Protective effect of Lespedeza cuneata ethanol extract on Bisphenol A-induced testicular dysfunction in vivo and in vitro

Biomedicine & Pharmacotherapy 102 (2018) 76–85

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Protective effect of Lespedeza cuneata ethanol extract on Bisphenol A-induced testicular dysfunction in vivo and in vitro

T

Bongkyun Parka, Jeong Eun Kwona, Se Min Choa, Chang Won Kima, Da Eun Leea, ⁎ Young Tae Koob, Sang Hun Leeb, Hyun Myung Leea, Se Chan Kanga, a b

Department of Oriental Medicine Biotechnology, Kyung Hee University, Yongin 17104, Republic of Korea Kwang-Dong Pharmaceutical Co., Ltd., Seoul 06650, Republic of Korea

A R T I C LE I N FO

A B S T R A C T

Keywords: Lespedeza cuneata BPA-induced testicular dysfunction Anti-oxidation Anti-apoptosis Prevention testicular dysfunction

Purpose: Bisphenol A (BPA) has been regarded as a possible risk factor for reproductive health. We examined potential reproductive health benefits of Lespedeza cuneata ethanol extract (LCE). Previously, Lespedeza cuneata showed many therapeutic effects. However, the protective effect of LCE on BPA-induced testicular dysfunction and its mechanisms have not been precisely studied. Methods: Mice were randomly divided into six groups (n = 7). Sperm counts and motility were measured by light microscope. Testosterone, total cholesterol, triglycerides, HDL, LDL-cholesterol, glucose, free fatty acids, hs-CRP, Angiotensinogen, Angiotensin II, GOT, GPT, TBARS, GSH, CAT, and SOD1 were measured in mouse serum. The potential protective effects of the LCE on mouse sertoli cells were evaluated. Results: Oral administration of LCE in BPA-exposed male mice restored testis weight, sperm count, motility, and testosterone levels by inhibiting markers in serum. In addition, treatment with LCE in BPA-treated TM4 sertoli cells recovered cell viability by attenuating Bax expression and activating caspase 3 and PARP. Conclusions: These results indicate that LCE prevented BPA-induced testicular dysfunction and cell viability in BPA-treated TM4 sertoli cells. Our study also suggests that LCE has the potential to protect male reproductive health.

1. Introduction Bisphenol A (BPA) is a high production volume chemical used in the manufacture of polycarbonate plastic and is widely used as a dental sealant and as a coating for water pipe walls, food packaging, and plastic bottles [1,2]. Over 8 billion pounds of BPA are produced annually worldwide, with a 6–10% growth rate per year; about 100 tons of BPA are released into the atmosphere every year [3,4]. BPA has estrogen-like activity that changes endocrine function through various pathways, and many previous studies have shown that BPA exposure is associated with damage to sperm DNA and a reduction in semen quality among men from an infertility clinic [5]. In addition, increased circulating levels of BPA are associated with coronary heart disease (CHD) as well as many cardiovascular risk factors such as hypertension, obesity, and diabetes, and metabolic syndrome [6–9]. There is also a study indicating that exposure to BPA in the coronary artery could lead to

atherosclerosis [10]. Increasing evidence demonstrates that BPA-induced damage is related to oxidative stress [11–14]. A recent study showed BPA-induced biochemical toxicity in mouse testicular mitochondria to be a function of oxidative stress, and found that several antioxidants can prevent against BPA-induced toxicity [15]. Lespedeza cuneata is an aggressive, warm-season perennial legume which was introduced from Asia for use in hay erosion, foraging of poor soils, and regulation of erosion along roadsides [16,17]. L. cuneata grows on exposed ground and grassy lowlands and has been used as a traditional herbal medicine for asthma, abscesses, breast cancer, and protection of liver and kidney function [18] Several previous studies have suggested that it has therapeutic effects on diabetes, low stamina, and amblyopia [19]. Its bioactive substances include β-sitosterol, quercetin, kaempferol, pinitol, avicularin, juglanin and trifolin, and others [20]. The roots and leaves of L. cuneata also have minerals, amino acids, vitamins, and flavonoids, indicating anti-oxidative and

Abbreviations: LCE, Lespedeza cuneata ethanol extract; BPA, bisphenol A; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; PARP, Poly (ADP-ribose) polymerase; hs-CRP, highsensitivity C-reactive protein; GOT, glutamic oxaloacetic transaminase; GPT, glutamic pyruvic transaminase; TBARS, Thiobarbituric acid reactive substances; CAT, catalase; SOD1, superoxide dismutase 1; HDL-cholesterol, high density lipoprotein; LDL-cholesterol, low density lipoprotein; FFA, free fatty acid ⁎ Corresponding author at: Department of Oriental Medicine Biotechnology, Kyung Hee University, 1732, Deogyeong-daero, Giheung-gu, Yongin-si, Gyeonggi-do, 17104, Republic of Korea. E-mail address: [email protected] (S.C. Kang). https://doi.org/10.1016/j.biopha.2018.03.045 Received 28 September 2017; Received in revised form 9 March 2018; Accepted 9 March 2018 0753-3322/ © 2018 Published by Elsevier Masson SAS.

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2.3. Sperm counts and motility

anti-inflammatory effects [17,21,22]. The ethanol extract of L. cuneata inhibited skin aging and photo-aging in UVB-induced hairless mice through antioxidant activity [23]. However, the effect of L. cuneata ethanol extract (LCE) on BPA-induced oxidative stress and apoptosis in mouse sperm quality and sertoli cells has not been demonstrated. In this study, we examined the effect of L. cuneata ethanol extract (LCE) on BPA-induced oxidative stress and apoptosis in vivo and in vitro. The results showed that BPA decreased sperm quality and induced oxidative stress, hyperlipidemia, and hypertension in vivo. In addition, BPA induced apoptosis in vitro by increasing expression of Bax, cleaved caspase 3, and PARP, which can cause cell death. However, this situation was inhibited by treatment with LCE. LCE prevented changes in sperm quality and sertoli cell growth by attenuating BPA-induced oxidative stress, hyperlipidemia markers, hypertension markers, and apoptosis. Therefore, these data might help to elucidate the effect of L. cuneata on BPA-induced reproductive toxicity in vitro and in vivo.

The right epididymis of each animal was isolated and cleared. The cauda epididymides were chopped into small pieces and incubated for 30 min in DMEM medium at 37 °C. Sperm cells were counted as previously described [24]. In brief, 10 μl of epididymal sperm was diluted with 390 μl of DMEM. Approximately 10 μl of the diluted sperm suspension was transferred to each counting chamber of the hemocytometer, and sperm numbers were counted under a light microscope at 200×. The sperm count was expressed as values ×107 per gram of cauda epididymides. Sperm were taken, placed on a pre-cleaned and warmed (at 37 °C) glass slide, and observed under a light microscope at 40× for motility and any abnormalities. A total of 100 sperm/sample were analyzed for motility and abnormalities by a technician blinded to the treatment groups. Each sample was analyzed three times and the average value was used. 2.4. Serum analysis

2. Material and methods

At sacrifice, samples of whole blood were collected by cardiac puncture, and blood was allowed to clot for 30 min. Serum was then separated via centrifugation at 1500 g for 10 min. Markers associated with testicular reproduction present in the serum were measured using enzyme linked immunosorbent assay (ELISA) kits for testosterone, total cholesterol, triglycerides, HDL, LDL-cholesterol, glucose, free fatty acids, hs-CRP, Angiotensinogen, Angiotensin II, GOT, GPT, TBARS, GSH, CAT, and SOD1 (Bio legend, Inc., San Diego, CA). All ELISA procedures were performed according to the manufacturers’ protocols.

2.1. Reagents Unless otherwise indicated, all chemicals used in this research were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). DC protein assay was obtained from Thermo Fisher Scientific (Waltham, MA). Antibodies against Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (BCL-2), caspase 3 and Poly (ADP-ribose) polymerase (PARP) were purchased from Cell Signaling Technology (Beverly, MA). Enhanced chemiluminescence (ECL) was purchased from Thermo Fisher Scientific (Waltham, MA). Mouse hs-CRP, Angiotensinogen, Angiotensin II, GOT, GPT, TBARS, GSH, CAT, and SOD1 Enzyme-linked immunoassay (ELISA) MAX™ standards were obtained from Bio legend, Inc. (San Diego, CA). Lespedeza cuneata was extracted with the various concentrations of ethanol (0∼100%) at room temperature for 24 h in a shaker, Dried berries from Saw Palmetto (Serenoa repens) were milled and extracted with 100% ethanol. The ethanol was removed under a vacuum to create an extract, and the filtered extracts were concentrated and powdered under reduced pressure. The powder was lyophilized and stored at 4 °C.

2.5. Cell culture and cell viability assay Mouse sertoli cell TM4 cells were purchased from the American Type Culture Collection (VA, USA) and incubated with high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C with a 5% CO2 atmosphere in a humidified incubator. The effects of LCE on cell viability were determined by the MTT colorimetric method. Cells (1 × 104 cells/well) were plated in a 96-well plate and treated with LCE or BPA for 24 h. At the end of treatment, 100 μl MTT solution (5 mg/ml) was added and incubated for another 4 h. The medium was removed and 100 μl dimethyl sulfoxide (DMSO) was added to dissolve the insoluble formazan. Absorbance was measured at 550 nm using an Infinite-M200 spectrophotometer (Tecan, Männedorf, Switzerland).

2.2. Animals and treatment Animal experiments were conducted in accordance with the current ethical regulations for animal care and use at Kyung Hee University (KHUASP(SE)-16-014). Forty-two ICR mice, clean-grade healthy, male, 8 weeks old, weighing 34–36 g were purchased from Korea Laboratory Animal Co. (Daejeon, Korea) and were housed for 7 days prior to the experiment. They were housed in solid-bottomed plastic cages designed to allow easy access to standard laboratory food and water. Mice were kept in sanitary ventilated animal rooms with a controlled temperature (25 ± 1 °C) and regular light cycle (12 h light: 12 h dark). The mice were randomly divided into six groups: the solvent control group (CTR); the 10 mg/kg BPA treated group; the 10 mg/kg BPA + 100mg/kg SPE (Saw Palmetto extract) group; and the 10 mg/kg BPA + 25, 50 and 100 mg/kg LCE (Lespedeza cuneata ethanol extract) group. Mice in the BPA + LCE groups were given LCE orally 30 min before intraperitoneal administration of BPA. At the same time, mice from the solvent-control and BPA treated groups were injected with distilled water in normal saline not containing LCE. After 30 min, mice in the BPA-treated group and the BPA + SPE and LCE groups were treated with BPA diluted in corn oil by gavage. All mice received their respective treatment for 12 weeks. 24 h after the last treatment, mice from all groups were anesthetized by CO2. The testes and epididymides were quickly removed and weighed. The testicular index calculated testicular weight to body weight. The right epididymides were separated for sperm quality, and the left testes were divided for RNA isolation.

2.6. Western blot analysis TM4 cells were seeded in 6-well plates (1.5 × 106 cells/well) and co-treated with LCE (50, 100 of 200 μg/ml) and BPA (300 μM) for 24 h. The cells were washed with PBS and lysed with RIPA lysis buffer (Invitrogen, CA, USA). Cell lysates (30 μg) were separated with a 10% SDS-PAGE gel and transferred to nitrocellulose membranes. The membranes were blocked in skim milk dissolved in TBST buffer for 1 h and then incubated with primary antibodies overnight at 4 °C. The membranes were washed five times with TBST buffer and incubated in 5% skim milk/TBST with secondary antibody for 2 h at room temperature. Target proteins were visualized using an enhanced chemiluminescence method and ImageSaver6 software. 2.7. UPLC and mass spectrometry analysis Chromatographic analysis was performed on a Waters Acquity IClass UPLC system (Waters Corporation, Milford, MA, USA). The separation was performed on a Waters Acquity UPLC®BEH C18 (2.1 × 100 mm, 1.7 μm) column. The mobile phase consisted of (A) 0.1% formic acid in purified water and (B) acetonitrile. The linear gradient elution was optimized as follows: 10–15% B (0–14 min), 77

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and 15% for each dose of LCE compared to the BPA-only group. Unlike sperm motility, sperm abnormalities increased about 84% in the BPAonly group compared to the control group. However, oral administration of LCE at 25, 50, and 100 mg/kg markedly attenuated the sperm abnormalities by 12%, 19%, and 23.8%, respectively, compared to the BPA-only group. LCE dose at 50 mg/kg had the most protective effect on BPA-decreased sperm count and motility compared to SPE. Previous studies have reported that testosterone levels are an indicator of testes function, as testosterone is important for spermatogenesis in sertoli cells [25,26]. The present data showed that the testosterone level in BPAonly treated group was about 23.1% lower than the control group, while oral-administration of LCE increased the testosterone levels by 14.4, 28.1, and 7%, by dose, compared to the BPA-only group.

15–15% B (14–17 min), 15–95% B (17–22 min), 95–95% B (22–26 min). The flow rate was 0.4 ml/min. The column and autosampler were maintained at 30 and 10 °C, respectively. The monitoring UV wavelength was set at 254 nm. Mass spectrometry was performed on a Waters Vion™ IMS-QTof mass spectrometer (Waters Corporation, Milford, MA, USA). Ionization was achieved using electrospray in the positive mode (ESI+). The desolvation gas was set to 800 l/h at 350 °C, the cone gas set to 50 l/h, the source temperature set to 150 °C. The cone voltage was set to 40 V. The energies for collision-induced dissociation (CID) were set at 6 and 20–40 eV respectively for fragmentation information. 2.8. Statistical analyses Representative data from three independent experiments are presented as means ± standard error of the mean (SEM). Comparisons between control and experimental values were calculated by two-way ANOVA. Analyses were performed with Graphpad Prism 5. Statistical significance was defined as p < 0.05.

3.2. Effect of LCE on the level of oxidative and anti-oxidative stress enzymes in BPA-stimulated male mice To determine whether LCE inhibited oxidative stress enzyme in BPAstimulated male mice, male mice were orally administered 25, 50, or 100 mg/kg of LCE and a positive control, SPE. An ELISA assay showed that TBARS, an oxidative stress enzyme, was significantly increased by 59% in the BPA-only group compared to the control group. However, TBARS was markedly inhibited in a dose dependent manner by LCE (Fig. 2A). Oral administration of LCE at 50 and 100 mg/kg was also more effective in comparison with treatment SPE. The levels of the anti-oxidative enzymes GSH, CAT, and SOD1 were decreased in the BPA-only treated group compared to the control group. However, serum levels of GSH, CAT, and SOD1 were significantly restored by oral administration of LCE at 25, 50, and 100 mg/kg as much as SPE treatment, suggesting the potential efficacy of LCE in combating oxidative stress (Fig. 2B–D).

3. Results 3.1. Effect of LCE on body and testis weight gain and testicular sperm quality in BPA-stimulated male mice We investigated whether LCE affected the restoration of body and testis weight as well as testicular sperm quality in BPA-stimulated male mice. Table 1 demonstrated that there was no difference in the initial body weight between groups (P > 0.05). At the end of the experiment, the final body weight was significantly increased in the BPA-only group. Moreover, oral administration of LCE did not affect changes in final body weight compared to the BPA-only group. The comparative testis weight was also about 12.0% lower in the BPA-only group compared to the control group. However, oral administration of LCE at 50 and 100 mg/kg and of SPE, a positive control, restored the relative testis weights to levels similar to these seen in the control group. In addition, induction of BPA significantly reduced testicular index in BPA-treated group, compared to CTR group. Treatment of LCE (50 and 100 mg/kg, p.o.) and SPE 100 mg/kg reasonably increased BPA-decreased testicular index, compared to BPA-induced group. Based on these data, we examined whether LCE regulated the sperm quality and level of testosterone in BPA-stimulated male mice. As shown in Table 2, the BPA-only group had 21% lower sperm counts than the control group. Oral administration of each dose of LCE and SPE remarkably increased the sperm count 26%, 27%, 8%, and 18%, respectively, compared to the BPA-only group. In addition, we investigated sperm motility, abnormalities, and morphology in the epididymal sperm of control and experimental animals. Sperm motility and morphology were reduced about 40% in the BPA-only group (Table 2 and Fig. 1). However, sperm motility was significantly restored by about 24%, 39%,

3.3. Effect of LCE on lipid metabolism and glucose homeostasis in BPAstimulated male mice Changes in serum parameters among the six groups are shown in Table 3. The levels of total cholesterol (TC), triglycerides (TG), LDLcholesterol, glucose, and free fatty acids (FFA) were significantly increased and the level of HDL-cholesterol was markedly decreased in the BPA-only group compared to the control group. Oral administration of LCE at 25, 50, and 100 mg/kg and SPE significantly suppressed the concentration of glucose about 11%, 24%, 25%, and 17%, respectively, compared to the BPA-only treated group. Moreover, TC, TG, and LDLcholesterol were significantly inhibited in a dose dependent manner of LCE. Oral administration of LCE also restored the BPA-decreased levels of HDL-cholesterol and FFA in a dose-dependent manner. Taken together, these findings suggest that LCE restored sperm quality and testosterone levels by decreasing the levels of glucose, TG, TC, and LDLcholesterol while improving the levels of HDL-cholesterol and FFA. In addition, LCE shows a tendency to be more effective than SPE to regulate lipid metabolism and glucose homeostasis.

Table 1 Effect of LCW on body and testis weight in BPA-stimulated mice. Body weight (g) Initial CTR BPA (10 mg/kg) BPA + SPE (100 mg/kg) BPA + LCE (25 mg/kg) BPA + LCE (50 mg/kg) BPA + LCE (100 mg/kg)

34.5 35.9 35.8 35.4 35.7 34.4

± ± ± ± ± ±

Testis weight (g/kg body weight)

Testicular Index x 10−3

4.5 4.0 4.5 3.9 4.2 4.3

1.271 1.103 1.192 0.997 1.198 1.211

Final 0.8 1.0 0.6 0.6 0.5 0.4

35.3 38.7 38.5 39.8 39.6 39.7

± ± ± ± ± ±

0.6 1.2# 0.9 0.4 0.9 0.6

± ± ± ± ± ±

0.8 1.1# 1.2* 1.0 0.5* 1.3*

Values are shown as means ± S.E.M. n = 7 for all groups. Data are the means ± S.E.M representative of three independent experiments. # P < 0.05, significantly different from the control group. * P < 0.05, significantly different from the BPA group.

78

± ± ± ± ± ±

0.211 0.152# 0.185* 0.241 0.102* 0.231*

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Table 2 Effect of LCE on testosterone levels and sperm count, motility, and abnormalities in BPA-stimulated mice.

CTR BPA (10 mg/kg) BPA + SPE (100 mg/kg) BPA + LCE (25 mg/kg) BPA + LCE (50 mg/kg) BPA + LCE (100 mg/kg)

Sperm count (106/ml)

Sperm motility (%)

Abnormal sperm (%)

Testosterone (mg/dL)

101.2 ± 2.5 80.1 ± 3.4# 98.2 ± 2.7* 107.7 ± 3.4* 108.3 ± 2.6* 86.2 ± 4.7*

72.1 43.5 69.8 57.4 71.8 51.3

13.2 84.9 56.7 74.2 68.4 64.7

1.08 0.83 5.31 0.97 2.95 0.89

± ± ± ± ± ±

3.5 2.0# 2.3* 1.9* 2.0* 3.9*

± ± ± ± ± ±

1.6 4.7# 3.5* 3.9* 2.6* 3.5*

± ± ± ± ± ±

0.2 0.1# 1.1* 0.1* 1.1* 0.02*

Values are shown as means ± S.E.M. n = 7 for all groups. Data are the means ± S.E.M representative of three independent experiments. # P < 0.05, significantly different from the control group. * P < 0.05, significantly different from the BPA group.

extract) for 24 h. LCE was cytotoxic to TM4 cells at a concentration exceeding 200 μg/ml (Fig. 3A). Next, we examined the protective effect of various ethanol percentages of LCE against BPA-induced cell cytotoxicity. As shown in Fig. 3B, 24h co-treatment of TM4 cells with BPA and 30% ethanol-extracted LCE significantly restored the BPA-decreased cell viability compared to BPA-only treated TM4 cells; this effect was dependent on the concentration of LCE. In addition, we investigated the effect of different concentrations (0–200 μg/ml) of 30% ethanol-extracted LCE on restoring cell viability in BPA-only treated TM4 cells. An MTT assay showed that BPA-decreased cell viability was markedly increased after treatment with 100 and 200 μg/ml of 30% ethanol-extracted LCE, when compared to BPA-only treated TM4 cells (Fig. 3C). In the following experiments, TM4 cells were treated with 50, 100, and 200 μg/ml of LCE. Various apoptosis pathways are involved in BPA-induced testis cell death including the intrinsic mitochondrial pathway, endoplasmic reticulum stress pathway, and the extrinsic pathway triggered through death receptors [29,30]. The intrinsic mitochondrial pathway may be activated by BPA, resulting in increased expression of Bax, which can inhibit Bcl-2 expression. Activation of caspase 3 and Poly (ADP-ribose)

3.4. Effect of LCE on hypertension and liver function in BPA-stimulated male mice It is widely acknowledged that the levels of TC, TG, LDL-cholesterol, and glucose are increased in BPA-damaged coronary arteries and livers, leading to hypertension and abnormal liver function [27,28]. We investigated whether LCE attenuates hypertension and liver function-related genes using ELISA. As shown in Table 4, LCE inhibited the production of High-sensitivity C-reactive protein (hs-CRP), angiotensinogen, angiotensin II, glutamate oxaloacetate transaminase (GOT), and glutamate pyruvate transaminase (GPT) in a dose dependent manner, indicating the possible efficacy on LCE in improving hypertension and liver function. Moreover, the doses of LCE (50 and 100 mg/kg) was as more effective as SPE (100 mg/kg), used as a positive control. 3.5. Protective effect of LCE on BPA-induced apoptotic cell death in TM4 sertoli cells We examined the effect of LCE on cytotoxicity in sertoli cells using an MTT assay at the indicated LCE concentration (100% ethanol

Fig. 1. Effect of LCE on epididymal sperm morphology in BPA-stimulated mice. (A) Control group; (B) mice treated with BPA (10 mg/kg); (C) mice cotreated with BPA (10 mg/kg) and SPE (100 mg/kg); (D) mice co-treated with BPA (10 mg/kg) and LCE (25 mg/kg); (E) mice co-treated with BPA (10 mg/ kg) and LCE (50 mg/kg); and (F) mice co-treated with BPA (10 mg/kg) and LCE (100 mg/kg). The tailless black arrow indicates an abnormality in the spermatozoon and the black arrow indicates a normal spermatozoon. Photomicrograph (×400) showed that more frequent abnormal tails were reduced and total sperm density was restored.

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Fig. 2. Effect of LCE on oxidative stress in BPA-stimulated mice. (A) TBARS, (B) GSH, (C) CAT, and (D) SOD1 were quantified with serum murine ELISA assays. Data are the means ± S.E.M from three independent experiments. # P < 0.05, significantly different from the control male mice group. * P < 0.05, significantly different from the BPA-induced male mice group.

compound in the chromatograms that have been identified based on retention times, parent ion [M + H]+, MS/MS fragmentation pattern, and calculated molecular formula of each peak, and by matching above data with that in related references and database (UNIFI Library, ChemSpider, and Massbank). Peak 1, at m/z 595.1655 [M + H]+, was assigned to vicenin 2 since the MS2 fragmentation pattern gave fragments at m/z 577.1558 [(M + H)-18]+, 457.1147 [(M + H)-120-18]+, 379.0814 [(M + H)2(18)-2(30)-120]+, and 325.0717 [(M + H)-2(120)-30]+ [32]. Peak 7, at m/z 611.1607 [M + H]+, was assigned to rutin since the 2 MS fragmentation pattern gave fragments at m/z 465.1040 [(M + H)146(Rha)]+ and 303.0492 [(M + H)-146(Rha)-162(Glc)]+ [33]. Peaks 4 (m/z 595.1662 [M + H]+), 5 (m/z 565.1556 [M + H]+), and 8 (m/z 579.1709 [M + H]+) yielded the similar MS2 productions with vitexin (m/z 433, 415, 397, 379, 367, 337, 313, 283) [34], it was then tentatively proposed as vitexin hexose (peak 4, m/z 433.1133

polymerase (PARP) was also induced by BPA, and suppression of each activity attenuated BPA-induced cell death [31]. We investigated whether LCE had the ability to inhibit BPA-induced apoptosis in TM4 cells. BPA increased apoptotic cell death in TM4 cells by increasing expression of BAX and activating caspase 3 and PARP. However, cotreatment with BPA and LCE significantly inhibited expression of Bax and activation of caspase 3 and PARP and restored Bcl-2 expression proportional to the LCE concentration, compared to BPA-treated cells. These findings indicated that LCE had a protective effect against the BPA-induced intrinsic pathway of apoptosis in TM4 cells (Fig. 4). 3.6. Constituents analysis We analyzed the chemical profile from LCE using UPLC-Qtof-MS, there were five compounds (Vicenin 2, Vitexin hexose, Vitexin pentose, Rutin, and Vitexin methyl-pentose) found in the LCE. Table 5 shows 5Table 3 Effect of LCE on serum lipid levels in BPA-stimulated mice.

CTR BPA (10 mg/kg) BPA + SPE (100 mg/kg) BPA + LCE (25 mg/kg) BPA + LCE (50 mg/kg) BPA + LCE (100 mg/kg)

Total-cholesterol (mg/dL)

Triglycerides (mg/dL)

HDL-cholesterol (mg/dL)

LDL-cholesterol (mg/dL)

Glucose (mg/dL)

FFA (mEq/L)

183.2 170.5 191.4 164.8 153.3 149.7

61.1 ± 1.5 129.6 ± 8.3# 104 ± 2.5* 115.4 ± 10.5 104.5 ± 4.5* 88 ± 6.9*

100.2 ± 1.8 73.4 ± 1.9# 103 ± 1.9* 82.75 ± 2.3* 128.8 ± 8.9* 130.9 ± 11.1*

10 ± 0.5 13 ± 1.1# 10.7 ± 0.2* 10 ± 0.8* 10 ± 0.2* 9 ± 1.3*

68 ± 3.8 138 ± 5.1# 114 ± 3.1* 123 ± 9.9* 105 ± 9.5* 103 ± 6.8*

2.6 ± 0.01 3.47 ± 0.02# 2.22 ± 0.11* 2.58 ± 0.11* 2.60 ± 0.06* 2.74 ± 0.05*

± ± ± ± ± ±

2.2 6.2 2.7 5.8 3.0* 11.1*

Values are shown as means ± S.E.M. n = 7 for all groups. Data are the means ± S.E.M representative of three independent experiments. # P < 0.05, significantly different from the control group. * P < 0.05, significantly different from the BPA group.

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Table 4 Effect of LCE on hypertension and liver function in BPA-stimulated male mice.

CTR BPA (10 mg/kg) BPA + SPE (100 mg/kg) BPA + LCE (25 mg/kg) BPA + LCE (50 mg/kg) BPA + LCE (100 mg/kg)

hs-CRP (ng/ml)

Angiotensinogen (ng/ml)

Angiotensin II (pg/ml)

GOT (IU/L)

GPT (IU/L)

14.6 39.5 28.4 30.5 27.9 24.2

1.6 7.2 2.5 4.2 4.1 3.1

238.1 513.8 288.2 406.9 357.4 332.3

65.3 ± 10.2 349.2 ± 10.1# 204.6 ± 12.3* 219.7 ± 10.9* 217 ± 4.3* 214.4 ± 9.5*

85.3 ± 2.4 341.2 ± 11.2# 150.4 ± 11.1* 137.9 ± 11.1* 130.9 ± 10.4* 111.4 ± 11.4*

± ± ± ± ± ±

1.7 1.1# 5.9* 7.8* 6.6* 5.5*

± ± ± ± ± ±

0.9 0.5# 1.3* 0.9* 0.7* 0.5*

± ± ± ± ± ±

8.6 5.1# 3.7* 3.8* 6.2* 1.4*

Values are shown as means ± S.E.M. n = 7 for all groups. Data are the means ± S.E.M representative of three independent experiments. # P < 0.05, significantly different from the control group. * P < 0.05, significantly different from the BPA group.

Fig. 3. Effect of LCE on cell viability in BPA-treated TM4 cells. Protective effect of LCE on cell cytotoxicity in BPA-stimulated TM4 cells. (A) TM4 cells were treated with 100% LCE in a concentration dependent manner for 24h. Cell viability was measured by MTT assay. (B) TM4 cells were co-treated with BPA (300 μM) and the indicated ethanol concentration of LCE for 24 h. (C) TM4 cells were treated with the indicated concentration of 30% LCE for 24 h. Data are the means ± S.E.M from representative of three independent experiments. # P < 0.05, significantly different from untreated cells. * P < 0.05, significantly different from BPA-treated cells.

[(M + H)-162]+), vitexin pentose (peak 5, m/z 433.1133 [(M + H)132]+), and vitexin methyl-pentose (peak 8, m/z 433.1132 [(M + H)146]+) [35]. Based on the UPLC-QTOF-MS profiling results, rutin content analysis in LCE was carried out using HPLC. As a result, the retention time of rutin and vicenin were confirmed to be 9.90 min and 5.32 min, and identified as 0.90 mg/g and 1.36 mg/g, respectively (Fig. 5).

motility, and testosterone by reducing BPA- induced oxidative stress, obesity or hypertension-associated serum markers, and liver function markers in male mice. In vitro, LCE attenuated apoptotic cell death in BPA-treated TM4 sertoli cells by moderating Bax and Bcl-2 expression as well as activation of caspase 3 and PARP.BPA is one of the most dominant endocrine-disrupting chemicals and can be found in plastic water bottles, food containers, various household products, and thermal paper. Daily BPA intake in adults has been estimated at 0.4–1.5 μg/kg· bw/day [36]. This continuously ingested BPA could lead to disturbances in the male reproductive system, as spermatogenesis is dependent on a well-organized hormonal environment [37]. In addition, BPA has been demonstrated to cause adverse effects on body weight and the weight of organs that are sensitive to estrogens or androgens by disturbing the interactions of endogenous reproductive hormones. In our study, BPA significantly increased body weight in male mice.

4. Discussion In this study, we evaluated the in vivo and in vitro protective effects of LCE in BPA-stimulated male mice and TM4 sertoli cells. There data revealed that restoration of sperm counts, motility and testosterone by LCE could be helpful in the treatment of spermatic damage and male reproductive disorders in adults. In vivo, LCE restored the sperm count, 81

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Fig. 4. Effect of LCE on expression of apoptotic genes in BPA-stimulated TM4 cells. (A) TM4 cells were co-treated with the indicated concentration of LCE and BPA (300 μM) for 24 h. The protein expression of Bax, Bcl-2, caspase, and PARP was analyzed by Western blot analysis. β-actin and each normally-formed MAPK were used as a loading control. (B) The relative intensities were expressed as the ratio of Bax and Bcl-2 to β-actin. (C) The relative intensities were expressed as the ratio of cleaved caspase 3 and caspase 3 to β-actin. (D) The relative intensities were expressed as the ratio of cleaved PARP and PARP to β-actin. Representative data from three independent experiments are shown and quantitated. Values are the mean ± S.E.M of three independent experiments. #, P < 0.05, significantly different from the untreated group, * P < 0.05, significantly different from the BPA-treated group.

Table 5 Identified or tentatively identified compounds in LCE by UPLC-QTOF-MS2 under positive ion mode. No.

RT (min)

Observed m/z

Calculated m/z

Mass accuracy (ppm)

MS2 fragments m/z

Molecular formula

Identification

1 2 3 4

3.72 5.55 5.79 7.40

595.1655 449.1082 565.1550 595.1662

595.1657 449.1078 565.1551 595.1657

−0.4 0.7 −0.2 0.8

C27H30O15 C21H20O11 C26H28O14 C27H30O15

Vicenin 2a Unknown Unknown Vitexin hexose

5

8.07

565.1556

565.1551

0.7

C26H28O14

Vitexin pentose

6 7 8

8.56 8.71 8.98

535.1448 611.1607 579.1709

535.1446 611.1606 579.1708

0.4 0.0 0.1

C25H26O13 C27H30O16 C27H30O14

Unknown Rutin a Vitexin methyl-pentose

9

12.56

535.1446

535.1446

−0.1

577.1558b, 481.1131, 457.1147, 379.0814, 325.0717 329.0672, 299.0550 379.0814, 325.0716 433.1133, 415.1033, 379.0837, 367.0823, 337.0708, 313.0714, 283.0620 433.1133, 415.1032, 379.0812, 337.0708, 313.0713, 283.0599 433.1132, 415.1032, 379.0836, 313.0713, 283.0598 465.1040, 303.0492 433.1132, 397.0904, 379.0811, 337.0707, 313.0713, 283.0598 287.0559

C25H26O13

Unknown

a b

Confirmed by standard compounds. Low energy fragments are underlined.

changes in the body weight of Sprague–Dawley rats exposed to high doses of BPA administered orally during the gestation and lactation periods [39]. Rubin et al., showed an increase in body weight of female offspring of mothers exposed to BPA in drinking water from day 6 of pregnancy through the lactation period [40]. These differences might

Although there were no significant changes in body weight, serum parameters associated with adipocyte growth was significantly inhibited by the oral-administration of LCE in a dose dependent manner. Some previous studies have demonstrated variation in body weight, with the outcomes being controversial [38]. Kwon et al., found no 82

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Fig. 5. HPLC chromatogram of L. cuneata ethanol extract. (A) extract, 254 nm; (B) rutin, 254 nm; (C) extract, 335 nm; (D) Vicenin-2, 335 nm.

several non-enzymatic antioxidants all help the testis by counteracting oxidative stress [48–50]. However, a number of previous studies have reported that environmental toxins such as BPA, phthalates, and alkylphenols disturb the pro or anti-oxidant balance in testes and suppress testicular function [51]. In animal experiments, moreover, treatments, including exposure to BPA, that inhibit the intratesticular concentration of testosterone diminish the testicular expression of antioxidant enzymes such as GSH, catalase, and SOD [52–54]. In the present study, treatment with 100 mg/kg BPA significantly increased TBARS, a marker of oxidative stress, and inhibited antioxidant enzymes. However, the oral administration of LCE recovered the antioxidant enzymes by inhibiting oxidative enzymes, suggesting that LCE has potent anti-oxidative activity. Previous animal experiments have indicated that BPA exposure may affect the development of hyperlipidemia by enhancing oxidative stress and damaging glucose homeostasis and insulin sensitivity in adult mice. Marmugi et al., (2012) showed that BPA exposure (5, 50, 500 μg/kg/ day) elevated total cholesterol, triglycerides, and LDL-cholesterol in adult mice [27]. However, Dodge et al., demonstrated that oral administration of BPA (0.1, 1 and 10 mg/kg/day) for 4 days in ovariectomized rats (6 months old) did not change the total cholesterol [55]. These differences may be explained by the exposure time, doses, and experimental animals, which can result in an age-dependent BPA metabolism or different doses and administration of BPA. Our results showed that BPA exposure (10 mg/kg) for 5 weeks significantly increased total and LDL-cholesterol, triglycerides, FFAs, and glucose, indicating hyperlipidemia and glucose tolerance. However, oral administration of LCE markedly attenuated these parameters, and enhanced the level of HDL-cholesterol. In addition, it has been reported that increased LDL-cholesterol and triglycerides due to BPA exposure could affect the cardiovascular environment, possibly leading to the development of cardiovascular disorders such as atherosclerosis and hypertension [56]. Chronic increases in biomarkers such as high sensitivity C-reactive protein (hs-CRP), angiotensinogen, and angiotensin II are also critical predictors for an elevated risk of vascular diseases. hsCRP upregulates complement, reduces endothelial nitric oxide synthase expression and nitric oxide synthesis [57], and induces expression of cellular adhesion molecules [58]. Moreover, the inflammatory state in

be related to the exposure time, dose, administration, or animal strains used. Moreover, it has been previously demonstrated testis weight is usually influenced by the number of sertoli cells [41]. Our data revealed that testicular weight was significantly reduced in male mice exposed to BPA. These results may be a consequence of BPA binding to the ERβ receptor in sertoli cells. BPA could change the morphology of these cells by binding to the receptors, and could also reduce the number of cells, consequently modifying testicular weight and tubular organization [42]. We showed that testicular weight was significantly restored by the oral administration of LCE. Although we did not confirm an interaction between BPA and estrogen receptors in LCE treatment, we have recognized that this is necessary for further studies. Several studies have estimated the effects of BPA exposure in vivo in adulthood. Oral administration of BPA to adult Sprague–Dawley rats at concentrations as low as 20 μg/kg bw/day resulted in a reduction in daily sperm production of up to 40% [43]. Wu et al., also reported spermatid abnormalities in adult Sprague–Dawley rats injected with 40 mg/kg bw/day for 10 days. Finally, there was a significant reduction in epididymal sperm motility and sperm count in male rats [44]. In our study, the oral administration of LCE was associated with a protective effect on BPA-decreased sperm counts and sperm motility and the increased percentage of abnormal sperm. Mendiola et al., showed that BPA exposure decreased plasma testosterone in adult male rats [45]. Our data demonstrated that BPA significantly decreased testosterone by 23.1% compared to the control group, but oral administration of LCE restored plasma testosterone levels in BPA-stimulated male mice. These data indicate that LCE restored sperm counts and motility and reduced sperm abnormalities by increasing testosterone levels to restore hormone balance after BPA exposure. Reactive oxygen species (ROS) are produced during normal testicular function and play a critical role in regulating the function of the testis. Although ROS are known to be damaging effectors, low levels of ROS have a beneficial role in normal testicular function, developing germ cells and spermatids [46,47]. However, the increased levels of ROS can be harmful to testicular function and spermatogenesis. To overcome this, therefore, the testis should be provided with a potent antioxidant system that protects it from the damaging effects of ROS. The glutathione family of proteins, superoxide dismutase, catalase, and 83

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the vascular wall is induced by angiotensin II, which upregulates ROS, inflammatory cytokines, and adhesion molecules. Angiotensin II, partially produced by the vessel wall, induces the secretion of IL-6, followed by enhanced angiotensinogen synthesis in the liver [59]. Regulation of these biomarkers could be a strategy to treat vascular diseases. Our data demonstrated that BPA induced hs-CRP, angiotensinogen, and angiotensin II in male mice. However, oral administration of LCE significantly attenuated the levels of these biomarkers, indicating that LCE could be an important candidate for treating vascular diseases such as hypertension and atherosclerosis. Sertoli cells are the second cell type in the seminiferous epithelium and are essential for spermatogenesis. Although some in vitro studies showed that BPA-induced apoptosis of sertoli cells [60,61], we did not find a protective effect of LCE on apoptosis in BPA-treated sertoli cells. In our in vitro study, TM4 sertoli cells that were co-treated with BPA and LCE demonstrated a significant restoration of cell viability in DNA damage, along with alteration in Bcl-2 and Bax expression and activation of caspase 3 and PARP. Our data demonstrated a protective role of LCE in BPA-induced apoptotic cell death through upregulation of Bcl-2 expression and inhibition of Bax expression as well as activation of caspase 3 and PARP. In conclusion, the present study demonstrated that BPA exposure caused a significant increase in DNA damage to spermatocytes. The BPA-damaged spermatocytes were correlated with increased oxidative stress, hyperlipidemia, and hypertension. These changes were effectively prevented by LCE treatment. These results suggest that LCE restored testis weight, sperm count and motility, and testosterone levels in BPA-treated male mice through the attenuation of TBARS levels, total and LDL-cholesterol, triglycerides, glucose, FFAs, hs-CRP, angiotensinogen, and angiotensin II. In addition, oral administration of LCE to treat BPA-induced testicular dysfunction was as effective as SPE administration we used for a positive control. In vitro, LCE acted as a protective regulator against BPA-induced sertoli cell apoptosis by inhibiting Bax expression and activation of caspase 3 and PARP. The present data provide insight into the inhibitory activity of LCE and suggest a potential pharmacological candidate for male sperm health.

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