2, CCNA1) increased significantly after SFN administration. According to previous studies, c-Kit is involved in the differentiation of spermatogonia, is a marker of differentiated spermatogonia type A1-B, and plays a key role in survival and proliferation [28]. Overexpression of c-Kit induced spermatogonial differentiation and indirectly promoted the expression of Stra8, thus accelerating spermatogenesis and meiosis [29]. SPO11 is highly expressed in testicular germ cells and is a specific protein for meiosis, initiating meiosis recombination by mediating double strand breaks (DSBs) [30,31]. Stra8 is expressed at the onset of meiosis in germ cells [32]. OVOL2, a family of zinc finger transcription factors, are expressed in spermatocytes at the pachytene stage and are suggested to be critical regulators of pachytene progression in male germ cells [33]. SYCP2 is required for synaptonemal complex assembly and chromosomal synapsis during male meiosis [34]. CCNA1 is localized in late meiotic spermatocytes and it plays an important regulatory role in cell-cycle [35]. Thus, it seems that SFN promotes the differentiation of spermatogonial cells by protecting the expression of genes that play a role in spermatogonial differentiation. Notably, we observed that the ability of 5 mg/kg SFN prevented spermatogenesis dysfunction caused by ionizing radiation is better than 10 mg/kg SFN.

Chromatin compaction is a vital step in sperm maturation and fertilization ability [36]. It plays a key role in sperm head shape and is expected to affect sperm function [37]. Prm1 replaces histones in testicular tissue and plays a crucial role in the compaction of chromatin, which is necessary to maintain strong sperm quality [38]. We can conclude that SFN promotes sperm maturation and maintains its chromatin density during spermatogenesis by protecting the expression of Prm1 gene. Our results showed a significant increase in genes associated with spermatogenesis after SFN administration.

Nrf2 is an important transcription factor that regulates redox responses and protects against oxidative stress by upregulating levels of antioxidant genes and enzymes [39]. Up-regulation of Nrf2 was shown to inhibit radiation-induced skin damage in mice [40]. This suggests that the precise interaction between the Nrf2-mediated antioxidant defense system and radiation may play a key role in the occurrence and development of irradiation-induced testicular injury. In this study, Nrf2 was not significantly upregulated in tisticular tissue of mice after irradiation. Notably, Nrf2 was significantly activated in SFN-treated mice. Thus, we hypothesize that the up-regulation and activation of Nrf2 in the testis with SFN treatment after radiation is an important response in attempting to overcome the radiation-induced oxidative stress. This suggests that targeting Nrf2 activation holds promise as an achievable protective measure against IR-induced testicular injury.

SFN is an Nrf2 activator and has been shown to activate Nrf2 and downstream antioxidant gene expression in the kidney, skin, and other tissues or organs [41]. The results of this study showed that SFN significantly reduced the IR-induced increase in ROS production, MDA and the expression of 3-NT and 4-HNE related indicators of oxidative stress. In addition, SFN significantly up-regulated the expression of Nrf2 and its downstream antioxidant factors SOD1, NQO1 and HO-1, which efficiently neutralizes ROS and detoxifies toxic chemicals and therefore inhibits ROS-mediated NF-κB activation. The results suggest SFN can alleviate irradiation-induced testicular damage by activating Nrf2 antioxidative signals and inhibiting NF-κB inflammatory response.

4. Conclusion

In conclusion, our identification of SFN as a potent drug against ionizing radiation, which activates Nrf2-dependent antioxidative pathways and inhibits NF-κB involved inflammatory response to protect testis from the irradiation-induced injury, unveils therapeutic opportunities of irradiation-induced injury therapies.

Acknowledgments

Yuanshuai Ran performed major experiments of this study, completed all data analysis and visualization procedures, and wrote the original manuscript, as well as completed the revision procedure and response to reviewer's comments. Nengliang Duan assisted with part of the experiments. Zhixiang Gao assisted with data collection. Boxin Xue, Xiaolong Liu, Yulong Liu conceived and supervised the research. All authors read and approved the final manuscript.

Disclosure statement

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

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Correction Statement

This article was originally published with errors, which have now been corrected in the online version. Please see Correction (http://dx.doi.org/10.1080/13510002.2024.2327255)

Additional information

Funding

This study was funded by the Projects of State Key Laboratory of Radiation Medicine and Protection (GZK1201901), Youth Excellence Projects of Suzhou Health (GSWS2019042, GSWS2020031) and Youth Excellence Projects of CNNC (CNNC-2020), Occupational health research project in Jiangsu Province (JSZJ20233201).