Protective Effects of Sulforaphane on Exercise-Induced Organ Damage via Inducing Antioxidant Defense Responses

Regular exercise is beneficial to maintain a healthy lifestyle, but the beneficial effects are lost in the case of acute exhaustive exercise; this causes significant inflammation, oxidative stress along with organ damage. Recently, sulforaphane (SFN), an indirect antioxidant, has drawn special attention for its potential protective effect against inflammation and oxidative stress. However, no studies have been performed regarding acute exhaustive exercise-induced organ damage in association with SFN administration. Therefore, the aim of this study was to investigate the effects of SFN on acute exhaustive exercise-induced organ damage and the mechanisms involved. To perform the study, we divided mice into four groups: Control, SFN, exercise, and SFN plus exercise. The SFN group was administered orally (50 mg/kg body wt) 2 h before the running test. We measured plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH), and acute exhaustive exercise significantly increased these biomarkers. In addition, the mRNA expression of pro-inflammatory cytokines, IL-6, IL-1β, and TNF-α, were significantly increased in the liver of exercise group. However, the SFN plus exercise group showed a significant reduction in the expression of cytokines and blood biomarkers of tissue damage or cell death. Furthermore, we measured mRNA expression of Nrf2, heme oxygenase (HO)-1, and antioxidant defense enzymes expression, i.e., superoxide dismutase (SOD1), catalase (CAT), and glutathione peroxidase (GPx1) in the liver. The expression of all these biomarkers was significantly upregulated in the SFN plus exercise group. Collectively, SFN may protect the liver from exhaustive exercise-induced inflammation via inducing antioxidant defense response through the activation of Nrf2/HO-1 signal transduction pathway.


Introduction
Over the decades, an enormous amount of research regarding physical exercise and its effects on health has been carried out. Regular physical exercise is the utmost important tool to maintain a healthy lifestyle. It has both therapeutic and preventive effects against chronic diseases [1] such as obesity and its metabolic complications [2], type 2 diabetes [3], cardiovascular disease, hypertension [4], cancer [5], depression, anxiety, stress, dementia, etc. [6]. Regular physical exercise increases the production of reactive oxygen species (ROS). However, the response to this ROS depends on the intensity or load and duration of exercise [7]. Despite the individual variation, exercise load is further associated with physiological stress [8]. During exercise, oxygen demand is increased in the body by up to 15-fold, basic region-leucine zipper (bZIP) transcription factors, is the key factor regulating the antioxidant response [28]. Nrf2 activity can be regulated by dietary phytochemicals and other chemoprotective agents [29]. In the homeostatic condition, the activity of Nrf2 signaling pathway remains suppressed in the cytoplasm by the repressor protein Keap1 (Kelch-like erythroid cell-derived protein with CNC homology-associated protein 1), localized near the plasma membrane, which targets Nrf2 for proteasomal degradation [30]. Furthermore, in response to the pathological condition associated with oxidative damage by ROS, Nrf2 readily translocates into the nucleus and interacts with specific ARE, followed by stimulating phase 2 enzyme gene expression [31][32][33].
The electron transport chain (ETC) in the mitochondria is the major site of ROS production. Naturally endogenous antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) are the first-line defense system, while the integrated action of these enzymes resists redox disturbance in the cells [34]. Glutathione (GSH) plays a crucial role in the antioxidant defense system, demonstrating unique antioxidant properties. The liver is the major site for GSH synthesis (90% of circulating GSH is synthesized in the liver) and under oxidative stress it is mobilized to other organs through the circulation [35]. Reduced GSH is utilized by GPx to H 2 O 2 and converted to oxidized GSH and water. This reaction is important for the multicomponent defense system within the cells [36].
In this study, we examined the effect of SFN on exercise-induced inflammation, as well as pro-inflammatory cytokine gene expression in the liver. This includes a mechanism involving a signal transduction pathway via alteration of antioxidant enzyme gene expression by potential induction of Nrf2 and its dependent phase 2 detoxification enzyme HO-1. Nrf2 activation by SFN was previously observed in a cell culture study (in vitro) [36] and in this report we detail whether these effects can be reproduced in the body (in vivo).

Animals
Thirty-six male C57BL/6 mice (seven weeks old) were purchased from the Takasugi Experimental Animals Supply (Kasukabe, Japan) and three to four mice were caged together (27 × 17 × 13 cm) under a temperature-controlled environment for two weeks with a 12:12 h light-dark cycle (08:00 to 20:00 light period and 20:00 to 08:00 was dark period). All the mice had free access to a standard chow diet (MF Oriental Yeast, Tokyo, Japan) and water until the experiment started. Mice were divided into four groups: Two for sedentary; control (CON, n = 9), SFN (n = 9), and two for exercise (Ex, n = 9) and SFN + Ex (n = 9). The experimental procedures followed the Guiding Principles for the Care and Use of Animals in the Academic Research Ethical Review Committee of Waseda University and were approved (2019-A109).

Exhaustive Exercise Protocol
One week before the exhaustive running exercise, all mice (including sedentary) were accustomed to a motorized treadmill (Natsume, Kyoto, Japan) running at 15 m/min for 10 min (0% incline). SFN (LKT Laboratories, St. Paul, MN, USA, product code S8044) was administered orally (50 mg/kg body wt), solubilized in distilled water with a volume of 150 µL on the day of the experiment at around 9 am. The uphill treadmill running (7% incline) was started 2 h after feeding. The dose and waiting hours were selected based on the previous literature [37,38]. The Ex and SFN + Ex groups of mice were subjected to run at 15 m/min for 10 min, followed by 20 m/min for 10 min, and finally, 24 m/min continued until exhaustion ( Figure 1). The state of exhaustion was defined as the inability to continue running despite tapping on the back of the mouse several times or repositioning by hand on the treadmill [39,40].

Sample Collection
The sedentary groups were sacrificed 2 h after oral ingestion and the exercise group was sacrificed immediately after exhaustion under light anesthesia with the inhalant isoflurane (Abbott, Tokyo, Japan). Blood samples were collected rapidly from the abdominal aorta using heparin under inhalant mild anesthesia, and centrifuged at 1500× g for 15 min at 4 • C to collect the plasma. The liver sample was immediately collected and stored in liquid nitrogen, and all the other samples were stored at −80 • C.

Real-Time (RT) Quantitative Polymerase Chain Reaction (qPCR)
Total RNA was extracted from the liver using the TRizol (Thermo Fisher, Rockford, IL, USA) extraction reagent, according to the maker's protocol. The NanoDrop system (NanoDrop Technologies, Wilmington, DE, USA) was used to confirm the concentration and purity of extracted RNA by the ratio of A260/280. Total RNA was reverse transcribed to cDNA using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) according to the provided instructions. PCR was performed using the Fast 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) and Fast SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The thermal profile for all genes consisted of one denaturing cycle at 95 • C for 10 min, 40 cycles consisting of denaturing at 95 • C for 3 s and annealing and elongation at 60 • C for 15 min. 18S mRNA was used as the housekeeping gene. All data were normalized using the ∆∆CT method and stated as a fold change relative to the values of the control group. Specific sequences of primers used for gene amplification are given in Table 1. Table 1. Specific primer sequences for RT-qPCR.

Target Gene
Accession Number (Size, bp)

Statistical Analysis
Data are expressed as the mean ± standard error (SE). A two-way analysis of variance (ANOVA) was performed to identify the main effect of SFN administration and/or exercise. To determine the significant difference among means, Tukey's post-hoc test was performed. Statistical significance was defined as p < 0.05, using IBM SPSS, V 25.0 (IBM Corp., Armonk, NY, USA).

Effect of SFN on Plasma Levels of Exercise-Induced AST, ALT, and LDH
Figure 2A-C presents plasma AST, ALT, and LDH levels among the sedentary and exercise groups in the absence or presence of SFN. AST or glutamic oxaloacetic transaminase (GOT), and ALT or glutamic pyruvic transaminase (GPT) are two important markers for hepatocellular damage. Exercise with or without SFN treatment significantly increased AST, ALT, and LDH levels in the plasma, compared with the control and/or sedentary groups (p < 0.001). However, there was a significantly less induction in all three biomarkers in the Ex + SFN group compared to the exercise group alone (p < 0.05). LDH is an important marker, that increases during prolonged exercise [39,41]. In addition to that, measuring AST and ALT, and assessing LDH gives a clear diagnosis of liver injury.  Figure 2 (A-C) represent aspartate aminotransferase (AST); alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) levels in plasma. ### p < 0.001, ## p < 0.01, # p < 0.05 as compared to the sedentary control. *** p < 0.001, * p < 0.05. Interaction of SFN and Ex written as "Interaction". Data are presented as mean ± SE.

Effect of SFN on Exercise-Induced Pro-Inflammatory Cytokine Expression
We analyzed the mRNA expression of pro-inflammatory cytokines IL-6, TNF-α, and IL-1β using RT-qPCR in the liver to identify the effect of SFN on pro-inflammatory cytokine levels after the endurance running exercise. Results are expressed as the relative differences from sedentary control as the baseline. While IL-6 mRNA expression was increased by 3.4-fold after exercise, the Ex + SFN group showed less increase in the mRNA expression (1.6-fold) ( Figure 3A); however, the interaction was not significant. Figure 3B,C showed results for TNF-α and IL-1β mRNA expression levels. The exercise group showed increased mRNA expression by 2.3-and 5.1-fold, respectively, while the Ex + SFN group showed less expression, showing changes of 1-and 3.2-fold, respectively.

Effect of SFN on Antioxidant Defense System Gene Expression
SOD1, CAT, and GPx1 are the prime endogenous defense enzymes in the redox balance system against ROS. As shown in Figure 4A-C, the mRNA expression of SOD1, CAT, and GPx1 enzymes was significantly upregulated by 2-, 1.5-, and 2.5-fold with exercise in the SFN group. A slightly reduced expression was observed only in the exercise group for CAT and GPx1, but not for SOD1. However, no significant difference was observed in the level of those enzymes between the exercise and control groups.

Effect of SFN on Nrf2/HO-1 Expression
Nrf2 is the pivotal transcription factor for regulating the cellular antioxidant system. In Figure 5A, Nrf2 mRNA expression was increased by 1.5-fold with SFN in the exercise group, which differs significantly as compared to the exercise and/or sedentary SFN groups. Interestingly, as shown in Figure 5B, phase 2 enzyme HO-1 gene expression was increased by 12.6-fold in the Ex + SFN group, which is around a three-times larger expression than the nonsupplement exercise group. In addition, the interaction effect of both expression levels was significant (p < 0.05).

Discussion
Here, we investigated the protective effect of SFN treatment, which induces the antioxidant defense system through the Nrf2/HO-1 signaling pathway, on acute exhaustive exercise-induced inflammation. Previously, we have conducted an in vitro study looking at the anti-inflammatory role of SFN (using LPS to mimic exercise-induced inflammation) by inducing the Nrf2/HO-1 signal transduction pathway [19]. This study was designed to confirm the signal transduction mechanisms in vivo responsible for inducing gene expression of pro-inflammatory cytokines and its attenuation via altering the mRNA expression of Nrf2/HO-1 and endogenous antioxidant defense enzymes. However, no study was found describing the effect of SFN administration on exercise-induced organ damage via inducing this signal transduction mechanism. To the best of our knowledge, this is the first report analyzing the protective effect of SFN on exercise-induced liver damage. Therefore, as an initial screen we analyzed different organ damage markers in the plasma and found significant values for the presented data.
Exhaustive exercise can improve our neural functions and learning behavior [42]. However, acute exhaustive exercise can accelerate oxidative stress and inflammation significantly due to the increased oxygen consumption. This can further downregulate the expression of endogenous antioxidant defense mechanisms [43]. From past research, controversial evidence was found, regarding antioxidant supplementation that modulates endurance capacity and prevents tissue damage [39,40,[44][45][46][47]. As a natural antioxidant, SFN has drawn great attention because of its potential contribution to chemo-preventive activities through multiple signaling pathways [48]. Despite knowing that SFN is a very strong inducer of phase 2 enzymes, very little is known about the protective effect of SFN on acute exercise-induced inflammation or organ damage. Cellular damage biomarkers can be identified in the plasma after a single bout of exhaustive exercise [14,16,49,50]. Different enzymes are released into the bloodstream due to tissue damage in several organs such as liver, kidney, or muscle. AST and ALT are commonly used as pathological damage markers for liver tissue, and the elevated levels of these markers represent loss of the cell membrane's functional integrity [51]. Acute exercise causes significant injury in the liver, increasing the expression of these enzymes [52]. Our study shows a consistently increased level in the nonsupplement exercise group, while the SFN with exercise group showed a reduced level of AST and ALT. Previous studies have reported that pretreatment with SFN attenuates liver injury by reducing serum levels of AST, ALT, and LDH through the Nrf2/ARE pathway [53,54]. A recent study by Lee et al. indicated that SFN may suppress lethal hepatic injury by modulating inflammatory pathways and may contribute to the treatment of liver diseases [55]. In addition to that, LDH in the blood is a sensitive indicator for both liver and muscle damage, and also sometimes for different types of cancer [56]. In the sedentary SFN group, all the markers (AST, ALT, and LDH) were slightly increased in comparison to control, but the difference between groups was not significant. We presume that without exercise, chronic administration of SFN might cause hepatotoxicity. In addition, the effect of brief stimulation of Nrf2 signaling pathway by SFN also provides long-lasting protection from radical-induced diseases [57]. Furthermore, creatinine kinase (CK) is another useful marker to identify exercise-induced injury. Elevated levels of both CK and LDH after exercise are considered as markers of muscle injury; however, an elevated level of LDH and normal CK level is considered as a liver injury marker [58].
Pro-inflammatory cytokines and chemokines that are released into the circulation after a single bout of exercise, cause substantial damage to the liver [59,60]. Among the cytokines, IL-6 is a multifunctional one that exerts both pro-and anti-inflammatory effects, depending on classic or trans-signaling modes [61]. TNF-α and IL-1β are vital mediators that can regulate acute phase protein (APP) synthesis, also responsible for acute hepatic inflammation [62,63]. The regulatory factor nuclear factor kappa-light-chain-enhancer of B cells (NF-κB) can be activated after a single bout of exercise [64]. In response to exercise, circulating pro-inflammatory cytokines activate the inhibitor of NF-κB (IκB) kinase complex (IKK). After activation, NF-κB with its attached subunits p65 and p50 translocate into the nucleus from the cytoplasm through the phosphorylation of IκB, and induces pro-inflammatory gene expression [65]. Antioxidant supplementation can suppress the inflammatory response and attenuate the expression of circulating pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α) [66,67]. SFN administration can suppress the expression of the NF-κB signaling pathway and via inducing the Nrf2 signal transduction pathway [68], can reduce mRNA expression of IL-6 and IL-1β in liver tissue [69]. In our study, pro-inflammatory cytokine (IL-6, IL-1β, and TNF-α) expression in the exercise group was increased significantly in the liver tissue, while the SFN-supplemented exercise group showed reduced expression of these pro-inflammatory cytokines. Nrf2 signal activation has also been reported to downregulate the overproduction of pro-inflammatory cytokines [70]. In our previous study, we found that SFN can prevent LPS-induced inflammation through the Nrf2/HO-1 signaling pathway [19]. Since endotoxins pass into the systemic circulation via the liver, we assume that SFN is a promising compound for the downregulation of pro-inflammatory cytokines circulating in the liver during high-intensity exercise.
The antioxidant enzymes, SOD1, CAT, and GPx1 are actively involved in reducing ROS. Previous research activities supported that a single bout of acute exhaustive exercise dramatically increases significant production of ROS from activated neutrophils, thereby suppressing antioxidant enzymes activity, i.e., GPx1, SOD1, and CAT in the liver tissue, which further impairs the free radical scavenging system [71][72][73]. In this study, we analyzed antioxidant enzyme (cytosolic SOD, CAT, and cytosolic and mitochondrial GPx1) mRNA expression in the liver tissue to identify whether SFN administration has any effect on these enzymes during exhaustive exercise. Previous studies have shown that SFN pretreatment can upregulate the SOD and GPx enzyme activities to reduce oxidative stress, which is associated with the Nrf2/HO-1 signaling pathway [53]. After exhaustive exercise, in the liver, the elevated xanthine oxidase (XO) activity is responsible for excessive production of superoxide radical [74]. Here, we found that SFN supplementation in the exercise group increases expression of SOD and GPx1 enzymes. Decreased CAT and GPx level might make liver more susceptible to oxidative stress [74]. However, our data show that SFN administration can alter the expression of these enzymes, thereby protecting liver from oxidative stress. Another study reported that treatment with SFN can significantly increase mRNA expression of SOD1 and CAT and other Nrf2 target genes to protect cells from oxidative stress [75]. Based on previous records, antioxidant enzyme expression increases in muscle after exhaustive exercise rather than liver or kidney [40,76]. Therefore, antioxidant supplementation or a combination of several major antioxidants may attenuate exercise-induced ROS production and contribute to endurance capacity [77]. Additional evidences from other studies have shown that multiple doses of SFN have a significant protective effect against muscle damage through the activation of Nrf2-dependent antioxidant/detoxification phase 2 enzymes [78,79]. Moreover, providing a SFN-enriched diet for 45 days, reduced exhaustive exercise-induced oxidative damage of the liver by increasing antioxidant enzymatic activities [80].
Nrf2 is an important transcription factor that binds to ARE regions in the promoters of genes during stressed conditions and induces antioxidant defense enzymes and phase 2 enzymes in tissues [81,82]. Indeed, transcriptional upregulation of Nrf2 inhibits pro-inflammatory cytokine expression [19,83]. Application of phytochemicals holding anti-inflammatory or anti-oxidative properties can trigger Nrf2 activation [70], also enhancing endurance capacity [78]. Our results are consistent with the previous studies that Nrf2 gene expression in the liver tissue was upregulated in the exercise group with SFN, which upregulated the phase 2 enzyme HO-1. Furthermore, multiple Nrf2-induced genes can be activated by treatment with SFN [84]. SFN can readily undergo a conjugation reaction in the liver after absorption [85], consequently inducing phase 2 enzyme and antioxidant enzyme expression via the Nrf2 signaling pathway. This mechanism could explain the protective role of SFN in exercise-induced inflammation and oxidative stress via the signal transduction mechanism of Nrf2/HO-1 activation.
Our study has several limitations; we measured gene expression of selected cytokines and enzymes, since we aimed to report on the mechanism of signal transduction. Although there are multiple published data on cell culture studies, SFN may regulate phase 2 detoxification and antioxidant enzyme expression; however very little is known about the in vivo studies. Therefore, we performed a single dose oral administration of SFN and presented findings on the protective effect of SFN on acute exhaustive exercise-induced inflammation, without directly measuring ROS levels. Our results also support that a single dose of SFN may protect liver damage by inducing phase 2 antioxidant enzyme expression via Nrf2 activation; however, SFN treatment showed no effect on the endurance capacity between the exercise groups. The dose we used in the present study (50 mg/kg body weight SFN), was a level not easily obtained by consuming an SFN-rich food. Further investigations are required to determine whether a similar effect may be observed through consuming SFN-rich foods.

Conclusions
Natural antioxidants have unique scavenging properties against free radicals. Much research has been carried out, regarding antioxidant supplementation to modulate exercise-induced stress and inflammation. Despite the somewhat paradoxical effects, exercise is important for our longevity and for living a sound life. Here, we presented data on the protective role of SFN on acute exercise-induced liver damage. Collectively, we summarized that SFN is not directly involved in the ROS reduction process, but it induces a cellular defense system through the induction of antioxidant enzymes via the upregulation of the Nrf2/HO-1 signal transduction pathway, thereby reducing oxidative stress and inflammation.
Author Contributions: R.T.R., S.M., and K.S. designed the study and the experiments; R.T.R. and S.M. carried out the experiments; R.T.R. analyzed the data and revised the statistical analyses; R.T.R. and S.M. prepared and edited the manuscript; all authors approved it. All authors have read and agreed to the published version of the manuscript.
Funding: This research was supported by the Waseda University Grant for Special Research Projects 2019Q-056. This study was also made possible by a Grant-in-Aid for MEXT-Supported Program for the Strategic Research Foundation at Private Universities 2015-2019 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (S1511017).