Background
Liver fibrosis is a common process resulting from chronic liver damage caused by different etiologies, and over repair leads to liver cirrhosis, which imposes a substantial economic burden because of the high mortality rate. However, increasing evidence has indicated the reversibility of liver fibrosis and early cirrhosis [
1]. Thus, the control of liver fibrosis is the key to avoiding cirrhosis. Extracellular matrix (ECM) is mainly produced by hepatic stellate cells (HSCs), and the activation of HSCs is a key step in liver fibrosis. Generally, HSCs are considered the key target of antifibrotic treatments [
2]. However, the mechanisms underlying liver fibrosis and HSC activation remain unclear, and further studies investigating new molecular mechanisms are still needed.
Generally, oxidative stress is a common phenomenon resulting from liver injury that plays a key role in the pathogenesis of liver fibrosis [
3]. When oxidative stress is triggered by various harmful stimuli, the accumulation of excess reactive oxygen species (ROS) leads to the lipid peroxidation (LPO) of unsaturated fatty acids in the biological membrane and the generation of byproducts such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), which result in structural and functional damage to the cell [
4]. Inhibition of oxidative damage effectively prevents or even reverses the process of liver fibrosis in multiple animal models [
5,
6]. HSCs are activated during liver injury caused by various etiologies [
2]. Oxidative stress is a common phenomenon resulting from liver injuries. Furthermore, ROS and lipid peroxidation products such as 4-HNE or MDA have been shown to trigger and perpetuate HSC activation via redox-sensitive signaling pathways [
7,
8]. For example, the classic mitogen-activated protein kinase (MAPK) signaling pathway has been considered one of the essential pathways involved in HSC activation, and this pathway is redox-sensitive and triggered by several growth factors. Platelet-derived growth factor-BB (PDGF-BB) is well known for its ability to strongly activate HSCs upon binding to specific transmembrane receptors in subjects with liver fibrosis [
9]. In addition to the pathways described above, novel pathways have also received attention. The Wnt/β-catenin signaling pathway has emerged as a fundamental growth control pathway that is typically involved in regulating development or the maintenance of most types of stem cells throughout the animal kingdom [
10]. However, according to recent studies, this pathway responds to oxidative stress and prevents HSC activation through a specific mechanism during liver fibrosis [
11]. GSK-3β is a key kinase in the Wnt/β-catenin pathway. ROS or 4-HNE can trigger GSK-3β inactivation in a manner dependent on ser9 phosphorylation and subsequently activate the downstream signaling cascade to promote HSC activation and proliferation [
12]. The detailed mechanisms by which ROS or 4-HNE activate MAPK and Wnt/β-catenin pathways remain unclear and unknown proteins may be involved in this process. Interestingly, as shown in our previous study, the levels of the antioxidant protease glutathione
S-transferase A3 (GSTA3) is decreased in fibrotic kidneys [
13]. GSTA3 belongs to the GST α-class, which converts lipid peroxides to glutathione conjugates Moreover, GSTA3 is major as a cytosolic protein that is expressed at high levels in the liver, kidney and adrenal gland [
14,
15]. New advances confirmed that GSTA3 knockout mice exhibit increased oxidative damage and GSTA3 is generally considered to exhibit hydroperoxidase activity in vivo [
16]. However, the pathophysiological role of GSTA3 in liver fibrosis has not been studied. Considering the crucial role of oxidative stress in HSC activation and liver fibrosis, we speculate that GSTA3 may play a unique role in liver fibrosis.
Our previous studies confirmed that fluorofenidone [1-(3-fluorophenyl)-5-methyl-2-(1
H)-pyridone; AKF-PD], which has undergone phase I clinical trials, reduces intracellular ROS accumulation and possesses potent antifibrotic properties because it inhibits HSC activation partially by suppressing MAPK signaling pathways [
17‐
20]. Thus, we speculate that AKF-PD may regulate the levels of certain proteins to mediate oxidative stress and subsequently inhibit HSC activation.
Therefore, the present study was designed to verify whether GSTA3 is involved in liver fibrosis, elucidate the underlying mechanisms and then prove whether AKF-PD reduces liver fibrosis by regulating GSTA3.
Discussion
Liver fibrosis is a common pathological process leading to cirrhosis and eventual liver failure [
22]. However, the pathogenesis of liver fibrosis is complicated, and the mechanism remains unclear. In fact, regardless of whether the cause is viral infection, fat- or alcohol-related liver disease, cholestasis, or a metabolic or toxic etiology, HSC activation is a common event that leads to ECM remodeling after evolving to chronic injury [
2].
Oxidative stress represents a common link among different etiologies of persistent liver injury. This injury occurs when oxidative stress-related molecules, including ROS and end-products of LPO, exceed the cellular antioxidant defense capabilities. MDA and 4-HNE are important end-products of LPO, and their concentrations reflect the level of LPO [
23]. GST family members are important components of the cellular antioxidant defenses and serve as antioxidant enzymes that scavenge end-products of LPO. GSTA3 is a special member of the α-GST subfamily. The antioxidant response elements in the proximal promoter region of the GSTA3 gene are involved in its antioxidant activity [
24]. Indeed, oxidative damage is aggravated in GSTA3 knockout mice following exposure to CCl
4 [
16]. In the present study, the expression of GSTA3 was dramatically decreased and the level of oxidative stress increased in rat fibrotic livers and activated HSCs. Only one previous report described the phenomenon in which GSTA3 was decreased and LPO products were significantly increased in rat HSCs [
25]. Another report clarified that GSTA3 reduced 4-HNE levels in hepatocytes and regulated the signaling pathways that protect against oxidative stress [
26]. Most likely, GSTA3 expression was decreased, resulting in a redox imbalance that lead to excess ROS and LPO accumulation. Both ROS and products of LPO initiate and perpetuate HSC activation [
27‐
29]. Thus, GSTA3 is involved in HSC activation and liver fibrosis by regulating oxidative stress.
PDGF-BB was reported to trigger HSC activation in a manner dependent on ROS generation via NADPH oxidase [
30,
31]. PDGF-BB induced ROS production in HSCs in the present study, consistent with the previous report. Since an antioxidant response element is located in the proximal promoter region of the rat and human GSTA3, GSTA3 expression may be triggered by ROS. In contrast, PDGF-BB downregulated GSTA3 expression in HSCs. Thus, we speculate that the expression of the GSTA3 gene is likely regulated by PDGF-BB through a specific mechanism. Of course, further studies are needed to identify the mechanism.
HSCs can transdifferentiate into a myofibroblast-like phenotype and acquire a fibrogenesis capacity. The process of HSC activation consists two major phases: initiation and perpetuation [
2]. This process was substantially enhanced by GSTA3 knockdown, and the effect was even more obvious after an incubation with PDGF-BB. Correspondingly, overexpression of GSTA3 inhibited HSC activation and FN production, even after PDGF-BB treatments. Notably, recent advances have identified GSTA3 as a novel adipocyte differentiation-associated protein [
32]. However, no investigation has indicated a causal relationship between GSTA3 and HSC differentiation. Our results are the first to show that GSTA3 negatively regulates HSC activation and fibrogenesis. This study unequivocally identified GSTA3 as one factor inducing HSC activation.
Various etiologies leading into liver fibrosis drive the differentiation of HSCs into a myofibroblast-like phenotype through various signaling networks. MAPK signaling is among the best characterized pathways involved in the HSC phenotype switch, and this pathway is activated by both ROS and 4-HNE [
33]. On the other hand, ROS and LPO also activate Wnt signaling, which has been reported to play a specific role in HSC activation and proliferation in hepatic fibrosis. When the canonical Wnt signaling pathway is activated, Wnt proteins bind to the transmembrane receptor complex and inhibit GSK-3β and its detachment from the scaffold protein; this process promotes further β-catenin accumulation in the cytoplasm and transport to the nucleus, thereby initiating the expression of the downstream target genes c-myc, c-Jun, cyclin D1, which mediate cell differentiation and proliferation [
10]. Our previous study confirmed that the cyclin D1 participates in the mechanism regulating HSC proliferation [
21]. However, PDGF-BB did not significantly induce the transport of β-catenin from the cytoplasm to the nucleus (Additional file
2: Figure S1). Although PDGF-BB is the strongest mitogen in HSCs, it is not a canonical Wnt signaling ligand. Actually, only a very small portion of cellular GSK3 and β-catenin are involved in the Wnt/β-catanin pathway [
34]. GSK3 actually participates in multiple signaling pathways. In HSCs, GSK-3β is constitutively active and maintains the HSCs in quiescent state [
11]. In the present study, GSTA3 inhibited the inactivation of GSK-3β. Thus, GSTA3 suppressed GSK-3β-mediated activation of HSCs. On the other hand, GSTA3 reduced ROS accumulation and inhibited MAPK signaling in HSCs. The MAPK signaling pathway is critical for liver fibrosis [
35]. Collectively, GSTA3 suppressed HSC activation by regulating the activity of the MAPK and GSK-3β signaling pathways, and partially through the negative regulation of oxidative stress in HSCs. Since both the MAPK and GSK-3β signaling pathways are critical for HSC activation, the significant effects of GSTA3 on these pathways are sufficient to clarify its unique role in the activation of HSCs. Although GSTA3 affected intracellular ROS accumulation and its common downstream pathways, we were unable to exclude the possibility that GSTA3 may affect intracellular signal transduction mediated by other pathways. In particular, according to recent evidence, several other GSTs regulate cell signaling pathways through direct protein–protein interactions [
36]. Of course, further studies are needed to clarify the mechanisms.
An effective treatment targeting the cause of liver fibrosis reduced and even reversed liver fibrosis in previous studies. Antiviral therapy targeting viral hepatitis has been reported to reverse cirrhosis in some patients [
37]. However, the development of an optimal antifibrotic treatment for numerous nonviral liver diseases is more difficult to achieve. Hence, an effective antifibrotic drug might ameliorate the symptoms of these patients. To date, no antifibrotic drug has been approved for liver fibrosis. Approaches designed to attenuate oxidative damage as therapeutic strategies for liver fibrosis are still being investigated [
38]. Our previous studies have shown the potent ability of AKF-PD to ameliorate fibrosis in multiple organs [
18‐
21]. Furthermore, AKF-PD inhibits intracellular ROS accumulation, suggesting that AKF-PD possesses antioxidant activity [
18]. In the present study, AKF-PD reduced ROS accumulation in HSCs and decreased LPO levels in vivo. Based on the important role of oxidative stress in liver fibrosis, we searched for a key target protein responsible for the antioxidant activity of AKF-PD. GSTA3 is an inducible antioxidant enzyme [
39,
40]. Surprisingly, AKF-PD effectively increased GSTA3 expression in fibrotic livers and activated HSCs. Even after GSTA3 knockdown in HSCs, AKF-PD fully restored expression of GSTA3 and effectively inhibited HSCs activation. Obviously, powerful up-regulation of AKF-PD in expression of GSTA3 offsets the effects of GSTA3 knockdown on HSCs. In a word, GSTA3 is a special target of AKF-PD and is at least partially responsible for its anti-fibrotic ability. Further mechanistic studies of the downstream signaling pathways of ROS showed that AKF-PD suppressed the activation of the MAPK and GSK-3β signaling pathways. Strategies targeting the MAPK and GSK-3β signaling pathways exert anti-fibrotic effects on animal models of fibrosis [
41,
42]. Collectively, AKF-PD relieved liver fibrosis partially by upregulating GSTA3 expression and negatively regulating oxidative stress and its downstream pathways.
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