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

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Fig.1

Fig.1

a-SMA staining 1 µM immunofluorescence

×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

Fig.7

immunofluorescence

immunofluorescence

×400

×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:

Thanks

P-CDC2 ：细胞周期蛋白依赖性激酶，在细胞周期调控中起着调控 G2 至 M 期的作用。正常情况下，在 G2 后期， CDC2 与细胞周期蛋白 B1(Cyclin B1) 结合形成激酶复合物，从而促使细胞从 G2 期进入 M 期，该复合物在 G2-M 期转换中是必须的。但在 HS-173 作用下，检测 cyclin B1 表达下调，限制了激酶复合物的形成，因此细胞暂停在 G2/M 期。