Black soybeans protect human keratinocytes from oxidative stress‐induced cell death

Abstract Black soybeans are functional foods containing a variety of bioactives such as isoflavones, carotenoids, tocopherols, phenolic acid as well as anthocyanins. Here, we examined whether Cheongja#3 black soybean extract has a protective effect on oxidative stress‐induced cell death in human keratinocytes HaCaT. First, we identified fat‐soluble bioactives in three varieties of soybean extracts (Saedanbaek, Daechan, and Cheongja#3). In particular, black soybean Cheongja#3 had high amounts of lutein than other varieties. We demonstrated that Cheongja#3 extract reduced intracellular reactive oxygen species levels in HaCaT cells. Furthermore, Cheongja#3 protected cells from hydrogen peroxide (H2O2)‐induced oxidative stress and triggered cell death determined by cell viabilities and apoptotic caspase activities. Next, we identified the underlying mechanism is due to increased Nrf2 antioxidant system by Cheongja#3, thus increasing the expression of heme oxygenases (HO)‐1. These results indicated that Cheongja#3 soybean extract has protective role against oxidative stress by upregulating the Nrf‐2 antioxidant system in human keratinocyte HaCaT cells.

Nuclear transcription factor erythroid-2-like factor 2 (Nrf2) is one of the major antioxidant systems that protect cells from oxidative stress (Motohashi & Yamamoto, 2004). Under normal condition, Nrf2 action is blocked by Keap1. Responding to extracellular stimuli such as UV light, oxidative stress, and hypoxia, Nrf2 is released from Keap1, thus translocating to the nucleus to bind to its downstream genes containing antioxidant response element (ARE) consensus sequence (Seo & Jeong, 2015). Heme oxygenase-1 (HO-1) is an antioxidant enzyme which contains an ARE site, and functions in the degradation of heme to bilirubin, carbon monoxide, and iron (Seo et al., 2011). Previous studies have reported that natural products from food sources induce antioxidant activity through upregulation of Nrf2-mediated HO-1 expression (Hseu et al., 2012;Nguyen et al., 2013;Seo et al., 2011).
Soybeans are rich in proteins, carbohydrates, dietary fiber, and phytochemicals. Isoflavones are unique components in soybeans and offer a variety of health benefits against obesity, cancer, diabetes, kidney diseases, osteoporosis, and cardiovascular disease (Anderson & Major, 2002;Anderson, Smith, & Washnock, 1999). In particular, black soybeans have been reported to contain even more nutrients including anthocyanins in their seed coat (Liao, Chen, & Yang, 2005). Their biological activities including antioxidative and anti-inflammatory effects help to reduce the risk of cancer and metabolic disorders (Ganesan & Xu, 2017). Cheongja#3 is a cultivar of black soybean which is well known to contain high amounts of anthocyanins as well as tocopherols (Lee et al., 2009;Lee, Park, et al., 2015). Several previous studies have shown that Cheongja#3 had antiobesity effects in cells, mice, and humans (Jeon, Lee, & Cheon, 2015;Kim et al., 2015;Lee, Sorn, Park, & Park, 2016), as well as neuroprotective effects (Bhuiyan, Kim, Ha, Kim, & Cho, 2012;Kim, Chung, et al., 2012). However, there has been a lack of information on the protective effects of Cheongja#3 with respect to oxidative damage in human keratinocyte HaCaT. Here, we tested the effect of Cheongja#3 on reducing oxidative stress-induced cell death and examined underlying mechanism of such action in HaCaT cells.

| Preparation of soybean extract
Three soybean cultivars (Saedanbaek,Daechan,Cheongja#3) were newly developed by National Institute of Crop Science as previously reported Lee et al., 2009;Min et al., 2015), and all soybeans were provided form the National Institute of Crop Science. These soybeans were grounded into powder at 500 g for 5 min, respectively. Forty grams of each powdered soybean was extracted in 500 ml of 40% ethanol solution (EtOH) for 24 hr. After repeating three times, the solutions were filtered and freeze dried.

| Cell culture
HaCaT cells were kindly provided from Dr. Ji-Hong Lim (Department of Integrated Biosciences, Konkuk University) and cultured in DMEM containing 10% FBS and antibiotics (100 units/ml penicillin, 100 μg/ ml streptomycin, and 250 ng/ml amphotericin B) and incubated at 5% CO 2 and 37°C in a humidified air.

| Ultra performance liquid chromatographic (UPLC) analysis
Fat-soluble micronutrients were extracted by using the slightly modified Folch method (Folch, Lees, & Sloane Stanley, 1957) and analyzed by previously reported UPLC method (Delpino-Rius et al., 2014). The UPLC (ACQUITY UPLC I-Class, Waters Co., Milford, MA, USA) system was equipped with a BEH C18 column (1.7 μm, 2.1 × 50 mm, Waters Co.), binary pump delivery system, autosampler, and photodiode array detector. The mobile phase A was acetonitrile/methanol (7:3, v/v), and the mobile phase B was water. Each sample was injected into the BEH C18 column (1.7 μm, 2.1 × 50 mm). The gradient conditions are described in Table 1. γ-Tocopherol (at 292 nm) and lutein (at 450 nm) were quantified by each standard curve. Each peak was confirmed by retention time and its unique spectrum. The interassay coefficient of variation (CV) was under 4% (n = 10), and the intraassay CV was under 4% as well (n = 10).

| DPPH radical scavenging assay
The ability of Cheongja#3 to scavenge free radicals was determined by the DPPH assay (Blois, 1958). Various concentrations of Cheongja#3 extract were dissolved in 40% EtOH and then mixed with equal volume of 0.2 mM DPPH solution (in EtOH). The mixtures were incubated at 37°C for 30 min. The absorbance was read at 517 nm (Spectramax M2e, Molecular devices, Sunnyvale, CA, USA).

| Cell viability assay
Cell viability was measured by the MTT colorimetric assay (Mosmann, 1983). Briefly, cells were seeded in 96-well plates and incubated overnight.
Next, cells were pretreated with various concentrations of Cheongja#3.
After 24 hr, cells were exposed to 500 μM of H 2 O 2 for 24 hr. Five mg/ml of MTT solutions was added into the medium at a final concentration of 0.5 mg/ml, and incubated for 4 hr. All medium were removed and DMSO solution was added into each well to resuspend the MTT formazan. The absorbance was measured at 540 nm (Spectramax M2e, Molecular devices).

| Preparation of cytosolic and nuclear fraction
To separate cytosolic and nuclear fraction in cells, we followed previous study (Park et al., 2017). Cells were pretreated with Cheongja#3 18,000 g, 10 min, 4°C, and then supernatant as nuclear protein was transferred into fresh tubes for immunoblotting.

| Immunoblotting
As previously reported (Lee, Han, et al., 2016), samples were lysed with a RIPA buffer containing 25 mM Tris-HCl at pH7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS and protease inhibitors. The supernatants were collected by centrifugation, and equal amounts of protein were mixed with 4× sample buffer (250 mM of Tris-Cl at pH 6.8, 8% SDS, 40% glycerol, 8% β-mercaptoethanol, and 0.01% bromophenol blue). Boiled samples were loaded into SDS-PAGE gels and transferred onto PVDF membrane (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk for 1 hr at room temperature and reacted with primary antibodies overnight at 4°C. The PVDF membranes were then incubated with a horseradish peroxidase-conjugated secondary antibody for 1 hr at room temperature. The protein band was developed using the enhanced chemiluminescence substrate.

| Caspase-3 activities assay (Colorimetric analysis)
Caspases-3 activities were analyzed using colorimetric assay kit provided by Cayman chemicals, and all experiments were preformed according to the manufacturer's instruction. Briefly, cells were pretreated with Cheongja#3 for 24 hr and further incubated in H 2 O 2 (500 μM) for 24 hr.
After harvesting the cells, cells were lysed with lysis buffer provided in the kit. After centrifuge, supernatant was reacted with 1 M DTT and 4 mM DEVD-p-NA substrate for 2 hr at 37°C. The absorbance was read at 405 nm using microplate reader (Spectramax M2e, Molecular devices).

| Statistical analysis
All experiments were performed in triplicate and expressed as mean ± SD. Data were analyzed using two-tailed unpaired student's t test and considered as significant when p value under 0.05.

| Fat-soluble micronutrients contents in three soybean extracts
In

| Antioxidant activities of Cheongja#3
We have evaluated free radical scavenging activities using DPPH assay in these three varieties of soybean extracts. It was found at 100 μg/ml compared to vehicle-treated cells) (Figure 1c). When treated with 1,000 μg/ml of Cheongja#3 extract, the ROS levels were similar to that of 100 μg/ml treatment in HaCaT cells (data not shown), suggesting enough concentration of antioxidant activities. From these results, we can conclude that the Cheongja#3 extract has strong antioxidant activities than the other varieties.

| Protective effects of Cheongja#3 on oxidative stress-induced apoptosis
Cheongja#3 extract had no cytotoxicity in the tested dose range and JNK protein expression, whereas phosphorylated ERK 1/2 protein levels were not affected by Cheongja#3 extracts (Figure 3c).

| Activation of Nrf-2-mediated HO-1 by Cheongja#3
We further examined the mechanism of how Cheongja#3 extract attenuates H 2 O 2 -induced apoptosis. Nrf-2 protein expressions were increased in Cheongja#3-treated cells in both total cell lysate ( Figure 4a) and nucleus fraction of cells (Figure 4b). In addition, HO-1, which is a gene downstream of Nrf2, was also increased by treatment of Cheongja#3 ( Figure 4b). As expected, H 2 O 2 slightly increased Nrf2 protein expression. We confirmed further increases of Nrf2 and HO-1 protein expressions in Cheongja#3-treated cells ( Figure 4c).  (Jeon et al., 2015; and neuroprotective effects (Bhuiyan et al., 2012;Kim, Chung, et al., 2012). Additionally, γ-tocopherol and lutein have been identified in Cheongja#3 extracts by UPLC. Tocopherols and carotenoids are well known for their antioxidant functions in prevention of various chronic diseases (Agarwal & Rao, 2000;Valentin & Qi, 2005). We believe that these fat-soluble micronutrients in Cheongja#3 create a synergistic effect with the antioxidant functions of black soybeans in Cheongja#3 soybean extract than that is not found in other soybean varieties (Lee, Park, et al., 2015).

| D ISCUSS I ON
Many scientific literatures have reported that oxidative stress certainly can induce apoptosis in cells (Simon, Haj-Yehia, & Levi-Schaffer, 2000). Upon initiating apoptosis, cytochrome C is released from mitochondria and triggers cleavage of caspases (Bergmann, Yang, & Srivastava, 2003 the major cell constituents of the epidermis in human and mice (Li et al., 2018;Zhang et al., 2017). Current study demonstrated that Cheongja#3, which is rich in antioxidants, and had a protective effect on oxidative stress, suggesting a promising functional food against skin diseases.
In summary, we demonstrated that Cheongja#3 extract had γ-tocopherol and lutein in addition to anthocyanin, which is F I G U R E 4 Effects of Cheongja#3 on antioxidant Nrf2 protein levels. (a) Cells were treated with 10, 100 μg/ml of Cheongja#3 for 24 hr. Nrf2 protein levels were assessed in total cell lysate using Western blotting (b). Nrf2 and HO-1 protein levels were analyzed in the nucleus cell lysate using Western blotting (c) Cells were treated with Cheongja#3 extracts. Afterward medium containing extract was removed and then treated with H 2 O 2 for another 24 hr. The Nrf2 and HO-1 protein levels were determined by Western blotting Actin well-studied previously. Cheongja#3 enhanced free radical scavenging activities and reduced intracellular ROS levels. It had protective effects on oxidative stress-induced apoptosis by attenuating cleaved caspases and phosphorylated JNK and p38.
Furthermore, they increased nucleus Nrf2 protein levels, thus proving its mechanism of antioxidant activities. Collectively, Cheongja#3 black soybean has biological function against oxidative stress in human keratinocytes.

ACK N OWLED G M ENTS
This work was supported by Rural Development Administration, Republic of Korea (PJ011885).

E TH I C A L S TATEM ENT
This work does not involve any human or animal studies.

CO N FLI C T O F I NTE R E S T
None declared.