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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

Hepatic fibrosis was attenuated in NOX1KO and NOX4KO mice after CCl4 injury.Livers were obtained from WT, NOX1KO, and NOX4KO mice by 12 intragastric administrations with CCl4 for 6 weeks, twice a week. (A) Representative images of Sirius red staining, immnohistochemistry stainings of desmin and α-SMA are shown. Original magnification X4 and X10. (B) Immunoblotting of α-SMA in liver tissues. (C) Liver function was assessed by ALT and AST. (D) Quantification of morphometric analysis of the sirius red staining and immunochemistry of desmin and α-SMA. (E) Hepatic mRNAs of fibrogenic genes were measured in WT, NOX1KO and NOX4KO mice after CCl4 treatment by way of quantitative real-time PCR. HPRT was used as an internal control. The data are shown as fold mRNA induction compared with control mice. *P <0.05, **P < 0.01.
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pone.0129743.g001: Hepatic fibrosis was attenuated in NOX1KO and NOX4KO mice after CCl4 injury.Livers were obtained from WT, NOX1KO, and NOX4KO mice by 12 intragastric administrations with CCl4 for 6 weeks, twice a week. (A) Representative images of Sirius red staining, immnohistochemistry stainings of desmin and α-SMA are shown. Original magnification X4 and X10. (B) Immunoblotting of α-SMA in liver tissues. (C) Liver function was assessed by ALT and AST. (D) Quantification of morphometric analysis of the sirius red staining and immunochemistry of desmin and α-SMA. (E) Hepatic mRNAs of fibrogenic genes were measured in WT, NOX1KO and NOX4KO mice after CCl4 treatment by way of quantitative real-time PCR. HPRT was used as an internal control. The data are shown as fold mRNA induction compared with control mice. *P <0.05, **P < 0.01.

Mentions: To investigate the roles of NOX1 and NOX4 in hepatic fibrosis, liver fibrosis was induced by CCl4 in knock-out and WT mice and was assessed by morphometric analysis quantification of Sirius red staining. Hepatic fibrosis was significantly decreased in NOX1KO and NOX4KO mice compared with WT mice after CCl4 injections (Fig 1A and 1C). We next investigated the activation of HSCs in NOX1KO, NOX4KO and WT mice after treatment with CCl4. Fig 1A showed that fewer Desmin+α-SMA+ HSCs in livers of CCl4-treated NOX1KO and NOX4KO mice, compared to WT mice. In concordance, western blotting results showed that the expression of α-SMA was higher in fibrotic liver from WT mice than in NOX1KO and NOX4KO mice (Fig 1B).


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)

Hepatic fibrosis was attenuated in NOX1KO and NOX4KO mice after CCl4 injury.Livers were obtained from WT, NOX1KO, and NOX4KO mice by 12 intragastric administrations with CCl4 for 6 weeks, twice a week. (A) Representative images of Sirius red staining, immnohistochemistry stainings of desmin and α-SMA are shown. Original magnification X4 and X10. (B) Immunoblotting of α-SMA in liver tissues. (C) Liver function was assessed by ALT and AST. (D) Quantification of morphometric analysis of the sirius red staining and immunochemistry of desmin and α-SMA. (E) Hepatic mRNAs of fibrogenic genes were measured in WT, NOX1KO and NOX4KO mice after CCl4 treatment by way of quantitative real-time PCR. HPRT was used as an internal control. The data are shown as fold mRNA induction compared with control mice. *P <0.05, **P < 0.01.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4519306&req=5

pone.0129743.g001: Hepatic fibrosis was attenuated in NOX1KO and NOX4KO mice after CCl4 injury.Livers were obtained from WT, NOX1KO, and NOX4KO mice by 12 intragastric administrations with CCl4 for 6 weeks, twice a week. (A) Representative images of Sirius red staining, immnohistochemistry stainings of desmin and α-SMA are shown. Original magnification X4 and X10. (B) Immunoblotting of α-SMA in liver tissues. (C) Liver function was assessed by ALT and AST. (D) Quantification of morphometric analysis of the sirius red staining and immunochemistry of desmin and α-SMA. (E) Hepatic mRNAs of fibrogenic genes were measured in WT, NOX1KO and NOX4KO mice after CCl4 treatment by way of quantitative real-time PCR. HPRT was used as an internal control. The data are shown as fold mRNA induction compared with control mice. *P <0.05, **P < 0.01.
Mentions: To investigate the roles of NOX1 and NOX4 in hepatic fibrosis, liver fibrosis was induced by CCl4 in knock-out and WT mice and was assessed by morphometric analysis quantification of Sirius red staining. Hepatic fibrosis was significantly decreased in NOX1KO and NOX4KO mice compared with WT mice after CCl4 injections (Fig 1A and 1C). We next investigated the activation of HSCs in NOX1KO, NOX4KO and WT mice after treatment with CCl4. Fig 1A showed that fewer Desmin+α-SMA+ HSCs in livers of CCl4-treated NOX1KO and NOX4KO mice, compared to WT mice. In concordance, western blotting results showed that the expression of α-SMA was higher in fibrotic liver from WT mice than in NOX1KO and NOX4KO mice (Fig 1B).

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