Fruits and vegetables are the major sources of antioxidants in the human body. Oxidative stress in cells induces the accumulation of reactive oxygen species (ROS), which plays an important role in aging, disease, developmental defects, cancer, and the reproductive system (Azab et al., 2016). Cells produce free radicals when oxygen reacts with cellular organic compounds or is exposed to ionizing radiation. In addition, free radicals are produced in cells during mitochondrial respiration (Dröge, 2002). Recent advances in medical technology have increased human life expectancy, and antioxidants are progressively being used to prevent aging and disease and increase immunity (Obrenovich et al., 2011). Members of the Brassica oleracea family include cabbage, broccoli, cauliflower, kale, Brussels sprouts, collard greens, Savoy cabbage, kohlrabi, and gai lan, which are antioxidant-rich foods and are natural sources of antioxidants (Al Talebi et al., 2023; Hossain et al., 2022). Broccoli (Brassica oleracea var. italica) is an abundant source of vitamins A and C, fiber, calcium, potassium, iron, and antioxidants, and it contains various bioactive compounds, including glucosinolates, indole-3-carbinol, and sulforaphane, which positively affect human health (Syed et al., 2023). Lv et al. reported that the sulforaphane content in broccoli sprouts (BS) was 1.12-3.58 times higher in the sprouts than in the seeds (Lv et al., 2020), and sulforaphane-rich sprouts possessed high amounts of antioxidant activity-inducing bioactive phytochemicals (Becker et al., 2017). Numerous molecules and chemicals can induce the production of reactive oxygen species (ROS). Lipopolysaccharide (LPS) induces the elevation of intracellular ROS levels and inflammation in cells (Sul and Ra, 2021). Our previous study reported the anti-inflammatory effects, and not the antioxidative effect, of the aqueous BS extract (BSE) in RAW 264.7 cells and testes (Park, 2023). LPS has been widely used to induce oxidation and inflammation in various cell types (Lin et al., 1979; Mokuno et al., 1994; Mohanta et al., 2023).

Cisplatin is a well-known chemotherapeutic drug used to treat numerous human cancers, including lung, ovarian, testicular, bladder, and lymphoma. Cisplatin interferes with DNA repair mechanisms, DNA damage, apoptosis, and necrosis (Santos et al., 2007; Gandin et al., 2023). Additionally, many studies have described cell damage caused by cisplatin-induced oxidative stress. A study on rats suggested that cisplatin exposure results in testicular toxicity and decreased sperm concentration and motility, and one of the key molecular mechanisms is increased ROS production in testes; hence, cisplatin has been extensively used for inducing oxidative stress to evaluate the antioxidative effects of chemical, drug, and natural extracts (Atessahin et al., 2006; Karimi et al., 2018; Tian En et al., 2020). Male reproductive system disorders, such as low sperm motility, germ cell damage, and hormone imbalance due to chemical exposure, smoking, and oxygen stress during aging, eventually lead to infertility (Rotimi et al., 2023). In this study, we investigated the antioxidative effects of alcoholic BSE in RAW 264.7 cells and evaluated whether the BSE extract could inhibit oxidative stress induced by cisplatin in neonatal testes using an organ culture system as an ex vivo model.

Preparation of alcoholic BSE

BS powder was purchased from K-Food Ltd. (Gyeonggi-do, Republic of Korea). Thereafter, 5 g of this powder was mixed with 95 mL of 70% ethanol for 2 h by ultrasonication, filtered, and freeze-dried to obtain a powder (the dry matter was approximately 0.5 g). Ethanolic BSE was dissolved in the cell culture medium to produce 5% BSE (50 mg/mL) for further experimentation. Water extraction was performed as previously described (Park, 2023).

DPPH and ABTS assays

The radical scavenging activity was measured using a DPPH scavenging photometric assay. The aqueous and ethanolic BSE and the positive control, ascorbic acid, were dissolved in methanol (dilution: ascorbic acid, 10-500 μg/mL; BSE, 1-50 mg/mL). The plates were incubated for 30 min at 25℃, and the absorbance was measured using a microplate reader (Bioteck Epock, Winooski, VT, USA) at 517 nm. The percentage of DPPH activity was calculated as follows:

DPPH scavenging percentage (%) = (control [A0.] - sample [A1.]/control [A0.]) × 100

The free radical scavenging activities of BSE and ascorbic acid were measured using the ABTS radical cation decolorization assay. The ABTS assay was performed as previously described (32300192). ABTS scavenging activity was calculated as follows:

ABTS scavenging percentage (%) = (control [A0.] - sample [A1.]/control [A0.]) × 100

Cell culture and treatment

RAW 264.7 cells (a mouse macrophage cell line) were purchased from the Korean Cell Line Bank (Seoul, Jongno-gu, Republic of Korea). RPMI medium with 10% fetal bovine serum and 1% penicillin and streptomycin (Nalgene Nunc International, Rochester, NY, USA) were added to the culture, and the cells were maintained in a 5% CO2 incubator at 37℃. The cells were seeded in 6-well plates at a density of 5 × 105 cells/well for 24 h. LPS (Sigma Aldrich, St. Louis, MO, USA) dissolved in DPBS (1 mg/mL stock). The cells were first pretreated with BSE for 1 h. Thereafter, the cells were induced with 1 μg/mL LPS for 24 h and harvested for further experiments.

CellROX staining and flow cytometry

ROS generation was investigated in RAW 264.7 cells treated with LPS and BSE using CellROX staining. Briefly, cells were seeded in 6-well plates (5 × 105 cells/well) with glass coverslips for 24 h. Cells were pretreated with BSE (0.5-1 mg/mL in medium) for 24 h, then treated with LPS (1 μg/mL) for another 24 h. CellROX staining was performed as previously described. CellROX intensity was measured from cell images obtained using a microscope (Olympus IX73, Tokyo, Japan), Motic Image Advanced software (Kowloon, Hong Kong), and Image J software (Park et al., 2020a). JC-1 staining (Biotium Inc., Fermont, CA, USA) was performed to measure the mitochondrial membrane potential (∆Ψm) in each sample after treatment with LPS and BSE. Briefly, cells were harvested after culturing with LPS and BSE and washed with PBS. The staining was performed as described previously (Park et al., 2020a). The stained cells were analyzed using flow cytometry (CytoFLEX, Beckman Coulter, Inc., Miami, FL, USA), and images were obtained using a microscope (Olympus IX73).

Preparation and culture of mouse testis fragments (MTFs)

Two-day-old male pups and their mothers were obtained from Dae Han BioLink Co. (Daejeon, Republic of Korea) and maintained for several days. Five-old male pups were dissected for testicular organ cultures. The mice were housed under constant conditions of 40-60% humidity, 12 h light:dark cycle, and 20-25℃. The Institutional Animal Care and Use Committee (IACUC) of Sangji University (protocol) approved the ethical approval for the study protocol (#2021-23). MTFs were cultured as described previously (Park et al., 2020b). Further, ROS generation was induced in the testes by cisplatin, and the cisplatin concentration was determined according to our previous study (Park et al., 2022). In brief, MTFs were cultured with 5 μg/mL cisplatin and 1 mg/mL BSE for five days.

Isolation of RNA and qPCR analysis

RNA was extracted from MTFs and RAW 264.7 cells using a Qiagen RNeasy Mini Kit (Qiagen, Cat: 74106) with on-column DNase treatment (Qiagen, Cat: 79254), according to the manufacturer’s instructions. cDNA was synthesized, and qPCR analyses were performed according to the protocol described in our previous study (Park et al., 2020b). The qPCR data were analyzed using the CT method, and Gapdh was used as the control gene. The mean and standard error of the mean (SEM) fold change values were plotted. The primer sequences are listed in Table 1.

Table 1. Primer list.

Gene	Forward primer	Reverse primer
Nrf2 (m)	5’-TCTCCTCGCTGGAAAAAGAA-3’	5’-AATGTGCTGGCTGTGCTTTA-3’
Gsr (m)	5’-TGGTGGAGAGTCACAAGCTG-3’	5’-TGCCAACTGAATTTACCCTCA-3’
HO-1 (m)	5’-TGATGCTGGTGACAACCACG-3’	5’-CAGAATTGCCATTGCACAACTC-3
Cat (m)	5’-CACTGACGAGATGGCACACT-3’	5’-CAAACACCTTTGCCTTGGAG-3’
Ddx4 (m)	5’-CCGCATGGCTAGAAGAGATT-3’	5’-TTCCTCGTGTCAACAGATGC-3’
Gapdh (m)	5’-GTGTCTCCTGCGACTTCA-3’	5’-GGTGGTCCAGGGTTTCTTA-3’

Western blotting

Proteins were prepared using ice-cold RIPA buffer (Thermo Fisher Scientific, Wilmington, DE, USA) containing protease inhibitors (Roche, Indianapolis, IN, USA). The BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA; #23 277) was used for protein quantification. Total protein (40 μg) was loaded onto a 4-20% acrylamide gel (Bio-Rad, Rockford, IL, USA). Proteins were then transferred onto a PVDF membrane and incubated with the primary antibody (anti-O-HdG, sc-66036, Santacruz Biotech, USA) for 16 h at 4℃. After washed twice and incubated secondary antibody (anti-mouse IgG) and a horseradish peroxidase (HRP) for 1 h. ECL substrate (Thermo Scientific; No. 34580) was used for detecting the protein band images, and images were collected by iBright^TM Imaging Systems (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Histology and immunostaining

MTFs were fixed with 4% paraformaldehyde for 6 h at 4℃. Histological analysis and tissue immunostaining were performed as previously described (Park et al., 2020b). For immunohistochemistry, sectioned slides were deparaffinized and rehydrated using xylene and ethanol (90-100%). Antigens were retrieved in 10 mM sodium citrate buffer, and the samples were boiled for 10 min. Samples were then blocked with the blocking buffer (0.01% Triton X-100 and 1% BSA) for 30 min at 25℃ and incubated with anti-O-HdG antibody (Santa Cruz Biotech, CA, USA) for 24 h at 4℃. Thereafter, the samples were incubated with a secondary antibody (Alexa Fluor 594 donkey anti-mouse IgG) for 1 h at 25℃. Finally, the tissues were incubated with 1 μg/mL 6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific) in PBS for 5 min, and the coverslips were covered with mounting solution (DAKO, Carpinteria, CA, USA; S3025). Samples were analyzed using a Nikon E-800 fluorescence microscope (Nikon, Tokyo, Japan).

Statistical analysis

All data are represented as the mean ± SEM of at least three independent experiments and evaluated using one-way analysis of variance (ANOVA), Tukey’s honest significance test was used for post-hoc analysis. The SPSS statistical package ver. 15.0 for Windows (IBM Corp., Armonk, NY, USA) was used for the data analysis. Values of *p < 0.05 were considered statistically significant, and for multiple comparisons, statistical differences among the groups are showed using ‘a, b, and c’.

Antioxidant activity of BSE

The antioxidant activities of the alcoholic and aqueous extracts were compared with those of ascorbic acid, a strong antioxidant, using the DPPH and ABTS assays. According to the DPPH results, there was no significant difference in scavenging activity between the water and alcoholic extracts at 1-2 mg/mL, whereas the scavenging activity was statistically higher in the alcoholic extract than in the water extract at 10-20 mg/mL. The ABTS assay showed that the alcoholic extract had slightly higher scavenging activity than the water extract at 10 mg/mL, not at 50 mg/mL. Therefore, we chose the alcoholic extract for further studies (Fig. 1A and 1B). We then verified the antioxidant effects of the alcoholic BSE using an in vitro cell culture system. LP) is an oxidative agent (Laskin et al., 2011). Hence, cells with LPS-induced oxidative stress were treated with 0.5-1 mg/mL BSE, and the antioxidative effect of BSE was evaluated using CellROX staining (Fig. 1C). The results showed that the CellROX intensity of LPS-treated cells increased and was suppressed by BSE treatment.

Figure 1.Antioxidative effect of alcoholic and water extracts of broccoli sprouts. (A) DPPH and (B) ABTS assays with 10–500 μg/mL and 1–50 mg/mL of aqueous and alcoholic extracts of BSE, respectively. *p < 0.05, EtOH extract groups are compared to water extract groups. Ascorbic acid groups as positive controls. (C) Detection of ROS levels using CellROX dye staining in RAW 264.7 cells. The graph represents the CellROX intensity (%) as the mean and the standard error of the mean, calculated by image analysis. Scale bar = 100 μm. Statistical differences among the groups are showed using ‘a and b’.

BSE increased the expression of antioxidation-related genes in LPS-induced oxidative stress

Next, to confirm the antioxidative effects of BSE on LPS-treated RAW 264.7 cells, the expression levels of antioxidant-related genes Nrf2, Gsr, HO-1, and catalase were measured in BSE- and LPS-treated cells. The expression of all these genes was significantly upregulated in both BSE- and LPS-treated cells compared to those in the LPS-treated and control groups (Fig. 2A). Similar to the gene expression data, the expression of Nrf2 protein was significantly increased in LPS-induced cells treated with 0.5-1 mg/mL BSE (Fig. 2B). These results indicate that BSE acts as an antioxidant in RAW 267.4 cells.

Figure 2.Expression of antioxidative markers in the RAW 264.7 cell culture. (A) Gene expression of antioxidation-related genes in LPS- and BSE-treated RAW 264.7 cells. (B) Expression of Nrf2 protein in RAW 264.7 cells after treatment with BSE or LPS. The graph shows the mean and the standard error of the mean as log2 scale. Statistical differences among the groups are showed using ‘a, b, and c’.

BSE inhibited mitochondrial dysfunction in cells with LPS-induced oxidative stress

We then investigated whether BSE suppresses ROS-mediated mitochondrial dysfunction in LPS-treated RAW 264.7 cells using the cell-permeable voltage-sensitive fluorescent mitochondrial dye JC-1, fluorescent microscopy, and flow cytometry analysis (Fig. 3). LPS caused significant mitochondrial membrane depolarization, visualized as green fluorescence (JC-1 aggregates); however, BSE inhibited the LPS-induced depolarization of the mitochondrial membrane (Fig. 3A and 3B).

Figure 3.BSE suppresses the cisplatin-induced loss of mitochondrial membrane potential in RAW 264.7 cells. (A) Mitochondrial membrane potential was assessed using JC-1 dye staining. Scale bar = 100 μm. (B) Flow cytometry plots of JC-1 dye staining of LPS− or LPS+ BSE-treated RAW 264.7 cells. The graph shows the relative ratios of the JC-1 monomer and its mean, and the statistical differences among the groups are showed using ‘a, b, and c’.

BSE suppressed the cisplatin-induced reduction in antioxidant activity in testicular tissue

Based on the in vitro results (Fig. 2, 3), we aimed to evaluate the antioxidative effect of alcoholic BSE on five-day neonatal testes cultures. Cisplatin induces oxidative stress in various cell types (Nizami et al., 2023). Herein, the expression levels of antioxidation-related genes such as Nrf2 and HO-1, and germ cell marker Ddx4 were investigated in each sample (Control, 5 μg/mL cisplatin, 1 mg/mL BSE, and 1 mg/mL BSE with 5 μg/mL cisplatin groups). The expression of Nrf2, HO-1, and Ddx4 in the cisplatin-treated group was significantly decreased, and BSE treatment prevented the downregulation of these genes (Fig. 4A). Histological analysis was performed using H and E staining. Testicular tissues of all groups, except the cisplatin-treated group, showed clear testicular tubules. In both BSE- and cisplatin-treated groups, although the cells in the tubules were not similar to those in the control groups, testicular tubules and barriers were clearly observed (Fig. 4B). To verify the qPCR results, levels of the oxidative stress marker 8-hydroxyguanosine (O-HdG) were observed in the testicular tissue from each sample by immunostaining (Fig. 4B). Strong O-HdG-positive signals were detected in cisplatin-treated MTFs, and not in both BSE- and cisplatin-treated groups. These results indicated that BSE protects MTFs from cisplatin-induced oxidative damage.

Figure 4.BSE inhibits the cisplatin-induced oxidative stress in in vitro cultured MTFs. (A) Gene expression of antioxidation-related genes, Nrf2, HO-1, and germ cell marker, Ddx4, in cultured MTFs using quantitative PCR. The graph shows the mean and the standard error of the mean as log2 scale. Statistical differences among the groups are showed using ‘a, b, and c’. (B) Immunostaining of O-HdG and DAPI in cultured MTFs. Scale bar = 100 μm.

This study described the antioxidative effect of alcoholic BSE in RAW 264.7 cells and cultured testis organs derived from rodents. First, we evaluated the in vitro antioxidant activity of BSE using the DPPH and ABTS methods. After comparing the antioxidant effects of aqueous and alcoholic extracts of BSE, the results showed that the alcoholic extract had relatively higher antioxidant activity. Many studies have demonstrated that various factors, such as the extraction method, temperature, time, and solvent type, can affect the antioxidant and physiological activities of the extract (Venkatesan et al., 2019a; Venkatesan et al., 2019b).

Several studies have reported that organic solvent extracts contain more bioactive substances than water extracts. For example, Venkatesan et al. reported that among the Pinus densiflora bark extracts extracted using various methods by different solvents such as water, ethanol, methanol, isopropanol, acetonitrile, and acetone, antioxidant activity and total phenol content (TPC) were significantly higher in extracts from 20% ethanol, 40% ethanol, and 20% acetonitrile compared to that in the aqueous extract (Venkatesan et al., 2019a). Another study reported that the methanolic extract of seaweed (Padina tetratromatica) exhibited higher bioactivity and antioxidant potential than in the aqueous extract (Sobuj et al., 2021).

According to our results, the gene expression of Nrf2, Gsr, HO-1, and catalase, which are related to antioxidant activity, was increased by 1 mg/mL BSE treatment in the LPS-induced oxidative stress models. Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor that responds to oxidative stress by binding to the antioxidant response element (ARE) in the promoter region of Nrf2 target genes, and the Nrf2/ARE pathway is activated in cells via phytochemicals such as sulforaphane, which is a major component of broccoli (Vomhof-Dekrey and Picklo, 2012). Nrf2 activation increases the expression of mitochondrial antioxidants, including superoxide dismutase 1 and 2 (SOD1 and 2) and glutathione peroxidase and reductase (GPx and GR, respectively). These genes are enhanced in cells for scavenging and modulating ROS levels (Dai et al., 2020). HO-1, NAD(P)H: quinone oxidoreductase (NQO1), SOD, and glutathione peroxidase (GSH-Px) have the potential to remove ROS and other harmful substances and facilitate antioxidative stress, anti-apoptosis, and anti-inflammatory effects (Cui et al., 2019; Dai et al., 2020).

Many studies have investigated the Nrf2/HO-1 pathway to validate the antioxidant effects of chemicals or natural extracts in various cell types, including H9c2 cardiomyocytes, umbilical vein endothelial cells, and RAW 264.7 macrophages (Niu et al., 2018; Sun et al., 2023). Studies have evaluated the antioxidative activity of compounds using LPS-treated RAW 264.7 cells as an oxidative stress model (Duan et al., 2022; Mantilla-Rojas et al., 2022).

As shown in Fig. 3, LPS induced mitochondrial dysfunction in the RAW 264.7 cell line, and BSE restored the LPS-induced mitochondrial dysfunction. Mitochondrial dysfunction induces ROS generation and oxidative stress. Raza et al. reported that ROS production, oxidative stress, mitochondrial respiratory dysfunction, and apoptotic cell death increased in LPS-treated HepG2 (human hepatoma) cells (Raza et al., 2016). Yu et al. described that 2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside (TSG) inhibited LPS-induced mitochondrial dysfunction via activation of mitochondrial biogenesis via activation of the HO-1 gene (Yu et al., 2017). Oxidative phosphorylation within the inner mitochondrial membrane involves four multi-subunit enzyme complexes, and the main source of ROS production in the mitochondria is electron leakage from respiratory complexes I, II, and III of the respiratory chain (Ashrafi and Schwarz, 2013). Sulforaphane is a key bioactive molecule in broccoli and increases the expression of mitochondrial biogenesis-related genes such as peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC1α) and Nrf2 in the hippocampus of mice (Shimizu et al., 2022).

Cisplatin induces mitochondrial oxidative stress in various organs and cells, and cisplatin-induced oxidative stress models have been used to evaluate the effects of antioxidants (Mohanmed et al., 2023; Zare et al., 2023). Therefore, in this study, cisplatin-induced oxidative stress was studied in testicular organ cultures in vitro. This model was reported in our previous study (Park et al., 2022).

Our results suggest that BSE prevents cisplatin-induced oxidative stress in MTFs. A recent study reported that an aqueous extract of broccoli improved spermatogenesis in mouse testes via upregulation of ADP ribosylation factor like GTPase 4A (Arl4α) (Jazayeri et al., 2021). Another study described that treatment of spermatozoa with 5 μM sulforaphane, which is the major component of broccoli extract, protects against oxidative stress during the freeze-thaw process, and these results strongly support our results.

In summary, our study demonstrated the antioxidative effect of an alcoholic extract from broccoli sprouts using the RAW 264.7 macrophage cell culture. Additionally, BSE prevented cisplatin-induced oxidative stress in the testicular organ culture model. These results suggest the potential of BSE as an anti-aging agent and supplement for male fertility.

None.

Conceptualization, H-J.P., W-Y.L.; methodology, H-J.P.; formal analysis, H-J.P., W-Y.L.; investigation, H-J.P., W-Y.L., H-W.S.; resources, H-J.P.; data curation, H-J.P., writing-original draft preparation, H-J.P.; writing–review and editing, H-J.P., W-Y.L.; supervision, H-J.P.; project administration. H-J.P.; funding acquisition, H-J.P.

This research was supported by Sangji university Research Fund, 2022. This study was supported by the graduate school of Sangji University. This Research was also supported by “Regional Innovation Strategy (RIS)” through the National Research Foundation of Korea (NRF), Funded by the Ministry of Education (MOE) (2022RIS-005).

All experiment were conduced in accordance with the guidelines established by the Institutional Animal Care and Use Committee of Sangji University approved protocol (#2021-23).

Not applicable.

Not applicable.

Not applicable.

No potential conflict of interest relevant to this article was reported.