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1 Effect of Berberine on Testosterone secretion from Testicular TM3 cell lines in hypoxia condition. Yi Jing Chen 1 , Ju-Wen Yu 1 , Hea-Wen Tzou 1 , Yuan-Hao Chang 1 , Ya-Wen Jeng 2 and Shyi-Wu Wang 2 * 1 Graduate Institute of Biomedical Sciences, 2 Department of Physiology and Pharmacology, College of Medicine, Chang-Gung University, Kweisan, Taoyuan, Taiwan, ROC Key words: hypoxia, berberine, Leydig TM3 cells, testosterone *Correspondence : Shyi-Wu Wang, Ph. D. Department of Physiology and Pharmacology Chang Gung University Taoyuan 33333, Taiwan, Republic of China. Tel No.: 886-3-211-8800 EXT 5253 Fax No.: 886-3-3288448

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1

Effect of Berberine on Testosterone secretion from

Testicular TM3 cell lines in hypoxia condition.

Yi – Jing Chen1, Ju-Wen Yu

1, Hea-Wen Tzou

1, Yuan-Hao Chang

1, Ya-Wen Jeng

2 and

Shyi-Wu Wang2*

1Graduate Institute of Biomedical Sciences,

2Department of Physiology and

Pharmacology, College of Medicine, Chang-Gung University, Kweisan,

Taoyuan, Taiwan, ROC

Key words: hypoxia, berberine, Leydig TM3 cells, testosterone

*Correspondence : Shyi-Wu Wang, Ph. D.

Department of Physiology and Pharmacology

Chang Gung University

Taoyuan 33333, Taiwan,

Republic of China.

Tel No.: 886-3-211-8800 EXT 5253

Fax No.: 886-3-3288448

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Abstract

Berberine is an isoquinoline alkaloid isolated from herb plants, such as Cortex

phellodendri (Huangbai) and Rhizoma coptidis (Huanglian). Huanglian and Huangbai

have used as “heat-removing” agents. In addition, berberine has been reported to have

anti-inflammatory effects both in vivo and in vitro. Hypoxia is a major stimulator to

cause the pathogenesis of many diseases such as heart disease, neuron death,

cardiovascular diseases, and cancer. Tumors hypoxia has been indicated to be strongly

associated with tumor malignant progression, propagation, and resistance to therapy.

Therefore, hypoxia has become a central issue in tumor physiology and cancer

treatment. However, the effect of berberine on male reproduction in hypoxia condition

is still not known. The purpose of this study is to investigate the effect of berberine in

hypoxia condition on testosterone secretion from TM3 cells, a kind of rat Leydig cell

lines. In 12-well plate, TM3 cells (4×104 /ml) were incubated with berberine (10, 20,

50 g/ml) under normoxia (21% O2) or hypoxia (1% O2) for 1 hour and treated with

or without hCG (0.5 IU/ml) for 16 hours. Media were collected later, centrifuged, and

stored in –20℃ for testosterone EIA. Meanwhile, different activators/inhibitors of

signal transduction with berberine and hCG were added into the media. Stimulatory

effect of berberine on testosterone release from TM3 cells was observed under

hypoxia condition. Significant inhibitory effect of nifedipine (L-type calcium channel

blocker), U73122 (PLC inhibitor), and helenaline (NF-B inhibitor) were found in

TM3 cells under hypoxia condition. In contrast, AG490 (JAK-2 inhibitor), forskolin

(activator of adenylyl cyclase), SQ22536 (adenylyl cyclase inhibitor), and

GF109203X (PKC inhibitor) did not affect the testosterone secretion from TM3 cells

under hypoxia condition. Our results showed that, under hypoxia condition, the

stimulatory effect of berberine on testosterone secretion from Leydig TM3 cells

maybe mediated through phospholipase C, calcium channel, and NF-B pathways.

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Introduction

Berberine is an isoquinoline alkaloid (Zhou and Mineshita, 2000) existed

in Hydrastis Canadensis (goldenseal), Cortex phellodendri (Huangbai), and

Rhizoma coptidis (Huanglian) (Ikram 1975). Extracts from these plant root or

skin are used for disease treatment, e.g. lumbago, rheumatism, and fever

(Yesilada and Kupeli 2002). Huanglian has been used as a herb in India and

China for more than 3000 years (Shirwaikar, Shirwaikar et al. 2006). As a herb,

huanglian has been employed as prophylactic drug, and for GI disorder (Lin,

Chung et al. 2005). Studies indicated that berberine poses the inhibitory effect

on cyclooxygenase-2 activity and lipoxygenase (Misik, et al., 1995),

interleukin-8 increase in inflammatory enteric disease (Nielsen, et al., 1987),

and colitis induced by trinitrobenzene sulfonic acid (Zhou and Mineshita,

2000). Also, the edema in hind paw in mice induced by serotonin can be

inhibited by berberine in vivo (Kupeli, et al., 2002). Moreover, studies showed

that berberine can be used in treatment hepatic injury (Hwang, et al., 2002),

thyroxine induced ventricular hypertrophy (Yang, et al., 2006),

hypercholesterolemia (Kong, et al., 2004), decreased bone mineral density (Wu,

et al., 1999; Li, et al., 2006), parasite infection (Subbaiah and Amin, 1967;

Stermitz, et al., 2000; Hawrelak J, 2003; Kong, et al., 2004),

erectile-dysfunction (Chiou, et al., 1998; Drewes, et al., 2003), tumor

metastasis (Hoshi, et al., 1976; Fukuda, et al., 1999; Marinova, et al., 2000;

Mitani, et al., 2001; Peng, et al., 2006; Yang, et al., 2006).

Studies showed that hypoxia decreased thyrotrophs and thyroid cells

(Gonsly, 1985), promoted glucocorticoid (Raff, et al., 2003) and erythropoietin

secretion (Wang, et al., 1996), inhibited aldosterone secretion (Raff, et al.,

1997;Raff and Bruder, 2006), disturbed the hypothalamic-pituitary-gonadal

function in male (Wang AC, 1990), lowered LH/FSH (Semple et al., 1981;

Farias et al., 2008) and testosterone secretion(Semple et al., 1984). Also,

Obstructive sleep apnea is considered to relate to intermittent hypoxia

condition. In these patients, low testosterone concentration is a common

syndrome (Santamaria, Prior, and Fleetham. 1988; Hu et al., 1998; Luboshitzky

et al., 2002; Gambineri, Pelusi, and Pasquali. 2003; Luboshitzky et al., 2003;

Luboshitzky et al., 2005; Zhuravlev et al., 2009). This hypogonadism has been

related to age and obesity (Gambineri, Pelusi, and Pasquali. 2003; Luboshitzky

et al., 2002; Luboshitzky et al., 2005). However, Coste et al. (2006) indicated

that there is no significant relationship between hypoxia and hypogonsdism. In

addition, our lab has demonstrated that hypoxia stimulated testosterone

secretion in Leydig TM3 cells (Hwang et al., 2007) and increased testosterone

secretion in TM3 cells was found in intermittent hypoxia (Hwang et al., 2009).

In the present study, mouse Leydig cells were used to investigate the

effects of berberine on (1) testosterone secretion, (2) signal transduction, and (3)

the involvement of basic regulatory pathways in regulation of HIF-1, VEGF,

and ERK1/2 expression under normoxia and hypoxia condition.

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Materials and Methods

Antibodies and reagents. Human chorionic gonadotropin (hCG) and mouse VEGF were purchased

from Sigma (St. Louis, MO). PD-98059 was purchased from Tocris Cookson

(Westwoods Business, Park Ellisville, MO). Anti-VEGF (1:200 dilution),

anti-phospho (p)-extracellular signal-regulated kinases 1 and 2 (ERK1/2;

1:1,000 dilution), anti--actin (1:8,000 dilution), and

anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:500 dilution)

were purchased from Santa Cruz (Watsonville, CA). The horseradish

peroxidaseconjugated IgG, goat anti-rabbit IgG (1:6,000 dilution), and goat

anti-mouse IgG (1:8,000 dilution) were purchased from ICN Pharmaceuticals

(Aurora, OH).

Cell culture. TM3 Leydig cells, a nontumorogenic cell line derived from mouse testis,

were obtained from the Culture Collection and Research Center (Food Industry

Research and Development Institute, Taiwan, Republic of China). This cell line

responds to LH by increasing testosterone production and secretion through

mechanisms similar to those encountered in freshly isolated cells. Cells were

cultured in 75-cm2 flasks (Falcon, Franklin Lakes, NJ) in a 1:1 mixture of

Ham’s F-12 and DMEM (Sigma) that contained 15 mM HEPES, 0.12%

NaHCO3 supplemented with 0.45% glucose, 5% horse serum, 2.5% FCS

(Kibbutz Beit, Haemek, Israel), and 100 IU/ml potassium penicillin G + 100

g/ml streptomycin sulfate (Sigma). Cells were cultured at 37°C in either a

humidified atmosphere of normoxic conditions (95% air-5% CO2) or in a

modular incubator chamber (Billups-Rothenberg, Del Mar, CA) and flushed

with hypoxic gas (95% N2-5% CO2).

ELISA of Testosterone Concentrations of testosterone in collected media were determined by

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ELISA (Enzyme-linked Immunosorbent Assay). Briefly, 0.1 ml of capture

antibodies (R & D systems, Minneapolis, MN, USA) were coated on the

polystyrene microtiter plates (NUNC, U16 Maxisorp type, Rochester, NY, USA)

and incubated at room temperature overnight. The plates were blocked the next

day and then incubated for 1 h. Then, 0.1 ml standard/sample was added and

incubated for 2 h. After washing 3 times, 0.1 ml of detection antibody (R & D

systems) was applied for 2 h. One hundred micorliters streptavidin horseradish

peroxidase (R & D systems) and then 0.1 ml tetramethylbenzidine substrate

(Clinical Science Products Inc., Mansfield, MA, USA) followed this incubation.

The reaction was stopped using 2 N sulfuric acid and optical density (OD)

reading was taken at 450 nm (BioTek, Winooski, VT, USA). All samples were

run in duplicates. The results were expressed as concentration of testosterone

(pg/ml) read from standard curves. The detection range, sensitivity, the

intraassay and interassay coefficients of variation are:7.8-500 pg/ml, 6 pg,

4.4%, and 7.7%, respectively.

Effect of hypoxia on the expressions of ERK1/2. Mouse TM3 Leydig cells (106 cells/10 ml) were seeded in 10-cm dishes

and then incubated with or without hCG at 1 IU/ml for 1 or 16 h in a normoxic

or hypoxic condition. At the end of incubation, cytoplasmic proteins were

extracted from the cells and used to determine the expression of VEGF, HIF-1,

p-ERK1/2, and p-p38 through Western blot.

Statistical analysis.

The data were expressed as means ± SE. The treatment means were

tested for homogeneity using ANOVA, and the difference between specific

means was tested for significance using Duncan’s multiple-range test or

Student’s t-test (Steel & Torrie, 1960). A difference between two means was

considered to be statistically significant when the P value was <0.05.

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Results

Effect of glycyrrhizin and berberine on testosterone secretion under

normoxia

Significant increases of testosterone were found in TM3 cells treated with

berberine (10~50 g/ml) or hCG (0.5 IU/ml) (Fig. 1).

Signal pathways in TM3 after berberine treatment

Increased testosterone secretion were found in TM3 cells treated AG490

(Fig 3), Nifedipine (Fig 4), SQ22536 (Fig 5), U73122 (Fig 6), GF109203X (Fig

7), Helenaline (Fig 8), and berberine.

No change in testosterone secretion was found in TM3 cells after forskolin

(Fig. 2). Also, these activators/inhibitors had no effect on testosterone secretion

from TM3 cells treated with berberine and hCG (Fig. 9).

Effect of berberine on testosterone from TM3 under hypoxia condition

Significant increase of hCG on testosterone secretion from TM3 under

hypoxia were found at 0.5 IU/ml (Fig. 10A) and 16 h (Fig. 10B). Increased

testosterone secretion were observed in TM3 treated with berberine (10-20

g/ml) only or with hCG (0.5 IU/ml) (Fig. 11A).

Signal pathway in TM3 after berberine treatment under hypoxia condition

No difference in testosterone concentration was found between berberine

only and berberine + forskolin (10-6

M) (Fig. 11B), AG490 (10-6

M) (Fig. 12A)

treatment under hypoxia condition. Decreased testosterone was observed in

TM3 with berberine + nifedipine (Fig. 12B), U73122 (Fig. 13B), helenaline

(Fig. 14B) compared to berberine treatment only in hypoxia. However,

increased testosterone was found in TM3 with berberine + SQ22536 compared

to berberine only (Fig. 13A). Decreased media testosterone was found only in

10 g/ml berberine + GF109203X compared to berberine only in hypoxia (Fig.

14A).

Effect of berberine on HIF-1, VEGF, and ERK1/2 expression in TM3 under

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hypoxia condition

No difference was found in VEGF in expression in TM3 between

berberine only or with hCG under hypoxia condition (Fig. 15A).

Augmented HIF-1 expression was found in TM3 with berberine only. In

contrast, lower expression of HIF-1 was observed after berberine and

hCG treatment (Fig. 15B). Enhanced p-ERK1/2 expression was found in

TM3 with berberine only or berberine + hCG treatment (Fig. 16A). Also,

stimulated p-p38 expression with berberine only or berberine + hCG

treatment (Fig. 16B)

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Discussion

In the present studies, we found that: (1) the stimulatory effect of berberine on

testosterone secretion from TM3 might be through calcium ion channel ,

adenylyl cyclase, phospholipase C, protein kinase C, and NF-B; (2) under

hypoxia condition, the stimulatory effect of berberine on TM3 cells is part

through adenylyl cyclase, protein kinase C, phospholipase C, NF-B, HIF-1,

ERK1/2, and p38.

Previous studies have indicated that regulation of MAPK is associated with the

action of berberine in tumors or cells. Berberine inhibits the expression of

inflammatory cytokines in ARPE-19 cells, a human retinal pigment epithelial

cell line, through down-reugulation of the ERK1/2, JNK, and p38 pathways

(Wang, et al., 2012). Yan et al. (2012) showed that berberine promotes recovery

of dextran sulfate sodium (DSS)-induced colitis and decreases proinflammatory

cytokine production through MAPK. Also, in human colonic carcinoma cells,

berberine reduces cAMP-induced chloride secretion, which is mediated by

stimulation of ERK1/2 and p-38 (Alzamora, et al., 2011). Berberine

hydrochloride attenuates LPS-induced COX-2 expression and p38 activation in

small intestinal mucosa cells (Feng, et al., 2011). In addition, through the

activation of ERK1/2 and p38, berberine chloride induced the anti-Leishmanial

activity in macrophages (Saha, et al., 2011). In lung tumor, administration ob

berberine inhibits cell cycle progression and tumorgenesis through Akt, CREB

and MAPK pathways (James, et al., 2011). Zhang, et al. (2011) demonstrated

berberine moderates glucose and lipid metabolism through the activation of

AMPK, p38, JNK and PPAR. In our study, we found that berberine, in

normoxia or hypoxia condition could regulate testosterone secretion from TM3

cells through the activation of either ERK1/2, JNK, or p38. This finding is in

agreement with these previous studies.

In conclusion, our results demonstrate (1) glycyrrhizin stimulated testosterone

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secretion from TM3 cells at least in part through the adenylyl cyclase, JAK-2,

calcium ion channel, and protein kinase C; (2) the stimulatory effect of

berberine on testosterone secretion from TM3 is via calcium ion channel ,

adenylyl cyclase, phospholipase C, protein kinase C, and NF-B; (3) under

hypoxia condition, glycyrrhizin stimulates testosterone secretion from TM3

cells through JAK-2, calcium ion channel, HIF-1, and ERK1/2; (4) the

stimulatory effect of berberine on TM3 cells is part through adenylyl cyclase,

protein kinase C, phospholipase C, NF-B, HIF-1, ERK1/2, and p38.

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Acknowledgments

This study was supported by the grant (CMRPD10041 and CMRPD10042) from

Chang-Gung Memorial Hospital, Taipei, Taiwan, ROC.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the

content and writing of the article.

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17

TM3 cells

Tes

tost

ero

ne

(p

g/m

l)

0

200

400

600

800

1000

1200

1400

1600

hCG(0.5 IU/ml) - + - - - + + + (3 hrs)

Ber (g/ml) (1 hrs)- - 10 20 50 10 20 50

*** ** ***

#####

Figure 1. Effect of berberine on testosterone secretion from TM3 cells treated with or

without hCG (0.5 IU/ml) under normoxia condition.

TM3 cells

Tes

tost

eron

e (p

g/m

l)

0

200

400

600

800

1000

1200

1400

1600

hCG (0.5 IU/ml)

Ber ( g/ml)

Forskoline (10-6

M)

(3 hrs)

(1 hrs)

- + - - - - - -

0 0 10 20 50 10 20 50 - - - - - + + +

(1 hrs)

Figure 2. Effect of berberine on testosterone secretion from TM3 treated with or

without forskolin (10-6

M; adenylyl cyclase activator). n=4.

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18

TM3 cells

Tes

tost

eron

e (p

g/m

l)

0

200

400

600

800

1000

1200

1400

1600

hCG (0.5 IU/ml)

Ber ( g/ml

AG490 (10-6

M)

(3 hrs)

(1 hrs)

- + - - - - - -

0 0 10 20 50 10 20 50 - - - - - + + + (1 hrs)

Figure 3. Effect of berberine on testosterone secretion from TM3 treated with or

without AG490 (10-6

M; JAK-2 tyrosine kinase inhibitor). n=4.

TM3 cells

Tes

tost

eron

e (p

g/m

l)

0

200

400

600

800

1000

1200

1400

1600

hCG (0.5 IU/ml)

Ber ( g/ml

Nifedipine (10-4

M)

(3 hrs)

(1 hrs)

- + - - - - - -

0 0 10 20 50 10 20 50 - - - - - + + + (1 hrs)

Figure .4 Effect of berberine on testosterone secretion from TM3 treated with or

without nifedipine (10-4

M; L-type calcium channel inhibitor). n=4.

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19

TM3 cellsT

esto

ster

on

e (p

g/m

l)

0

200

400

600

800

1000

1200

1400

1600

hCG (0.5 IU/ml)

Ber ( g/ml

SQ22536 (10-5

M)

(3 hrs)

(1 hrs)

- + - - - - - -

0 0 10 20 50 10 20 50 - - - - - + + + (1 hrs)

Figure 5. Effect of berberine on testosterone secretion from TM3 treated with or

without SQ22536 (10-5

M; adenylyl cyclase inhibitor). n=4.

TM3 cells

Test

oste

rone

(pg/

ml)

0

200

400

600

800

1000

1200

1400

1600

hCG (0.5 IU/ml)

Ber ( g/ml)

U73122 (10-4

M)

(3 hrs)

(1 hrs)

- + - - - - - -

0 0 10 20 50 10 20 50 - - - - - + + + (1 hrs)

Figure 6. Effect of berberine on testosterone secretion from TM3 treated with or

without U73122 (10-4

M; phocpholipase C inhibitor). n=4.

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TM3 cells

Test

oste

rone

(pg/

ml)

0

200

400

600

800

1000

1200

1400

1600

hCG (0.5 IU/ml)

Ber ( g/ml)

GF109203X (10-6

M)

(3 hrs)

(1 hrs)

- + - - - - - -

0 0 10 20 50 10 20 50 - - - - - + + + (1 hrs)

Figure 7. Effect of berberine on testosterone secretion from TM3 treated with or

without GF109203X (10-6

M; protein kinase C inhibitor). n=4.

TM3 cells

Tes

tost

eron

e (

pg/m

l)

0

200

400

600

800

1000

1200

1400

1600

hCG (0.5 IU/ml)

Ber ( g/ml)

Helenaline (10-6

M)

(3 hrs)

(1 hrs)

- + - - - - - -

0 0 10 20 50 10 20 50 - - - - - + + + (1 hrs)

Figure 8. Effect of berberine on testosterone secretion from TM3 treated with or

without helenaline (10-6

M; NF-B inhibitor). n=4.

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21

TM3 cellsT

esto

ster

on

e (p

g/m

l)

0

200

400

600

800

1000

hCG (0.5 IU/ml)

Ber (50 g/ml)

Forskoline (10-6

M)

AG490 (10-6

M)

Nifedipine (10-4

M)

SQ22536 (10-5

M)

U73122 (10-4

M)

GF109203X (10-6

M)

Helenaline (10-6

M)

(3 hrs)

(1 hrs)

- + - + + + + + + + +

- - + + + + + + + + +- - - - + - - - - - -- - - - - + - - - - -

- - - - - - + - - - -- - - - - - - + - - -

- - - - - - - - + - -- - - - - - - - - + -- - - - - - - - - - +

Figure 9. Effect berberine (50 g/ml) on testosterone secretion from hCG-treated

TM3 cells with or without activators/inhibitors.

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22

A.

TM3 cellsT

esto

ster

one

(pg/

ml)

0

2000

4000

6000

8000

10000

hCG(IU/ml) 0 0.1 0.2 0.5 1 2 (16hrs)

B.

TM3 cells

Tes

tost

eron

e (p

g/m

l)

0

1000

2000

3000

4000

5000

hCG 0.5 IU/ml 0 3 6 16 24 (hrs)

Figure 10. Effect of hCG on testosterone secretion from TM3 under hypoxia condition

in different dosage of hCG(A) and time course (B).

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23

A.

TM3 cellshypoxia

Tes

tost

eron

e (p

g/m

l)

0

200

400

600

800

1000

hCG(0.5IU/ml) - - - - + + + + (16h)

Ber(g/ml) 0 10 20 50 - 10 20 50 (1h)

B.

TM3 cellshypoxia

Tes

tost

eron

e (p

g/m

l)

0

200

400

600

800

1000

hCG (0.5 IU/ml)

Ber (g/ml)

Forskolin (10-6

M)

(16 hrs)

(1 hrs)

- + - - - - - -

0 0 10 20 50 10 20 50 - - - - - + + + (1 hrs)

Figure 11. Effect of berberine on testosterone secretion from TM3 treated with hCG

(A) and forskolin (B) under hypoxia condition.

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24

A.

TM3 cellshypoxia

Tes

tost

eron

e (p

g/m

l)

0

200

400

600

800

1000

1200

1400

hCG (0.5 IU/ml)

Ber (g/ml)

AG490 (10-6

M)

(16 hrs)

(1 hrs)

- + - - - - - -

0 0 10 20 50 10 20 50 - - - - - + + + (1 hrs)

B.

TM3 cellshypoxia

Tes

tost

ero

ne

(pg

/ml)

0

2000

4000

6000

8000

10000

hCG (0.5 IU/ml)

Ber (g/ml)

Nifedipine (10-4

M)

(16 hrs)

(1 hrs)

- + - - - - - -

0 0 10 20 50 10 20 50 - - - - - + + + (1 hrs)

Figure 12. Effect of berberine on testosterone secretion from TM3 treated with hCG

and (A) AG490 or (B) nifedipine under hypoxia condition.

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25

A.

TM3 cellshypoxia

Tes

tost

eron

e (p

g/m

l)

0

5000

10000

15000

20000

25000

30000

35000

hCG (0.5 IU/ml)

Ber (g/ml)

SQ22536 (10-5

M)

(16 hrs)

(1 hrs)

- + - - - - - -

0 0 10 20 50 10 20 50 - - - - - + + + (1 hrs)

B.

TM3 cellshypoxia

Tes

tost

eron

e (p

g/m

l)

0

10000

20000

30000

40000

50000

60000

hCG (0.5 IU/ml)

Ber (g/ml)

U73122 (10-4

M)

(16 hrs)

(1 hrs)

- + - - - - - -

0 0 10 20 50 10 20 50 - - - - - + + + (1 hrs)

Figure 13. Effect of berberine on testosterone secretion from TM3 treated with hCG

and (A) SQ22536 or (B) U73122 under hypoxia condition.

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26

A.

TM3 cellshypoxia

Tes

tost

erone

(pg/m

l)

0

5000

10000

15000

20000

hCG (0.5 IU/ml)

Ber (g/ml)

GF109203X (10-6

M)

(16 hrs)

(1 hrs)

- + - - - - - -

0 0 10 20 50 10 20 50 - - - - - + + + (1 hrs)

B.

TM3 cellshypoxia

Tes

tost

eron

e (p

g/m

l)

0

2000

4000

6000

8000

10000

12000

hCG (0.5 IU/ml)

Ber (g/ml)

Helenaline (10-6

M)

(16 hrs)

(1 hrs)

- + - - - - - -

0 0 10 20 50 10 20 50 - - - - - + + + (1 hrs)

Figure 14. Effect of berberine on testosterone secretion from TM3 treated with hCG

and (A) AG490 or (B) nifedipine under hypoxia condition.

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27

A.

β-Actin

VEGF

43 KDa

55 KDa

TM3 cells

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

hCG 0.5 IU/ml - + - - - + + + (1 hrs)

20110930N=1

5 x 104/ml

pass. 4hypoxia

Ber (g/ml) (1 hrs)- - 10 20 50 10 20 50

VEGF

/ A

ctin

(flod

relat

ive t

o co

ntro

l)β-Actin

VEGF

43 KDa

55 KDa

TM3 cells

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

hCG 0.5 IU/ml - + - - - + + + (1 hrs)

20110930N=1

5 x 104/ml

pass. 4hypoxia

Ber (g/ml) (1 hrs)- - 10 20 50 10 20 50

VEGF

/ A

ctin

(flod

relat

ive t

o co

ntro

l)

B.

β-Actin

HIF-1α43 KDa

72 KDa

TM3 cells

0.0

0.5

1.0

1.5

2.0

2.5

hCG 0.5 IU/ml - + - - - + + + (1 hrs)

20110930

N=1

5 x 104/ml

pass. 4

hypoxia

Ber (g/ml) (1 hrs)- - 10 20 50 10 20 50

HIF

-1A

ctin

(fol

d re

lativ

e to

con

trol)

β-Actin

HIF-1α43 KDa

72 KDa

TM3 cells

0.0

0.5

1.0

1.5

2.0

2.5

hCG 0.5 IU/ml - + - - - + + + (1 hrs)

20110930

N=1

5 x 104/ml

pass. 4

hypoxia

Ber (g/ml) (1 hrs)- - 10 20 50 10 20 50

HIF

-1A

ctin

(fol

d re

lativ

e to

con

trol)

Figure 15. Effect of berberine on (A) VEGF and (B) HIF-1 expression in TM3

treated with hCG (0.5 IU/ml) under hypoxia condition.

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28

A.

TM3+Ber+hCGHypoxia

ER

K1/

2 /

-act

in

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

ERK 1ERK 2

Ber(g/ml) - - 10 20 50 10 20 50 (1h)

hCG(0.5 IU/ml) - + - - - + + + (3h)

p-ERK1/2

β-actin

TM3+Ber+hCGHypoxia

ER

K1/

2 /

-act

in

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

ERK 1ERK 2

Ber(g/ml) - - 10 20 50 10 20 50 (1h)

hCG(0.5 IU/ml) - + - - - + + + (3h)

p-ERK1/2

β-actin

B.

0

1

2

3

4

5TM3+Ber+hCGHypoxia

p-P3

8 /

-acti

n

Ber(g/ml) - - 10 20 50 10 20 50 (1h)

hCG(0.5 IU/ml) - + - - - + + + (3h)

p-P38

β-actin

0

1

2

3

4

5TM3+Ber+hCGHypoxia

p-P3

8 /

-acti

n

Ber(g/ml) - - 10 20 50 10 20 50 (1h)

hCG(0.5 IU/ml) - + - - - + + + (3h)

p-P38

β-actin

Figure 16. Effect of berberine on (A) ERK and (B) p38 expression in TM3 treated

with hCG (0.5 IU/ml) under hypoxia condition.