Selenium Protects Against Cadmium-Induced Hepatotoxicity via Regulation of Lipid Metabolism and Inflammatory Pathways

Abstract

Background:

Cadmium (Cd), a widespread environmental pollutant, poses significant risks to human health due to its high bioaccumulation potential and prolonged biological half-life. Selenium (Se) has been reported to exert protective effects against Cd-induced organ toxicity; however, the underlying molecular mechanisms, particularly those associated with lipid metabolism and inflammatory regulation, remain insufficiently elucidated.

Methods:

The hepatoprotective effects of Se, administered as selenomethionine (SeMet) and Se-enriched Cardamine enshiensis extract (CE), were investigated against Cd-induced hepatic injury using both in vitro (L-02 hepatocytes) and in vivo (C57BL/6J mice) models.

Results:

SeMet significantly attenuated Cd-induced cytotoxicity, lipid accumulation, and metabolic dysregulation in L-02 cells. In Cd-exposed mice, treatment with SeMet or CE significantly mitigated hepatic injury, steatosis, and inflammation, as evidenced by normalized serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), and total cholesterol (TC) levels, improved hepatic histoarchitecture, and reduced lipid droplet deposition. Integrated lipidomic and transcriptomic analyses demonstrated that Se supplementation restored Cd-perturbed polyunsaturated fatty acid metabolism, downregulated lipogenic genes (SCD1, Pparγ, Fasn), and suppressed pro-inflammatory mediators (Cxcl2, Ccl2).

Conclusion:

Se confers hepatoprotection against Cd toxicity not only through its classical antioxidant activity but also through coordinated modulation of lipid metabolic pathways and inflammatory signaling. This study provides mechanistic insights into Se-mediated defense against Cd-induced hepatotoxicity and highlights the therapeutic potential of Se-enriched phytochemicals for mitigating the adverse effects of environmental Cd exposure.

Graphical abstract

Keywords

cadmium / selenium / chemical and drug induced liver injury / lipid metabolism / inflammation

1. Introduction

Cadmium (Cd), a widespread and bioaccumulative environmental toxicant, poses severe threats to human health owing to its prolonged biological half-life and inefficient metabolic clearance [1]. The liver and kidneys, as primary sites of xenobiotic metabolism, are particularly vulnerable to both acute and chronic Cd exposure, making them major targets of Cd-induced toxicity [2, 3, 4]. Extensive evidence indicates that Cd exerts multi-organ toxicity through diverse mechanisms, including oxidative stress, DNA damage, endoplasmic reticulum stress (ERS), apoptosis, autophagy, and reproductive system disorders [5, 6]. Among these, oxidative stress and inflammation are considered key pathogenic events in Cd-mediated tissue injury, despite Cd lacking redox activity as a non-Fenton metal [7, 8]. Although significant advances have been made in delineating the molecular basis of Cd toxicity, effective therapeutic strategies to counteract its deleterious effects remain scarce. Therefore, elucidation of the molecular mechanisms underlying Cd-induced hepatic and renal injury and the development of targeted interventions represent critical research imperatives.

Selenium (Se), an essential micronutrient, is vital for maintaining human health due to its key roles in antioxidative defense, anti-inflammatory regulation, and immune modulation [9, 10, 11, 12]. Accumulating evidence supports the protective effects of Se against Cd toxicity, with exogenous Se supplementation reported to attenuate Cd-induced cellular and tissue damage through multiple mechanisms [13]. One of the primary protective pathways involves the formation of Se–Cd complexes, which promote Cd detoxification and excretion from the body [14, 15, 16]. Furthermore, Se mitigates Cd-induced oxidative stress and ERS in renal tissues, as well as oxidative damage and programmed necrosis in the liver [17]. Se exerts its antioxidant and anti-inflammatory functions through incorporation into selenoproteins, including glutathione peroxidase (GPx) and thioredoxin reductase (TrxR), which collectively maintain redox homeostasis and suppress inflammatory signaling cascades [18, 19]. Moreover, Se has been reported to attenuate Cd toxicity through activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway [20]. Recent studies have demonstrated that various forms of Se, including sodium selenite, selenomethionine (SeMet), nano-Se, and Se derived from Cardamine hupingshanensis, effectively alleviate Cd-induced toxicity in multiple organs such as the liver, kidneys, spleen, brain, and heart in both in vivo and in vitro models [20, 21]. Despite these advances, the precise molecular mechanisms underlying the protective actions of Se, particularly its modulation of lipid metabolic pathways and suppression of inflammatory processes in Cd-induced hepatotoxicity, remain insufficiently elucidated.

The significance of this study is derived from the considerable public health burden associated with environmental cadmium exposure, for which no pharmacological intervention with an established safety and efficacy profile is currently available. Clinical management remains largely dependent on chelation therapy, which is frequently limited by off-target toxicity and organ-related adverse effects. This situation emphasizes the need to identify safe and effective alternative strategies and to advance beyond the conventional antioxidant framework toward a precise characterization of the molecular pathways responsible for selenium-mediated protection. Based on previous observations [21, 22], the aim of this study is to elucidate the molecular mechanisms by which selenium, particularly in the form of a Cardamine enshiensis–derived extract enriched in SeCys₂ (CE), protects against cadmium-induced hepatotoxicity, using integrated lipidomic and transcriptomic analyses to define its regulatory effects on polyunsaturated fatty acid metabolism, lipogenic gene networks, and inflammatory signaling pathways.

2. Materials and Methods

2.1 Cell Culture

The L-02 hepatocyte line (kindly provided by Prof. Jiangfeng Wu, China Three Gorges University) was cultured in DMEM/F-12 (Gibco, 11320082, Grand Island, CA, USA) supplemented with 10% fetal bovine serum (FBS; Vivalcell, C04001-050X10, Shanghai, China) and 1% penicillin/streptomycin (Gibco, 15140-122, Grand Island, CA, USA). Cells were maintained at 37 °C in a humidified incubator containing 5% CO₂. The L-02 cells used in our laboratory have undergone recent STR profiling authentication, and the validation report has been uploaded as an attachment. Additionally, PCR testing confirmed the absence of mycoplasma contamination. As detailed in Section 2.1, the forward and reverse primer sequences employed for PCR-based mycoplasma identification in this study were 5^′-GGGAGCAAACAGGATTAGATACCCT-3^′ and 5^′-TGCACCATCTGTCACTCTGTGTTAACCTC-3^′, respectively.

2.2 Cell Viability Assay

L-02 cells were seeded in 96-well plates at a density of 1 \times 10⁴ cells per well and cultured until reaching 60–70% confluency. To assess single-agent cytotoxicity, cells were treated with increasing concentrations of SeMet (0–100 µM; TargetMol, T1658, Boston, MA, USA) or CdCl₂ (0–200 µM; Macklin, C805625, Shanghai, China) for 24 hours (h). For combination studies, cells were pretreated with SeMet for 24 h, followed by 24 h exposure to CdCl₂. Cell viability was measured using the Cell Quanti-Blue assay kit (BioAssay Systems, CQBL-05K, Grand Island, CA, USA), and absorbance was recorded at 570 nm and 600 nm after a 3 h incubation with the detection reagent. The protocol was followed in accordance with the manufacturer’s guidelines.

2.3 Observation of Cellular Growth Morphology

L-02 cells were plated in 6-well plates at 2 \times 10⁵ cells per well in 2 mL of culture medium. Cells were divided into four treatment groups: control (serum-free medium), Se (0.1 µM SeMet), Cd (40 µM CdCl₂), and Se+Cd (0.1 µM SeMet + 40 µM CdCl₂). Once cells reached 60–70% confluency, Se and Se+Cd groups were pretreated with SeMet for 24 h. Following, Cd and Se+Cd groups were exposed to CdCl₂ for another 24 h. After treatment, the medium was removed, and the cells were washed twice with phosphate-buffered saline (PBS). Cells were then fixed with 500 µL of 4% paraformaldehyde per well for 15 minutes, washed twice with PBS, and stained with 500 µL of crystal violet for 10 minutes in the dark. Following two further PBS washes, cell morphology was assessed via microscopic imaging.

2.4 Animal Model and Experimental Design

C57BL/6J mice (sourced from Hua Lian Ke, Wuhan, China) were housed under standardized conditions at the Central Hospital of Enshi Tujia and Miao Autonomous Prefecture, with temperature maintained at 20–26 °C, relative humidity at 40–70%, chamber pressure at 20–50 Pa, and a 12 h/12 h light-dark cycle. Animals had ad libitum access to food and water. All experimental procedures were approved by the Institutional Animal Ethics Committee (Approval No.: 202101001). A chronic Cd exposure model was established by intraperitoneal injections of CdCl₂ (2 mg/kg) [21, 23] every 48 h for 4 weeks. Se supplementation was administered weekly via oral gavage at 7.5 µg per 20 g body weight, a dose extrapolated from human pharmacokinetics based on a maximum tolerable intake of 400 µg/day [24]. Mice were randomly assigned to six groups (n = 10 per group) [21, 25]: Control (vehicle), Se (SeMet only), CE (Se-rich CE extract only), Cd (CdCl₂ only), Se+Cd (SeMet plus CdCl₂), and CE+Cd (CE extract plus CdCl₂). One week before Cd exposure, Se and Se+Cd groups received a single SeMet pretreatment, while CE and CE+Cd groups were pretreated with CE extract to prime Se-dependent biochemical pathways. Dosing volumes were adjusted every 4 days according to body weight.

Throughout the study, mice were closely monitored for behavioral indicators (locomotor activity, fur condition), physiological parameters (food and water intake, body weight), and general clinical signs.

2.5 Sample Collection and Processing

Mice were anesthetized via intraperitoneal injection of 1.25% tribromoethanol (0.4 mL/10 g body weight). Adequate anesthesia was confirmed by the absence of a withdrawal reflex upon hind paw pinching. The animals were then euthanized by cervical dislocation, and death was verified by complete cessation of respiration and heartbeat, along with pupil dilation. Following euthanasia, blood was collected by cardiac puncture into heparinized tubes. Serum was obtained by centrifugation at 2000 \timesg for 15 min at 4 °C and stored at –20 °C. Tissues were rinsed with ice-cold saline, blotted dry, and weighed. Serum samples were analyzed using an automated biochemical analyzer to measure alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), globulin (GLO), total protein (TP), total cholesterol (TC), and triglycerides (TG) [26].

2.6 Se Quantification

The selenium content of the Cardamine extract was quantified by inductively coupled plasma–mass spectrometry (ICP–MS; Thermo Fisher Scientific, iCAP TQe, Serial No. ICAPTQe00081, Waltham, MA, USA). Calibration curves were generated using a series of working standard solutions prepared by serial dilution of a multi-element standard stock solution with ultrapure water. An internal standard solution was continuously introduced online into all calibration standards and sample solutions to correct for instrumental drift and matrix effects. Before analysis, Cardamine extract samples were sequentially diluted with ultrapure water (100-fold and 1000-fold) and analyzed directly under the same instrumental conditions.

2.7 Quantitative Real-Time PCR (qPCR) Analysis

Total RNA was extracted from liver tissues using the EASYspin Tissue & Cell RNA Kit (Aidlab, RN07, Beijing, China) following the manufacturer’s protocol. RNA concentration and purity were evaluated spectrophotometrically. One microgram of total RNA was reverse-transcribed into first-strand cDNA using ToloScript All-in-One RT EasyMix for qPCR (TOLOBIO, 22107, Shanghai, China). qPCR was performed in triplicate on a CFX96 Touch Real-Time PCR Detection System using 2\timesQ3 SYBR qPCR Master Mix (TOLOBIO, 22204, Shanghai, China). Each 20 µL reaction contained 10 µL SYBR Green Master Mix, 0.8 µL each of forward and reverse primers (10 µM), 2 µL cDNA template, and 6.4 µL nuclease-free water. Thermal cycling was conducted with an initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Relative gene expression levels were normalized to the housekeeping gene Gapdh and calculated using the 2^{-Δ⁢Δ⁢Ct} method.

2.8 Statistical Analysis

All data are expressed as mean \pm Standard Error of the Mean (SEM) and analyzed using GraphPad Prism 9 (GraphPad Software, Boston, MA, USA). Statistical differences among groups were evaluated using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test for multiple comparisons.

3. Results

3.1 Se Mitigates Cd-Induced Hepatotoxicity by Restoring Lipid and Metabolic Balance

To investigate the protective role of SeMet against Cd-induced cytotoxicity in L-02 cells, the IC₅₀ of CdCl₂ was first determined to be 139.1 µM (Fig. 1a), while 0.1 µM was identified as the non-toxic concentration of SeMet (Fig. 1b). SeMet treatment significantly reduced Cd-induced cytotoxicity in a dose-dependent manner, with stronger protection observed at higher Cd concentrations (Fig. 1c,d). Furthermore, SeMet ameliorated Cd-induced metabolic dysregulation, as reflected by decreased levels of hepatic injury markers (AST and ALT) and lipid metabolites (TG and TC) (Fig. 1e–h). Treatment with 0.1 µM SeMet also significantly reduced lipid accumulation in L-02 cells exposed to 140 µM CdCl₂ (Fig. 1i,j).

3.2 Se Supplementation Protects Against Cd-Induced Liver Injury and Steatosis in Mice

To investigate the hepatoprotective effects of Se against Cd-induced toxicity, selenomethionine (SeMet) and Se-enriched Cardamine extract (CE) were administered in a mouse model. Cd exposure caused significant hepatic inury, characterized by a rough liver surface and firm texture, which were significantly alleviated by Se supplementation (Fig. 2a,b). Histopathological assessment using H&E staining revealed that Se supplementation significantly reduced Cd-induced hepatocellular ballooning and inflam matory cell infiltration, while Oil Red O staining demonstrated a significant attenuation of hepatic lipid accumulation (Fig. 2c,d). Serum biochemical analyses showed that Cd exposure significantly elevated the activities of liver injury markers (ALT, AST, and the AST/ALT ratio) [27, 28] whereas Se supplementation restored these parameters to near-normal levels (Fig. 2e–g). In comparison, markers of hepatic synthetic function (ALB, GLO, and TP) showed no significant changes across the experimental groups (Fig. 2h–j). Moreover, Cd exposure disrupted hepatic lipid metabolism, as indicated by increased TC and TG levels, both of which were significantly reduced following Se supplementation (Fig. 2k,l). These findings indicate that Se supplementation mitigates Cd-induced hepatotoxicity by preserving liver architecture and alleviating lipid accumulation.

3.3 Se Counteracts Cd-Induced Hepatotoxicity by Modulating Lipid Homeostasis and Inhibiting Inflammatory Responses

To elucidate the molecular mechanisms underlying the protective effects of Se against Cd-induced hepatotoxicity, a comprehensive analysis of hepatic lipid metabolism was performed on liver tissues from control, Cd-exposed, and CE+Cd-treated mice (Table 1). Cd exposure resulted in significant perturbations of polyunsaturated fatty acids (PUFA) metabolic pathways compared with controls, reflecting a disruption of hepatic lipid homeostasis. The CE supplementation effectively restored these Cd-induced metabolic alterations. Furthermore, hepatic lipidomic profiling revealed that CE supplementation significantly modulated liver lipid metabolite composition in Cd-exposed mice (Fig. 3a–c, Supplementary Fig. 1a–d). Quantitative analysis showed significant reductions in specific phospholipid and TG species in the CE+Cd group relative to Cd-exposed animals, including phosphatidylethanolamine (PE) species (16:1_22:6, 18:0p_22:5, and 36:7e), phosphatidylcholine (PC) derivatives (18:1_14:0, 36:1e, 44:4e, and 45:2), and triacylglycerol (TG) molecules (6:0_14:3_18:2, 11:0_12:2_18:2, and 12:0e_6:0_22:6) (Fig. 3d–k). Transcriptomic analysis revealed significant enrichment of the cytochrome P450 pathway in CE-treated mice compared with Cd-exposed animals (Fig. 3l, Supplementary Fig. 1e–g), suggesting a key role of Se in regulating PUFA metabolism. Gene expression profiling demonstrated that Cd exposure significantly upregulated major lipogenic regulators (SCD1 and Ppar\gamma) and moderately increased the expression of Fasn, Ppar\alpha, and Cpt1\alpha, whereas CE treatment effectively reversed these alterations (Fig. 3m–q). Moreover, CE administration significantly reduced Cd-induced elevations of pro-inflammatory cytokines (Cxcl2 and Ccl2) (Fig. 3r,s).

4. Discussion

This study employed the distinctive biomarker levels reported in the Enshi population, serum selenium (416.977 µg/L) and urinary cadmium (3.848 µg/L), as references for human exposure, allowing reverse calculation of corresponding doses for animal experiments [29, 30]. This approach is consistent with strategies that employ cadmium-based physiologically based pharmacokinetic (PBPK) modeling to establish recommended reference concentrations for diseases such as chronic kidney disease and osteoporosis [31]. Deriving exposure doses from human biomarkers not only demonstrates the feasibility of translating environmental epidemiological data into toxicological studies but also provides a basis for the future development of precise selenium-cadmium combined exposure kinetic models.

Ferroptosis is a novel iron-dependent form of programmed cell death driven by lipid peroxidation [32]. Selenium and its associated selenoproteins are key inhibitors of ferroptosis [33]. Previous study has shown that cadmium can induce ferroptosis through nuclear translocation of the antioxidant transcription factor Nrf2 [34]. In comparison, inhibition of the TXNRD1/Nrf2 pathway, an essential component of mammalian antioxidant defense, via agents such as auranofin enhances Nrf2 activation [35]. Our previous work demonstrated that selenium effectively counteracts ferroptosis induced by RSL3 through targeting TXNRD1 [36]. This study further shows that selenium supplementation (CE) exerts anti-ferroptotic effects by regulating PUFA metabolism. Consistent with previous reports indicating that selenium mitigates doxorubicin-induced ferroptosis by reducing key phospholipids involved in lipid peroxidation, such as PE and PC [24], our data reveal that CE treatment significantly restored cadmium-induced PUFA metabolic disturbances, reversing aberrant changes in specific PEs (e.g., 16:1_22:6, 18:0p_22:5) and PC derivatives.

Further analysis indicates that activation of the PPAR\gamma/SCD1 pathway during the early phase of Cd exposure represents a central mechanism driving further lipid metabolic disorders. Cadmium exposure upregulates PPAR\gamma and SCD1 expression, resulting in significant PUFA accumulation, consistent with the classical regulatory cascade in which PPAR\gamma transcriptionally controls SCD1 [37], and SCD1 mediates triglyceride accumulation [38]. We propose that under sustained cadmium stress, the PUFA pool generated by the PPAR\gamma/SCD1 axis becomes unstable and is readily converted into lipid peroxidation products, including 15-HETE, 4-HNE, and MDA. These oxidized lipid derivatives have been reported to act as endogenous ligands for PPAR\gamma [39], leading to a self-amplifying cycle of “From PPAR\gamma activation to PUFA accumulation to lipid peroxidation and back to PPAR\gamma reactivation” that exacerbates lipid metabolic imbalance. CE treatment effectively suppressed the overactivation of PPAR\gamma and SCD1, interrupting this cycle. Lipidomic profiling further revealed that cadmium exposure significantly disrupted membrane phospholipid side-chain integrity, an early indicator of hepatocyte injury [40]. SCD1-mediated lipid reprogramming also directly activates inflammatory signaling pathways, such as NF-\kappaB, promoting chemokine release [41], which explains the observed upregulation of Cxcl2 and Ccl2 in cadmium-exposed livers. By modulating upstream lipid metabolic networks, particularly restoring PUFA homeostasis and inhibiting SCD1, CE not only repaired the membrane lipid environment but also blocked the pathological progression from lipotoxicity to inflammation at its origin.

Different chemical forms of selenium display distinct biological activities [42]. The major selenium species present in the Se-enriched CE used in this study were selenocysteine (SeCys₂) [43]. Organic selenium generally exhibits higher bioavailability and tissue retention compared to inorganic forms [44] and demonstrates greater efficacy in regulating lipid metabolism–related genes, such as PPAR\gamma, and in mitigating hepatic steatosis [26]. In this study, both CE and selenomethionine interventions significantly reduced serum TC and TG levels. These results are consistent with previous reports showing that selenium nanoparticles ameliorate metabolic-associated steatohepatitis (MASH) and that sodium selenite improves alcoholic fatty liver disease [26, 45], supporting the role of selenium supplementation in counteracting cadmium-induced inflammatory injury through modulation of lipid metabolism.

5. Limitations and Future Directions

Although this study demonstrates selenium’s protective effects against cadmium toxicity via regulation of lipid metabolism and inflammatory responses, several limitations remain in the molecular mechanisms and experimental design. First, the intervention period was four weeks. While significant protective effects were observed, this relatively short duration may have limited the evaluation of long-term outcomes under chronic cadmium exposure. Second, as a water extract, CE represents a complex mixture. Although previous studies indicate that its primary active component is organic selenium, and SeMet was included as a control in this study, other coexisting phytochemicals may exert synergistic effects. To clarify selenium’s specific contribution to CE’s protective effects, future studies should incorporate more refined control groups, including purified SeCys₂, selenium-enriched CE, and selenium-depleted CE, to systematically identify the independent and combined actions of each component.

No specific therapeutic agents currently exist for cadmium exposure. Based on the significant antagonistic effects of CE observed here, we plan to implement a human intervention study in cadmium-polluted regions. Participants will receive selenium preparations derived from Cardamine enshiensis, with dynamic monitoring of blood selenium metabolites, cadmium body burden, and hepatic and renal function markers. This study aims to validate the “selenium–cadmium antagonism” mechanism in humans and provide evidence-based strategies for health interventions in populations exposed to environmental heavy metals.

6. Conclusions

Se-enriched CE effectively mitigates cadmium-induced hepatotoxicity at environmentally relevant exposure levels. The protective mechanism involves a dual pathway: CE inhibits overactivation of the PPAR\gamma/SCD1 signalling axis, correcting PUFA metabolic disorders and restoring membrane phospholipid homeostasis in hepatocytes; simultaneously, by remodelling the lipid microenvironment, it suppresses the release of lipid peroxidation driven inflammatory mediators (Cxcl2, Ccl2) and potential ferroptotic processes. These findings elucidate the molecular basis by which selenium counteracts heavy metal toxicity through a “metabolism immunity” network and provide experimental support for the application of naturally selenium-enriched plant resources in mitigating environmental cadmium exposure at physiologically relevant doses.

Availability of Data and Materials

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Funding

Natural Science Foundation of Hubei Province(2025AFD137)