MitoQ alleviates carbon tetrachloride-induced liver fibrosis in mice through regulating JNK/YAP pathway

Abstract

Background

Liver fibrosis is a pathological wound-healing response caused by chronic liver damage. Mitochondria regulate hepatic energy metabolism and oxidative stress. Accumulating evidence has revealed that increased mitochondrial oxidative stress contributes to the activation of fibrogenesis. However, the roles and underlying mechanisms of mitochondrial oxidative stress in liver fibrosis remain unknown.

Methods and results

In this study, C57BL/6 mice were used to establish a model of liver fibrosis via oral gavage with CCl₄ treatment for 8 weeks. Furthermore, intervention experiments were achieved by CCl₄ combined with the intraperitoneal injection of mitoquinone mesylate (mitoQ). We demonstrated that the chronic CCl₄ exposure resulted in severe hepatic fibrogenesis and significantly promoted the production of reactive oxygen species (ROS) and mitochondrial abnormalities. Besides, JNK/YAP pathway was also activated. By contrast, the administration of mitoQ markedly inhibited the expression of pro-fibrogenic transforming growth factor-β as well as type I collagen. The antifibrotic effects of mitoQ were also confirmed by hematoxylin and eosin staining and Sirius red staining. Moreover, mitoQ substantially reduced CCl₄-induced mitochondrial damage and the release of ROS. Further studies suggested that this protection against liver fibrosis was mechanistically related to the inhibition of phosphorylation of JNK and the nuclear translocation of YAP.

Conclusion

In conclusion, these findings revealed that mitoQ attenuated liver fibrosis by inhibiting ROS production and the JNK/YAP signaling pathway. Selective targeting JNK/YAP may serve as a therapeutic strategy for retarding progression of chronic liver disease.

1. Introduction

Liver fibrosis characterized by the deposition of extracellular matrix (ECM) is a reversible wound-healing response triggered by acute or chronic liver injury.^1,2 Hepatic stellate cell (HSC) is the primary source of ECM in liver upon injury, and their activation represents a critical event in fibrosis.³ Cirrhosis is the terminal stage of progressive liver fibrosis, which is estimated to affect 1–2% of global population and results in over 1 million deaths annually worldwide.^4,5 However, there is still a huge unmet medical need for anti-fibrotic therapies to prevent liver disease progression and hepatocellular carcinoma (HCC) development. In recent years, researches have shown that oxidative stress plays an important role in the occurrence and development of liver fibrosis, and many of the factors that cause liver injury are associated with the increase of oxidative level or the decrease of antioxidant capacity in varying degrees.⁶ Therefore, the relationship between oxidative stress and liver fibrosis’ pathological mechanism is attracting more and more attention.

Mitochondrial oxidative damage contributes to a range of pathologies including steatosis and insulin resistance (IR), diabetes, nonalcoholic steatohepatitis (NASH), or various stages of alcoholic steatohepatitis.^7–10 Previous study has demonstrated that liver tissues from patients with obesity and NASH have ultrastructural damage to the mitochondria,^11,12 involving in the mechanism of high mitochondrial levels of reactive oxygen species (ROS) and ROS-mediated mitochondrial DNA (mtDNA) damage.^10,13 Low and intermediate levels of ROS are physiologically important as cell signaling molecules, whereas high ROS concentrations that preclude cell clearance cause oxidative stress, mitochondrial dysfunction, and cellular and DNA damage that can result in inflammation and cell death.^14,15 More importantly, ROS are believed to play an important role in inducing the proliferation of HSCs, the migration of HSCs, collagen accumulation, and the formation of liver fibrosis.^16,17 It implies that ROS generation may be considered as one of the mechanisms of CCl₄-induced liver fibrosis.¹⁸

The roles for mitochondrial ROS-induced MAPK signaling pathways in the development of NAFLD, NASH, fibrosis, and IR have been investigated in animal models—sustained activation and mitochondrial translocation of c-Jun N-terminal kinase (JNK) mediate liver injury.¹⁹ Phosphorylated JNK translocate to mitochondria where it binds to MOM and MIM scaffold proteins to inhibit MRC, increasing ROS formation.^19,20 Furthermore, reducing production of mitochondrial ROS and inhibiting interactions between phosphorylated JNK and mitochondria reduce liver injury in mouse models.¹⁹ Besides, recent studies have shown that JNK is an upstream molecule of YAP and regulate the activation of YAP.^21,22 For example, one study demonstrated that the JNK inhibitor (SP600125) blocked mechanical-tension-induced YAP activation.²² Importantly, activation of YAP promotes fibrosis in multiple organs, including the liver, kidney, skin, and lung.^23–25 Mechanistic studies have revealed that YAP drives HSC activation and fibrotic programs regulated by Hedgehog signaling,^26,27 and it is an important regulator of biology in liver diseases.²⁸ Targeting YAP in fibroblasts has been shown to attenuate fibrosis.^29,30 However, the mechanism link between mitochondrial oxidative stress and JNK/YAP pathway in liver fibrosis remains largely unclear.

Mitoquinone mesylate (mitoQ), mitochondria-targeted antioxidant, comprises coenzyme Q10 and TPP cations, which makes it several hundred-fold more potent than an untargeted antioxidant with respect to the prevention of mitochondrial oxidative damage.³¹ MitoQ plays a protective role in various diseases, such as neurodegenerative disease, diabetic kidney disease, and liver fibrosis.^32–35 Rehman et al.³³ demonstrated that mitoQ ameliorates liver fibrosis in vivo and directly inhibits HSC activation in vitro. However, its exact role and mechanism remain unclear, especially whether mitoQ is related to JNK/YAP signaling pathway in the process of suppressing CCl₄-induced liver fibrosis.

This study was designed to investigate the roles of mitochondrial oxidative stress on HSC activation driven by chronic CCl₄ exposure and to determine the potential mechanisms by investigating the roles of mitochondrial oxidative stress in JNK/YAP pathway. We found that CCl₄ administration induced the mass production of ROS and mitochondrial damage. Moreover, JNK/YAP pathway was significantly activated by chronic CCl₄ exposure, thereby increasing HSC activation. More importantly, our results revealed that the inhibition of mitochondrial oxidative stress effectively prevented mitochondrial damage and the occurrence of CCl₄-induced liver fibrosis. Mechanistically, mitoQ significantly inhibited the activation of HSCs through regulating the phosphorylation levels of JNK and nuclear transcription of YAP. Therefore, our study demonstrates that mitochondrial oxidative damage involves in liver fibrosis and may be attributed to the modulation of JNK/YAP pathway, which underline the hepatoprotective effects of mitoQ against chronic liver disease.

2. Materials and methods

2.1 Materials

CCl₄ (purity ≥99%) was purchased from Shanghai Macklin Biochemical Co., Ltd. MitoQ (purity ≥98.0%) was purchased from Med Chem Express (Monmouth Junction, NJ). Horseradish peroxidase (HRP)-conjugated goat anti-mouse/rabbit IgG SABC kit (SA1020) and diaminobenzidine (DAB) chromogenic reagent kit (AR1027-3) were purchased from Boster Biological Technology, Wuhan, China. Primary antibodies used in this study include anti-COL1A1 (CST, #91144), α-SMA (CST, #19245s), JNK (Santa Cruz, sc-7345), p-JNK (CST, #4668), LATS1 (CST, #3477), YAP (CST, #4912) and YAP (Santa Cruz, sc-101199), p-YAP (CST, #4911), transforming growth factor-β (TGF-β, CST, 3711s), and TGF-β (LBI1E12). Monoclonal anti-β-actin, peroxidase-conjugated goat anti-rabbit and goat anti-mouse IgG secondary antibodies, and protease inhibitor cocktail were purchased from Sigma-Aldrich Chemical Co. Alexa Fluor 568 goat anti-mouse IgG (H + L), Alexa Fluor 488 goat anti-mouse IgG (H + L), Alexa Fluor® 568 goat anti-rabbit IgG (H + L), Alexa Fluor® 488 Goat Anti-Rabbit IgG (H + L), Hoechst 33343 are from Life Technologies (Carlsbad, CA). NE-PER nuclear and cytoplasmic extraction reagents kits, BCA protein assay kit, and SuperSignal® West Pico chemiluminescent substrate kit were purchased from Pierce Biotechnology, Inc. (Rockford, IL). All other chemicals were of analytical grade from commercial supplier.

2.2 Animals and treatments

Male C57BL/6 mice were purchased from the Experimental Animal Center of Shandong University. The animals were kept in an air-conditioned room at 23–25 °C with a 12 h dark/light cycle for 1 week prior to the initiation of the experiments. All the animals received appropriate care during the study with free access to food and water. Fifty male C57BL/6 mice were randomly divided into five groups to obtain the time-response model (n = 10 mice/group): (i) control group, (ii) 2-week CCl₄ group, (iii) 4-week CCl₄ group, (iv) 6-week CCl₄ group, (v) 8-week CCl₄ group. Mice receive escalating doses of CCl₄ (50/50 vol. mixed with corn oil) three times per week via oral gavage for 8 weeks, starting with 0.437 ml/kg (first to third dose, week 1), 0.656 ml/kg (fourth dose, week 2), 0.875 ml/kg (fifth to ninth dose, week 2–4), and 1.25 ml/kg (10th–24th dose, week 4–8). Control mice received the same volume of corn oil via oral gavage. Then, animals were sacrificed every 2 weeks. Furthermore, another 40 C57BL/6 mice were divided into four groups for mitoQ intervention experiment (n = 10 mice/group): (i) control group, the mice were oral gavage with corn oil for 8 weeks; (ii) 2 mg/kg mitoQ group, (iii) CCl₄ group, the mice were oral gavage with CCl₄ for 8 weeks, (iv) CCl₄ + 2 mg/kg mitoQ group, the mice were administered both mitoQ and CCl₄ for 8 weeks. MitoQ was dissolved in 5% polyethylene glycol 400, 5% Tween 80 and was given intraperitoneally to animals of intervention groups. The mice were euthanized, and liver samples were removed and snap-frozen in liquid nitrogen for storage at −80 °C. An additional section was fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. Serum and liver tissue of all groups were collected for further analysis. All experiments were carried out in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and followed the principles in the “Use of Animals in Toxicology”. The use of animals was approved by the Ethics Committee of Shandong University Institute of Preventive Medicine, and Ethics Committee number is 20190201.

2.3 Serum analyses and histological examination

The blood samples were kept at room temperature for 1 h. After centrifugation at 3,000 g for 15 min, serum was collected to measure the levels of alanine transaminase (ALT) and aspartate transferase (AST) with kits according to the manufacturer’s instructions. Furthermore, a segment of liver was fixed in 4% neutral buffered formalin solution at least 24 h. Then, the liver tissues were dehydrated with a sequence of ethanol solutions, embedded in paraffin wax, and sectioned at 4-μm thickness. At last, the liver tissue sections were stained with hematoxylin and eosin (H&E) for morphological evaluation; Sirius Red staining were used to assess the degree of liver fibrosis and then observed to evaluate histopathological changes under light microscopy. Quantification of the liver damage area and percentage of collagen per field with Sirius Red staining used Image J.

2.4 Immunohistochemical staining

The liver tissues embedded in paraffin were sliced into 4–6-μm pieces, deparaffinized, and serially dehydrated in ethanol. The sections were boiled for 3 min while immersed in EDTA-Tris (pH 9.0) for antigen retrieval to nuclear staining and then treated with hydrogen peroxide for 30 min to inactivate the endogenous peroxidase. The sections were incubated overnight at 4 °C with primary antibody α-SMA. The sections were subsequently incubated with the appropriate biotinylated secondary (goat anti-rabbit /mouse IgG) for 30 min at room temperature, stained with DAB, and counterstained with hematoxylin. The positive areas were colored a brownish yellow and relative α-SMA expression was quantified by Image J.

2.5 Immunofluorescence staining of livers

Paraffin sections of mice livers (5 μm) were firstly deparaffinized and hydrated. Then, antigen retrieval was performed by submerging the sections in citrate buffer and microwaving for 10 min. For immunostaining, the sections were treated with 10% serum, followed by overnight incubation with primary antibodies. The next day, sections were incubated with fluorescent secondary antibodies for 4 h at room temperature. Nuclei were stained with Hoechst 33343 and mounted with SlowFade Antifade Mountant. Finally, stained sections were observed and photographed with Zeiss fluorescence microscope. The relative fluorescence intensity was quantified by Image J.

2.6 Tissue preparation and immunoblot analysis

Total proteins were extracted from mouse liver using RIPA lysis buffer. Tissue homogenates were centrifuged at 12,000 g for 15 min, and then the supernatants were kept for western analysis of target protein. Mitochondrial proteins were extracted from mice livers using the tissue mitochondria isolation kit. The cytosolic and nuclear fractions were prepared as previously described with mild modification.³⁶ Briefly, mice liver was homogenized in the hypotonic buffer (1.5 mM MgCl2, 10 mM KCl, 1 mM dithiothreitol, 10 mM HEPES, 1 mM PMSF, 1 mM Na3VO4, 5 mM NaF, 1% cocktail protein protease inhibitor, pH 7.9). The homogenates were centrifuged at 1,000 × g for 10 min, and the supernatants were collected as the cytosolic fraction. The pellet was lysed in another buffer (1.5 mM MgCl2, 5 mM HEPES, 0.2 mM EDTA, 0.5 mM DTT, 400 mM NaCl, 26% glycerol, 1% cocktail protein protease inhibitor, pH 7.9) and then centrifuged at 24,000 g for 20 min. The supernatant was collected as the nuclear protein fraction. Equivalent amounts of protein samples were separated by SDS-PAGE electrophoresis and transferred to polyvinylidene fluoride(PVDF) membranes. Then the membranes were blocked in nonfat milk for 1 h at room temperature, incubated with primary antibody overnight at 4 °C, and incubated with HRP-conjugated secondary antibody at the room temperature. Subsequently, the membranes were detected by chemiluminescence kit. Finally, the immunoreactive bands of proteins were scanned with Agfa Duoscan T1200 scanner, and the digitize data were quantified as integrated optical density using Image-Pro Plus software.

2.7 mtDNA copy number

mtDNA in the cytosolic fraction was analyzed by quantitative polymerase chain reaction (qPCR). Briefly, liver tissue was lysed with RIPA lysis buffer, homogenized, and incubated on ice for 15 min. The lysates were centrifuged at 13,000 rpm at 4 °C for 15 min. The cytosolic mtDNA from supernatant was detected by DNeasy blood and tissue kit according to manufacturer’s protocol. The relative expression of the genes was detected using the SYBR Premix Ex Taq II kit via real-time fluorescent qPCR. qPCR was carried out in a 20 μl reaction volume containing 10 μl LightCycler 480 SYBR Green I Master (Roche, Shanghai, China), 1 μl forward primer, 1 μl reverse primer, 2 μl DNA, and 6 μl RNase Free dH2O. PCR was carried out in LightCycler 480II qPCR system (Roche). Threshold cycles were determined and used for quantification of initial inputs. The hepatic mtDNA copy number was assessed by determining the copy number of the mtDNA relative to the copy number of the nuclear DNA (18 S rRNA) using quantitative PCR as previously described. qPCR was performed in MicroAmp optical 96-well plates using StepOne PCR system (Applied Biosystems). Primer sequences were:

mtDNA forward—5′-CACCCAAGAACAGGGTTTGT-3′, mtDNA reverse—5′-TGGCCATGGGTATGTTGTTAA-3′,

18 s rRNA forward—5′-TAGAGGGACAAGTGGCGTTC-3′, 18 s rRNA reverse—5′-CGCTGAGCCAGTCAGTGT-3′.

2.8 Statistical analysis

All data were expressed as the mean ± standard deviation. Multiple statistical comparisons between CCl₄ groups and control groups were performed by one-way analysis of variance using SPSS software, version 20.0. P value <0.05 was considered statistically significant.

3. Results

3.1 CCl₄ induces a severe fibrosis in mice liver

Chronic CCl₄ exposure caused severe liver fibrosis, as indicated by the markers of hepatocellular damage and liver fibrogenesis. As shown in Figure 1a, histological analysis with H&E staining showed that liver sections from the CCl₄ groups exhibited various degrees of several alterations, including hepatocellular vacuolation, extensive necrosis, and mild inflammatory cell filtration. Sirius staining suggested that CCl₄ exposure mice had an obvious increase in collagen deposition and fibrosis compared with control group (Fig. 1a). Consistent with the severity of liver fibrosis detected by Sirius staining, immunofluorescence staining and immunoblot analysis demonstrated that expression of TGFβ and collagen1 were also significantly increased (Fig. 1a and e). The results demonstrated that 8-week CCl₄ exposure caused a progressive liver fibrosis.

Fig. 1.

CCl₄ induces a severe fibrosis in mice liver. a) Histopathological analysis of representative HE-stained liver samples of CCl₄-treated mice with b) the liver damage area quantified (n > 5). Scale bars = 50 μm. Collagen deposition was evaluated by Sirius Red staining with c) percentage of collagen per field quantified (n > 5). Scale bars = 200 μm. The expression of TGF-β was detected by immunofluorescence staining (magnification ×200) with d) relative fluorescence intensity quantified (n > 5). Scale bars = 100 μm. e) Western blots showing the fibrosis protein expression in livers from control or CCl₄ treatment mice. The data of target proteins in the experiment groups were normalized to β-actin and then expressed as the mean percentage of the control (100%), n = 6. The data are representative of three independent experiments. ^*P < 0.05 compared with control group; ^#P < 0.05 compared with 4-week group.

3.2 CCl₄ induces production of ROS and mitochondrial damage in mice

Excessive ROS interferes with the normal function of liver-specific cells and presumably plays a role in the pathogenesis of liver fibrosis.^37,38 To assess the effect of CCl₄ exposure on liver oxidative stress, we determined the ROS levels using the fluorogenic dye Dihydroethidium(DHE). As shown in Figure 2a, CCl₄ challenge led to a significant increase in ROS production, as shown by the strong red fluorescence observed under a fluorescence microscope. Additionally, excessive activation of ROS leads to mitochondrial damage. We next tested the cytoplasmic mtDNA content, which is potential sensitive biomarker to mitochondrial damage. The results showed that the cytoplasmic mtDNA content gradually increased with the aggravation of liver fibrosis (Fig. 2c).

Fig. 2.

The changes of production of ROS and mitochondrial damage in livers of mice treated with CCl₄. a) Representative microphotographs of DHE staining in liver sections in each group with b) relative fluorescence intensity quantified (n > 5). Scale bars = 100 μm. c) The cytoplasmic mtDNA contents (n = 6). The data are representative of three independent experiments. ^*P < 0.05 compared with control group; ^#P < 0.05 compared with 4-week group.

3.3 CCl₄ induces upregulation of JNK/YAP pathway and HSCs activation in mice

Oxidative stress-induced mitochondrial damage has been demonstrated to drive the activity of JNK signaling.¹⁹ Therefore, we investigated whether chronic CCl₄ exposure induced JNK signaling. As shown in Figure 3a, CCl₄ challenge resulted in increased JNK and p-JNK protein levels. In addition, we test downstream signaling of JNK, and the result showed that CCl₄ led to a significant increase in YAP nuclear transcription, but the protein levels of LATS1 and p-YAP were decreased (Fig. 3a).

Fig. 3.

The changes of JNK/YAP pathway and HSCs activation in livers of mice treated with CCl₄. a) Immunoblot analysis for JNK, p-JNK, LATS1, YAP, and p-YAP in mice liver lysates. The data of target proteins in the experiment groups were normalized to β-actin or Lamin B, then expressed as the mean percentage of the control (100%), n = 6. b) The HSCs activation was detected by α-SMA immunohistochemical staining with relative expression quantified (n > 5). Scale bars = 100 μm. The data are representative of three independent experiments. ^*P < 0.05 compared with control group; ^#P < 0.05 compared with 4-week group.

The activation of HSCs is a key event for liver fibrosis. In normal liver, quiescent HSCs secrete a minute amount of α-SMA, which is upregulated shortly after liver fibrosis. Therefore, α-SMA usually acts as an HSC activation marker.^39,40 In this study, immunohistochemistry staining revealed no obvious positive staining in the normal control, but the number of α-SMA positive cells was significantly increased in the CCl₄ group in a time-dependent manner (Fig. 3b). In summary, the results suggested that CCl₄ chronic exposure induced the upregulation of JNK/YAP pathway and the activation of HSCs.

3.4 MitoQ ameliorates liver fibrosis damage in mice challenged with CCl₄

Based on above findings, we explored whether mitochondrial oxidative damage was an early event during the development of liver fibrosis. To this end, mice were treated with mitoQ, an inhibitor of mitochondrial oxidative stress. As shown in Figure 4a, the fresh mice livers in the model group were obviously swollen, hard in texture, dark red in color, rough and uneven on the surface, and some had granular protrusions with blunt edges. By contrast, inhibition of mitoQ apparently reversed these changes. In the meantime, treatment with mitoQ significantly attenuated the increase in ALT and AST activities and decreased the areas of liver extensive necrosis and mild inflammatory cell filtration in CCl₄-treated mice (Fig. 4b and c). In particular, the activity of serum ALT and AST in mice treated with mitoQ was markedly reduced to the margin level of normal mice. Sirius staining showed that mitoQ-treated mice had a marked reduction in collagen deposition and fibrosis compared with CCl₄ exposure group (Fig. 4a). Consistent with histological observation, immunofluorescence and immunoblot analysis further revealed mitoQ significantly attenuated CCl₄-induced liver fibrosis by the expression of TGFβ and collagen I (Fig. 4c and g). Taken together, these results support that suppression of mitochondrial oxidative stress attenuates CCl₄-induced liver fibrosis.

Fig. 4.

Biochemical assessment, histopathological and liver fibrosis analysis of mice in intervention experiments with mitoQ. a) Observation of fresh liver tissue. b) Serum ALT and AST activities in mitoQ intervention experiment. c) Histopathological analysis of representative HE-stained liver samples of mitoQ-treated mice with d) the liver damage area quantified (n > 5). Scale bars = 50 μm. Collagen deposition was evaluated by Sirius Red staining with e) percentage of collagen per field quantified (n > 5). Scale bars = 200 μm. The expression of TGF-β was detected by immunofluorescence staining (magnification ×200) with f) relative fluorescence intensity quantified (n > 5). Scale bars = 100 μm. g) Western blots showing the fibrosis protein expression in livers from mitoQ intervention mice. The data of target proteins in the experiment groups were normalized to β-actin and then expressed as the mean percentage of the control (100%), n = 6. The data are representative of three independent experiments. ^*P < 0.05 compared with control group; ^#P < 0.05 compared with CCl₄ group.

3.5 MitoQ suppresses upregulation of JNK/YAP pathway and prevents HSCs activation in CCl₄-induced mice

Given the protective role of mitoQ in liver fibrosis, we next sought to further explore possible mechanism. As shown in Figure 5a, CCl₄ challenge resulted in increased phosphorylation JNK protein levels, which was significantly suppressed by mitoQ. YAP activity is regulated by phosphorylation, which causes YAP inactivation and nuclear to cytoplasmic translocation. Activation of YAP is thus associated with its translocation from the cytoplasm into the nucleus.⁴¹ We found that mitoQ led to a significant increase in the protein levels of LATS1 and p-YAP compared with CCl₄-treated liver. In addition, western blotting results showed that fibrous livers had elevated levels of nuclear YAP and decreased levels of cytoplasm YAP. Importantly, mitoQ significantly restored the levels of nuclear YAP and cytoplasm YAP (Fig. 5a). Immunofluorescence result further confirmed that mitoQ effectively blocked CCl₄-induced YAP nuclear localization (Fig. 5b). α-SMA, the marker of HSCs activation, was also reduced by mitoQ treatment by the results of immunohistochemistry staining and western blotting (Fig. 5c). These results provided possible evidence that mitoQ inhibit the activation of HSCs by regulating JNK/YAP pathway.

Fig. 5.

The changes in JNK/YAP pathway and HSCs activation in mice liver with mitoQ treatment. a) Immunoblot analysis for JNK, p-JNK, LATS1, YAP, and p-YAP in mice liver lysates. The data of target proteins in the experiment groups were normalized to β-actin or Lamin B, then expressed as the mean percentage of the control (100%), n = 6. b) YAP into the nucleus in mice liver sections by immunofluorescence analysis with YAP-nucleated cells per picture quantified (n > 5). YAP, red; Hoechst 33343, blue. Scale bars = 20 μm. c) The HSCs activation was detected by α-SMA immunohistochemical staining with relative expression quantified (n > 5) and immunoblot analysis in mice liver lysates. Scale bars = 100 μm. The data of target proteins in the experiment groups were normalized to β-actin and then expressed as the mean percentage of the control (100%), n = 6. The data are representative of three independent experiments. ^*P < .05 compared with control group; ^#P < .05 compared with CCl₄ group.

3.6 MitoQ restores ROS generation and ameliorates mitochondrial damage in CCl₄-induced mice

As shown in Figure 6a, we found that mitoQ administration markedly decreased CCl₄-induced ROS generation, as shown by no apparent fluorescence under a fluorescence microscope, and the quantitative data showed similar fluorescence intensity between control and mitoQ-treated mice liver (Fig. 6b). Furthermore, mitoQ significantly suppressed the CCl₄-induced increase in the cytoplasmic mtDNA production (Fig. 6c), suggesting mitochondrial damage was relieved.

Fig. 6.

The changes in production of ROS and mitochondrial damage in mice liver with mitoQ treatment. a) Representative microphotographs of DHE staining in liver sections in each group with b) relative fluorescence intensity quantified (n > 5). Scale bars = 100 μm. c) The cytoplasmic mtDNA contents (n = 6). The data are representative of three independent experiments. ^*P < 0.05 compared with control group; ^#P < 0.05 compared with CCl₄ group.

4. Discussion

Previous studies have demonstrated that mitochondrial oxidative stress plays critical roles in the development of liver fibrosis.^33,42 Accumulating evidence has also demonstrated that the activation of HSCs is the key cause of liver fibrosis.^3,43 ROS, which are produced by mitochondria, induce the transdifferentiation of HSCs and increase the production of collagen; again, antioxidants prevent these effects,⁴⁴ but the underlying mechanism remains to be elucidated. In this study, we used chronic CCl₄ exposure-driven in vivo fibrosis models to mimic liver fibrosis in chronic liver diseases. We found that CCl₄ challenge significantly increased the excretion of ROS and mitochondrial damage in mice liver and promoted the activation of JNK/YAP signaling pathway and HSCs. However, mitoQ attenuated CCl₄-driven mitochondrial oxidative damage in mouse livers. More importantly, mitoQ effectively suppressed CCl₄-induced the increase in the phosphorylation level of JNK and the significant nuclear transcription of YAP and eventually inhibited the activation of HSCs. These results clearly demonstrated that mitoQ ameliorated the CCl₄-driven liver fibrosis in mice liver, suggesting that the protective effect of mitoQ against chronic liver diseases might be related to the regulation of JNK/YAP pathway.

Mitochondria play a pivotal role in energy metabolism and are the major site of ROS generation through the electron transport chain.^45,46 Oxidative stress is a redox state caused by an imbalance between the production and detoxification of ROS.⁴⁷ As previously shown by others’ studies, chronic liver diseases are associated with increased oxidative stress.^48,49 In this study, we found that chronic CCl₄ challenge led to a significant increase in ROS production, as shown by the strong DHE red fluorescence. Additionally, excessive activation of ROS leads to mitochondrial damage revealed by the result of increased cytoplasmic mtDNA content. Considering that elevated oxidative stress in liver fibrosis is one of the major mechanisms leading to HSC activation and that mitochondria are central sites where ROS are produced,⁵⁰ the present study evaluated the effects of the mitochondria-targeted antioxidant mitoQ on oxidative stress. Our study confirmed that mitoQ effectively inhibited ROS generation and mitochondrial damage in chronic CCl₄ exposure-induced liver fibrosis, consistent with the study by Vilaseca et al.⁵¹

A previous study reported that excessive ROS may directly damage mtDNA and activate the MAPK signaling pathway.⁵² Specifically, ROS oxidizes Trx to dissociate from ASK-1 for its activation, resulting in the activation of JNK and p38 pathways.⁵³ In addition, MAPK signaling pathway, especially JNK, is activated in response to different stresses, and this signaling pathway has important roles in the development of liver diseases and injuries, such as NAFLD, ALD, viral hepatitis, fibrosis, inflammation, carcinogenesis, and drug-induced hepatotoxicity.^19,54 In the current study, we found that CCl₄-treated mice liver exhibited increased protein levels of JNK and phosphorylated JNK. Furthermore, mitoQ significantly suppressed the phosphorylation of JNK, suggesting that mitochondrial oxidative damage resulted in JNK activation in liver fibrosis injury. JNK signaling is considered as a major modulator of liver fibrosis.⁵⁵ And one study confirmed that JNK1 promotes cholestasis- and CCl₄-induced liver fibrosis,⁵⁶ which is similar to our research. Therefore, it is demonstrated that mitochondrial oxidative stress might play a vital role in CCl₄-induced mice liver fibrosis by regulating the activation of JNK.

JNK contributes to YAP nuclear expression.²² YAP is a transcription coactivator known to be a key nuclear effector of HSCs activation.⁵⁷ Under active conditions, the phosphorylated form of YAP localizes in the cytoplasm and the dephosphorylated form of YAP localizes in the nucleus, where it interacts with other transcription factors.⁵⁸ In this study, we detected hardly any nuclear YAP expression in the livers of control mice. By contrast, CCl₄ administration significantly promoted YAP nuclear location and prevented the expression of phosphorylated form of YAP. Our further research found that the nuclear accumulation of YAP was completely blocked by mitoQ treatment in fibrosis liver. And the phosphorylation of YAP returned to control levels. Our findings are in agreement with previous studies, which reported that JNK and p38 MAPK signaling cascades regulated YAP nuclear expression. And their results demonstrated that JNK inhibitor SP600125 shifted YAP from a predominantly nuclear to predominantly cytoplasmic localization.^21,22,59 Therefore, studies from our lab and others have revealed that mitochondrial damage-induced JNK activation did promote nuclear expression of YAP in CCl₄-induced liver fibrosis. More importantly, as shown by recent researches that YAP played a vital role in regulating HSCs activation.^25,27 Continued activation of stellate cells during chronic liver damage causes excessive matrix deposition and the formation of pathological scar tissue leading to fibrosis.²⁵ Furthermore, transforming growth factor β1 is closely related to HSCs activation and is considered as one of the most important markers of HSCs activation.^60,61 We found that CCl₄ exposure significantly increased protein levels of TGFβ, which suggesting HSCs activation. Besides, increased TGFβ expression in CCl₄-challenged mice was abolished by mitoQ treatment. Liver-specific YAP gene knockout improved IR-induced liver fibrosis and liver dysfunction.⁶² These results suggest that inactivation of JNK/YAP pathway contributes to the suppression of HSCs activation by mitoQ treatment.

In conclusion, this study demonstrated the novel beneficial effects of mitoQ on liver fibrosis using chronic CCl₄ exposure model. The mechanism underlying its effects involve restoring mitochondrial oxidative damage, thereby inhibiting phosphorylation of JNK and nuclear localization of YAP, which eventually results in decreased collagen fiber production in HSCs and liver fibrosis regression (Fig. 7). These results suggest that the mitochondrial-targeted antioxidant mitoQ is an attractive agent for preventing liver fibrosis injury.

Fig. 7.

MitoQ improves CCl₄-induced liver fibrosis in mice by inhibiting JNK/YAP pathway. TAZ is a YAP paralog in mammals similarly regulated by the Hippo pathway.⁶³ CCl₄ challenge significantly increased the excretion of ROS and mitochondrial damage in mice liver and promoted the activation of JNK/YAP signaling pathway and HSCs. By contrast, mitoQ attenuated CCl₄-driven mitochondrial oxidative damage, thereby effectively suppressed CCl₄-induced the increase in the phosphorylation level of JNK and the significant nuclear transcription of YAP and eventually inhibited the activation of HSCs and liver fibrosis injury.

Funding

This work was supported by Key Research and Development Plan of Shandong Province (2018GSF118013) and National Natural Science Foundation of China (No. 82173552).

Conflict of interest. The authors declare no conflict of interest.

Authors’ contributions

Fuyong Song conceived of the study. Shulin Shan performed data collection, data analysis. Zhaoxiong Liu produced the figures and finally wrote the initial draft, with overall guidance from Fuyong Song. All authors reviewed and edited the manuscript.