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Scientific Reports , 2013-12-11
HS-173, a Novel PI3K Inhibitor, Attenuates the Activation of Hepatic Stellate Cells in Liver Fibrosis
陈卓
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Background
HSCs are recognized as the primary cellular source of ECM, and play a critical role in the development and maintenance of liver fibrosis.
Hepatic fibrosis represents the final common pathway of virtually all types of chronic liver diseases. This progressive pathological process is characterized by the accumulation of extracellular matrix (ECM) proteins
Activated HSCs with a myofibroblastic phenotype have a high proliferative index, and these cells release profibrogenic cytokines, and consequently produce ECM-related molecules such as alpha-smooth muscle actin (a-SMA), collagen, and tissue inhibitors of metalloproteinases (TIMPs). HSCs are major cellular targets for preventing the progression of liver fibrosis.
In the liver, phosphatidylinositol 3-kinase (PI3K) represents an important signaling molecule that controls many cellular functions including proliferation, survival, adhesion, and migration.
Inhibition of PI3K signaling in HSCs suppresses extracellular matrix (ECM) deposition, type I collagen synthesis, and reduce the expression of profibrogenic factors.
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Blocking PI3K activity with LY294002 has been found to inhibit HSC proliferation and collagen gene expression through the interruption of key downstream signaling pathways including ones involving Akt and P70S6K. Therefore, the interruption of PI3K signaling could inhibit the key components of HSC activation and proliferation, and may represent a target for treating hepatic fibrosis.
Liver fibrosis
HSCs
PI3K
LY294002/HS-173
This article mainly evaluated the anti-fibrotic effect of HS-173 along with the mechanisms underlying these processes in liver fibrosis.
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Background
HS-173 : ethyl6-(5-(phenylsulfonamido)pyridin-3-yl)imidazo[1,2-a]pyridine-3-carboxylate 乙烷 6-(5-( 苯磺酰胺基 ) 吡啶 -3- 基 ) 咪唑 [1,2-a] 吡啶 -3- 羧酸盐LY294002 : 是一种常用的 phosphatidylinositol 3-kinase (PI3K) 抑制剂。可以通透细胞,特异性抑制 PI3K ,抑制 PI3K/Akt 信号途径,包括常见的抑制 Akt 磷酸化等。
HSC-T6 :大鼠肝星状细胞系
LX2 :人肝星状细胞系
HSC cell cycle arrest
p-cdc2 and cyclin B1 expression
PI staining flow cytometry Fig.2A
immunofluorescence Fig.2B
HSC-T6
HS-173 induces cell cycle arrest in the G2/M phase
HS-173 inhibits the expression of profibrotic mediators and ECMdegradation modulators
Fibronetin / vimentin
collagen I/ TIMP-1
collagen IV / MMP-2
Fig.4A
Fig.4B
Fig.4C
immunofluorescence
Western blotting
immunofluorescence
HSC-T6
LX-2
Cytotoxic effects (Cell viability) Fig.1AMTT assay
HSC proliferationBrdU staining
Fig.1B
HSC activation (a-SMA) Fig.1C
TUNEL staining HSC apoptosis
Fig.3A
Western blotting cleaved caspase-3 /Bcl-2 Fig.3B
JC-1 stainingFig.3Cmitochondria potential
HS-173 induces HSC apoptosis
HS-173 inhibits the Proliferation and activation of HSCs
HS-173 blocks PI3K/Aktsignaling by decreasing p-Akt and p-P70S6K
p -Akt and p-P70S6K Western blotting
Western
Fig.5A
Fig.5B
HS-173
p -Akt and p-P70S6KHS-173/ LY294002
In vitro
HSC-T6
immunofluorescence
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Fig.1
Fig.1
HS-173 inhibits the proliferation and activation of HSCs
Fig.1
MTT assay
BrdU staining
DAPI
1 µM
×400
Fig.2
Fig.2
HS-173 induces cell cycle arrest in the G2/M phase
Fig.2
PI staining flow cytometry
immunofluorescence 5 µM
G1 SG2/M
SubG1
×800
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Fig.3
HS-173 induces HSC apoptosis
TUNEL staining
Western blotting
JC-1 staining
5 µM
HSC-T6
1 µM
×800
×200
Fig.4
HS-173 inhibits the expression of profibrotic mediatorsand ECM degradation modulators in HSCs
immunofluorescence
immunofluorescence
Western blotting
1 µM
×400
×400
Fig.5
HS-173 blocks PI3K/Akt signaling by decreasing the expression phosphorylation of Akt and P70S6K in HSCs
Western blotting Western blotting
CCl 4 -induced liver fibrosis in mice
H&E and MT staining Liver fibrosis
AST/ALT in mice serum
Fig.6A
Fig.6B
HS-173 improves liver fibrosis
HS-173 inhibits ECM accumulation and PI3K/Akt signaling
TGF-β1/a-SMA/ collagen I /MMP-2/TIMP-1
p-Akt /p-P70S6K
immunostaining in tissues
p-Akt+collagen I/p-P70S6K/+ vimentin
immunofluorescence
immunofluorescence
Fig.7A
Fig.7B
Fig.7C
In vivo
Fig.6
HS-173 improves CCl 4 -induced liver fibrosis in mice animal model
H&E and MT staining
×200
Aspartate transaminase Alanine transaminase
Serum
collagen
Fig.7
HS-173 inhibits ECM accumulation and PI3K/Akt signaling in mice with CCl 4 -induced liver fibrosis animal model
immunostaining in tissues
×400
HS-173 ameliorates liver fibrosis in vitro and in vivo by promoting HSC apoptosis and inhibiting the expression of fibrotic mediators by blocking the PI3K/Akt pathway in vitro and in vivo.
Conclusion: