The experimental procedures received authorization from both the Administration of Affairs Concerning Experimental Animals of the Ministry of Science and Technology of China (2025) and the Institutional Animal Care and Use Committee of the College of Animal Science and Technology at Yangzhou University. When adult animals needed to be euthanized, humane methods were employed under standard conditions to minimize any potential discomfort or distress.

C57 mice (6–7 weeks old) were housed under controlled environmental conditions at a temperature of 21 °C ± 2 °C and relative humidity of 60%–70%, with a 12 h light/dark cycle. Animals had free access to standard chow and water throughout the experiment. Mice were randomly assigned to receive either vehicle (sterile saline) or cadmium (Cd) in the vehicle solution via oral gavage. To determine the optimal Cd exposure concentration for inducing hepatic injury, mice were administered CdCl₂ at concentrations of 1.5, 3, 10, 18, 50, 90, 250, 450, and 1,200 μg/L (0.8 mL/day) for 5 days. Each group consisted of three mice, with an additional control group (n = 3) receiving sterile saline. Among these groups, exposure to 18 μg/L CdCl₂ resulted in the most pronounced hepatic injury. Based on this finding, subsequent experiments were designed to evaluate the time-dependent effects of Cd exposure. Mice were divided into four groups: pre-exposure (day 0), 3-day exposure (18 μg/L, 0.8 mL/day), 5-day exposure (18 μg/L, 0.8 mL/day), and 7-day exposure (18 μg/L, 0.8 mL/day), with three mice per group. To further assess tissue-specific responses to Cd exposure, mice were treated with high, medium, and low concentrations of CdCl₂ (0.8 mL/day) for 5 days, with three mice per group. Following exposure, mice were deeply anesthetized with sodium pentobarbital (100 mg/kg body weight, intraperitoneally; Butler Co., Columbus, OH). Blood was collected via abdominal aorta transection, and organs and tissues were immediately harvested for further analysis.

The serum biochemical parameters of mice were measured by using an animal biochemical analyzer (SMT-120VP, Seamaty, CHINA) and animal biochemical reagent plate (Seamaty, CHINA). The concentrations of Aspartate aminotransferase (AST), Blood urea nitrogen (BUN), Glucose (GLU), Total cholesterol (CHOL), Triglycerides (TG), High-density lipoprotein cholesterol (HDL-C), White Blood Cell Count (WBC), Hemoglobin (HGB), Platelet Count (PLT), Lymphocyte Count (Lymph#), Red Cell Distribution Width (RDW), Mean Platelet Volume (MPV), in serum were measured in the present study.

Treated hepatocytes were first dewaxed in xylene for 10 min, after which the reagent was removed using 100% ethanol. The cells were then rehydrated through a graded ethanol series (95%, 85%, and 75%) for 5 min each. Hematoxylin was applied for a 3-min stain, followed by a 2-min rinse in distilled water. The sections were subsequently subjected to brief differentiation in 1% hydrochloric acid alcohol for 2 s and then washed in distilled water for 15 min. A drop of neutral gum was used to mount the slides. Finally, the prepared sections were examined and imaged under a microscope.

The immunohistochemistry procedure followed the approach described by Soundararajan et al. (2022). Slides were incubated at 60 °C for 30–60 min, then dewaxed in xylene and rehydrated through a graded ethanol series. Antigen retrieval was performed in citrate buffer (pH 6.0) using microwave heating, followed by cooling and PBS washes. Endogenous peroxidase activity was blocked with 3% H₂O₂, and sections were blocked with 5% goat serum. Sections were incubated overnight at 4 °C with primary antibody (PBS for control), followed by secondary antibody incubation at 37 °C. Signal was developed with DAB, and sections were counterstained with hematoxylin. Finally, sections were dehydrated, cleared in xylene, mounted with neutral resin, and observed under a microscope.

Liver sections were dewaxed in xylene (2 × 10 min) and rehydrated through graded ethanol solutions (100%, 95%, 85%, and 75%) before rinsing in distilled water. Antigen retrieval and permeabilization were performed using proteinase K (37 °C, 30 min) followed by PBS washes. Sections were incubated with TUNEL reaction mixture (TdT:Biotin-dUTP = 1:9) at 37 °C for 60 min in the dark, followed by PBS washes and treatment with reaction stop solution. Streptavidin-HRP was then applied at 37 °C for 30 min, followed by additional PBS washes. Chromogenic development was performed with DAB for 30 s to 5 min, terminated with distilled water, and sections were counterstained with hematoxylin, differentiated in 1% acid alcohol, rinsed, dehydrated through graded ethanol, cleared in xylene, and mounted with neutral resin. Stained sections were examined and photographed under a light microscope (Han et al., 2022).

The wound healing assay mimics cell migration as it occurs in vivo and serves as a straightforward, cost-effective approach for evaluating migratory behavior in vitro. In this method, a deliberate gap—referred to as a “scratch”—is created on a confluent cell monolayer. Cells located at the borders of this gap progressively move inward, leading to closure of the scratched area. Images of the cells were taken at 0 and 24 h after the scratch was made (Wang et al., 2023).

Liver samples were first fixed with glutaraldehyde followed by 1% osmium tetroxide. After fixation, the tissues were dehydrated and embedded in Epon resin to prepare ultrathin sections measuring approximately 200–400 Å. These sections were then stained sequentially with uranyl acetate and lead citrate before being examined under a digital electron microscope.

Following collection, the samples were immersed in 2.5% glutaraldehyde and fixed overnight at 4 °C. Afterward, they were rinsed and post-fixed with osmium acid, then subjected to a graded acetone dehydration process. The tissues were embedded in epoxy resin to produce ultrathin sections, which were subsequently stained, examined, and imaged using a transmission electron microscope.

After total RNA was isolated from the samples, mRNA molecules were captured and purified using oligo (dT) magnetic beads, then randomly sheared with a fragmentation reagent. PCR products ranging from 200 to 300 bp were separated and collected from a 6% polyacrylamide Tris-borate-EDTA gel to complete library construction. The resulting libraries were sequenced on an Illumina HiSeq 2500 platform, and the reads were aligned to the corresponding mRNA reference database. Differentially expressed genes were identified through Chi-square testing, followed by GO and KEGG analyses for functional enrichment.

Quantitative primers for the differentially expressed genes were designed using Primer Premier 6.0, with β-actin selected as the internal control. The qPCR reaction mixture consisted of 1 μL cDNA, 0.4 μL of each primer (10 μmol/L), 0.4 μL Rox reference dye II, 10 μL SYBR Green real-time PCR Master Mix, and 7.8 μL ddH₂O. The amplification protocol included an initial denaturation at 95 °C for 15 s, followed by 40 cycles of 5 s at 95 °C and 34 s at 60 °C. After the reaction, melting curve analysis was performed, and relative expression levels were calculated using the 2^−ΔΔCT method. For protein expression analysis, lysates from BMECs were subjected to SDS-PAGE, transferred to nitrocellulose membranes (Millipore, United States of America), and incubated with primary antibodies, including a rabbit monoclonal anti-TGFB2 (19999-1-AP, Sanying, Wuhan, China) or a mouse monoclonal anti-β-actin (66009-1-IG, Proteintech, China).

Statistical analyses were performed using SPSS 18.0, and the results were visualized with GraphPad Prism V5.0. A P-value <0.05 was considered statistically significant.

Serum biochemical analysis revealed that CdCl₂ exposure markedly disrupted hepatic function. Compared with the control group, mice treated with CdCl₂ exhibited significant elevations in serum glucose (GLU), aspartate aminotransferase (AST), tri-glycerides (TG), and high-density lipoprotein cholesterol (HDL-C) levels (Figures 1A–D). Among these, AST—a key clinical indicator of hepatocellular injury—showed a pronounced increase, strongly suggesting the occurrence of acute hepatocellular damage. The abnormal increases in GLU, TG, and HDL-C further indicate that Cd exposure substantially impaired hepatic metabolic processes. In contrast, blood urea nitrogen (BUN) and total cholesterol (CHOL), which primarily reflect renal function and lipid metabolism, respectively, did not differ significantly between the control and treated groups (Figures 1E,F). These findings suggest that, under the present experimental conditions, the toxic effects of Cd were predominantly localized to the liver.

In addition, to evaluate whether Cd exposure elicited a systemic inflammatory response, peripheral white blood cell (WBC) and lymphocyte (Lymph) counts were examined. The results showed that CdCl₂ treatment significantly increased both parameters (Figures 1G,H), indicating the activation of a typical inflammatory response in the organism.

At the hematological level, mice exposed to CdCl₂ exhibited a significant decrease in hemoglobin (HGB), platelet (PLT), and mean platelet volume (MPV) levels compared with the control group (Figures 2A,B,D). In addition, red blood cell distribution width (RDW) was also markedly reduced following CdCl₂ exposure (Figure 2C). To further validate Cd-induced hepatic injury at the histological level, TUNEL staining was performed on liver tissues. Minimal apoptotic signals were observed in the control group, whereas extensive brown-positive staining was evident in the CdCl₂-treated group, indicating that cadmium exposure markedly induced large-scale hepatocellular apoptosis (Figure 2E). To confirm the local inflammatory status of the liver, immunohistochemical (IHC) analysis was conducted to detect the expression of the inflammatory marker pentraxin-3 (PTX3). As shown in Figure 2F, PTX3 expression was markedly upregulated in the liver tissues of CdCl₂-treated mice, confirming at the protein level that severe inflammatory responses occurred in hepatic tissue following cadmium exposure.

Hematoxylin and eosin (H&E) staining provided a direct visualization of Cd-induced histopathological alterations in liver tissues. As shown in Figure 3J, liver sections from control mice displayed normal lobular architecture, well-organized hepatic cords, and morphologically intact hepatocytes. In contrast, CdCl₂-treated mice exhibited pronounced pathological changes, including disordered hepatic cord arrangement, extensive hepatocellular necrosis, and marked inflammatory cell infiltration. Scanning electron microscopy (SEM) revealed that hepatocytes in the control group exhibited a smooth surface and well-defined morphology (Figure 3A), whereas those in the CdCl₂-treated group displayed rough and uneven surfaces, loosened intercellular junctions, and severely distorted cellular morphology (Figure 3B). Transmission electron microscopy (TEM) analysis demonstrated that hepatocytes in the control group possessed well-defined and intact organelles (Figure 3C). In contrast, hepatocytes from the CdCl₂-treated group showed swollen mitochondria, dilated and fragmented endo-plasmic reticulum, and blurred intracellular structures, indicating profound subcellular damage (Figure 3D).

We further performed in parallel in a Wound healing assay using the mouse hepatocyte cell AML-12 to validate the in vivo findings. Consistent with the results observed in liver tissues, SEM analysis revealed that CdCl₂-treated AML-12 cells changed from a normal adherent and well-spread morphology to a contracted and rounded shape, with numerous folds and microvilli appearing on the cell surface (Figures 3E,F). TEM observations similarly demonstrated that CdCl₂ exposure caused severe destruction of intracellular organelles in AML-12 cells (Figures 3G,H). To determine whether these structural alterations further impaired the cells’ migratory capacity—which is critical for hepatic self-repair—we performed a wound-healing assay to simulate the regenerative process following tissue injury. As shown in Figure 3I, control AML-12 cells exhibited robust migratory activity and almost completely closed the scratch area within 48 h, whereas CdCl₂-treated cells displayed markedly reduced migration, resulting in a significantly lower wound closure rate compared with the control group.

In this study, the effects of cadmium exposure on inflammation-associated molecules were systematically investigated from three perspectives—dose, exposure duration, and tissue specificity. In the dose-dependent study, mice were administered varying concentrations of CdCl₂, and the mRNA levels of key inflammatory mediators were quantified by qPCR. The results revealed that multiple pro-inflammatory cytokines were markedly upregulated within the medium-to-high dose range, but their expression patterns exhibited nonlinear dynamics. Specifically, Both TNF-α and IL-1 showed pronounced expression peaks at particular exposure doses of CdCl₂ (Figures 4A,C), whereas IL-6 was predominantly induced at higher doses (Figure 4E). In addition, ID1 and AMPK, both associated with inflammatory and stress responses, were elevated mainly at high concentrations, while TGF-β1 did not show a monotonic increase and instead fluctuated or declined at low-to-moderate doses (Figures 4B,D,F). These findings indicate that Cd-induced inflammation follows a threshold- or peak-type response pattern, rather than a simple dose-dependent enhancement. In the time-course experiment, mice were treated with CdCl₂ (18 μg/L, 0.8 mL/day), and liver samples were collected at days 3, 5, and 7 post-exposure. Line-plot analyses of qPCR data demonstrated that the expression of key inflammatory cytokines—TNF-α, IL-1, and IL-6—progressively increased over time, reaching maximal levels at either day 5 or day 7 (Figures 4G–L). This temporal pattern suggests that Cd-induced hepatic inflammation represents a progressive and sustained pathological process, rather than a transient stress response. Finally, to explore tissue specificity, we compared inflammatory responses across major organs, including the heart, liver, spleen, lung, and kidney. Total RNA was extracted from each tissue, and the expression levels of TNF-α and IL-6 were quantified. Remarkably, both cytokines exhibited several-to dozens-fold higher mRNA expression in the liver compared with other organs (Figures 4M,N).

The initiation and precise regulation of inflammatory responses rely on complex molecular networks, among which microRNAs (miRNAs) act as pivotal post-transcriptional regulators. By targeting multiple inflammation-related genes, miRNAs play central roles in either amplifying or suppressing inflammatory signaling. To elucidate the upstream regulatory mechanisms underlying Cd-induced hepatic inflammation, we performed high-throughput miRNA sequencing on liver tissues from control and CdCl₂-treated mice. Correlation heatmap analysis revealed strong intragroup consistency and clear inter-group separation, indicating high reproducibility and distinct expression profiles between treatment conditions (Figure 5A). Furthermore, principal component analysis (PCA) demonstrated that samples from the control and CdCl₂-treated groups clustered into two well-separated groups, confirming that cadmium exposure induced a global alteration in the hepatic miRNA expression landscape (Figure 5B).

Based on high-quality sequencing data, we identified a set of differentially ex-pressed miRNAs (DE-miRNAs) that exhibited significant changes following Cd expo-sure. A total of 47 DE-miRNAs were detected, of which 13 were significantly upregulated and 34 were downregulated (Figure 5C). The hierarchical clustering heatmap clearly illustrated distinct expression patterns between the control and CdCl₂-treated groups (Figure 6). Moreover, the top 10 most abundantly expressed miRNAs in each group were ranked and visualized to highlight dominant miRNA species under different conditions (Figure 7A). To further explore the biological significance of these DE-miRNAs, their predicted target genes were subjected to Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. GO enrichment analysis indicated that the target genes were mainly associated with cellular processes, action potential, and sodium ion transmembrane transport (Figures 7C, 9B). In parallel, KEGG pathway analysis revealed a marked enrichment of these target genes in the MAPK signaling pathway (Figures 7B, 9A).

To identify the most critical regulatory nodes within the complex target gene network, we constructed a protein–protein interaction (PPI) network based on the predicted target genes and performed centrality analysis. The PPI network visualization (Figure 8) revealed intricate interaction relationships among the target proteins. By calculating the centrality coefficients of each node (Figure 9C), we identified the top 10 hub genes, including Grb2, Egfr, Ctnnb1, Traf6, Rad9a, Vamp2, Ptch1, Rbx1, Ikbkb, and Cdk2 (Figure 9D). These genes participate in diverse but essential biological processes: Grb2 functions as an adaptor protein integrating multiple signaling pathways; Egfr regulates cell proliferation, differentiation, and survival; Ctnnb1 encodes β-catenin, a core component of the classical cadherin adhesion complex; Traf6 mediates inflammatory and immune signaling through K63-linked polyubiquitin chain formation; Rad9a regulates cell-cycle checkpoints; Vamp2 controls vesicle fusion and neuro-transmitter release; Ptch1 governs cell growth, differentiation, and tissue development; Rbx1 participates in protein ubiquitination and SCF complex assembly; Ikbkb is a key modulator of the NF-κB pathway and inflammatory responses; and Cdk2 plays a central role in cell-cycle progression. These genes occupy key “traffic hub” positions within the regulatory network, and their dysregulation may exert decisive effects on overall network stability and biological function.