The nicotinamide adenine dinucleotide phosphate oxidase (NOX) homologues NOX1 and NOX2/gp91(phox) mediate hepatic fibrosis in mice

Abstract

Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is a multicomponent enzyme that mediates electron transfer from nicotinamide adenine dinucleotide phosphate to molecular oxygen, which leads to the production of superoxide. NOX2/gp91(phox) is a catalytic subunit of NOX expressed in phagocytic cells. Several homologues of NOX2, including NOX1, have been identified in nonphagocytic cells. We investigated the contributory role of NOX1 and NOX2 in hepatic fibrosis. Hepatic fibrosis was induced in wild-type (WT) mice, NOX1 knockout (NOX1KO) mice, and NOX2 knockout (NOX2KO) mice by way of either carbon tetrachloride (CCl(4) ) injection or bile duct ligation (BDL). The functional contribution of NOX1 and NOX2 in endogenous liver cells, including hepatic stellate cells (HSCs), and bone marrow (BM)-derived cells, including Kupffer cells (KCs), to hepatic reactive oxygen species (ROS) generation and hepatic fibrosis was assessed in vitro and in vivo using NOX1 or NOX2 BM chimeric mice. Hepatic NOX1 and NOX2 messenger RNA expression was increased in the two experimental mouse models of hepatic fibrosis. Whereas NOX1 was expressed in HSCs but not in KCs, NOX2 was expressed in both HSCs and KCs. Hepatic fibrosis and ROS generation were attenuated in both NOX1KO and NOX2KO mice after CCl(4) or BDL. Liver fibrosis in chimeric mice indicated that NOX1 mediates the profibrogenic effects in endogenous liver cells, whereas NOX2 mediates the profibrogenic effects in both endogenous liver cells and BM-derived cells. Multiple NOX1 and NOX2 components were up-regulated in activated HSCs. Both NOX1- and NOX2-deficient HSCs had decreased ROS generation and failed to up-regulate collagen α1(I) and transforming growth factor β in response to angiotensin II.

Conclusion: Both NOX1 and NOX2 have an important role in hepatic fibrosis in endogenous liver cells, including HSCs, whereas NOX2 has a lesser role in BM-derived cells.

Fig. 1. Expression of NOX1 and NOX2 are upregulated in hepatic fibrosis

Livers were obtained from WT mice after 12 times of intraperitoneal injection of CCl₄ (n=8) or 3 weeks of BDL (n=10). (A, B) Hepatic mRNA levels of NOX1 and NOX2 were measured after treatment with (A) CCl₄ or (B) BDL using qPCR. (C) Double immunofluorescence staining for NOX1 (red fluorescence) and α-SMA (HSC), F4/80 (KC), or CD31 (SEC) (all green fluorescence). (D) Double Immunofluorescence staining for NOX2 (red fluorescence) and α-SMA (HSC), F4/80 (KC) or CD31 (SEC) (all green fluorescence). The liver sections were from (C, D) CCl₄-treated mice. Arrow head indicates double positive cells. Data are representative of three independent experiments. *P<0.05, **P<0.01. Original magnification, ×200 (C,D).

Fig. 2. Hepatic fibrosis is attenuated in NOX1KO and NOX2KO mice after CCl₄ treatment or BDL

Livers were obtained from WT mice after 12 times of intraperitoneal injection of CCl₄ (n=8) or 3 weeks of BDL (n=10). (A) Representative images of Sirius Red staining in WT, NOX1KO, and NOX2KO mice after 12 CCl₄ injections or 3 weeks of BDL are shown. Hepatic fibrosis was assessed by morphometric analysis of the Sirius Red-stained area in the liver or by the measurement of hepatic hydroxyproline content in (B) CCl₄-treated mice or (C) BDL mice. Statistical significance relative to WT CCl₄-treated mice or WT BDL mice: *P < 0.05, **P < 0.01. Original magnification, ×100 (A).

Fig. 3. Activation of HSCs and expression of profibrogenic genes are decreased in NOX1KO and NOX2KO mice after CCl₄ treatment or BDL

(A) Representative images of α-SMA immunohistochemical staining in WT, NOX1KO and NOX2KO mice after 12 intraperitoneal injections of CCl₄ (n=8) or 3 weeks of BDL (n=10) are shown. Activation of HSCs was assessed by (B) morphometric measurement of α-SMA stained area in the liver or (C) immunoblotting for α-SMA. (D, E) Hepatic mRNA levels of collagen α1(I), α-SMA, TGFβ, and TIMP1 were measured in WT, NOX1KO, and NOX2KO mice after (D) 12 CCl₄ injections (n=8) or (E) 3 weeks of BDL (n=10) by quantitative real-time PCR. Statistical significance relative to WT CCl₄-treated mice or WT BDL mice: *P < 0.05, **P < 0.01. Original magnification, ×50 (A).

Fig. 4. Lipid peroxidation is reduced in NOX1KO and NOX2KO mice compared to WT mice after CCl₄ treatment or BDL

(A) Representative images of 4-hydroxynonenal (4-HNE) immunofluorescent staining in WT, NOX1KO, and NOX2KO mice after 12 intraperitoneal injections of CCl₄ (n=8) or 3 week of BDL (n=10) are shown. (B) Hepatic malondialdehyde levels were measured using thiobarbituric acid reactive substances (TBARS) assay in WT, NOX1KO and NOX2KO mice after 12 CCl₄ injections (n=8) or 3 weeks of BDL (n=10). Statistical significance relative to WT CCl₄-injected mice or WT BDL mice: *P < 0.05, **P < 0.01. Original magnification, ×200 (A).

Fig. 5. Chimeric mice with NOX1- or NOX2-deficient endogenous liver cells show decreased hepatic fibrosis after CCl₄ treatment

After the lethal irradiation, BMT was performed to generate chimeric mice as follows: WT mice with WT BM, WT mice with NOX1KO or NOX2KO BM, NOX1KO mice with WT or NOX1KO BM, and NOX2KO mice with WT or NOX2KO BM. Liposomal clodronate was injected intraperitoneally to deplete resident KCs after 2 weeks of BMT. Mice underwent 10 times of intraperitoneal CCl₄ injections from 8 weeks after BMT. (A and D) Representative images of Sirius Red staining are shown (original magnification, ×100). (B and E) Liver injury was assessed by measurement of serum ALT levels. (C and F) Hepatic fibrosis was assessed by measurement of hepatic hydroxyproline content. Statistical significance relative to WT mice with WT BM: *P < 0.05, **P < 0.01.

Fig. 6. Expression of NOX components in isolated liver cell fractions

mRNA levels of NOX1, NOX2, NOX4, p22^phox, p40^phox, p47^phox, p67^phox, NOXO1, NOXA1, and Rac1 were evaluated by quantitative real-time PCR in (A) isolated liver cell fractions from WT mice including hepatocytes, KCs, SECs, and HSCs, and in (B) quiescent (1 day in culture) HSCs, in vitro activated (7 days in culture) HSCs and in vivo activated (isolated from WT mice after 3 weeks of BDL) HSCs. Ribosomal 18S was used as an internal control. Data were from 2 independent experiments performed in triplicates. Statistical significance compared to quiescent HSCs: *P < 0.05, **P < 0.01.

Fig. 7. Both NOX1 and NOX2 mediate ROS production and fibrogenic responses in HSCs

(A) HSCs from WT, NOX1KO and NOX2KO mice were loaded with redox-sensitive dye CMH₂DCFDA (10 μM) for 20 minutes. Cells were then washed twice and subsequently stimulated with angiotensin II (10⁻⁶ M). Fluorescent signals were quantified continuously for 30 minutes using a fluorometer. (B) mRNA expression of collagen α1(I), TGF-β, TIMP1, and α-SMA were measured in WT, NOX1KO, and NOX2KO mice 24 hours after angiotensin II (10⁻⁶ M) stimulation by qPCR. Data were from 2 independent experiments performed in triplicates. *P < 0.05, **P < 0.01.