lab metting on friday 2011.03.11
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Baicalein protects HT22 murine hippocampal neuronal cells
against endoplasmic reticulum stress-induced apoptosis
through inhibition of reactive oxygen species production and
CHOP induction
Ji Hyun Choi, A Young Choi , Hana YoonWonchae Choe , Kyung-Sik Yoon , Joohun Ha, Eui-Ju Yeo and
Insug Kang
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Aims or objectives
- To investigate the effects of Baicalein on apoptotic celldeath of HT22 cells induced by thapsigargin(TG) and
brefeldin A (BFA)
- To measure the production of ROS, MMP and
expression and cleavage of ER-stress associated
proteins such as caspase -12,-3, poly(ADP-ribose)polymerase, CHOP, P38, JNK, and ERK
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Introduction
- Baicalein (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one) is an effective flavonoids found in Scutellaria
baicalensis
- It shows cytoprotective and anti-inflammatory actions
through radical quenching and anti-oxidative properties- ER stress-induced apoptosis observed in diabetes,
Alzheimers disease, Parkinson disease, and cerebral
ischemia
- ER-stress causes the expression, cleavage, phosphorylation
and activation of different types of unfolded and apoptotic
protein like caspase -12,-3, poly(ADP-ribose) polymerase,
CHOP, P38, JNK, and ERK
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Materials and methods
Cell line:
HT22 mouse hippocampal neuronal cell
Antibodies: Antibodies against CHOP, GRP78, XBP-1, ATF-6, p38,
JNK1, ERK, and eIF2a, HRP-conjugated anti-goat and
anti-rabbit secondary antibodies
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Methods
A. MTT assay
B. Western blot analysis
C. Flow cytometry for DNA contentD. RNA interference (siRNA)
E. Measurement of ROS production
F. Immunoprecipitation
G. Measurement of MMP
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Results and discussions
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Baicalein protects HT 22 hippocampal neuronal cells
against TG- and BFA-induced cell death
MTT assay:
Baicalein reduced cell death
in adose dependent manner
Flow cytrometry:
- Absence ofBaicalein apoptotic
DNA sub-G1 increase from 6.95%
(control) to 63.84%(TG) and 55.27%
(BFA).-Baicalein alone increase 6.95 % to
9.79%
- Combine Baicalein +TG reduces
63.84 to 28.96% and Baicalein+
BFA reduces 55.27 to 26.20%
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Baicalein inhibits the cleavage of caspase-12/-3 and
poly(ADP-ribose) polymerase
Western blot:- Alone TG and BFA increase the expression ofcaspase-12, -3 and cleavage of
PARP
- Treatment with Baicalein reduce their expression and cleavage
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Baicalein regulates unfolded protein responses
Western blot:
- TG and BFA alone
increase the expression
of CHOP, GRP78,
cleavage ofXBP-1, ATF6
and phosphorylation ofelF2
- Baicalein reduce their
expression ,cleavage and
phosphorylation
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Involvement of p38, JNK and ERK to ER-stress
Inhibitor:
SB203580= p38 inhibitorSP600125=JNK inhibitor
PD98059=ERK inhibitor
Results:A. SB203580, SP600125 significantly and
PD98059 less significantly inhibit TG
and BFA induced cell death
B. Knockdown ofcaspase-12 and CHOP
by siRNA reduce the TG and BFA
induced cell death
C + D. SP and PD inhibitor show strong
effect on CHOP production but SB
has noeffect on CHOP production
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Baicalein reduces ER stress-induced ROS
accumulation in HT22 neuronal cells
CellularROS levels were measured by flowcytometry using 2,7-dichlorofluorescein
diacetate (DCF-DA) staining
Result: Baicalein reduces the cellularROS level therefore it has the
anti-oxidant activity
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Anti-oxidants:
N-acetyl cysteine (NAC)
Catalase
Results:
B. NAC and catalase reduce the ROS
level more effectively than Baicalein
C. NAC reduce the TG andBF
A inducedapoptotic death
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Results:
D+ E. NAC block the expression of CHOP and cleavage of caspase-12 in TG and
BFA induced cell
F+G. NAC reduce the phosphorylation of elF2
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Baicalein reduces ER stress-induced MMP in
HT22 neuronal cells
Results:
- MMP is an indicator of mitochondrial damage due to ER-stress examined by
3,3-dihexyloxacarbocyanine iodide (DiOC 6)
- NAC reduce the MMP reduction more effectively than Baicalein
- Rotenone used as an inhibitor of mitochondrial complex I as positive control
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Conclusion
Baicalein reduc e TG and BFA induced unfoldedprotein response such as expression of CHOP and
GRP78, spliced form of XBP-1, cleavage of ATF-6, and
phosphorylation of MAPKS and elF2
Baicalein alone slightly induce MMP reduction and
apoptosis in HT22 cells
Pretreatment with baicalein attenuated TG and BFA
induce ROS accumulation So, Baicalein exerts an anti-oxidative effect against
TG and BFA induced mitochondrial dysfunction and
apoptosis
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Caspase activity assay
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
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