(-)-EPIGALLOCATECHIN-3-GALLATE (EGCG) EHNAHCES OSTEOGENESIS IN
A BONE MARROW MESENCHYMAL STEM CELL LINE
Chung-Hwan
1,3
Chen ;
Mei-Ling
2,3
Ho ;
Je-Ken
1,3
Chang
; Shao-Hung
,3
Hung
; Gwo-Jaw
1,3
Wang
Departments of Orthopaedics1, and Physiology2, School of Medicine,
Orthopaedic Research
3
Center
Kaohsiung Medical University, Kaohsiung, TAIWAN
INTRODUCTION:
Green tea is one of the most popular beverages in the world. Among the catechins, (-)epigalloncatechin -3-gallate (EGCG) has received by far the most attention(1). Previous
studies verified the beneficial effects of catechins in decreasing serum lipid, reducing
blood pressure, antitumorigenic and antibacterial effects. Recent surveys have
reported to reduce the risk of having a hip fracture with higher bone mineral density
(BMD) by habitual tea drinkers (2). However, the effective components and the action
mechanisms of tea on bone remodeling remain unclear. In this study, we attempt to
elucidate the osteogenic effects of a green tea catechin, EGCG, on a stem cell line by
screening the mRNA expressions of some osteogenic markers Furthermore, the effects
of EGCG on the osteocalcin protein production, ALP activity and mineralization are
also evaluated. Besides, we evaluate the effect of EGCG on cell proliferation and
apoptosis.
MATERIALS AND METHODS:
The cloned pluripotent mesenchymal cells, D1, were maintained in Dulbecco Modified
Eagle Medium (DMEM) (3). D1 cells were treated with EGCG at concentration of 1
µmol/L or 10 µmol/L for 48 hours respectively. The mRNA expressions of Cbfa1/Runx2,
osterix, osteocalcin (OC) and alkaline phosphatase (ALP) were examined by RT-PCR.
Protein production of OC was evaluated by ELISA after treatment for 4 and 7 days. ALP
activity will be assayed by chemiluminescent method after EGCG treatment for 4, 7 and
14 days. The degree of mineralization effected by EGCG was determined by von Kossa
stain at the 3rd and 4th week. Cell proliferation was assayed by thymidine
incorporation after treated for 24 hours. Cells were stained with tryphan blue for cell
count after treated for 24 hours. Apoptotic effect of EGCG was evaluated by TUNEL
stain after treated for 24 hours.
RESULTS:
First the mRNA expression of osterix, ALP, and OC have shown a markedly increase
with the addition of different concentrations (1 µmol/L or 10 µmol/L) of EGCG, 66 %
( P<0.05) and 137% (P<0.01) for osterix, 44% ( P<0.05) and 109% (P<0.01) for ALP, and
40% ( P<0.05) and 44% ( P<0.05) for OC, respectively. (Fig.1) Second, with respect to
the control and the increase of EGCG concentrations, ALP activities increased by 202%
and 278% (P<0.01) on the 4th day, 161% and 183% (P<0.01) on the 7th day, and 134%
and 148% (P<0.01) on the 14th day (Fig.2). Third, OC production was increase the
addition of different concentrations (1 µmol/L or 10 µmol/L) of EGCG, 96% and 135% on
the 4th day and 71% and 590% on the 7th day(Fig.4). Fourth, On the 3rd week, different
concentration of EGCG, 1 and 10 µmol/L, increased mineralization by 5% and 29%, with
respect to the control, respectively. The differences extend on the 4th week to 25% and
44%, with respect to the control, by addition of EGCG, 1 and 10 µmol/L (Fig.4). Fifth, a
24-hour treatment of EGCG decreased thymidine incorporation by 12% (1 µmol/L) and
24% (10 µmol/L) (P<0.05) (Fig.5). Sixth, cell count after EGCG treated for 24 hours
decreased by 30% (1 µmol/L) and 30% (10 µmol/L) (P<0.05) (Fig.6). Seventh, no obvious
apoptotic effect of EGCG on D1-cell cultures after treated for 24 hours.
**
250
**
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*
200
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150
100
50
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O
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EGCG co n cen tratio n (µmo l/L)
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1
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light unit /mg
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* : P<0.05
**: P<0.01
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Fig 1. mRNA
Fig
2
ALP
activity
40
30
0
1µ
mol/L
10 µ
mol/L
20
10
Fig 3. EGCG increases Mineralization
DISSCUSSIONS:
Our results illustrated that the effective concentration of EGCG is at
the range of 1 to 10 µmol/L. Previous report indicated that one cup of
green tea drinking could accumulate the circulating level of EGCG to
1 µmol/L. Our results indicated that EGCG acts on bone marrow
mesenchymal cells by modulating the differentiation through
augmenting the gene expressions of Cbfa1/Runx2, osterix, ALP and
OC. Our results further demonstrated that EGCG can increase the
potential of terminal osteoblastic differentiation by increasing OC
protein production, ALP activity, which eventually leads to
mineralization confirmed by von Kossa stain. EGCG’s stimulatory
effects on OC protein production, ALP activity and mineralization in
D1-cell cultures further confirmed its post-transcriptional influences
on osteogenesis. However, a 24-hour treatment of EGCG inhibited
thymidine incorporation of D1-cells and decreased cell count. No
obvious apoptotic effect of EGCG on D1-cell under the concentration
of 1 µmol/L and 10 µmol/L. These results demonstrated that long-term
treatment of EGCG increases the expressions of osteogenic genes
and OC protein production, elevates ALP activity and eventually
stimulates mineralization, in spite of its inhibitory effect on
proliferation. The osteogenic effect of EGCG may rely on inducing
osteogenic differentiation, not via increasing proliferation.
Elucidations on the detail mechanisms of EGCG in mesenchymal
stem cells and osteoblasts, both in mice and in humans, require
further investigation.
REFERENCES:
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days
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2. Wang GJ, Cui Q & Balian G, Clin Orthop: 295-310, 2000.
3. Hegarty VM et.al., Am J Clin Nutr: 1003-1007, 2000.