Steroidogenic Effects of Korean Rice Bran Extract

Original Article DOI: 10.31083/jomh.v16i4.282

STEROIDOGENIC EFFECTS OF KOREAN RICE BRAN EXTRACT ON MOUSE NORMAL LEYDIG CELLS VIA MODULATION OF STEROIDOGENESIS-RELATED ENZYMES Hyun Joo Chung1,2,*, Jin-Wook Kim1, Chae-Eun Lee1,2, Soon Chul Myung1,2 1Department of Urology, Chung-Ang University College of Medicine, Seoul, Republic of Korea 2Bio-Integration Research Center for Nutra-Pharmaceutical Epigenetics, Chung-Ang University, Seoul, Republic of Korea *Corresponding Author: Soon Chul Myung: [email protected]

Submitted: 16 June 2020. Accepted: 15 September 2020. Published: 13 October 2020.

ABSTRACT Background and objective Many plant extracts have various effects on diseases. In particular, some medicinal plants have been shown to have steroidogenic effects in men. However, the effects of Korean rice bran extract (RBE) on steroidogenesis in men have not been reported previously. Accordingly, in this study, we investigated the effects of RBE (Oryza sativa L.) on mouse normal Leydig cells (TM3), which produce testosterone with increasing levels of key enzymes (e.g., StAR, CYP11A1, and CYP17A1) for male steroidogenesis. Material and methods Using mouse normal Leydig cells (TM3), we determined whether RBE had steroidogenic effects by eval- uating changes in the levels of steroidogenesis-related enzymes, such as StAR (steroidogenic acute regulatory protein), CYP11A1, and CYP17A1, using quantitative real-time PCR, western blotting analysis, and ELISA method. Results RBE significantly enhanced mRNA and protein levels of StAR, CYP11A1, and CYP17A1, thereby enhanc- ing synthetic testosterone levels in mouse Leydig TM3 cell supernatants. These findings indicated that RBE increased the levels of steroidogenic enzymes to modulate steroidogenesis.

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Conclusion RBE enhanced mRNA and protein levels of key enzymes including StAR, CYP11A1, and CYP17A1 for male steroidogenesis. These changes could induce testosterone production by TM3 cells in vitro. Thus, RBE may be used as a food additive or medicinal plant for the treatment of diseases characterized by insufficient testosterone (e.g., late-onset hypogonadism). Keywords: Korean rice bran extract; Oryza sativa L.; medicinal plant; steroidogenesis; late-onset hypogonadism

INTRODUCTION decreases in testosterone. However, oral adminis- Testosterone is largely synthesized from Leydig tration of testosterone or direct injection of artificial cells found in testis. Firstly, cholesterol is used as testosterone may result in side effects and it is not substrate for steroidogenesis.1 In aging men, the generally considered highly sustainable as a general levels of blood testosterone decrease owing to the healthcare measure.7 Thus, we have attempted the reduced ability of Leydig cells to produce testoster- identification of natural plant compounds in food one.2 The rate-limiting step during testosterone bio- items and food supplements that reduce side effects synthesis is the process of transferring the substrate at least and increase testosterone biosynthesis in cholesterol from the outer mitochondrial membrane Leydig cells effectively.8 into the inner mitochondrial membrane, thereby ini- Oryza sativa L. has been used as a food addi- tiating the steroidogenesis.3 In previous studies, it tive and has anti-oxidative and antimicrobial effects was reported that StAR (steroidogenic acute regu- on mold and yeast.9 Moreover, administration of latory protein) plays critical roles during this step rice bran extract (RBE) in mice improves sleep effi- by facilitating mitochondrial cholesterol transfer ciency and sleep onset.10 In RBE, the most abundant and that StAR protein expression levels affect tes- phenolic compounds or polyphenols are found as tosterone production in Leydig cells, strongly.4 The plant metabolites in plant tissues.11 In vitro studies substrate cholesterol is metabolized by P450scc of plant extracts have shown that these preparations (cytochrome P450 cholesterol side-chain cleavage are promising because of their high antioxidant and enzyme) into pregnenolone which is further metab- antimicrobial activities. Rice bran phenolic extract olized by CYP11A1 and HSD3B in the mitochondria (RBPE) also protects the liver by inactivating the and CYP17A1 and HSD17B in the smooth endoplas- endotoxin/Toll-like receptor 4/nuclear factor-κB mic reticulum to produce testosterone.2 pathway and by repressing inflammatory responses; However, in aged Leydig cells, several cel- therefore, intake of RBPE or brown rice may be an lular changes, including reducing luteinizing effective approach for mitigating alcoholic liver hormone-stimulated cAMP production and down- injury.12 regulating StAR, the downstream steroidogenic Oryza sativa L. is consumed as common enzyme CYP11A1 (mitochondria), and CYP17A1 foodstuff by people in South Korea. We have been (smooth endoplasmic reticulum) have been shown interested in studying the testosterone-increasing to collectively decrease overall testosterone synthe- activity of this food, with a focus on the effects sis through the steroidogenic pathway.5,6 in TM3 mouse Leydig cells. We hypothesize that Therefore, a clinically strategic approach would RBE may have potential applications in alleviating be to block deterioration of natural aging-related late-onset hypogonadism (LOH) or spermatogenesis

e46 Steroidogenic effects of Korean rice bran extract in aging men. However, definitive reports of the assay kit (Cell Counting Kit-8; cat. no. CK04; steroidogenic capacity of RBE in vitro have not Dojindo Molecular Technologies, Inc., Rockville, IL, yet been published. Accordingly, in this study, we USA), according as the manufacturer’s protocols. All evaluated the steroidogenic effects of RBE in TM3 experiments were performed in triplicate. (mouse Leydig) cells through analyses of mRNA, protein, and testosterone levels. Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction TM3 cells were seeded in six-well plates MATERIALS AND METHODS (5.0×10 5 cells/well) and cultured for 24 h before Plant Materials treatment. The fresh medium was changed and they Fresh Korean RBE (Oryza sativa L.) was pur- were treated with or without RBE (0, 1, 10, 25, or chased from a local market in Korea. All samples 50 µg/mL). Total RNA was isolated from the cell were washed several times and dried in an oven pellets using an RNeasy mini kit (Qiagen, Germany) at 40ºC for 3 days. The plant materials were put according to the manufacturer’s instructions, cDNA through a grinder to yield fine powder. was made by a high-capacity cDNA reverse transcription kit (QuantiTect Reverse Transcription Preparation of Plant Extracts kit; Qiagen). Quantitative Real-Time Reverse For plant extract preparation, dry plant materi- Transcription Polymerase Chain Reaction (qRT- als were isolated twice by adding 10-fold volume of PCR) was performed with a Rotor-Gene SYBR water for 3 h by refluxing and then they were filtered Green PCR kit (Qiagen) on a Rotor-Gene Q PCR (Whatman, Little Chalfont, UK). After filtration, machine (Qiagen) with a two-step cycling protocol, aqueous extracts were concentrated under reduced as follows: the denaturation step, at 95°C for 5 s and pressure using a rotary vacuum evaporator (Eyela annealing/extension step at 60°C for 10 s (40 cycles). CCA-1111; Tokyo Rikakikai Co., Ltd., Tokyo, Japan) Cycle threshold (Ct) values were within 0.1 among and lyophilized (Bondiro; IlShin BioBase, Seoul, triplicates. Primers for qRT-PCR were designed to Korea) for obtaining powder. target StAR, CYP11A1, CYP17A1, and 18s rRNA and the results were quantified by the −ΔΔCt2 method TM3 Cell Culture after normalization to 18s rRNA expression as an TM3 (mouse Leydig) cells were purchased from internal control. The primer sequences were as ATCC (American Type Culture Collection; ATCC follows: No. CRL–1714; ATCC, Manassas, VA, USA) and they were cultured in DMEM/F12 medium contain- StAR-forward ing 2.5 mM l-glutamine, 0.5 mM sodium pyruvate, (5′-TCTCTAGTGTCTCCCACTGCATAGC-3′), 1.2 g/L sodium bicarbonate, 15 mM HEPES (92.5%), StAR-reverse 5% horse serum, and 2.5% fetal bovine serum, sup- (5′-TTAGCATCCCCTGTTCGAGCT-3′), plemented with antibiotics (100 U/mL penicillin and CYP11A1-forward 100 µg/mL streptomycin) at 37°C cell incubator. (5′-ACATGGCCAAGATGGTACAGTTG-3′), Every 2 days, the new medium was supplied. CYP11A1-reverse (5′-ACGAAGCACCAGGTCATTCAC-3′), TM3 Cell Viability Assay CYP17A1-forward In order to measure the effects of RBE on (5′-CTCCAGCCTGACAGACATTCTG-3′), TM3 cell viability, we used an 3-(4,5-dimethylthi- CYP17A1-reverse azol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (5′-TCTCCCACCGTGACAAGGAT-3′),

e47 Steroidogenic effects of Korean rice bran extract

18s rRNA-forward before experiment. The medium was discarded and (5′-GAGGCCCTGTAATTGGAATGAG-3′), and the cells were treated in RBE (0, 1, 10, 25, and 50 18s rRNA-reverse µg/mL) for another 48 h. TM3 cell supernatants (5′-GCAGCAACTTTAATATACGCTATTGG-3′). were frozen before use in experiments or the fresh cell supernatants were used directly to Enzyme- The experiments were done in triplicate. Linked Immunosorbent Assay method for detecting testosterone (ELISA). ELISA was performed using Western Blot Assay a Parameter Testosterone Assay kit (KGE010; R&D TM3 cell were reacted with cell lysis buffer Systems, Inc.) according to the manufacturer’s (Cell Signaling Technology, Danvers MA, USA instructions. Detection was carried out by measur- #9803). Protein amount is normalized with BCA ing the absorbance at 450 nm with microplate reader. protein assay kit (Thermo Scientific, Rockford, IL. All experiments were conducted in triplicate. USA, #23225). Next, 20 µg cell lysate was sepa- rated by 10% SDS-PAGE (SDS-polyacrylamide Statistical Analysis gel electrophoresis) and electro-transferred onto All experiments were repeated three times NC (nitrocellulose) membranes, as described else- and all data are presented as means ± standard where. The blotted membranes were then washed deviations. To identify significant differences (SD) in TBST(Tris-buffered saline: 20 mM Tris-HCl, pH between groups, we used one-way analysis of vari- 7.6, containing 137 mM NaCl and 0.05% [v/v] Tween ance followed by the Fisher’s least significant differ- 20), blocked with 5% skimmed milk for 1 h at room ence (LSD) multiple comparison test in GraphPad temperature with shaking, reacted with primary Prism 5 software (GraphPad, La Jolla, CA, USA). antibodies (1:1000) at 4°C for 16 h with shaking. Results with P values of less than 0.05 (*, p<0.05) The primary antibodies were used with anti-actin were considered statistically significant. (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-47778), anti-StAR (Abcam, USA; ab203193), anti-CYP11A1 (Abcam, USA; ab175408), and anti- RESULTS CYP17A1 (Abcam, USA; ab115022). Subsequently, RBE Did Not Affect Cell Viability in TM3 Cells the membranes were rinsed with TBST buffer sev- To examine whether RBE affected the viabil- eral times (10 min each) and then reacted with sec- ity of TM3 cells, they were incubated with varying ondary antibodies (1:5000) labeled with HRP at room concentrations of RBE (0, 1, 5, 10, 25, or 50 µg/mL) temperature for 1 h with shaking. The membranes for 24 h (Figure 1A) and 48 h (Figure 1B). MTT were washed with TBST buffer several times (10 min assays resulted that RBE did not significantly affect each) and the signals were detected by an ECL solu- cell viability (Figure 1). tion (enhanced chemiluminescence western blot kit: EzWestLumi plus; ATTO Corporation, Motoasakusa, RBE Induced StAR, CYP11A1, and CYP17A1 Tokyo, Japan) and quantified using an ImageQuant mRNA Expression of Steroidogenesis-Related LAS 4000 mini machine (GE Healthcare). Genes in TM3 Cells Next, RBE was treated with TM3 cells at dif- Enzyme-Linked Immunosorbent Assay Method for ferent concentrations for 24 h and changes were Detecting Testosterone assessed in mRNA levels by qRT-PCR. The ste- In order to detect synthetic testosterone con- roidogenesis-related genes StAR, CYP11A1, and tents in TM3 cell supernatants, at first TM3 cells CYP17A1 were upregulated by 18.46, 31.2, and were seeded in cell dishes and cultured for 24 h 30.8 times, respectively, following treatment with

e48 Steroidogenic effects of Korean rice bran extract

(A) (B) 180 24hr 160 48hr 140 140 120 120 100 100 80 80 60 60 40 40 abilty (Percent of Control)

abilty (Percent of Control) 20 20 Vi 0 0 Cell Cell Vi 01155110022555500 015102550 Korean Rice Bran Extract (ug/ml)Korean Rice Bran Extract (ug/ml)

FIGURE 1 Viability of TM3 (mouse Leydig) cells treated with RBE. TM3 Cells were treated with 0, 1, 5, 10, 25, and 50 μg/mL of RBE for 24 h (A) and 48 h (B). All experiments were conducted in triplicate.

1 µg/mL RBE (Figure 2A–2C), compared with with 1 μg/mL and 25 μg/mL RBE (Figure 4). These that in the control group. Thus, these findings indi- findings suggested that RBE enhanced testosterone cated that RBE activated steroidogenic enzymes levels directly involved in testosterone synthesis at specific concentrations in vitro and highlighted in vitro. the role of RBE in male steroidogenesis in mouse Leydig cells. DISCUSSION RBE Increased StAR, CYP11A1, and CYP17A1 Rice (Oryza sativa L.) is a staple food in Asian Protein Expression of Steroidogenesis-Related countries, including South Korea. Rice bran, as Genes in TM3 Cells the most abundant and valuable by-product of the TM3 cells were then treated with 0, 1, 10, 25, rice milling process, is a unique source of bioactive or 50 µg/mL RBE for 48 h and protein expression phytochemicals with potential applications in var- was assessed by western blotting. Similar to the ious fields. Indeed, RBE possesses large amounts qRT-PCR data, we found that StAR, CYP11A1, of phytochemicals which can be extracted and used and CYP17A1 protein levels were increased fol- in foods, cosmetics, and pharmaceuticals.13 These lowing treatment with RBE (Figure 3A–3D). Thus, include liposoluble compounds (such as γ-oryzanol, these findings confirmed that RBE activated the tocopherols, tocotrienols, and phytosterols) and steroidogenic enzymes STAR, CYP11A1, and phenolic compounds. Many secondary metabo- CYP17A1 in vitro and further supported that RBE lites are phenolic compounds that are widely dis- affected male steroidogenesis in mouse Leydig cells. tributed in plant tissues14 and have been shown to have multiple bioactivities, such as anti-oxidative, RBE Enhanced Testosterone Synthesis antimicrobial,9,15 antiviral,16 and anti-inflammatory in TM3 Cells properties.12,17 Next, we evaluated changes in the levels of tes- However, the mechanisms mediating the ste- tosterone produced following treatment with RBE roidogenic effects of RBE in TM3 (mouse Leydig) for 48 h in mouse Leydig cells. Testosterone levels cells have not been thoroughly assessed. In this were significantly increased following treatment study, we found that RBE increased testosterone

e49 Steroidogenic effects of Korean rice bran extract

(A) StAR (B) Cyp11a1 20 20

15 15

10 10

expression (fold) 5 expression (fold) 5

0 0 mRNA mRNA 0110 25 50 0110 25 50 Korean Rice Bran Extract (ug/ml_24h) Korean Rice Bran Extract (ug/ml_24h)

expression (fold) 10

0 mRNA 0110 25 50 Korean Rice Bran Extract (ug/ml_24h)

FIGURE 2 mRNA levels of StAR, Cyp11a1, and Cyp17a1 in TM3 (mouse Leydig) cells following treatment with RBE. TM3 cells were reacted with 0, 1, 10, 25, or 50 μg/mL RBE for 24 h, and qRT-PCR was conducted with steroidogenic genes-specific primers to detect StAR (A), Cyp11a1 (B), and Cyp17a1 (C). Three mRNA levels were normalized to the expression of the 18s rRNA gene. The experiments were done in triplicate.

biosynthesis directly in Leydig cells in vitro. was increased at 48 h after treatment with RBE in Furthermore, RBE treatment effectively increased mouse Leydig cells. Thus, StAR expression was StAR, CYP11A1, CYP17A1, mRNA, and protein activated by BRE in vitro. StAR forms a complex levels in mouse Leydig cells and enhanced testos- with cholesterol and the outer mitochondrial mem- terone production. brane cholesterol-binding translocator protein in the Steroidogenesis in mouse Leydig cells has been inner membrane of mitochondria in Leydig cells reg- reported to involve several enzymes.2 Luteinizing ulates downstream steroidogenic enzymes (HSD3B hormone (LH) and the LH receptor together form and CYP11A1) in the mitochondria and HSD17B, the Luteinizing Hormone/Choriogonadotropin HSD3B, and CYP17A1 enzymes in smooth endo- Receptor (LHCGR) complex in response to LH plasmic reticulum (SER). It is showed that StAR, stimulation. Then, the LHCGR complex stimulates CYP11A1, and CYP17A1 expression levels were cAMP for using cholesterol in the cytosol as a sub- increased at the mRNA and protein levels follow- strate for steroidogenesis, resulting in induction of ing treatment with RBE in mouse Leydig cells. StAR, a cholesterol transporter.2,5,18–20 In our study, Accordingly, these findings suggested that RBE it was confirmed that StAR mRNA expression was enhanced CYP11A1 and CYP17A1 expression in the increased in a concentration-dependent manner at mitochondria and smooth endoplasmic reticulum. 24 h after RBE treatment, whereas StAR protein Finally, we also found that RBE treatment for 48 h

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(A) (B) STAR 0110 25 50 ug/ml RBE 5 STAR 4 3 CYP11A1 2

CYP17A1 AR/ ß-ACTIN ) 1 (ST 0

ß-ACTIN Relative Protein Epxression 0110 25 50 Korean Rice Bran Extract (ug/ml)

) 8 3 6 2 4

1A1 / ß-ACTIN) 1 2

(CYP1 0 0 (CYP17A1 / ß-ACTIN Relative Protein Epxression

0110 25 50 Relative Protein Epxression 0110 25 50 Korean Rice Bran Extract (ug/ml) Korean Rice Bran Extract (ug/ml)

FIGURE 3 Protein levels of STAR, CYP11A1, and CYP17A1 in TM3 (mouse Leydig) cells following treatment with RBE. TM3 cells were incubated with 0, 1, 10, 25, or 50 μg/mL RBE for 48 h and western blotting analysis was performed for detection of StAR, CYP11A1, and CYP17A1 (A). Total protein was normalized with β-actin as internal control and the data were quantified (B–D). The experiments were performed in triplicate.

800

750 )

700

650

600 estosterone (pg/ml T 550

500 0110 25 50 Korean Rice Bran Extract (ug/ml)

FIGURE 4 Testosterone levels in TM3 cell supernatants. TM3 cells were treated with 0, 1, 10, 25, or 50 μg/mL RBE for 48 h. TM3 cell supernatants were obtained and subjected to ELISA method. *p < 0.05. The experiments were performed three times.

e51 Steroidogenic effects of Korean rice bran extract increased testosterone levels. Thus, our results con- steroidogenesis-related enzymes (i.e., StAR, CYP11A1, firmed the steroidogenic effects of RBE in mouse and CYP17A1), which facilitate the production of tes- Leydig cells. tosterone in TM3 mouse Leydig cells. Therefore, RBE The pharmacological effects of many plant might be used as a food additive or medicinal plant for extracts have been reported; however, this is the the treatment of diseases characterized by insufficient first study supporting the potential steroidogenic testosterone. In further studies, the clinical efficacy of effects of RBE as a medicinal plant in aging males. RBE will need to be investigated. However, the bioactivities of many plant extracts have not been defined well, and information about toxicity and adverse effects is insufficient. Thus, it REFERENCES will be necessary to evaluate the safety, efficacy, 1. Miller WL. Molecular biology of steroid hormone and optimal dose of this medicinal plant prior to its synthesis. Endocr Rev 1988;9:295–318. https://doi. applications in the clinical setting. Moreover, it will org/10.1210/edrv-9-3-295 be important to perform studies of QC (quality con- 2. Beattie MC, Adekola L, Papadopoulos V, et al. trol), standardization, and validation through vari- Leydig cell aging and hypogonadism. Exp ous methods. Gerontol 2015;68:87–91. https://doi.org/10.1016/j. In this study, it was found that RBE treatment exger.2015.02.014 increased the expression of steroidogenesis-related 3. Stocco DM, Clark BJ. Regulation of the acute pro- enzymes and promoted testosterone production in duction of steroids in steroidogenic cells. Endocr Rev Leydig cells. Thus, our findings suggested that RBE 1996;17:221–44. https://doi.org/10.1210/er.17.3.221 4. Stocco DM. StAR protein and the regulation of may be an alternative medicinal plant extract for steroid hormone biosynthesis. Annu Rev Physiol improvement of diseases characterized by insuf- 2001;63:193–213. https://doi.org/10.1146/annurev. ficient testosterone (i.e., male infertility, LOH and physiol.63.1.193 male hypogonadism). Future studies are needed to 5. Culty M, Luo L, Yao ZX, et al. Cholesterol trans- improve its clinical efficacy. port, peripheral benzodiazepine receptor, and steroidogenesis in aging leydig cells. J Androl 2002;23:439–47. FUNDING 6. Decaroli MC, Rochira V. Aging and sex hormones This work was supported by the Korea Institute in males. Virulence 2017;8:545–70. https://doi.org/1 of Planning and Evaluation for Technology in 0.1080/21505594.2016.1259053 Food, Agriculture and Forestry (IPET) through 7. Grober ED. Testosterone deficiency and replace- Technology Commercialization Support Program, ment: Myths and realities. Can Urol Assoc J funded by Ministry of Agriculture, Food and Rural 2014;8(7–8 Suppl 5):S145–7. https://doi.org/10.5489/ cuaj.2309 Affairs (MAFRA) (grant no. 817021-3). 8. Chung HJ, Noh Y, Kim MS, et al. Steroidogenic This work was also supported by Basic effects of Taraxacum officinale extract on the lev- Science Research Program through the National els of steroidogenic enzymes in mouse leydig cells. Research Foundation of Korea (NRF) (grant no. Animal Cells Syst 2018;22:407–14. https://doi.org/1 NRF-2018R1D1A1B07049673). 0.1080/19768354.2018.1494628 9. Martillanes S, Rocha-Pimienta J, Gil MV, et al. CONCLUSION Antioxidant and antimicrobial evaluation of rice bran (Oryza sativa L.) extracts in a mayonnaise-type In summary, we found that RBE treat- emulsion. Food Chem 2020;308:125633. https://doi. ment enhanced the mRNA and protein levels of org/10.1016/j.foodchem.2019.125633

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