Download AMP-activated protein kinase induces apoptosis in LX2 cell

AMP-activated protein kinase induces apoptosis in
LX2 cells involving of Bax pathway
 Background
 Result
 Possible mechanism
 Next works
Background
 Hepatic stellate cells (HSCs) are a major
fibrogenic cell type which contributes to
extracellular matrix accumulation during chronic
liver diseases.
 HSCs also play a critical role in the resolution of
hepatic fibrosis, where activated HSCs take
place apoptosis.
 So inducing apotosis of HSCs is a potential
therapic strategy for hepatic fibrosis.
 Recent evidence has indicated that AMPactivated protein kinase (AMPK) can induce
apoptosis of several kinds of cells, such as
rat liver cells, MIN6 cells and human
neuroblastoma cells, but little is known
regarding this matter in HSCs.
AMPK
 AMPK is a serine/threonine
protein kinase, composed of
a catalytic subunit (α) and
two regulatory subunits
(βandγ) .
 AMPKα1 1-312 residues no
longer associates with
theβandγsubunits but
retaines significant kinase
activity .
 Mutation of thronine 172
within theαsubunit to an
asparitic acid residue within
this truncated protein prevent
its inactivation by protein
phosphatases.
Result
Adenovirus-mediated expression of AMPKα1 312
AMPKα1 312 should be expressed between 48h and 72h,
and the activity can sustain to 72 at least.
Expression of constitutively active AMPK induces apoptosis in LX2 cell
DNA ladder appeared after expression of
constitutively active AMPK
Lane1: Marker
Lane2: treated with Ad-CA-AMPK
Lane3: treated with Ad-Luc
Lane4: treated with H2O2
Lane5: blank control
Apoptotic peak appeared after expression of constitutively active AMPK
Blank control
Ad-Luc
Group
Control
Ad-Luc
Ad-CA-AMPK
apoptosis rate（％）
0.873±0.389
0.953±0.141
26.040±7.762*#
* vs to control; # vs to Ad-Luc
Ad-CA-AMPK
Bax was up-regulated with the over-expression of AMPK
Bcl-2 is expressed at a
low level
Pro-caspase-3 was activated and
bax was upregulated
Bax was knocked down by RNAi
RNAi for Bax
Cell phenotype changes after RNAi for Bax and infection of adenoviruses
Ad-CA-AMPK
RNAi + Ad-Luc
RNAi + Ad-CA-AMPK
Activation of caspase-3 induced by AMPK
recovered with RNAi for Bax
Possible mechanisms
AMPK
AMPK
P
P
JNK
ACC
P
c-Jun
Cyto c
Malonyl-CoA
Caspase-9
ba
x
β-oxidation
Caspase-3,6,7
Oxidative stress
Apoptosis
下步的工作
 进一步证实AMPK是不是通过JNK通路激活了Bax，诱导凋
亡。
 动物试验
1. 建模后尾静脉注射Ad-CA-AMPK,
2. Metformin，TZDs喂食动物,分析其对肝纤维化的影响。
 调查，统计分析： Metformin, TZDs在治疗糖尿病患者时是
否对与同时患有肝炎的人同样具有治疗作用。
Thank you！
Metformin:二甲双胍，
通过抑制糖原异生及糖原
的分解，可降低糖尿病时
的高肝糖生成率。
TZDs：噻唑烷二酮，也
称格列酮类药物，增强靶
组织对胰岛素的敏感性。
其中TRG（曲格列酮）可
引起肝损伤,同类药物还
有RSG(罗格列酮),
PIO(帕格列酮)。
Following chronic liver injury, HSCs proliferate and transform to a
myofibroblast-like phenotype secreting large amounts of extracellular
matrix proteins and tissue inhibitor of metalloproteinase（TIMP).
 Consequences of hepatic AMPK activation. The pharmacologic agents,
metformin and thiazolidinediones (TZDs), activate AMPK in the liver. In
addition, the deletion of SCD results in AMPK activation through an
undetermined mechanism. The activation of AMPK reduceslipogenesis
through three independent mechanisms. Activated AMPK phosphorylates and
inhibits the activity of ACC, which reduces malonyl-CoA formation. ChREBP
is phosphorylated by activated AMPK, which inhibits its entry into the nucleus,
thus suppressing L-PK and lipogenic gene expression. SREBP-1c expression
is reduced by activated AMPK through undefined mechanisms. The
cumulative result of AMPK activation, whether by drugs or through the
deletion of SCD, is a reduction in fatty acid synthesis, decreased malonyl-CoA
concentrations, and increased CPT-1 activity, resulting in increased fatty acid
oxidation.