Effects of crude adlay hull acetone extract on ... - Springer Link

Naunyn-Schmiedeberg’s Arch Pharmacol (2006) 374:141–152 DOI 10.1007/s00210-006-0094-x

ORIGINAL ARTICLE

Effects of crude adlay hull acetone extract on corticosterone release from rat zona fasciculata-reticularis cells Ling-Ling Chang & Alfred Wan-Song Wun & Chien-Te Hung & Shih-Min Hsia & Wenchang Chiang & Paulus S. Wang

Received: 22 December 2005 / Accepted: 7 July 2006 / Published online: 21 September 2006 # Springer-Verlag 2006

Abstract Adlay is a grass crop which has been used in traditional Chinese medicine and also as a nourishing food. It has been shown to posses anti-allergic, antimutagenic and hypolipemic effects. However, the effects and action mechanisms of crude adlay hull acetone extract (AHA) on adrenal zona fasciculata-reticularis (ZFR) cells

Ling-Ling Chang and Paulus S. Wang: These authors contributed equally to this work. L.-L. Chang (*) Department of Chemical Engineering, Chinese Culture University, Shih-Lin, Taipei 111, Taiwan, Republic of China e-mail: [email protected] A. W.-S. Wun Obstetrical and Gynecological Associates, P.A., Houston, TX, USA C.-T. Hung Division of Metabolism and Endocrinology, Taipei City Hospital, Taipei, Taiwan, Republic of China S.-M. Hsia : W. Chiang Graduate Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan, Republic of China P. S. Wang School of Medicine, Department of Physiology, National Yang-Ming University, Taipei, Taiwan, Republic of China P. S. Wang Department of Medical Research and Education, Taipei City Hospital, Taipei, Taiwan, Republic of China

are still unclear. This study explored the effects of AHA on corticosterone release. ZFR cells were incubated with AHA in the presence or absence of adrenocorticotropin (ACTH), 8-bromo-cyclic 3′: 5′- adenosine monophosphate (8-Br-cAMP), forskolin (FSK), 25-hydroxy cholesterol (25-OH-cholesterol), pregnenolone, progesterone or deoxycorticosterone. The concentrations of corticosterone or pregnenolone in the media were measured by radioimmunoassay (RIA). The cells were used to measure the expression of steroidogenic acute regulatory (StAR) protein by Western blot. The present data demonstrated that: (1) AHA inhibited ACTH-, 8-Br-cAMP-, forskolin-, 25-OH-cholesterol-, pregnenolone-, progesterone- or deoxycorticosterone-stimulated corticosterone release; (2) AHA (800 μg/ml) caused more pregnenolone release in control group, but not in 25-OH-cholesterol, trilostane or 25-OH-cholesterol+trilostane group; (3) kinetic study showed an uncompetitive inhibition model of AHA to P450 side chain cleavage enzyme (P450scc); (4) kinetic study showed a noncompetitive inhibition model of AHA to 11β-hydroxylase; and (5) AHA inhibited the expression of StAR protein. These results suggest that AHA acts directly upon rat ZFR cells to diminish corticosterone release. These results indicate the inhibitory mechanism of AHA mediates through an inhibition of the activities of the post-cAMP corticosterone synthesis enzymes, i.e. 3βHSD, 21-hydroxylase, 11β-hydroxylase, and inhibition of StAR protein expression.

Keywords cAMP . Crude adlay hull acetone extract . P450scc . 11β-hydroxylase . Zona fasciculata-reticularis cells

142

Introduction Adlay is a traditional oriental food supplement. Crude extract of adlay seed modulated expressions of leptin and TNF-alpha, and reduced food intake, body weight, and serum hyperlipidemia from obese rats (Kim et al. 2004). Adlay oil has also been reported to reduce leptin in adipose tissue and LDL levels in the rats (Huang et al. 2005). These studies intrigue the epidemic obesity population. Methanolic extract of adlay seed suppressed the expression of human lung cancer cell gene of COX-2 activity (Hung and Chang 2003). The methanol extract also had antiproliferative and chemopreventive effects on mouse lung cancer both in vitro and in vivo (Chang et al. 2003). The dehulled adlay suppressed the production of IgE against injecting ovalbumin in the mouse model (Shyu et al. 1998) and modulated the immune response of helper 1 and 2 T-cells (Hsu et al. 2003). The water extract of adlay seed has been demonstrated to increase COX-2, ERK 1/2, and PKC-alpha expressions which induce embryotoxicity and enhance uterine contractility during pregnancy in rats (Tzeng et al. 2005). These reports showed that different extractions of adlay seed gave various effects. The bran or hull from different cereals has been demonstrated as beneficial supplements. The bran oil has been shown to reduce human serum cholesterol and triglyceride levels (Kuriyan et al. 2005). Rye bran increased the epithelial cell apoptosis in transgenic mouse prostate tumor (Wikstrom et al. 2005). Oat bran decreased rat serum cholesterol and increased bile acid excretion (Drzikova et al. 2005). Intakes of whole grain and bran were associated with a decreasing trend of C-reactive protein and tumor necrosis factor-alpha receptor 2 in diabetic women (Qi et al. 2006). These studies indicate that bran/hull posseses lower serum cholesterol, anti-tumorgenesis, and anti-inflammation benefits. Methanolic extraction of adlay bran has been reported to suppress the progesterone biosynthesis in rat granulosa cells via inhibition of cAMP-PKA pathway, P450scc activity, StAR protein expression, and phosphorylation of ERK (Hsia et al. 2006). In the present study, rat zona fasciculata-reticularis (ZFR) cells were utilized to investigate the direct effects and mechanisms of crude adlay hull acetone extract (AHA) on corticosterone synthesis and release including the steroidogenesis of ZFR cells, the function of the cytochrome P450 side-chain cleavage enzyme (P450scc) and 11β-hydroxylase. These results suggest that the stimulatory effects of ACTH-, 8-Br-cAMP- or steroidogenic precursors on the biosynthesis of corticosterone are reduced by AHA. The mediated mechanisms of AHA on corticosterone release are due to: (1) inhibition

Naunyn-Schmiedeberg’s Arch Pharmacol (2006) 374:141–152

of the post-cAMP pathway; (2) affection of the steroidogenic enzymes after P450scc; (3) uncompetitive inhibition of P450scc; (4) noncompetitive inhibition of 11β-hydroxylase; and (5) reduction of StAR protein expression.

Materials and methods Animals Female Sprague-Dawley rats weighing 300–350 g (2 months old) provided by National Yang-Ming University were housed in a temperature-controlled room (22±1°C) with photoperiod of 14 h (light): 10 h (dark). The light came on at 0600 hours. Food and water were provided ad libitum. Rats were ovariectomized 4 days before the experiments. The use of the animals was approved by the institutional Animal Care and Use Committee of the National YangMing University. All animals received human care in compliance with the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals, published by the National Science Council, Taiwan, ROC. Reagents Bovine serum albumin (BSA), glucose, adrenocorticotropin (ACTH), 8-Br-cyclic AMP (8-Br-cAMP), forskolin (FSK), phenylmethylsulfonyl fluoride (PMSF), 25-hydroxy cholesterol (25-OH-cholesterol), pregnenolone, progesterone and deoxycorticosterone were purchased from Sigma Chemical (St. Louis, Mo., USA). The above steroids were dissolved in ethanol as stock solutions. When they were used to challenge cells, the stock solutions were diluted in KRBGA medium. We had tested that the dilute ethanol did not kill cells (data not shown). Sodium dodecyl sulphate (SDS), bromophenol blue and dithiothreitol were purchased from Research Organics (Cleveland, Ohio, USA). Proteinase inhibitor cocktail tablets were purchased from Boehringer Mannheim (Mannheim, Germany). Trilostane (4,5-epoxy17-hydroxy-3-oxoandrostane -2-carbonitrile), an inhibitor of 3β- hydroxysteroid dehydrogenase (3β-HSD), was provided by Sanofi-Synthelabo (Malvern, Pa., USA). [3H]-corticosterone and [3H]-pregnenolone were obtained from Amersham Life Sciences (Buckinghamshire, UK). The anti-pregnenolone antiserum was purchased from Biogenesis (Sandown, N.H., USA). The anti-steroidogenic acute regulatory protein antibody was provided by Dr. D.M. Stocco (Lin et al. 1998). The peroxidase-conjugated IgG fraction to mouse IgG and peroxidase-conjugated IgG fraction to rabbit IgG were purchased from ICN Pharmaceuticals (Aurora, Ohio, USA).


143

Preparation of crude adlay hull acetone extract (AHA)

Effects of AHA on corticosterone release

The crude adlay hull acetone extract was prepared and provided by Dr. W. Chiang (Graduate Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan, ROC). Adlay was purchased from a local farmer who planted Taichung Shuenyu No. 4 (TSC4) of Coix lachryma-jobi L. var. ma-yuen Stapf. in Taichung, Taiwan, in March and harvested it in July of the same year. The air-dried adlay hull (18 kg) was blended into powder form and screened through a 20-mesh sieve (aperture 0.94 mm). The adlay hull powder was rinsed in 60 l of nhexane 3 times for 2 days, then it was extracted 3 times with 60 l of acetone at room temperature for 2 days. The acetone extract was filtered through #1 filter paper (Whatman, Hillsboro, Ore., USA). The filtrate of acetone extract was adsorbed on the chromatography column (the column was packed with 70–230 mesh of silica gel), then the adsorbed product was eluted by 20% ethanol/n-hexane. The eluted product was concentrated to dryness in vacuum condition, and named as crude adlay hull acetone extract powder. For in vitro study, crude adlay hull acetone extract powder was dissolved in DMSO solution to prepare stocks. The final concentration of DMSO in the stock crude adlay hull acetone extract (AHA) was less than 0.1%. The concentration of stock AHA is 0.4 g/ml. When AHA was used to challenge cells, the stock AHA was diluted in KRBGA medium and sonicated. We had tested the dilute DMSO did not kill cells (data not shown).

The ZFR cells in 1 ml/tube KRBGA medium were preincubated for 1 h at 37oC under 95% O2 and 5% CO2. To determine the effect of AHA on adenyl cyclase, cells after preincubation were incubated for 30 min with 0.3 ml medium containing ACTH (0 or 10−9 M), 8-Br-cAMP (0 or 10−4 M) or forskolin (FSK, 0 or 10−5 M) combined with AHA (0 or 160–800 μg/ml). After incubation, the media were stored at −20°C for corticosterone analysis by RIA.

Preparation of zona fasciculata-reticularis (ZFR) cells for cell culture Rat adrenal glands were excised, then kept in an ice-cold 0.9 % (w/v) NaCl solution. The adipose tissues were removed. The encapsulated glands were separated by forceps into capsule (mainly zona glomerulosa) and inner zone (mainly zona fasciculata-reticularis) fractions. The fractions of inner zone from 10–20 adrenals were assigned as one dispersion, then the ZFR cells were prepared following (Purdy et al. 1991) procedures with minor modifications (Lo et al. 1998). The cells (5×104 cells/ml) were preincubated with KRBGA medium (KrebsRinger bicarbonate buffer with 3.6 mmol K+/l, 11.1 mmol glucose/l, 0.2% BSA ) for 1 h at 37°C in a shaker bath (50 cycles per min) aerated with 95% O2 and 5% CO2. The supernatant was decanted after centrifugation of the tubes at 200 g for 10 min. Finally, the cells were resuspended in fresh incubation medium for 30 min or 3 h. After incubation and centrifugation, the medium was stored at −20°C for corticosterone or pregnenolone RIA, and the cells were applied for Western blot.

Effects of AHA on steroidogenesis of ZFR cells To measure the effect of AHA on enzyme P450scc of ZFR cells, cells after preincubation were incubated for 30 min with AHA (0 or 160–800 μg/ml) in the presence or absence of 25-OH-cholesterol (10–5 M, substrate of P450scc) combined with trilostane (10–5 M, a blocker of 3β-HSD). After incubation and centrifugation at 200 g for 10 min, the supernatant was used to measure medium concentration of pregnenolone by RIA. To measure the effect of AHA on steroidogenetic enzymes other than P450scc of ZFR cells, cells after preincubation were incubated for 30 min with AHA (0 or 160–800 μg/ml) in the presence or absence of 25-OHcholesterol (10−5M), pregnenolone [10−5 M, substrate for 3β-hydroxysteroid dehydrogenase (3β-HSD)], progesterone (10−5 M, substrate for 21-hydroxylase) or deoxycorticosterone (10−5 M, substrate for 11β-hydroxylase). After incubation and centrifugation at 200 g for 10 min, the supernatant was used to measure medium concentration of corticosterone by RIA. Effects of AHA on kinetic performance of P450scc and 11β-hydroxylase For kinetic analysis of P450scc, ZFR cells (5×104 cells/ml) were incubated for 30 min with trilostane (10−5 M) or trilostane plus 800 μg/ml AHA in the presence of 25-OHcholesterol (5×10−7 to 10−3 M). For kinetic analysis of AHA on 11β-hydroxlase, cells after preincubation were incubated for 30 min with or without AHA (800 μg/ml) in the presence of deoxycorticosterone (10−8-10−4 M). After incubation and centrifugation at 200 g for 10 min, the supernatant was used to measure medium concentration of corticosterone or pregnenolone by RIA. The reaction constants of steroidogenic pathway The steroidogenic pathway from 25-OH-cholesterol to corticosterone from rat ZFR cells can be expressed as, k2progesteronek3 25 OH cholesterolk1 ! pregnenolone! ! k4 deoxycorti cos terone! corti cos terone:

144


From material balances of substrates and products, we can get the first differential equations of substrates and products,

solve equations (4), (5) and let the concentration of progesterone equal to zero to get the solution,

d ½25 OH cholesterol dt

½corti cos terone

¼ k1½25 OH cholesterol

ð1Þ

d ½pregnenolone ¼ k1½25 OH cholesterol dt k2½pregnenolone

ð2Þ

¼ ½deoxycorti cos teroneO 1 expk4t

Take the substrate progesterone (the initial concentration of progesterone ½progesteroneo ), solve equations (3)–(5) and let the concentration of pregnenolone equal to zero to get the solution, ½corti cos terone ¼

d ½progesterone ¼ k2½pregnenolone dt k3½progesterone

k4½progesteroneO 1 expk3t k4 k3 k3½progesteroneO 1 expk4t k4 k3

ð3Þ

ð7Þ

Take the substrate pregnenolone (the initial concentration of pregnenolone ½pregnenoloneo ), solve equations (2)– (5) and let the concentration of 25-OH-cholesterol equal to zero to get the solution,

d ½deoxycorti cos terone ¼ k3½progesterone dt k4½deoxycorti cos terone ð4Þ d ½corti cos terone ¼ k4½deoxycorti cos terone dt

ð6Þ

ð5Þ

Take the substrate deoxycorticosterone (the initial concentration of deoxycorticosterone ½deoxycorti cos teroneo ),

½corti cos terone

k3k4½pregnenoloneO 1 expk2t ¼ ðk3 k2Þðk4 k2Þ k2k4½pregnenoloneO expk3t 1 þ ðk3 k2Þðk4 k3Þ k2k3½pregnenoloneO 1 expk4t þ ðk4 k2Þðk4 k3Þ

ð8Þ

Take the substrate 25-OH-cholesterol (the initial concentration of 25-OH-cholesterol ½25 OH cholesterolo ), solve equations (1)–(5) and get the solution, ( ½corti cos terone ¼k1k2k3k4½25 OH cholesterolO

expk1t k1ðk2 k1Þðk3 k1Þðk4 k1Þ

expk2t expk3t k2ðk2 k1Þðk3 k2Þðk4 k2Þ k3ðk4 k3Þðk3 k1Þðk3 k2Þ ) expk4t þ k4ðk4 k1Þðk4 k2Þðk4 k3Þ

þ

Effects of AHA on steroidogenic acute regulatory protein expression The Western blotting method has been reported previously (Kau et al. 1999a; Lo et al. 2000). The ZFR cells (1.5×106– 3×106 cells) were incubated with medium containing AHA (160–800 μg/ml) or ACTH (10−7M) for 3 h. At the end of

ð9Þ

incubation, cells were washed twice with ice-cold saline and dissolved with 50 μl lysis buffer. The lysis buffer consisted of 1.5% Na-lauroylsacrosine, 2.5 mM Tris-base, 1 mM EDTA, 0.68% phenylmethylsulfonyl fluoride (PMSF) and 2% proteinase inhibitors, at pH 7.8. Cell mixtures were centrifuged for 10 min at 13,500 g. The protein concentration in the supernatant was determined by the Bradford method (Bradford 1976). Extracted proteins


were denatured by boiling for 10 min in SDS buffer (0.125 M Tris-base, 4% sodium dodecyl sulphate (SDS), 0.001% bromophenol blue, 12% sucrose and 0.15 M dithiothreitol) (Hu et al. 1991). The proteins in the samples were separated using 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at 75 V for 15 min and then at 150 V for 40 min using a running buffer. The proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (PerkinElmer Life Science Products, Boston, Mass., USA) using a Trans-Blot SD semi-drytransfer cell (170–3940; Bio-Rad, Hercules, Calif., USA) at 60 mA (for 8 mm×10 mm membrane) for 60 min in a transfer solution. The membranes were blocked by a 120min incubation in blocking buffer (TBS-T buffer containing 5% nonfat dry milk) and then washed four times in buffer (TBS-T buffer, containing 0.8% NaCl, 0.02 M Tris-base and 0.1% Tween-20, pH 7.6) for 10 min. These membranes were incubated with anti- steroidogenic acute regulatory protein antibody (1:1,000, rabbit) in 5% nonfat dry milk of TBS-T buffer overnight at 4°C. After four washes with TBS-T buffer of 10 min each, the membranes were incubated for 2 h with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG, 1:6,000 dilution) in 5% nonfat dry milk of TBS-T buffer. The membranes were washed four times with TBS-T buffer, and then the band for steroidogenic acute regulatory protein was visualized by chemiluminescence (ECL Western blotting

Basal (n=6)

*

1.0 40 35

ACTH (10-9 M, n=6)

++

0.0

detection reagents, Amersham Pharmacia Biotech, Buckinghamshire, UK). RIA of corticosterone The concentrations of corticosterone in media were determined by RIA as previously described (Chang et al. 2002; Lo et al. 1998). With this antiserum (PSW#4–9), a RIA was established for the measurement of media corticosterone levels. The sensitivity of corticosterone RIA was 5 pg/tube. The intra- and interassay coefficients of variation were 3.3% (n=5) and 9.2% (n=4), respectively. For studying the effect of AHA interfering with corticosterone RIA, the concentration of corticosterone in the medium containing different concentration of AHA (0 or 160–4,000 μg/ml) plus 300 pg of corticosterone was determined by RIA. RIA of pregnenolone The concentration of pregnenolone in media was determined by RIA as previously described (Kau et al. 1999b). The sensitivity of pregnenolone RIA was 16 pg/tube. The intra- and interassay coefficients of variation were 2.3 % (n=6) and 3.7 % (n=4), respectively. Statistical analysis Treatment means were tested for homogeneity using an analysis of variance, and difference between specific means was tested for significance using Duncan’s multiple range test or Student’s t- test (Steel and Torrie 1960). A difference between two means was considered statistically significant when P was less than 0.05.

30

0 20

++

8-Br-cAMP (10-4 M, n=6) 15

** ++

10 5

2

Forskolin (10-5 M, n=6)

**

5

Basal (n=6)

*

10

3

++

15

Corticosterone Release (ng/5x104 cells/30 min)

20

** ++

** ++

25

++

4

Corticosterone Release (ng/5x10 cells/30 min)

2.0

**

3.0

145

1

0 0

160

800

AHA (µg/ml)

Fig. 1 Effects of AHA (160 and 800 μg/ml) on the basal, ACTH (10−9 M) and 8-Br-cAMP (10−4 M)-stimulated corticosterone release from ZFR cells. *P