AMP-activated protein kinase induces apoptosis in LX2 cells involving of Bax pathway

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AMP-activated protein kinase induces apoptosis in
LX2 cells involving of Bax pathway
2

• Background
• Result
• Possible mechanism
• Next works

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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.

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• Recent evidence has indicated that AMP-activated
  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.

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AMPK

• AMPK is a serine/threonine protein kinase,
  composed of a catalytic subunit (a) and two
  regulatory subunits (ßand?) .
• AMPKa1 1-312 residues no longer associates with
  theßand?subunits but retaines significant kinase
  activity .
• Mutation of thronine 172 within theasubunit to an
  asparitic acid residue within this truncated
  protein prevent its inactivation by protein
  phosphatases.

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Result

• Adenovirus-mediated expression of AMPKa1 312

AMPKa1 312 should be expressed between 48h and
72h, and the activity can sustain to 72 at least.
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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
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Apoptotic peak appeared after expression of
constitutively active AMPK
Blank control
Ad-Luc
Ad-CA-AMPK
Group apoptosis rate()
Control 0.8730.389 Ad-Luc 0.9530.141 Ad-CA-AMPK 26.0407.762
vs to control vs to Ad-Luc
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Bax was up-regulated with the over-expression of
AMPK
Pro-caspase-3 was activated and bax was
upregulated
Bcl-2 is expressed at a low level
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Bax was knocked down by RNAi
RNAi for Bax
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Cell phenotype changes after RNAi for Bax and
infection of adenoviruses
RNAi Ad-Luc
RNAi Ad-CA-AMPK

• Ad-CA-AMPK

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Activation of caspase-3 induced by AMPK recovered
with RNAi for Bax
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Possible mechanisms
AMPK
AMPK
P
ACC
Cyto c
Malonyl-CoA
Caspase-9
Respiratory china
bax
ß-oxidation
Caspase-3,6,7
Oxidative stress
Apoptosis
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• ?????AMPK?????JNK?????Bax,?????
• ????
• 1. ????????Ad-CA-AMPK,
• 2. Metformin,TZDs????,????????????
• ??,???? Metformin, TZDs??????????????????????????
  ????

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Thank you!
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• Metformin????,??????????????,???????????????
• TZDs?????,????????,????????????????TRG(????)??
  ????,??????RSG(????), PIO(????)?

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• 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).

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• 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.