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Deficiency of NOX1 or NOX4 Prevents Liver Inflammation and Fibrosis in Mice through Inhibition of Hepatic Stellate Cell Activation.

Lan T, Kisseleva T, Brenner DA - PLoS ONE (2015)

Bottom Line: Furthermore, the mRNAs of proliferative and pro-fibrotic genes were downregulated in NOX1 and NOX4 knock-out activated HSCs (cultured on plastic for 5 days).Finally, NOX1 and NOX4 protein levels were increased in human livers with cirrhosis compared with normal controls.Thus, NOX1 and NOX4 signaling mediates the pathogenesis of liver fibrosis, including the direct activation of HSC.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California San Diego, La Jolla, California, United States of America; Vascular Biology Research Institute, Guangdong Pharmaceutical University, Guangzhou, China.

ABSTRACT
Reactive oxygen species (ROS) produced by nicotinamide adenine dinucleotide phosphate oxidase (NOX) play a key role in liver injury and fibrosis. Previous studies demonstrated that GKT137831, a dual NOX1/4 inhibitor, attenuated liver fibrosis in mice as well as pro-fibrotic genes in hepatic stellate cells (HSCs) as well as hepatocyte apoptosis. The effect of NOX1 and NOX4 deficiency in liver fibrosis is unclear, and has never been directly compared. HSCs are the primary myofibroblasts in the pathogenesis of liver fibrosis. Therefore, we aimed to determine the role of NOX1 and NOX4 in liver fibrosis, and investigated whether NOX1 and NOX4 signaling mediates liver fibrosis by regulating HSC activation. Mice were treated with carbon tetrachloride (CCl4) to induce liver fibrosis. Deficiency of either NOX1 or NOX4 attenuates liver injury, inflammation, and fibrosis after CCl4 compared to wild-type mice. NOX1 or NOX4 deficiency reduced lipid peroxidation and ROS production in mice with liver fibrosis. NOX1 and NOX4 deficiency are approximately equally effective in preventing liver injury in the mice. The NOX1/4 dual inhibitor GKT137831 suppressed ROS production as well as inflammatory and proliferative genes induced by lipopolysaccharide (LPS), platelet-derived growth factor (PDGF), or sonic hedgehog (Shh) in primary mouse HSCs. Furthermore, the mRNAs of proliferative and pro-fibrotic genes were downregulated in NOX1 and NOX4 knock-out activated HSCs (cultured on plastic for 5 days). Finally, NOX1 and NOX4 protein levels were increased in human livers with cirrhosis compared with normal controls. Thus, NOX1 and NOX4 signaling mediates the pathogenesis of liver fibrosis, including the direct activation of HSC.

No MeSH data available.


Related in: MedlinePlus

ROS production and gene expression were attenuated by GKT137831 in HSCs in response to LPS, PDGF and Shh stimulation.(A) HSCs isolated from WT mice were loaded with H2DCFDA (10 μM) for 20 min. Cells were then washed and subsequently induced with LPS (100 ng/ml), PDGF (10 ng/ml) and Shh (1 μg/ml) in the presence or absence of GKT137831 (5 μM). ROS production was assessed by fluorescent signals quantified continuously for 60 min using a fluorometer. (B) Chemokine genes were attenuated by GKT137831 in HSCs in response to LPS. HSCs isolated from WT mice were pretreated with GKT137831 (20 μM) for 30 min, and then treated with LPS (100 ng/ml) for 6 h. Chemokine genes were analyzed by quantitative real-time PCR. **P < 0.01, ***P < 0.001 vs control; #P < 0.05, ##P < 0.01 vs LPS. (C) Proliferative genes were attenuated by GKT137831 in HSCs in response to PDGF. HSCs isolated from WT mice were treated with PDGF (10 ng/ml) in the presence or absence of GKT137831 (20 μM) for 24 h. Proliferative genes were analyzed by quantitative real-time PCR. **P < 0.01 vs control; #P < 0.05, ##P < 0.01 vs PDGF. (D) Hedgehog genes were attenuated by GKT137831 in HSCs in response to hedgehog ligand Shh. HSCs isolated from WT mice were treated with Shh (1 μg/ml) in the presence or absence of GKT137831 (20 μM) for 24 and 48 h. Hedgehog genes were analyzed by quantitative real-time PCR. HPRT was used as an internal control. **P < 0.01, ***P < 0.001 vs control; ##P < 0.01, ###P < 0.001 vs Shh.
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pone.0129743.g005: ROS production and gene expression were attenuated by GKT137831 in HSCs in response to LPS, PDGF and Shh stimulation.(A) HSCs isolated from WT mice were loaded with H2DCFDA (10 μM) for 20 min. Cells were then washed and subsequently induced with LPS (100 ng/ml), PDGF (10 ng/ml) and Shh (1 μg/ml) in the presence or absence of GKT137831 (5 μM). ROS production was assessed by fluorescent signals quantified continuously for 60 min using a fluorometer. (B) Chemokine genes were attenuated by GKT137831 in HSCs in response to LPS. HSCs isolated from WT mice were pretreated with GKT137831 (20 μM) for 30 min, and then treated with LPS (100 ng/ml) for 6 h. Chemokine genes were analyzed by quantitative real-time PCR. **P < 0.01, ***P < 0.001 vs control; #P < 0.05, ##P < 0.01 vs LPS. (C) Proliferative genes were attenuated by GKT137831 in HSCs in response to PDGF. HSCs isolated from WT mice were treated with PDGF (10 ng/ml) in the presence or absence of GKT137831 (20 μM) for 24 h. Proliferative genes were analyzed by quantitative real-time PCR. **P < 0.01 vs control; #P < 0.05, ##P < 0.01 vs PDGF. (D) Hedgehog genes were attenuated by GKT137831 in HSCs in response to hedgehog ligand Shh. HSCs isolated from WT mice were treated with Shh (1 μg/ml) in the presence or absence of GKT137831 (20 μM) for 24 and 48 h. Hedgehog genes were analyzed by quantitative real-time PCR. HPRT was used as an internal control. **P < 0.01, ***P < 0.001 vs control; ##P < 0.01, ###P < 0.001 vs Shh.

Mentions: Liver fibrosis is characterized by secretion of inflammatory and fibrotic cytokines. Hedgehog (Hh) ligands, platelet derived growth factor (PDGF), and lipopolysaccharide (LPS) activate signaling pathways in HSCs to promote liver fibrosis. Therefore, we tested ROS production in HSCs treated with LPS, PDGF or the Hh ligand Shh. ROS production in HSCs is increased by LPS, PDGF and Shh (Fig 5A). GKT137831 prevented ROS generation in HSCs to the similar levels as HSCs treated with vehicle alone (Fig 5A), implying that NOX1 or NOX4 is required to generate the ROS by these ligands.


Deficiency of NOX1 or NOX4 Prevents Liver Inflammation and Fibrosis in Mice through Inhibition of Hepatic Stellate Cell Activation.

Lan T, Kisseleva T, Brenner DA - PLoS ONE (2015)

ROS production and gene expression were attenuated by GKT137831 in HSCs in response to LPS, PDGF and Shh stimulation.(A) HSCs isolated from WT mice were loaded with H2DCFDA (10 μM) for 20 min. Cells were then washed and subsequently induced with LPS (100 ng/ml), PDGF (10 ng/ml) and Shh (1 μg/ml) in the presence or absence of GKT137831 (5 μM). ROS production was assessed by fluorescent signals quantified continuously for 60 min using a fluorometer. (B) Chemokine genes were attenuated by GKT137831 in HSCs in response to LPS. HSCs isolated from WT mice were pretreated with GKT137831 (20 μM) for 30 min, and then treated with LPS (100 ng/ml) for 6 h. Chemokine genes were analyzed by quantitative real-time PCR. **P < 0.01, ***P < 0.001 vs control; #P < 0.05, ##P < 0.01 vs LPS. (C) Proliferative genes were attenuated by GKT137831 in HSCs in response to PDGF. HSCs isolated from WT mice were treated with PDGF (10 ng/ml) in the presence or absence of GKT137831 (20 μM) for 24 h. Proliferative genes were analyzed by quantitative real-time PCR. **P < 0.01 vs control; #P < 0.05, ##P < 0.01 vs PDGF. (D) Hedgehog genes were attenuated by GKT137831 in HSCs in response to hedgehog ligand Shh. HSCs isolated from WT mice were treated with Shh (1 μg/ml) in the presence or absence of GKT137831 (20 μM) for 24 and 48 h. Hedgehog genes were analyzed by quantitative real-time PCR. HPRT was used as an internal control. **P < 0.01, ***P < 0.001 vs control; ##P < 0.01, ###P < 0.001 vs Shh.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4519306&req=5

pone.0129743.g005: ROS production and gene expression were attenuated by GKT137831 in HSCs in response to LPS, PDGF and Shh stimulation.(A) HSCs isolated from WT mice were loaded with H2DCFDA (10 μM) for 20 min. Cells were then washed and subsequently induced with LPS (100 ng/ml), PDGF (10 ng/ml) and Shh (1 μg/ml) in the presence or absence of GKT137831 (5 μM). ROS production was assessed by fluorescent signals quantified continuously for 60 min using a fluorometer. (B) Chemokine genes were attenuated by GKT137831 in HSCs in response to LPS. HSCs isolated from WT mice were pretreated with GKT137831 (20 μM) for 30 min, and then treated with LPS (100 ng/ml) for 6 h. Chemokine genes were analyzed by quantitative real-time PCR. **P < 0.01, ***P < 0.001 vs control; #P < 0.05, ##P < 0.01 vs LPS. (C) Proliferative genes were attenuated by GKT137831 in HSCs in response to PDGF. HSCs isolated from WT mice were treated with PDGF (10 ng/ml) in the presence or absence of GKT137831 (20 μM) for 24 h. Proliferative genes were analyzed by quantitative real-time PCR. **P < 0.01 vs control; #P < 0.05, ##P < 0.01 vs PDGF. (D) Hedgehog genes were attenuated by GKT137831 in HSCs in response to hedgehog ligand Shh. HSCs isolated from WT mice were treated with Shh (1 μg/ml) in the presence or absence of GKT137831 (20 μM) for 24 and 48 h. Hedgehog genes were analyzed by quantitative real-time PCR. HPRT was used as an internal control. **P < 0.01, ***P < 0.001 vs control; ##P < 0.01, ###P < 0.001 vs Shh.
Mentions: Liver fibrosis is characterized by secretion of inflammatory and fibrotic cytokines. Hedgehog (Hh) ligands, platelet derived growth factor (PDGF), and lipopolysaccharide (LPS) activate signaling pathways in HSCs to promote liver fibrosis. Therefore, we tested ROS production in HSCs treated with LPS, PDGF or the Hh ligand Shh. ROS production in HSCs is increased by LPS, PDGF and Shh (Fig 5A). GKT137831 prevented ROS generation in HSCs to the similar levels as HSCs treated with vehicle alone (Fig 5A), implying that NOX1 or NOX4 is required to generate the ROS by these ligands.

Bottom Line: Furthermore, the mRNAs of proliferative and pro-fibrotic genes were downregulated in NOX1 and NOX4 knock-out activated HSCs (cultured on plastic for 5 days).Finally, NOX1 and NOX4 protein levels were increased in human livers with cirrhosis compared with normal controls.Thus, NOX1 and NOX4 signaling mediates the pathogenesis of liver fibrosis, including the direct activation of HSC.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California San Diego, La Jolla, California, United States of America; Vascular Biology Research Institute, Guangdong Pharmaceutical University, Guangzhou, China.

ABSTRACT
Reactive oxygen species (ROS) produced by nicotinamide adenine dinucleotide phosphate oxidase (NOX) play a key role in liver injury and fibrosis. Previous studies demonstrated that GKT137831, a dual NOX1/4 inhibitor, attenuated liver fibrosis in mice as well as pro-fibrotic genes in hepatic stellate cells (HSCs) as well as hepatocyte apoptosis. The effect of NOX1 and NOX4 deficiency in liver fibrosis is unclear, and has never been directly compared. HSCs are the primary myofibroblasts in the pathogenesis of liver fibrosis. Therefore, we aimed to determine the role of NOX1 and NOX4 in liver fibrosis, and investigated whether NOX1 and NOX4 signaling mediates liver fibrosis by regulating HSC activation. Mice were treated with carbon tetrachloride (CCl4) to induce liver fibrosis. Deficiency of either NOX1 or NOX4 attenuates liver injury, inflammation, and fibrosis after CCl4 compared to wild-type mice. NOX1 or NOX4 deficiency reduced lipid peroxidation and ROS production in mice with liver fibrosis. NOX1 and NOX4 deficiency are approximately equally effective in preventing liver injury in the mice. The NOX1/4 dual inhibitor GKT137831 suppressed ROS production as well as inflammatory and proliferative genes induced by lipopolysaccharide (LPS), platelet-derived growth factor (PDGF), or sonic hedgehog (Shh) in primary mouse HSCs. Furthermore, the mRNAs of proliferative and pro-fibrotic genes were downregulated in NOX1 and NOX4 knock-out activated HSCs (cultured on plastic for 5 days). Finally, NOX1 and NOX4 protein levels were increased in human livers with cirrhosis compared with normal controls. Thus, NOX1 and NOX4 signaling mediates the pathogenesis of liver fibrosis, including the direct activation of HSC.

No MeSH data available.


Related in: MedlinePlus