Effect of Berberine on Testosterone secretion from
Testicular TM3 cell lines in hypoxia condition.
Yi – Jing Chen1, Ju-Wen Yu1, Hea-Wen Tzou 1, Yuan-Hao Chang1, Ya-Wen Jeng2 and
Shyi-Wu Wang2*
1
Graduate 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
1
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.
2
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.
3
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
4
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.
5
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
6
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)
7
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
8
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.
9
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.
10
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16
TM3 cells
1600
##
###
Testosterone (pg/ml)
1400
1200
1000
800
600
400
***
**
***
-
-
-
+
+
+
(3 hrs)
10
20
50
10
20
50
(1 hrs)
200
0
hCG(0.5 IU/ml)
-
+
Ber (g/ml)
-
-
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
1600
Testosterone (pg/ml)
1400
1200
1000
800
600
400
200
0
hCG (0.5 IU/ml)
Ber ( g/ml)
0
-6
Forskoline (10 M) -
+
0
-
10
-
20
-
50
-
10
+
20
+
50
+
(3 hrs)
(1 hrs)
(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.
17
TM3 cells
1600
Testosterone (pg/ml)
1400
1200
1000
800
600
400
200
0
hCG (0.5 IU/ml)
Ber ( g/ml
AG490 (10-6 M)
0
-
+
0
-
10
-
20
-
50
-
10
+
20
+
50
+
(3 hrs)
(1 hrs)
(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
1600
Testosterone (pg/ml)
1400
1200
1000
800
600
400
200
0
hCG (0.5 IU/ml)
Ber ( g/ml
0
Nifedipine (10-4 M) -
+
0
-
10
-
20
-
50
-
10
+
20
+
50
+
(3 hrs)
(1 hrs)
(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.
18
TM3 cells
1600
Testosterone (pg/ml)
1400
1200
1000
800
600
400
200
0
hCG (0.5 IU/ml)
Ber ( g/ml
SQ22536 (10-5 M)
0
-
+
0
-
10
-
20
-
50
-
10
+
20
+
50
+
(3 hrs)
(1 hrs)
(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
1600
Testosterone (pg/ml)
1400
1200
1000
800
600
400
200
0
hCG (0.5 IU/ml)
Ber ( g/ml)
U73122 (10-4 M)
0
-
+
0
-
10
-
20
-
50
-
10
+
20
+
50
+
(3 hrs)
(1 hrs)
(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.
19
TM3 cells
1600
Testosterone (pg/ml)
1400
1200
1000
800
600
400
200
0
hCG (0.5 IU/ml)
Ber ( g/ml)
0
GF109203X (10-6 M) -
+
0
-
10
-
20
-
50
-
10
+
20
+
50
+
(3 hrs)
(1 hrs)
(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
1600
Testosterone (pg/ml)
1400
1200
1000
800
600
400
200
0
hCG (0.5 IU/ml)
Ber ( g/ml)
0
Helenaline (10-6 M) -
+
0
-
10
-
20
-
50
-
10
+
20
+
50
+
(3 hrs)
(1 hrs)
(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.
20
TM3 cells
Testosterone (pg/ml)
1000
800
600
400
200
0
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.
21
A.
TM3 cells
Testosterone (pg/ml)
10000
8000
6000
4000
2000
0
hCG(IU/ml)
0
0.1
0.5
0.2
1
2
(16hrs)
B.
TM3 cells
Testosterone (pg/ml)
5000
4000
3000
2000
1000
0
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).
22
A.
TM3 cells
hypoxia
Testosterone (pg/ml)
1000
800
600
400
200
0
Ber(g/ml)
0
hCG(0.5IU/ml) -
10
20
50
-
10
20
50
(1h)
-
-
-
+
+
+
+
(16h)
B.
TM3 cells
hypoxia
Testosterone (pg/ml)
1000
800
600
400
200
0
hCG (0.5 IU/ml)
Ber (g/ml)
-6
Forskolin (10 M)
-
+
-
-
-
-
-
-
0
-
0
-
10
-
20
-
50
-
10
+
20
+
50
+
(16 hrs)
(1 hrs)
(1 hrs)
Figure 11. Effect of berberine on testosterone secretion from TM3 treated with hCG
(A) and forskolin (B) under hypoxia condition.
23
A.
TM3 cells
hypoxia
Testosterone (pg/ml)
1400
1200
1000
800
600
400
200
0
hCG (0.5 IU/ml)
Ber (g/ml)
AG490 (10-6 M)
-
+
-
-
-
-
-
-
0
-
0
-
10
-
20
-
50
-
10
+
20
+
50
+
(16 hrs)
(1 hrs)
(1 hrs)
B.
TM3 cells
hypoxia
Testosterone (pg/ml)
10000
8000
6000
4000
2000
0
hCG (0.5 IU/ml)
Ber (g/ml)
0
Nifedipine (10-4 M) -
+
-
-
-
-
-
-
0
-
10
-
20
-
50
-
10
+
20
+
50
+
(16 hrs)
(1 hrs)
(1 hrs)
Figure 12. Effect of berberine on testosterone secretion from TM3 treated with hCG
and (A) AG490 or (B) nifedipine under hypoxia condition.
24
A.
TM3 cells
hypoxia
35000
Testosterone (pg/ml)
30000
25000
20000
15000
10000
5000
0
hCG (0.5 IU/ml)
Ber (g/ml)
SQ22536 (10-5 M)
-
+
-
-
-
-
-
-
0
-
0
-
10
-
20
-
50
-
10
+
20
+
50
+
(16 hrs)
(1 hrs)
(1 hrs)
B.
TM3 cells
hypoxia
Testosterone (pg/ml)
60000
50000
40000
30000
20000
10000
0
hCG (0.5 IU/ml)
Ber (g/ml)
-4
U73122 (10 M)
-
+
-
-
-
-
-
-
0
-
0
-
10
-
20
-
50
-
10
+
20
+
50
+
(16 hrs)
(1 hrs)
(1 hrs)
Figure 13. Effect of berberine on testosterone secretion from TM3 treated with hCG
and (A) SQ22536 or (B) U73122 under hypoxia condition.
25
A.
TM3 cells
hypoxia
Testosterone (pg/ml)
20000
15000
10000
5000
0
hCG (0.5 IU/ml)
Ber (g/ml)
0
GF109203X (10-6 M) -
+
-
-
-
-
-
-
0
-
10
-
20
-
50
-
10
+
20
+
50
+
(16 hrs)
(1 hrs)
(1 hrs)
B.
TM3 cells
hypoxia
Testosterone (pg/ml)
12000
10000
8000
6000
4000
2000
0
hCG (0.5 IU/ml)
Ber (g/ml)
0
Helenaline (10-6 M) -
+
-
-
-
-
-
-
0
-
10
-
20
-
50
-
10
+
20
+
50
+
(16 hrs)
(1 hrs)
(1 hrs)
Figure 14. Effect of berberine on testosterone secretion from TM3 treated with hCG
and (A) AG490 or (B) nifedipine under hypoxia condition.
26
A.
VEGF/Actin (flod relative to control)
VEGF
β-Actin
55 KDa
43 KDa
20110930
N=1
5 x 104/ml
pass. 4
hypoxia
TM3 cells
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
hCG 0.5 IU/ml
-
+
-
-
-
Ber (g/ml)
-
-
10
20
50
+
+
+
(1 hrs)
10
20
50
(1 hrs)
B.
HIF-1Actin (fold relative to control)
HIF-1α
β-Actin
72 KDa
43 KDa
20110930
N=1
5 x 104/ml
pass. 4
hypoxia
TM3 cells
2.5
2.0
1.5
1.0
0.5
0.0
hCG 0.5 IU/ml
-
+
-
-
-
Ber (g/ml)
-
-
10
20
50
+
+
+
(1 hrs)
10
20
50
(1 hrs)
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.
27
A.
p-ERK1/2
β-actin
TM3+Ber+hCG
Hypoxia
3.5
ERK 1
ERK1/2 / -actin
3.0
ERK 2
2.5
2.0
1.5
1.0
0.5
0.0
Ber(g/ml) -
-
10
hCG(0.5 IU/ml) -
+
-
20 50
10
20 50 (1h)
-
+
+
-
+ (3h)
B.
p-P38
β-actin
TM3+Ber+hCG
Hypoxia
5
p-P38 / -actin
4
3
2
1
0
Ber(g/ml)
hCG(0.5 IU/ml)
-
-
10
20
50
10
20
+
-
-
-
+
+
50 (1h)
+ (3h)
Figure 16. Effect of berberine on (A) ERK and (B) p38 expression in TM3 treated
with hCG (0.5 IU/ml) under hypoxia condition.
28