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 Yoon

Wonchae Choe , Kyung-Sik Yoon , Joohun Ha, Eui-Ju Yeo and
Insug Kang
 Aims or objectives
- To investigate the effects of Baicalein on apoptotic
cell death 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
 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,
Alzheimer’s 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
 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
 Methods
A. MTT assay
B. Western blot analysis
C. Flow cytometry for DNA content
D. RNA interference (siRNA)
E. Measurement of ROS production
F. Immunoprecipitation
G. Measurement of MMP
Results and discussions
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 of Baicalein 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%
 Baicalein inhibits the cleavage of caspase-12/-3 and
poly(ADP-ribose) polymerase

Western blot:
- Alone TG and BFA increase the expression of caspase-12, -3 and cleavage of
PARP
- Treatment with Baicalein reduce their expression and cleavage
Baicalein regulates unfolded protein responses

Western blot:
- TG and BFA alone
increase the expression
of CHOP, GRP78,
cleavage of XBP-1, ATF6α
and phosphorylation of
elF2α

- Baicalein reduce their

expression ,cleavage and
phosphorylation
Baicalein inhibits ER stress-induced activation of
MAPKs such as p38, JNK, and ERK

Western blot:
- Alone TG and BFA
increase the phosphorylation
of p38, JNK, and ERK

- Treatment with Baicalein

reduce the phosphorylation
of p38, JNK but it shows
small inhibitory effect on
ERK phosphorylation
 Involvement of p38, JNK and ERK to ER-stress

B. Knockdown of caspase-12 and CHOP

Inhibitor: by siRNA reduce the TG and BFA
SB203580= p38 inhibitor induced cell death
SP600125=JNK inhibitor
PD98059=ERK inhibitor C + D. SP and PD inhibitor show strong
effect on CHOP production but SB
Results: has no effect on CHOP production
A. SB203580, SP600125 significantly and
PD98059 less significantly inhibit TG
and BFA induced cell death
 Baicalein reduces ER stress-induced ROS
accumulation in HT22 neuronal cells

Cellular ROS levels were measured by flow

cytometry using 2’,7’-dichlorofluorescein
diacetate (DCF-DA) staining

Result: Baicalein reduces the cellular ROS level therefore it has the
anti-oxidant activity
Anti-oxidants:
N-acetyl cysteine (NAC)
C. NAC reduce the TG and BFA induced
Catalase
apoptotic death
Results:
B. NAC and catalase reduce the ROS
level more effectively than Baicalein
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 elF2α

 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
 Conclusion
• Baicalein reduc e TG and BFA induced unfolded
protein response such as expression of CHOP and
GRP78, spliced form of XBP-1, cleavage of ATF-6, and
phosphorylation of MAPKS and elF2α
• 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
Caspase activity assay