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. 2026 Jul 2;204(10):7549–7560. doi: 10.1007/s12011-026-05207-1

Arsenic Exposure Induces Hepatic Fibrosis and Triggers Early Inflammatory Responses by Inhibiting Hepatic Stellate Cell PINK1/Parkin-Mediated Mitophagy

Feiyang Wu 1, Yan Wang 2, Sheng Li 1, Zhiyong Xu 1, Xiaohui Wang 1, Haijing Yin 1, Teng Ma 1,✉, Li Wang 1,✉
PMCID: PMC13616000  PMID: 42393349

Abstract

Arsenic contamination poses a significant environmental health risk, yet the mechanisms underlying its induction of hepatic fibrosis remain incompletely elucidated.Our previous clinical studies in arsenic poisoning areas found that serum inflammatory factors (such as IL-6, IL-1β, TNF-α) levels were significantly elevated, but there were no significant differences in liver function and fibrosis markers, suggesting that the inflammatory response precedes detectable typical pathological alterations. Established theories suggest that persistent inflammation is a key factor in the initiation of hepatic fibrosis. PINK1, a key initiator of mitophagy, was found to be downregulated in the livers of arsenic-exposed mice.Based on this, we propose a scientific hypothesis: arsenic exposure induces a persistent inflammatory response by inhibiting PINK1/Parkin-mediated mitophagy, thereby driving hepatic stellate cells (HSCs) activation and the fibrosis process. Experimental validation demonstrated that arsenic exposure significantly inhibited the PINK1/Parkin pathway, impaired mitophagy, and upregulated the expression of fibrosis markers and inflammatory factors in both liver tissues and cells. Conversely, PINK1 overexpression reversed these pathological phenotypes. In summary, dysregulation of the PINK1/Parkin pathway is a pivotal mechanism connecting arsenic exposure to chronic inflammation and the initiation of hepatic fibrosis, providing new insights for early recognition and targeted intervention of arsenic-related liver diseases.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1007/s12011-026-05207-1.

Keywords: Arsenic, Hepatic fibrosis, Mitophagy, PINK1/Parkin pathway, Hepatic stellate cells

Introduction

Arsenic is a widely distributed environmental toxin that represents a considerable public health concern. Chronic exposure in humans, primarily through drinking water, diet, or occupational sources, poses significant health risks, with the liver serving as the principal target organ [1–2]. Epidemiological studies demonstrate that populations exposed to arsenic over the long-term are susceptible to liver dysfunction and the development of hepatic fibrosis [3]. However, the key molecular drivers mediating the progression from early inflammatory responses to advanced fibrosis remain poorly understood.

Previous studies indicate that long-term or high-dose exposure to arsenic disrupts liver structure and function through multiple mechanisms, including metabolic impairment, increased oxidative stress, sustained inflammatory activation, and enhanced hepatocyte apoptosis [4]. Metabolomic and proteomic analyses further reveal that arsenic perturbs hepatic lipid metabolism, leading to free fatty acid accumulation, increased triglyceride synthesis, and upregulation of inflammatory cytokines (TNF-α and IL-6), synergistically promoting hepatocyte injury, hepatic stellate cell activation, and extracellular matrix deposition, ultimately promoting hepatic fibrosis [5]. Among these mechanisms, oxidative stress and mitochondrial dysfunction have emerged as central mediators of arsenic-induced liver injury. Arsenic inhibits antioxidant enzyme activity, leading to excessive generation of reactive oxygen species (ROS) that damage mitochondrial membranes, impair ATP synthesis, and reduce mitochondrial membrane potential. This process is closely related to the Nrf2 antioxidant pathway and the p38 MAPK stress signaling pathway, whose dysregulation further regulates cell survival, inflammation, and fibrotic response [6–8]. Previous studies have largely focused on single mechanisms or terminal phenotypes, leaving the role of mitophagy as a core regulator of cellular homeostasis in arsenic-induced hepatic fibrosis. inadequately defined.

Maintenance of mitochondrial homeostasis is fundamental to liver physiology and pathology. The PINK1/Parkin pathway orchestrates selective mitophagy by sensing mitochondrial damage and initiating autophagy for clearance. Upon mitochondrial membrane depolarization, PINK1 stabilizes and accumulates on the outer mitochondrial membrane, recruiting Parkin. This triggers ubiquitination of damaged mitochondria, allowing their recognition by adaptor proteins such as p62, which interact with LC3-II to facilitate autophagosome formation, culminating in lysosomal degradation [9–10]. This regulatory mechanism not only acts as a gatekeeper to maintain mitochondrial homeostasis but also mitigates ROS overproduction, suppresses NLRP3 inflammasome activation, and modulates apoptosis signaling [11–12]. Although PINK1/Parkin-mediated mitophagy has been associated with hepatic fibrosis, the extent to which arsenic exposure disrupts this pathway to initiate fibrogenesis remains unclear. However, arsenic may lead to dysfunction of the PINK1/Parkin pathway, preventing timely clearance of damaged mitochondria and resulting in the upregulation of ROS-dependent inflammatory responses and fibrotic progression, potentially in coordination with dysregulated Nrf2 and MAPK pathways. A detailed investigation is therefore essential to delineate this relationship.

The study proposes the following hypotheses:1.Arsenic exposure inhibits PINK1/Parkin-mediated mitophagy in both in vivo and in vitro experimental models. 2.Arsenic exposure promotes the release of inflammatory factors by inhibiting the PINK1/Parkin-mediated mitophagy pathway, activating hepatic stellate cells (HSCs). 3.Restoring PINK1 expression reverses arsenic-induced hepatic stellate cell activation and the hepatic fibrosis phenotype.

Materials and Methods

Study Population

The study enrolled seventeen patients from an arsenic-endemic area in Inner Mongolia as the study group, and seventeen healthy, matched residents from the same region served as the control group. The control group was individually matched to the study group based on the following criteria: Case group definition: Patients were clinically diagnosed with chronic endemic arsenic poisoning, based on the Chinese health industry standard “Diagnostic Criteria for Endemic Arsenic Poisoning” (WS/T 211–2015). The main criteria included: (1) Long-term (> 6 months) consumption of drinking water with an arsenic concentration > 0.05 mg/L; (2) Presence of typical skin lesions such as hyperkeratosis and/or hyperpigmentation/depigmentation. We recruited 17 patients meeting these criteria from the arsenic-endemic area in Inner Mongolia. Control group definition and matching: The control group consisted of 17 healthy residents from the same administrative village sharing the same drinking water source. They also had a long-term (> 6 months) history of arsenic exposure through drinking water, but upon clinical examination, showed no skin or neurological manifestations of endemic arsenic poisoning, and their liver function parameters (ALT, AST) were within normal ranges. We performed 1:1 individual matching based on: (1) Age (± 5 years); (2) Sex; (3) Residence and drinking water source. Serum levels of AST, ALT, HA, LN, IL-6, IL-1β, and TNF-α were determined. Pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) were quantified using ELISA kits (Pumei Biotechnology Co., Ltd), whereas AST, ALT, HA, and LN levels were assessed by the Center for Disease Control and Prevention of Bayannur City, Inner Mongolia.The study was conducted in accordance with the Declaration of Helsinki, and approved by the Baotou Medical College Ethics Review Committee (approval number No.002), Baotou Medical College, 2021).

Simulation of Environmental Arsenic Exposure

Male C57BL/6J mice (8-weeks old) were obtained from SPF (Beijing) Biotechnology Co., Ltd. All experimental procedures and housing conditions were approved by the Animal Ethics Committee of Baotou Medical College (Approval No.002, 2021). Mice were randomly divided into four groups (n = 12 per group) and exposed to sodium arsenite (NaAsO₂, Sigma, Germany) in drinking water at concentrations of 0, 0.5, 5, and 50 mg/L for 9 months. Water and food intake, as well as body weight, were recorded weekly. At the conclusion of the exposure period, mice were anesthetized with 1% pentobarbital sodium via intraperitoneal injection, euthanized by cervical dislocation, and the liver was collected for further analyses.

To Arsenic dose conversion to human equivalent dose: Based on standard assumptions of daily water intake (5 mL for a 25 g mouse), the estimated mouse arsenic doses for the 0.5, 5, and 50 mg/L NaAsO₂ groups were approximately 0.04, 0.4, and 4.0 mg As/kg/day, respectively. These were converted to human equivalent doses (HED) using body surface area normalization, yielding approximately 0.003, 0.03, and 0.3 mg As/kg/day.

Cell Culture and Treatment

LX-2 human HSCs were obtained from Wuhan Saiweier Biotechnology Co., Ltd. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Gaithersburg, MD, USA) and 1% antibiotic solution (penicillin 100 IU/mL, streptomycin 100 µg/mL; Beyotime, Shanghai, China) in a humidified incubator at 37 °C with 5% CO₂. Upon reaching 80–90% confluence, cells were dissociated with trypsin, evenly seeded into well plates or dishes, cultured for three passages, and then harvested or used for further experiments.

Cell Proliferation Assay

Cell viability was evaluated using a Cell Counting Kit-8 (CCK-8) assay (Biosharp, Hefei, China). LX-2 cells were seeded at a density of 5 × 10³ cells per well in a 96-well plate and treated with varying concentrations of NaAsO2 for 48 h. After the incubation period, the old culture medium was discarded and replaced with an equal volume of fresh medium containing 10% CCK-8 reagent. The cells were then incubated at 37 °C for 2 h, and absorbance at 450 nm was measured using a microplate reader (Multiskan MK3, Thermo Fisher Scientific, Waltham, USA).

Quantitative Real-Time PCR (qRT-PCR) Analysis

Quantitative real-time PCR was performed using a PCR kit (Promega, Madison, Wisconsin, USA) and analyzed on a LightCycler 96 instrument (Roche, Basel, Switzerland). Relative gene expression of PINK1 and Parkin was calculated using the 2 − ΔΔCt method, with GAPDH as the housekeeping gene. The primers used were:

PINK1: Forward 5′-AGTCCATTGGTAAGGGCTGC-3′.

Reverse 5′-AAATCTGCGATCACCAGCCA-3′.

Parkin: Forward 5′-GTGCAGAGACCGTGGAGAAA-3′.

Reverse 5′-CTTCCAGCTGGTGGTGAGTC-3′.

GAPDH: Forward 5′-ACAACTTTGGTATCGTGGAAGG-3′.

Reverse 5′-GCCATCACGCCACAGTTTC-3′.

Western Blotting

Cells were collected, and protein concentrations were quantified using a BCA assay kit (Beyotime, Beijing, China). Proteins were separated by SDS-PAGE and transferred onto a PVDF membrane (Millipore, Burlington, USA). The membrane was blocked with 5% non-fat milk for 2 h, followed by overnight incubation at 4 °C with primary antibodies against Vimentin (1:1000; Huaan Bio, Hangzhou, China), α-SMA (1:1000; Huaan Bio, Hangzhou, China), PINK1 (1:1000; Huaan Bio, Hangzhou, China), PARKIN (1:1000; Huaan Bio, Hangzhou, China), p62 (1:1000; Huaan Bio, Hangzhou, China), and LC3 (Cell Signaling Technology, Danvers, Massachusetts, USA). The membrane was then incubated with enzyme-labeled secondary antibody (1:5000; Pumei Bio, Beijing, China) at room temperature for 1 h. Protein bands were visualized using ECL reagent (Pumei Bio, Beijing, China).

PINK1 Overexpression

For PINK1 overexpression, LX-2 cells were transfected with PINK1 overexpression plasmid (OE-PINK1) and its negative control plasmid (OE-PINK1-NC), both obtained from Pumei Biotechnology (Wuhan, China), using Lipofectamine 2000. Cells were seeded into a six-well plate, and the plasmid-containing DMEM was mixed with DMEM solution containing Lipofectamine 2000. The mixture was applied to the cells for 6 h, after which the cells were incubated in NaAsO2-containing medium for 48 h and subsequently harvested for downstream experimental procedures.

Histopathological Evaluation (HE and Masson Staining)

Fibrotic areas were analyzed using Image-Pro Plus software, and α-SMA immunohistochemistry was assessed based on the average optical density value. HE-stained slides were independently evaluated by two pathologists in a double-blind manner.

Statistical Analysis

All analyses were performed using GraphPad Prism version 10.1.2 (GraphPad Software, Inc., La Jolla, CA, USA). Experiments were independently repeated three times, and the results are expressed as mean ± standard deviation (SD). Intergroup differences were analyzed using one-way ANOVA, and pairwise comparisons between groups were conducted using Tukey’s test. Statistical significance was set at p < 0.05.

Results

Arsenic Exposure Induces Early Inflammatory Responses Prior to Detectable Liver Fibrosis Markers

The serum concentrations of inflammatory cytokines IL-6, IL-1β, and TNF-α were significantly elevated in arsenic-exposed patients compared with the control group (all p < 0.0001), as shown in Table 1. However, markers of liver injury (AST, ALT) and fibrosis (HA, LN) showed no significant differences between the groups.These population-based data indicate that chronic arsenic exposure can trigger a significant systemic inflammatory response before the onset of detectable serological changes indicative of liver injury or fibrosis, suggesting inflammation as an early event in arsenic-induced hepatotoxicity.

Table 1.

Comparison of Serum Inflammatory Cytokines, Liver Injury, and Fibrosis Markers Between Arsenic-Exposed and Control Groups

Detection Indicators Control group Patient group p-value
IL-6 (ng/L) 15.78 ± 0.90 23.61 ± 0.94 p < 0.0001
IL-1β (ng/L) 23.88 ± 1.67 42.64 ± 1.79 p < 0.0001
TNF-α (ng/L) 316.93 ± 17.7 482.43 ± 21.07 p < 0.0001
AST (U/L) 22.03 ± 2.27 32.13 ± 15.93 ns
ALT (U/L) 25.7 ± 4.79 36.02 ± 15.51 ns
HA (ng/mg) 109.49 ± 23.03 118.25 ± 25.14 ns
LN (ng/mg) 71.99 ± 13.59 82.04 ± 12.64 ns
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Liver Injury Induced by Arsenic Exposure is Associated with Increased Levels of Inflammatory Factors

Arsenic exposure via drinking water resulted in significant hepatic injury after 9 months compared with the control group receiving deionized water. (Fig. 1A). The liver organ coefficient(liver-to-body weight ratio) was lower in the arsenic-exposure group than in the control group. (Fig. 1B) ICP-MS confirmed a marked increase in arsenic content in the exposed mice compared with the control group (Fig. 1C). HE staining revealed disorganized hepatocytes and infiltration of inflammatory cells (Fig. 1D), while Masson staining demonstrated prominent fibrotic lesions (Fig. 1E). Immunohistochemistry showed upregulated α-SMA in the exposed group (Fig. 1F-G). Western blotting indicated that the expression levels of α-SMA and Vimentin were elevated in high-dose arsenic mice (Fig. 1H-J). ELISA results showed that the levels of inflammatory cytokines IL-6, IL-1β, and TNF-α were significantly elevated in arsenic-exposed mice in a dose-dependent manner compared to the controls (Fig. 1K-M). Collectively, long-term arsenic exposure successfully recapitulated key early pathological features of hepatic fibrosis in mice, including inflammatory infiltration, hepatic stellate cell activation (upregulated α-SMA), and collagen deposition, accompanied by a dose-dependent elevation in inflammatory factors. This providesdirectin vivo experimental evidence supporting the human findings.

Fig. 1.

Fig. 1

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Long-term exposure to sodium arsenite leads to liver injury in mice. (A) C57BL/6J mice were given water containing 0, 0.5, 5, or 50 mg/L sodium arsenite for 9 months. (B) Organ coefficient of mouse liver, n = 6 per group. (C) ICP-MS analysis of arsenic content in mouse liver, n = 3. (D) HE-stainedliver sections showing the degree of liver injury. (E) Masson-stained liver sections showing fibrotic lesions. (F-G) IHC analysis of α-SMA expression in mouse liver, n = 3. (H-J) Western blot analysis of α-SMA and Vimentin protein levels in mouse liver, n = 3. (K-M) ELISA detects the expression levels of IL-6, IL-1β, and TNF-α in mouse liver, n = 6. Data are presented as the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared to the control group

Arsenic Exposure Dysregulates the PINK1/Parkin-Mediated Mitophagy Pathway

Proteomic sequencing analysis revealed significant dysregulation of signaling pathways induced by arsenic exposure. Specifically, KEGG pathway enrichment analysis (Fig. 2A) demonstrated significant enrichment of pathways related to mitophagy and associated metabolic processes. Gene Ontology (GO) analysis (Fig. 2B) further revealed significant alterations in functional terms associated with organelle quality control and stress response at the biological process and molecular function levels. The volcano plot (Fig. 2C) visually illustrated the overall distribution of differentially expressed proteins between the arsenic-treated and control groups.Western blotting results indicated that arsenic exposure significantly downregulated PINK1 and Parkin protein expression, increased p62 protein expression, and elevated the LC3-II/LC3-I ratio (Fig. 2D-H). These results collectively indicate that chronic arsenic exposure significantly suppresses the PINK1/Parkin pathway and impairs mitophagic flux in the liver, identifying this as a potential key molecular link connecting arsenic exposure to subsequent inflammation and fibrosis.

Fig. 2.

Fig. 2

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Proteomic sequencing analysis in the mouse liver. (A-C) KEGG, GO, and volcano plots. (D-H) Western blot assessment of PINK1, Parkin, p62, and LC3Ⅱ/Ⅰ in mouse liver. Data are presented as the mean ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, compared with the control group

Arsenic Exposure Impairs LX-2 Cell Viability and Promotes Fibrosis and Inflammation

Effects of Arsenic on LX-2 Cell Viability, Fibrosis, and Inflammation Following 48 h of exposure to different concentrations of NaAsO₂, LX-2 cell viability was measured using CCK-8. A significant reduction in cell viability was observed at 2 µM in the treatment group compared with the control group (Fig. 3A). Therefore, concentrations of 0.5, 1, and 2 µM were used in subsequent experiments. Western blot results showed that the expression levels of α-SMA and Vimentin in the 2 µM NaAsO₂ exposure group were significantly higher than those in the control group(Fig. 3B-D). ELISA results showed the levels of inflammatory cytokines IL-6, IL-1β, and TNF-α increased in a dose-dependent manner following NaAsO₂ exposure (Fig. 3E-G). Furthermore, qRT-PCR revealed progressive downregulation of PINK1 and Parkin with increasing NaAsO₂ doses (Fig. 3H-I). Western blotting confirmed a decrease in PINK1/Parkin protein expression, increased p62 accumulation, and an elevated LC3-II/LC3-I ratio in the 2 µM NaAsO₂ exposure group (Fig. 3J-N). Thus, arsenic exposure directly inhibits the PINK1/Parkin pathway in HSCs, induces their transformation into an activated myofibroblast phenotype, and promotes the expression of inflammatory cytokines, recapitulating key pathological features observed in vivo. The results show that PINK1 was successfully overexpressed (Fig. 3O-P).

Fig. 3.

Fig. 3

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Effects of sodium arsenite on LX-2 cell viability, fibrosis, and inflammatory responses. (A) CCK-8 assay was performed to evaluate cell viability after 48 hours of NaAsO₂ treatment. (B-D) Western blot analysis of α-SMA and vimentin expression. (E-G) ELISA of IL-1β, IL-6, and TNF-α. (H-I) qRT-PCR was used to determine the expression levels of PINK1 and Parkin. (J-N) Western blot was used to detect the protein expression of PINK1, Parkin, p62, and LC3Ⅱ/Ⅰ. (O-P) PINK1 overexpression efficiency. Data are presented as the mean ± SD, n=3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with the control group.

PINK1 Overexpression Attenuates Arsenic-Induced Fibrosis and Modulates Mitophagy in LX-2 Cells

To investigate the role of the PINK1/Parkin pathway in arsenic-induced hepatic stellate cell activation, we overexpressed PINK1. PINK1 overexpression significantly decreased α-SMA and Vimentin protein levels compared with the control group (Fig. 4A, C-D). PINK1 and Parkin protein expression was elevated, p62 accumulation was reduced, and the LC3-II/LC3-I ratio decreased (Fig. 4B, E-H). ELISA analysis demonstrated corresponding reductions in IL-6, IL-1β, and TNF-α levels (Fig. 4I-K). Similarly, treatment with the mitophagy agonist CCCP decreased α-SMA and Vimentin expression (Fig. 4L, N-O), accompanied by upregulation of PINK1 and Parkin, downregulation of p62, and a reduced LC3-II/LC3-I ratio (Fig. 4M, P-S). ELISA results also confirmed decreased levels of IL-6, IL-1β, and TNF-α (Fig. 4T-V). These findings demonstrate that restoring PINK1 expression or pharmacologically enhancing mitophagy effectively reverses arsenic-induced HSC activation, inflammatory cytokine release, and the suppression of mitophagy, thereby confirming the central role of impaired PINK1/Parkin-mediated mitophagy in this pathological process.

Fig. 4.

Fig. 4

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PINK1 overexpression reverses sodium arsenite-induced fibrosis and regulates downstream proteins in LX-2 cells. (A-H) Western blot analysis of α-SMA, vimentin, PINK1, Parkin, p62, and LC3Ⅱ/Ⅰ following PINK1 overexpression. (I-K) ELISA of IL-6, IL-1β, and TNF-α after PINK1 overexpression. (L-M) Western blotting of α-SMA, Vimentin, PINK1, Parkin, p62, and LC3Ⅱ/Ⅰ following treatment with the mitophagy agonist CCCP. (T-V) ELISA quantification of IL-6, IL-1β, and TNF-α after CCCP treatment. Data are presented as the mean ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with the control group

Discussion

Chronic arsenic exposure is a recognized risk factor for liver injury, which can progress to fibrosis and even cirrhosis [13–15]. Although oxidative stress, metabolic disruption, and inflammation have been implicated [16–18], the precise molecular mechanisms initiating this process remain incompletely defined. Recent studies have highlighted mitophagy as an important mechanism for maintaining mitochondrial homeostasis [19], and it has increasingly been recognized for its role in liver pathology. The PINK1/Parkin pathway, in particular, is critical for the clearance of damaged mitochondria [20]. This study provides a novel mechanistic link by demonstrating that arsenic-induced inhibition of PINK1/Parkin-mediated mitophagy serves as an early event driving inflammatory activation and subsequent hepatic fibrosis.

Our initial population-based finding is pivotal: individuals chronically exposed to arsenic exhibited a significant elevation in systemic inflammatory cytokines (IL-6, IL-1β, TNF-α) without concurrent increases in conventional liver injury (AST, ALT) or fibrosis (HA, LN) serum markers. This clinically relevant observation suggests that a persistent, subclinical inflammatory state precedes detectable fibrotic sequelae. This hypothesis was rigorously tested in our experimental models. In mice, chronic arsenic exposure recapitulated key features of liver injury, including inflammatory cell infiltration, upregulation of pro-fibrotic markers (α-SMA, Vimentin), and collagen deposition. Crucially, these pathological changes were accompanied by a dose-dependent suppression of the PINK1/Parkin pathway, evidenced by decreased PINK1 and Parkin protein levels, accumulation of the autophagy adapter p62, and an altered LC3-II/LC3-I ratio—collectively indicating impaired mitophagy. Consistently, the protein level of OPTN, a mitophagy receptor acting downstream of the PINK1/Parkin axis, was also significantly decreased upon arsenic exposure (Fig S1), further corroborating impaired mitophagy.

PINK1, a kinase located on the outer mitochondrial membrane, senses organelle damage and recruits Parkin [21] to initiate mitophagy. Both proteomic and Western blot analyses confirm that arsenic exposure significantly downregulates PINK1 and Parkin, underscoring the critical regulatory role in arsenic-induced liver injury. The causal role of PINK1/Parkin dysregulation was established through gain-of-function experiments in LX-2 human hepatic stellate cells (HSCs). Arsenic treatment in these cells mirrored the in vivo findings, inhibiting the PINK1/Parkin pathway, activating HSCs (increased α-SMA and Vimentin), and stimulating pro-inflammatory cytokine secretion. These findings align with previous studies describing PINK1/Parkin regulation in other cell types [22–24] and further highlight the specific role of this signaling pathway in arsenic-induced liver injury and its potential as a therapeutic target. Previous studies indicate that arsenic promotes the transformation of HSCs into a myofibroblast phenotype by inducing ROS production and activating the NLRP3 inflammasome [25]. The observed inhibition of proliferation is likely associated with arsenic-induced cell cycle arrest and mitochondrial injury, which may further exacerbate cell damage by disrupting mitochondrial function and impairing PINK1/Parkin-mediated mitophagy. Importantly, overexpression of PINK1 or pharmacological enhancement of mitophagy significantly attenuated these arsenic-induced effects, effectively reversing HSC activation and cytokine production. This is consistent with reports showing that MitoQ enhances PINK1/Parkin-mediated mitophagy, alleviating hepatic stellate cell activation and liver fibrosis [26, 27]. Previous studies have shown that Resveratrol activates this pathway to promote mitophagy, reduce oxidative stress, and decrease hepatocyte apoptosis, exerting a hepatoprotective effect [28]. This rescue experiment provides direct evidence that the arsenic-induced fibro-inflammatory response is, at least in part, mediated through the suppression of the PINK1/Parkin-mitophagy axis.

In the mouse model, increasing arsenic exposure significantly elevated levels of inflammatory factors such as IL-6, IL-1β, and TNF-α, highlighting the central role of inflammation in arsenic-induced liver injury. Consistent with previous studies, these results demonstrate that arsenic regulates hepatic immune and inflammatory responses via the MAPK/Nrf2/NF-κB signaling pathway [29–30]. This study further demonstrates that arsenic triggers a synergistic upregulation of inflammatory factors that correlate positively with fibrosis-related proteins, supporting an inflammation-mediated mechanism of HSC activation and extracellular matrix (ECM) deposition. Moreover, arsenic-induced Th1/Th2/Th17 immune imbalance further contributes to the hepatic immune microenvironment [31]. The results of this study align with observations of elevated levels of IL-6, IL-8, and IL-12 reported in systematic reviews [32–34], emphasizing the potential of inflammatory factors as biological markers of arsenic exposure. The elevation of inflammatory factors not only reflects an arsenic toxicity response but may also drive the progression of liver fibrosis through complex signaling networks [35].

Chronic low-dose arsenic exposure (9 months in mice, 2 µM for 48 h in LX-2 cells) significantly downregulated PINK1/Parkin expression at both mRNA and protein levels, indicating functional exhaustion of this protective pathway. This contrasts with the transient activation of PINK1/Parkin reported in acute exposure studies [36–37], which likely represents an early compensatory response. We propose a biphasic model: acute arsenic exposure rapidly collapses mitochondrial membrane potential and activates PINK1/Parkin-mediated mitophagy as a protective mechanism, whereas chronic exposure leads to sustained downregulation, potentially through transcriptional repression or accelerated protein degradation. The upregulation of PINK1/Parkin in a sub-chronic arsenic-antimony co-exposure model [38] further highlights the importance of exposure duration and toxicant composition in modulating mitophagy. Notably, differences in cell type may also contribute to divergent findings. Hepatic stellate cells (HSCs) exhibit distinct redox and autophagic responses during quiescence-to-activation transition [39], which may account for the sustained PINK1/Parkin suppression observed in this study. Collectively, these results demonstrate that chronic arsenic exposure leads to suppression rather than activation of PINK1/Parkin-mediated mitophagy. This shift from protective mitophagy to mitophagic dysfunction represents a previously unrecognized mechanism in arsenic-induced liver fibrosis.

We propose a coherent mechanistic model (Fig. 5). Arsenic exposure leads to mitochondrial damage. Under physiological conditions, the PINK1/Parkin pathway is activated to clear these damaged organelles via mitophagy, maintaining cellular homeostasis. Our data demonstrate that arsenic disrupts this critical quality-control mechanism. The resulting accumulation of dysfunctional mitochondria perpetuates oxidative stress and, as supported by prior literature, likely activates innate immune sensors such as the NLRP3 inflammasome [12, 40]. This explains the persistent inflammation associated with arsenic exposure. Mitochondrial dysfunction and impaired mitophagy are key mechanisms underlying intracellular ROS accumulation and enhanced inflammatory responses [41–42]. This creates a pro-inflammatory microenvironment that promotes the transformation of quiescent HSCs into activated, collagen-producing myofibroblasts, initiating the fibrotic cascade. This model aligns with and extends previous knowledge by positioning defective mitophagy as a key upstream event that bridges initial arsenic insult to sustained inflammation and HSC activation. Consistent with previous findings in AFB1-induced liver injury, our study demonstrates the protective role of PINK1/Parkin-mediated mitophagy [43]. Studies on PTEN regulating autophagic activity show that modulating autophagy-related signals can affect arsenic-induced HSC fibrosis [44]. This study meticulously elucidates the mechanism by which arsenic exposure inhibits the PINK1/Parkin pathway-mediated mitophagy, enhances inflammation, and increases the expression of fibrosis-related proteins. This provides a new perspective for revealing the molecular mechanisms of arsenic-induced liver injury.

Fig. 5.

Fig. 5

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Chronic arsenic exposure downregulates the PINK1/Parkin pathway, impairing mitophagy and leading to the accumulation of damaged mitochondria. This accumulation triggers the release of inflammatory cytokines, which activate hepatic stellate cells and ultimately result in liver fibrosis. The red dashed line illustrates a positive feedback loop: the accumulation of damaged mitochondria further suppresses the PINK1/Parkin pathway, thereby establishing a self-perpetuating cycle that exacerbates pathological progression

This study has several limitations. First, the control group IL-1β and TNF-α values exceeded some reference values, and we cannot fully explain this discrepancy. Insufficient remaining serum samples precluded repeat measurements or hsCRP/arsenic level analysis. Future studies should address these issues. Second, the clinical sample size is modest, and the cross-sectional design warrants validation in larger longitudinal cohorts. Third, while we focused on the PINK1/Parkin pathway, arsenic toxicity is pleiotropic. The potential interplay with other stress pathways (e.g., ER stress) and their collective impact on mitochondrial homeostasis merit future investigation.

In conclusion, our integrated clinical and experimental data unveil a previously underappreciated mechanism for arsenic-induced hepatotoxicity. We identify the inhibition of PINK1/Parkin-mediated mitophagy as a critical early event that fosters a pro-inflammatory and pro-fibrotic hepatic milieu. These findings not only advance our understanding of arsenic pathogenesis but also highlight the PINK1/Parkin pathway as a potential therapeutic target. Strategies aimed at enhancing mitophagy flux could represent a promising avenue for intervening in the progression of arsenic-related liver fibrosis.

Conclusions

This study confirmed through in vivo and in vitro experiments that long-term arsenic exposure induces significant liver inflammation and fibrotic damage. The underlying mechanism involves suppression of the PINK1/Parkin-mediated mitophagy pathway, resulting in decreased clearance of damaged mitochondria, and exacerbated cellular dysfunction and inflammatory responses. Restoration of PINK1 expression or pharmacological activation of mitophagy effectively reversed arsenic-induced hepatic stellate cell activation and inflammatory cytokine production. These findings reveal the critical regulatory role of the PINK1/Parkin pathway in arsenic-induced liver fibrosis, suggesting that targeting this pathway to restore mitophagy may represent a promising strategy for preventing or treating arsenic-related liver injury. Future research should explore the therapeutic potential of existing mitophagy inducers (e.g., urolithin A, NAD+ boosters) and address challenges in developing PINK1-specific activators to advance this strategy toward clinical application.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (1.3MB, docx)

Acknowledgements

This work was supported by the Natural Science Foundation of China (82060605), Baotou medical college research projects for cultivation of public health and preventive medicine first-class discipline (GWGG-20250801), 2025 Baotou Medical College Annual Higher Education Special Funds (201-2025401607) and BaotouMedical College Zhenchuang Project (GWGG-20250822). We would also like to thank everyone who worked so hard on this whole experiment.

Author Contributions

All authors contributed to the conception and design of the study. Material preparation (Feiyang Wu); data collection and analysis (Feiyang Wu) and (Yan Wang); Methodology(Teng Ma) and (Sheng Li); Validation (Feiyang Wu) and (Yan Wang); Formal Analysis (Yan Wang) and (Haijing Yin); Investigation (Zhiyong Xu) and (Xiaohui Wang); Resources (Teng Ma) and (Li Wang); Draft writing of the manuscript (Feiyang Wu); Writing – Review & Editing (Teng Ma) and (Li Wang); Supervision (Sheng Li) and (Teng Ma); Funding Acquisition and Project Administration (Teng Ma). All authors have read and approved the final manuscript.

Funding

This research was funded by Baotou medical college research projects for cultivation of public health and preventive medicine first-class discipline (GWGG-20250822), Baotou Medical College Zhen chuang Project (BYJJ-ZCJH 202601), Baotou Medical College-2026 -Central University Basic Research Fund- New Master's and Doctoral Programs Project (BYJJ-GWZX202507) and Natural Science Foundation of China (82060605).

Data Availability

The data presented in this study will not be made available to the public prior to publication of the research results but can be obtained from the corresponding author upon reasonable request.

Declarations

Institutional review board statement

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Baotou Medical College Ethics Review Committee (approval number No.002), Baotou Medical College, 2021). The study was conducted according to the guidelines of the Declaration of Helsinki, and all animal experiments and housing conditions were approved by the Laboratory Animal Ethics Committee of Baotou Medical College (approval number No.002), Baotou Medical College, 2021).

Informed consent

Written informed consent has been obtained from the patient(s) to publish this paper.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Teng Ma, Email: 102018924@btmc.edu.cn.

Li Wang, Email: wangli1626@126.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (1.3MB, docx)

Data Availability Statement

The data presented in this study will not be made available to the public prior to publication of the research results but can be obtained from the corresponding author upon reasonable request.

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