Lab metting on Friday 2011.03.11

8/7/2019 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

Documents

Lab metting on Friday 2011.03.11