Inflammation disease is a process in which the immune system targets the body’s own tissues, resulting in inflammation; nonetheless, the immune system typically initiates protective mechanisms to promote tissue repair by producing a range of inflammatory mediators against infection, pathogens, irritants, toxic cellular components, and injury (Sherwood and Toliver-Kinsky, 2004). Research surrounding production of new alternatives to interfere with major inflammatory factors revealed that natural products are rich source of these inflammatory factor inhibitors (Azab et al., 2016). Consequently, many studies have reported the protective role of herbal extracts against oxidative stress and inflammatory-related diseases (Menghini et al., 2016; Li et al., 2020). Physical manifestations of inflammation after infection include redness, heat, pain, and swelling. Various immune cells, such as monocytes, macrophages, neutrophils, basophils, mast cells, and T- and B-cells, are involved in this immune response. Generally, inflammation involves cytokine signaling via interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) (Turner et al., 2014), alongside enzymes and proteins, such as cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), and matrix metalloproteinases (MMPs) (Prasad et al., 2016).

Lipopolysaccharide (LPS) is a major component of the outer membrane of gram-negative bacteria; exposure to LPS activates the innate immune system cells, including macrophages and neutrophils, to secrete pro-inflammatory cytokines such as IL-1β, IL-6, and inducible nitric oxide synthase (iNOS) via the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways (Jayakumar et al., 2021). The LPS-induced endotoxemia rodent model has been used for approximately 100 years to evaluate anti-inflammatory effects in various organs (Hwang et al., 2019; Wang et al., 2020). Additionally, researchers have used various models to investigate anti-inflammatory agents, such as in vitro immune cell cultures and in vivo murine models (Song et al., 2015; Wang et al., 2021). LPS is a potent activator of macrophages; further, LPS generates auto-amplificatory loops after the corresponding activation of monocytes/macrophages. Therefore, LPS triggers the secretion of various cytokines from macrophages, including IL-1, IL-6, and TNF-α (Rossol et al., 2011).

Brassica oleracea var. italica (broccoli), is a member of the cabbage family. It has been reported that broccoli is rich in nutrients such as vitamins A and C, fiber, isothiocyanates, and sulforaphane; further, these nutrients modulate cell proliferation and development, and stimulate the production of anti-inflammation related factors and antioxidants. As the life expectancy of the global population increases, there has been an increasing interest in natural products that possess anti-inflammatory and antioxidant effects to reduce the effects of aging. Further, Broccoli is consumed worldwide (Piao et al., 2010; Saw et al., 2011; Lafarga et al., 2018). Broccoli sprouts (BS) are a promising natural product for the prevention of diseases related to oxidative stress, inflammation, and cancer, with sprouts containing up to 100 times more chemoprotective compounds than fully grown broccoli (Lippman, 1989; Rychlik et al., 2015; Nandini et al., 2020). The sulforaphane biogenic precursor, glucoraphanin, was abundantly found in BS and was confirmed to be active in carcinogenesis rodent models (Fahey et al., 1997). In male reproductive system, Sulforaphane protects the male reproductive system from obesity-induced mice (Huo et al., 2019). Broccoli aqueous extract but, not the broccoli spouts is associated with improved spermatogenesis in mice testes (Jazayeri et al., 2021), and broccoli extracts protected rat sperm against oxidative damage during cryopreservation and improved male reproductive performance (Raeeszadeh et al., 2022). Despite the results of previous studies evaluating the effects of broccoli extracts, there is still lack of information on effects of broccoli sprouts extract on LPS-induced acute testis inflammation in mice. In these study, we investigated the effect of broccoli sprouts extract on the inflammatory damage of testis using LPS-induced acute inflammation model.

Preparation of BSE and HPLC

BS powder was obtained from KFood. Ltd. (Gyeonggi-do, Korea). For hot water extraction, 5 g of dry BS powder was heated by refluxing with 95 mL water; then, the mixture was cooled to room temperature, filtered, and freeze-dried. BSEs (hot water extractions) were ground to produce fine particles that were dissolved in cell culture medium to produce 5% BSE (50 mg/mL) as stock for experiment.

Additionally, the BSEs were analyzed by HPLC using a water system consisting of a 996 photodiode array detector (PDA). The column used was Phenomenex Luna C18 (ID 4.6 × 250 mm, 5 μm particle size) (Torrance, CA, USA). For the mobile solvent system, 0.1% formic acid distilled water (A channel) and 0.1% formic acid acetonitrile (B channel) were used. The slope profile proceeded as follows: 0-5 min, 5-20% B linear; 5-85 min, 20-30% B linear; 85-90 min, 30% B linear; 90-100 min, 30-80% B linear; 100-105 min, 80-100% B linear; and 105-110 min, 100-5% B linear. The flow rate (1.0 mL/min) was maintained throughout the analysis. For sample injection, 20 μL of BSE solution was injected at 5 mg/mL; additionally, 6.25-100 ppm sulforaphane (Sigma-Aldrich, St. Louis, MO, USA) dissolved in methanol was injected as a standard sample. The detection wavelength was adjusted to 254 nm.

Cell culture and cell viability assay

The mouse RAW 264.7 macrophage cell line were purchased from the Korean Cell Line Bank (Seoul, Jongno-gu, Korea). The macrophage cells were cultured in RPMI medium with 10% fetal bovine serum (Nalgene Nunc International, Rochester, NY, USA) and 1% penicillin/streptomycin (Gibco, Paisley, Scotland, UK) in a humidified atmosphere of 5% CO2 at 37℃. For cell viability assay, RAW 264.7 macrophage cells (2.5 × 10⁶ cells/plate) were seeded into a 96-well plate for 24 h; these cells were incubated with 0.01, 0.05, or 0.1% BSE for 24 h. Cell viability was determined using the EZ-Cytox assay kit (Wellbio, Seoul, Korea) according to the manufacturer’s instructions. The absorbance at 492 nm was measured using a BioTek Epoch Microplate reader (Winooski, VT, USA).

Nitrate scavenging ability assay

The NO assay was performed using the NO plus detection kit (iNtRON Biotechnology, Gyeonggi-do, Korea) following the manufacturer’s instructions. RAW 264.7 cells (5 × 10⁴ cells) were seeded onto 96 well plates. The following day, 1 μg/mL LPS with or without BSE at different concentrations (0.05 or 0.1%) were added to cells. After 24 h of incubation, 100 μL of supernatant and an equal amount of Griess reagent were mixed; the absorbance was measured at 540 nm. The image of RAW 264.7 cell was collected from microscope (Olympus IX73; Tokyo, Japan).

Animals and treatment

Six-week-old male Balb/c mice were obtained from Dae Han Bio Link Co. (Daejeon, Korea). Mice were maintained under constant conditions of 40-60% humidity, 12 h light:dark cycle, and at 20-25℃. After acclimatization for a week, the mice were randomly divided into four groups of six mice: control, 20 mg/kg LPS injected, 20 mg/kg BSE (BSE dissolved in PBS) orally injected for 2 weeks before LPS injection (20 mg/kg), and Dex administered at a dose of 5 mg/kg one day (24 h) before LPS injection. All experimental groups, except for the control group, received an LPS injection at a dose of 20 mg/kg via intraperitoneal injection 24 h before euthanization for analysis using Avertin. Body and testis weight were measured, and tissue samples were collected for further experiments. All mice were treated according to the protocols reviewed and approved by the Institutional Animal Care and Use Committee of the University of Sangji, and all procedures were carried out in accordance with the approved guidelines (No. 2022-23).

Isolation of RNA and qPCR analysis

Total RNA was extracted from testis tissue 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 from total RNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). The specific qPCR primers were obtained from Bioneer (Daejeon, Korea) and are described in Table 1. qPCR analyses were performed using the Real-Time system QuantStudio 1 (Applied Biosystems, Foster City, CA, USA) with EvaGreen^® qPCR supermix (Solis BioDyne, Tartu, Estonia). Denaturation and polymerase activation were performed at 94℃ for 1 min, followed by 40 cycles of the following: 94℃ for 10 s, 57℃ for 10 s, and 72℃ for 20 s. Data were analyzed using the C_T method, and GAPDH was used as the control gene. After normalization to GADPH levels, which were reflected in the ΔC_T values, the relative quantification (RQ) of the fold-change for each treatment compared with that of the reference control was determined using the following equation: RQ = 2(-ΔC_T)/2(-ΔC_T reference). The RQ mean and standard error of the mean (SEM) fold-change values were plotted.

Table 1. List of primers for qPCR.

Gene	Forward primer	Reverse primer
IL6	5’-TGATGCTGGTGACAACCACG-3’	5’-CAGAATTGCCATTGCACAACTC-3’
IL-1β	5’-ACCTTCCAGGATGAGGACATGA-3	5’-CTAATGGGAACGTCACACACCA-3
TNF-α	5’-CAGGCGGTGCCTATGTCTC-3’	5’-CGATCACCCCGAAGTTCAGTAG-3’
GADPH	5’-GTCGGTGTGAACGGATTTG-3’	5’-CTTGCCGTGGGTAGAGTCAT-3’

Western blot studies

Protein expression was determined by western blotting. Testis tissue lysates were prepared using ice-cold RIPA buffer (Thermo Fisher Scientific, Wilmington, Delaware, USA) containing protease inhibitors (Roche, Indianapolis, IN, USA). Total protein was quantified using a BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA; #23 277). Samples were loaded as equal volumes (40 μg) of protein into wells containing 4-20% acrylamide gels (Bio-Rad, Rockford, IL, USA). Following this assay, proteins were transferred from gels to PVDF membranes. Membranes were incubated with primary antibodies diluted in 1% bovine serum albumin (BSA) in TBS-Tween buffer (TBST, 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween-20) overnight at 4℃. The Cox2 and β actin primary antibodies are used (Cell signaling Technology Inc, Danvers, MA, USA). After three washes with TBST, the membrane was incubated for 1 h with a 1:30000 dilution of anti-mouse and anti-rabbit IgG and a horseradish peroxidase (HRP)-linked antibody (Thermo Scientific) in TBST containing 1% BSA. Pierce ECL Western Blotting Substrate (Thermo Scientific; No. 34580) was used for visualization and band images were collected using iBright^TM Imaging Systems (Thermo Fisher Scientific, Inc., Waltham, MA, USA). β-actin was used as the control for normalization.

Histology and immunostaining

Testis tissues were fixed with 4% paraformaldehyde overnight at 4℃. Histological analysis and tissue immunostaining were performed as described in our previous study (Jhun et al., 2022). For immunohistochemistry, Testis sections were deparaffinized and rehydrated using xylene and various concentrations of ethanol (90-100%). Antigens were retrieved in 10 mM sodium citrate buffer and the samples were boiled for 15 min. The tissues were blocked with blocking buffer (0.01% Triton X-100 and 1% BSA) for 60 min at 25℃ and tissues were incubated with PCNA antibody for 24 h at 4℃. Next, tissues were incubated with a secondary antibody (Alexa Fluor 594 donkey anti-mouse IgG) and 1:200 BSE (diluted in PBS) for 1 h at 25℃. The tissue was 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). The samples were analyzed using a Nikon E-800 fluorescence microscope (Nikon, Tokyo, Japan).

Sperm motility

Spermatozoa were collected from cauda epididymis of mice from each experimental groups, and placed in a dish containing M2 medium. Then, pierced using a needle to disperse the spermatozoam. The spermatozoa incubated in a M2 medium for 60 min at 37℃. 30 μL of the sperm suspension was placed in a counting chamber for assessment of sperm motility parameters by computer-assisted sperm analysis (CASA) using AndroVision (Minitub, Tiefenbach, Germany).

Statistical analysis

SPSS statistical package ver. 15.0 for Windows (IBM Corp., Somers, NY, USA) was used for data analysis. All data were expressed as the mean ± SEM of at least three independent experiments and evaluated using one-way analysis of variance (ANOVA). Values of *p < 0.05, and **p < 0.01 were considered statistically significant.

HPLC analysis of BSE

As sulforaphane is one of the major compounds of BSE, this was used as the standard compound in HPLC analysis of BSE (González et al., 2021). HPLC chromatograms were obtained from this analysis by monitoring the detector responses at 254 nm. As shown in Fig. 1A, the retention time of the main peak in sulforaphane was 14.7 min and allowed indication of sulforaphane content in hot water BSE. BSE samples also showed a peak at the same retention time as sulforaphane (14.7 min). Water BSE showed 0.43 ± 0.00% sulforaphane (Fig. 1B), and we used water BSE for further analysis.

Figure 1.HPLC chromatogram of sulforaphane as a standard compound alongside hot water broccoli sprout (BS) extract. (A) Sulforaphane, (B) hot water extract of BS.

Effect of BSE on cell viability RAW 364.7 macrophage and NO production

The cytotoxic effect of BSE was examined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which was used to measure cell viability. Cell viability was observed to be significantly higher when the cells were exposed to 0.01-0.1% BSE when compared to that of the control; additionally, a consistent increase in the number of cells was observed in the 0.01-0.1% BSE group (Fig. 2A). Further analysis determined whether BSE could reduce NO production in RAW 264.7 cells in response to LPS stimulation. The levels of NO in the culture media were determined using a NO assay kit. Cells treated with LPS alone showed a significant increase in NO production compared to that in controls; this increase was dramatically attenuated in cells that were pretreated for 1 h with 0.01-0.1% BSE (Fig. 2B). The morphology of RAW264.7 cells changed lie macrophage-like cells into dendritic-like cells after LPS treatment, but not in BSE treatment.

Figure 2.Effect of BSE on RAW 264.7 cell viability and production of NO in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. (A) The cell was treated with 0–0.1% BSE for 24 h. The corresponding graph represents the percentage of cell viability. Values of **p < 0.01 were considered statistically significant compared to Ctrl. (B) Cells were pretreated with BSE for 1 h and stimulated with 1 μg/mL LPS for 24 h. The levels of NO in cultured medium were quantified by Griess reagent. Values of **p < 0.01 were considered statistically significant compared to cells treated with LPS only.

Effects of BSE on LPS-induced inflammation on the testis

The effects of BSE on LPS-induced inflammation of mouse testes were assessed. Mice growth and their health conditions were observed. At eight weeks, there were no differences in both body weight and testis weight among the groups (Fig. 3A and 3B). Additionally, to evaluate the effect of BSE on spermatozoa motility. The results showed that BSE rescued the decrease in spermatozoa motility following the injection of LPS (Fig. 3C). Based on this result, PCNA immunostaining performed in order to evaluate the effect of BSE on proliferation of testicular cells in LPS- induced inflammation of mouse testes (Fig. 3D). The graph showing the number of PCNA-positive stained cells within seminiferous tubules in each group (Fig. 3E). the number of PCNA-positive cells were significantly reduced in LPS-injected mice, but the number of theses PCNA-positive cells were notably increased in the BSE + LPS experimental groups compared to LPS treated groups. As expected, the number of PCNA-positive cells in tubule were higher in Dexamethasone, anti-inflammatory drug treated groups with LPS compared to LPS only.

Figure 3.Effect of BSE on testis of LPS-induced inflammation model. (A) Body weight (BW), (B) testis weight of mice after administration of BSE and LPS. (C) Changes in sperm parameters, total motility (%). Data shows mean ± SEM. Values of *p < 0.05, **p < 0.01 were considered statistically significant compared to mice treated with LPS only. (D) Image of immunostaining of PCNA in testes from each group. (E) The number of PCNA-positive cells in tubule from each experiment group. Data are expressed as the mean ± SD. **p < 0.01.

BSE suppressed Pro-inflammatory response in testis of LPS-induced inflammation model.

Based on in vitro results (Fig. 2B), the mRNA expression levels of pro-inflammatory cytokines were assessed using qPCR analysis. mRNA expression levels of IL-6, IL-1β, and TNF-α in testis tissue were significantly increased in LPS-injected mice, and dose-dependently decreased in BSE-administered mice for 2 weeks and 24 h after LPS stimulation (Fig. 4A). The expression levels of Cox2 protein in the testis were also detected by immunoblotting (Fig. 4B). The Cox2 expression were obviously higher in the LPS-treated mice than in the BSE-treated groups with LPS stimulation. As expected, Dex markedly reduced gene and protein expression of pro-inflammatory cytokine in testes.

Figure 4.BSE suppressed the expression of pro-inflammatory genes and protein in testis of LPS-induced inflammation model. (A) Testis tissue samples were harvested from BSE-administrated mice for 2 weeks and then, 24 h post-intraperitoneal LPS injection (20 mg/kg). Expression of IL-6, IL-1β, and TNF- α were analyzed from testis tissue of each experimental groups by qPCR. The graph shows mean ± SEM of three independent experiments. Values of **p < 0.01 were considered statistically significant compared to mice treated with LPS only. (B) The protein levels of Cox-2 in testis were analyzed by immunoblot from each experimental group. Values of **p < 0.01 were considered statistically significant compared to cells treated with LPS only.

This study presents evidence for the anti-inflammatory activity of BSE and the possible mechanisms implemented by BSE to reduce the inflammatory response in testis in rodents. First, we performed detection of sulforaphane content in BSE by HPLC. In the current study, the morphology of RAW 264.7 cells was also different following LPS-stimulation when compared to the control or BSE treated groups. Expectedly, NO production was highly increased in LPS-stimulated RAW 264.7; further, these NO production levels were dramatically decreased in LPS-stimulated RAW cells after BSE treatment. Previously, Hwang et al. (2019) reported that broccoli florets were extracted with 80% methanol and fractionated with ethyl acetate; these corresponding extracts were observed to inhibit NO release, NF-κB inhibitor (IκB-α) degradation, and NF-κB activation in LPS-stimulated RAW 264.7 cells. These results indicated that the broccoli extract had potent anti-inflammatory effects, which is similar to the findings in the current study that instead utilized broccoli sprouts (Bozkurt et al., 2014). NO is a major biomarker of oxidative stress in the inflammatory response; further, oxidative damage and NO production increase in immune cells via iNOS overexpression (Seibert and Masferrer, 1994).

Many recent studies have demonstrated that BSE is a positive regulator of multiple diseases. Sulforaphane derived from BS has anti-cancer effects by inhibiting the absorption of chemical compounds into the body and acting as a natural Nrf2 inducer, thereby increasing antioxidation (Fahey et al., 1997; Bauman et al., 2016). Additionally, sulforaphane-rich BSE attenuates nasal allergic response to diesel exhaust particles (Heber et al., 2014). Similarly, our results also showed that BSE remarkably suppressed the LPS-induced acute inflammatory response in the testis of mice. The expression of pro-inflammatory gene such as TNFα, IL-1β, and IL-6 in testis were downregulated in LPS-induced inflammation model following BSE administration in mice. Many investigators have used LPS injection as an experimental model to induce testis inflammation and study the underlying molecular and cellular mechanisms of various molecules in mice. Recently, Qi et al. (2016) reported that sulforaphane inhibited LPS-induced inflammation in acute lung injury in rodents; consequently, sulforaphane inhibited the expression levels of iNOS and COX-2 genes, and the production of IL-6 and Prostaglandin E2 (PGE2) via the Nrf2/antioxidant response element (ARE) pathway.

Several studies have reported the effects of sulforaphane on male reproductive system. Sulforaphane suppressed the male reproductive toxicity associated with obesity mice model via inhibiting oxidative stress (Li et al., 2019), and also prevents testicular damage in mice exposed to Cadmium through activation of Nrf2/ARE signaling (Yang et al., 2016). There are few studies that describe the effect of broccoli extract on sperm motility. Raeeszadeh et al. (2022) also suggested that broccoli extract prevents oxidative damage of sperm during cryopreservation for improved fertility rates. In our study, LPS-injected mice exhibited a significantly decreased in the percentage of motile sperm compared with the other groups, but the motility of sperm in LPS-injected mice supplemented with BSE was significantly higher than in the LPS only. Dex was used as a positive control to determine the anti-inflammatory effect of BSE in the testis of LPS-injected mice. The gene expression levels of pro-inflammatory cytokines in the testis were reduced in BSE-administered mice with LPS injection, and the gene expression levels of several cytokines were similar to those in Dex-treated groups. In addition, BSE also prevents LPS-induced decreases in proliferation of testicular cells. Therefore, these results indicate that BSE can be a powerful natural anti-inflammatory supplement for improve male fertility.

BSE, which was extracted from hot water, suppressed NO production in RAW 264.7 cells and the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in inflammation-inducing mice testis. Additionally, BSE prevents the reduction of sperm motility and proliferation of testicular cells in LPS-induced inflammation mice. The results suggest the possibility of using BSE from natural sources as supplement for male fertility.

None.

Conceptualization, H-J.P.; methodology, H-J.P.; formal analysis, H-J.P. investigation, H-J.P.; resources, H-J.P.; data curation, H-J.P.; writing-original draft preparation, H-J.P.; writing - Review and editing, H-J.P.; supervision, H-J.P.; project administration, H-J.P.; funding acquisition, H-J.P.

This work was supported by the Regional Specialized Industry Development Plus Program (S3271756), funded by Ministry of SMEs and Startups (MSS, Korea).

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