Ethics statement

All procedures involving animals were approved by the Animal Ethics Committee of the Chinese Center for Parasitic Diseases Prevention and Control (Approval Code: IPD-2021–30) and the Animal Care and Use Committee of Inner Mongolia University (Permit No. SCXK (Inner Mongolia) 2020–0006). All measures were taken to minimize discomfort during handling

Isolation of Echinococcus protoscoleces

Mongolian jirds (Meriones unguiculatus) were infected with protoscoleces (PSCs) of E. multilocularis (Qinghai isolate, China) and maintained for six months post-infection. Animals were euthanized by cervical dislocation according to institutional guidelines, and metacestode tissues were collected aseptically from the peritoneal cavity. The harvested material was rinsed with sterile phosphate-buffered saline (PBS, pH 7.4) following established procedures [16,17].

After carefully removing surrounding host tissue with sterile forceps, the metacestode masses were cut into 1–2 mm pieces using a sterile scalpel to maximize PSC release. The fragments were passed through sterile 100-mesh and 40-mesh sieves (Biosharp, Beijing, China; BS-100-CS and BS-40-CS), and the resulting suspension was transferred into centrifuge tubes. PSCs were washed five times with PBS containing 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco, Grand Island, NY, USA). After each wash, tubes were allowed to stand for 5 min at room temperature to allow natural sedimentation, and the supernatant was aspirated gently. The cleaned PSCs were used for the subsequent in vivo and in vitro experiments.

Culture of Echinococcus Protoscoleces In Vitro

The cleaned PSCs pellet was resuspended in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Auckland, New Zealand) supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. Cultures were maintained at 37°C in a humidified atmosphere with 5% CO₂, and the medium was refreshed every 48 h. PSC viability and larval vesicle formation were monitored weekly using an Olympus IX71 inverted microscope (Tokyo, Japan) at ×100 magnification. PSCs showing normal motility and intact morphology were selected for infection of C57BL/6 mice and for in vitro induction of AML-12 cells.

Collection of PSC culture supernatant and stimulation of AML-12 cells

PSCs were cultured in DMEM supplemented with 10% FBS for 72 h. The culture supernatant was then collected, centrifuged at 1,000 × g for 10 min to remove cellular debris, and filtered through a 0.22 μm syringe filter. The resulting sterile supernatant was stored at −80°C until use. To determine the optimal stimulation conditions, a CCK-8 assay was performed to screen various dilution ratios (1:1, 1:2.5, 1:5, and 1:10) and time points (0, 6, 12, 24, and 48 h), ensuring that the selected conditions effectively activated the fibrotic response without inducing significant cytotoxicity. Based on these results, the supernatant was diluted 1:2.5 (v/v) in complete DMEM and applied to cells AML-12 cells for 72 h at 37°C in 5% CO₂.

Animal model and parasite infection

Eight-week-old female C57BL/6 mice (20–22 g) were obtained from Beijing Sibeifu Biotechnology Co., Ltd. (Beijing, China). Animals were maintained in specific pathogen-free (SPF) conditions with unrestricted access to standard chow and water. Twenty mice were randomly divided into four groups: Group 1: Uninfected control group; Group 2: Uninfected + corosolic acid group; Group 3: E. multilocularis-infected group; and Group 4: E. multilocularis-infected + corosolic acid group.

Mice in Groups 3 and 4 were infected with 1,500 cleaned PSCs via in situ intrahepatic surgical implantation, which closely mimics natural hepatic infection. Mice in Group 1 underwent sham surgery without PSC implantation. Beginning at 60 dpi, Groups 2 and 4 received intraperitoneal injections of corosolic acid (CorA), a pentacyclic triterpenoid reported to modulate hexokinase activity and glucose metabolism [18], at 10 mg/kg every 3 days for a total of 5 doses. All mice were kept under identical environmental conditions (22 ± 1°C, 12 h light/dark cycle), and the infection procedures were identical across groups to ensure consistency.

Blood lactate and hepatic histopathological analysis in mice

At 12 weeks post-infection, C57BL/6 mice were anesthetized with isoflurane (induction 3–5%; maintenance 1–2% in oxygen). Blood samples were collected by orbital enucleation, transferred to serum separation tubes, and centrifuged at 3,000 rpm for 15 min at 4°C. Serum lactate levels were quantified using an automated biochemical analyzer (BS-240VET; Mindray, Shenzhen, China) following the manufacturer’s instructions.

After blood collection, mice were euthanized by cervical dislocation. Liver tissues from Groups 1, 3, and 4 were fixed in 4% paraformaldehyde (Biosharp, Beijing, China) for 24 h at room temperature, embedded in paraffin, and sectioned at 3.0-5.0 µm. Histopathological changes were evaluated using hematoxylin-eosin (H&E) staining according to standard protocols [19], and hepatic fibrosis was assessed using Sirius Red and Masson’s trichrome staining as previously described [20]. H&E staining involved hematoxylin staining for 5 min (preceded by 1 min pretreatment with a high-concentration staining solution) and eosin staining for 15 s, with intermediate rinsing under running tap water for 30 min and subsequent dehydration via 95% ethanol and gradient ethanol before mounting in neutral balsam. For Sirius Red staining, sections were incubated in Sirius Red A solution at 65°C for 30 min, sequentially treated with modified Sirius Red B solution (2 min) and C solution (30 min), briefly rinsed with distilled water, then rapidly dehydrated in three anhydrous ethanol baths (5 s each), cleared in xylene for 5 min, and mounted with neutral balsam. Masson’s trichrome staining included overnight incubation in Masson A solution, 1 min staining with a 1:1 mixture of Masson B and C solutions, 6 min incubation in Masson D solution, 1 min staining in Masson E solution, 30 s treatment in Masson F solution (without intermediate washing), differentiation with 1% acetic acid, followed by dehydration through anhydrous ethanol, xylene clearing, and neutral balsam mounting.

RT-qPCR Analysis of TGF-β, α-SMA, and Hexokinase Isoenzymes Expression in AML-12 Cells and Mice Livers

To determine whether E. multilocularis-induced hepatic fibrosis is associated with lactate accumulation, mRNA expression levels of pro-fibrotic markers and members of the hexokinase family were measured in AML-12 cells and mouse liver tissues by reverse transcription quantitative PCR (RT-qPCR).

Total RNA from liver samples or AML-12 cells was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) with the procedure undertaken in strict accordance with the manufacturer’s protocol [20]. RNA concentration and purity were assessed with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). First-strand cDNA was synthesized from 1 μg of total RNA using the PrimeScript RT reagent kit with gDNA Eraser (Takara Bio, Otsu, Japan). The 20 μL reaction contained 1.0 μL PrimeScript RT Enzyme Mix I, 1.0 μL RT Primer Mix, 4.0 μL 5 × PrimeScript Buffer, and RNase-free ddH₂O to volume. Reverse transcription was carried out at 37°C for 15 min, followed by 5 s at 85°C.

RT-qPCR reactions were performed using TB Green Premix Ex Taq II (Takara Bio) on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). Each 20 μL reaction contained 10 μL TB Green Premix, 0.4 μL each of forward and reverse primers (10 μM), 2 μL cDNA template, and 7.2 μL nuclease-free water. Thermal cycling involved an initial denaturation at 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and annealing/extension at 60°C for 30 s. A melting curve was generated to verify amplification specificity.

The target genes included two key markers of hepatic fibrosis, transforming growth factor-β1 (Tgfb1), and alpha-smooth muscle actin (Acta2), as well as four hexokinase isoenzymes: hexokinase 1 (Hk1), hexokinase 2 (Hk2), hexokinase 3 (Hk3), and hexokinase 4 (Hk4). Glyceraldehyde-3-phosphate dehydrogenase (Gapdh), the housekeeping gene, served as the internal control. Relative expression levels were calculated using the 2^−ΔΔCt method [21]. All assays were run in triplicate. Primer sequences, designed using GenBank references, are listed in S1 Table.

Western blot analysis of fibrosis-related proteins and histone lactylation in AML-12 cells and mice livers

Western blot (WB) was performed to determine whether E. multilocularis-induced lactate accumulation alters the expression of fibrosis-related proteins and histone lactylation in AML-12 cells and liver tissues. The procedure for WB followed previously described methods [22,23]. Uncropped blots are shown in S1 Raw Gel.

Liver tissues or cultured cells were homogenized in RIPA lysis buffer (P0013B, beyotime, China) supplemented with protease and phosphatase inhibitors (78443, Thermo Fisher Scientific, USA); the lysates were centrifuged at 13,000 × g for 30 min at 4°C to collect cytoplasmic fractions, while nuclear proteins were isolated per standard protocols. Protein concentrations were determined using the BCA assay kit (Thermo Fisher Scientific, USA). Equal amounts of protein were mixed with sample buffer, denatured at 95°C for 10 min, separated by SDS–PAGE, and transferred onto PVDF membranes. Following transfer, membranes were blocked for 1 h at room temperature in 5% BSA dissolved in 1 × Tris-buffered saline with 0.1% Tween-20 (TBS-T), then incubated overnight at 4°C with primary antibodies against α-SMA, TGF-β1, HK3, pan-L-lactyllysine, H3K18la, H3K14la, and GAPDH (S2 Table for sources and dilutions). After three 5-min washes with TBS-T, membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated Goat Anti-Rabbit IgG H&L (1:10,000; Abcam, ab6721), followed by three additional TBS-T washes to remove excess antibody. Protein bands were visualized using the SuperSignal West Pico PLUS ECL chemiluminescent reagent (Thermo Fisher Scientific) and imaged with a Bio-Rad gel documentation system.

Immunofluorescence staining

Cellular immunofluorescence was performed as previously described [24]. AML-12 cells were seeded onto 12-well chamber slides and fixed in 4% paraformaldehyde for 15 min. Cells were permeabilized with 0.1% Triton X-100 for 10 min and blocked at room temperature for 30 min with 5% BSA prepared in 1 × PBST. Slides were then incubated overnight at 4°C with primary antibody against HK3 (1:200; Abclonal, A8428). After three rounds of washes with PBST, cells were incubated for 1 h at room temperature in the dark with Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody (1:500; Thermo Fisher Scientific, A32731). Nuclear staining was performed with 4,’6-diamidino-2-phenylindole (DAPI) for 10 min. After three additional rounds of washing with PBST, an anti-fade mounting medium was applied. Images were acquired using a fluorescence or confocal laser scanning microscope.

Tissue immunofluorescence was conducted using previously described procedures [25]. Mouse livers were snap-frozen in optimal cutting temperature (OCT) compound on dry ice and stored at −80°C. Sections were prepared at 7 μm thickness using a cryostat, baked, and rehydrated. Antigen retrieval was performed in citric acid buffer (pH 6.0), followed by permeabilization with 0.1% Triton X-100 in PBS and blocking with 5% goat serum in PBS containing 0.5% BSA for 30 min. Sections were incubated with primary antibodies against HK3 (1:200; Abclonal, A8428) and TGF-β (1:200; Abcam, ab315254), Collagen I (1:200; Servicebio, GB155707) and Collagen III (1:200; Servicebio, GB121323). After washing, slides were incubated with Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody (1:500; Thermo Fisher Scientific, A32731). Nuclei were counterstained with DAPI for 5 min. Sections were mounted with ProLong Gold and allowed to cure overnight at room temperature. Images were obtained using an LSM800 (ZEISS; Germany) confocal microscope.

Immunohistochemistry (IHC)

Paraffin-embedded tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval in sodium citrate buffer (10 mM, pH 6.0). Endogenous peroxidase activity was quenched with 3% H₂O₂, followed by blocking with 5% BSA. Sections were incubated with primary antibody against Collagen I and Collagen III (1:500) overnight at 4℃, followed by HRP-conjugated secondary antibody (1:500) for 1 h at room temperature. Signals were developed with DAB and counterstained with hematoxylin. Images were acquired under a light microscope.

Cleavage Under Targets and Tagmentation (CUT&Tag) analysis of mice livers using anti-H3K18la antibody

Motif analysis

Motif enrichment analysis was conducted using the findMotifsGenome.pl script in Hypergeometric Optimization of Motif EnRichment (HOMER; http://homer.ucsd.edu/homer/). Peak BED files and the corresponding genome FASTA files were used as input. HOMER extracted DNA sequences based on peak coordinates and compared them with known motif databases to identify significantly enriched transcription factor motifs.

Peak annotation

Peak annotation was performed with the ChIPseeker package. Annotated genomic features, including promoters, exons, introns, and intergenic regions, were quantified, and distribution plots were generated in R.

GO and KEGG enrichment analyzes

Gene Ontology (GO) enrichment analysis was performed to investigate the biological functions associated with peak-linked genes [28]. ClusterProfiler was used to identify enriched GO terms, with multiple-testing correction applied (Padj). KEGG pathway enrichment analysis was also carried out using ClusterProfiler, and pathways with q-values < 0.05 were considered significant [29].

Differential peak analysis

Differentially enriched peaks were identified through the following steps: 1. Peak files from all samples were merged using BEDTools; 2. Read counts for the unified peak set were quantified using BEDTools multicov; 3. DESeq2 was applied to calculate differential enrichment. Peaks with |log₂ fold change| > 1 and P < 0.05 were defined as significantly differential.

Co-immunoprecipitation (Co-IP)

AML-12 cells stimulated with E. multilocularis protoscolex culture supernatant were lysed in RIPA buffer containing PMSF (P0100, Solarbio, China) at 4 °C for 30 min, and total protein was collected by centrifugation (12,000 × g, 30 min, 4 °C); an aliquot was retained as input. The lysate was incubated overnight at 4 °C with either an IgG isotype control or an anti-HK3 antibody, followed by Protein A/G agarose (Thermo, 20421) for 3–4 h. After washing three times, the immunocomplexes were eluted in 1 × loading buffer, boiled, and collected as the IP sample. Input and IP samples were analyzed by SDS-PAGE and WB using antibodies against HK3, H3K18la, and H3K14la to determine whether lactylated histones (H3K18la and H3K14la) co-precipitate with HK3. Antibody sources and working dilutions are listed in S2 Table.

Transfection of small interfering RNA (siRNA)

HK3 siRNAs and non-targeting negative control siRNA (si-NC) were obtained from GenePharma Inc. (Suzhou, China). The sequences of the HK3 siRNAs were as follows (5’-3’): si-1 GGGCCAGGAUGUUGUGUAUTT; si-2, GCCCUCAGGUAGCUUAGAATT; si-3, GCAGUGUGCAGAUCAUCAATT; si-3, GCCAGGAUGUGGUCCAGUUTT. Among these, si-4 was selected for subsequent functional experiments based on its knockdown efficiency. Transient transfection was performed using Lipofectamine 3000 (Invitrogen, USA) at a final siRNA concentration of 10 μM according to the manufacturer’s instructions. Six hours after tranfection, the medium was replaced with complete medium and cells were cultured for an additional 48 h. Knockdown efficiency was confirmed by Western blot.

CorA treatment and lactate supplementation of AML-12 cells

To evaluate the effect of CorA on E. multilocularis-induced fibrosis, AML-12 cells were pre-treated with 10 μM CorA for 1 h, followed by stimulation with E. multilocularis culture supernatant, and maintained for 72 h. To assess the role of lactate independently of HK3, HK3-knockdown AML-12 cells were treated with 10 mM lactic acid (L6402, Sigma-Aldrich) for 72 h in the absence of E. multilocularis culture supernatant. To further examine whether CorA acts on the lactate-driven phenotype, HK3-knockdown cells were pre-treated with 10 μM CorA for 1 h, followed by the addition of 10 mM lactate acid, and maintained for 72 h.

Statistical analysis

Continuous variables are presented as mean ± standard deviation. Comparisons between two groups were performed using Student’s t-test, whereas analyzes involving three or more groups were conducted using two-way analysis of variance (ANOVA). Graphs were generated using GraphPad Prism 10.0 (Dotmatics, Inc., Boston, USA), while band intensities and all histopathological staining results were quantified and analyzed, respectively, using ImageJ/Fiji (macOS version; National Institutes of Health, Bethesda, MD, USA; https://imagej.net/Fiji). A P value < 0.05 was considered statistically significant.

E. multilocularis infection induces hepatic fibrosis in mice and promotes lactate accumulation

E. multilocularis infection is well known for triggering progressive hepatic fibrosis in host tissues. To characterize these pathological changes, liver sections from infected and control mice were examined using H&E and Masson’s trichrome staining. Infected mice displayed pronounced histopathological changes, including distinct parasitic cystic structures, extensive fibrotic septa, and dense inflammatory infiltrates surrounding lesion areas (Fig 1A). Quantitative analysis showed that fibrotic regions within HAE lesions were significantly expanded compared with control liver tissues (Fig 1A). In addition, immunofluorescence and immunohistochemistry staining for Collagen I and Collagen III were performed in the livers of HAE and NC mice. The results showed markedly increased Collagen I and Collagen III staining in the livers of AE mice compared with the control group, as detected by both immunohistochemistry and immunofluorescence (S1A and S1B Fig).

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Fig 1. Extensive fibrosis in the liver tissue of mice with hepatic alveolar echinococcosis (HAE).

A: Histopathological observations of liver tissue from control (NC) and HAE mice by hematoxylin–eosin (HE; top) and Masson’s trichrome (bottom) staining, with quantitative analysis of fibrosis area (n = 3; ****P < 0.0001, two-tailed unpaired t-test). Blue arrows indicate vesicular structures in HAE lesions, green arrows mark inflammatory cell bands, and red arrows denote fibrotic areas. B: Western blot analysis of fibrosis-related proteins (TGF-β1 and α-SMA), relative mRNA quantification, and statistical comparison of expression levels in liver tissue from NC and HAE mice (n = 3; ****P < 0.0001, two-tailed unpaired t-test). C: Lactate concentrations in liver tissues and serum of NC and HAE mice, and a line graph showing time-dependent changes in lactate levels in AML-12 hepatocytes co-cultured with E. multilocularis protoscolex culture supernatant for 72 h (n = 3; ****P < 0.0001, two-tailed unpaired t-test).

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To further corroborate the fibrotic response, we measured the expression levels of two key fibrosis-associated markers, TGF-β and α-SMA, by RT-qPCR and WB. Both markers were significantly upregulated in infected liver samples compared with the control group (Fig 1B), confirming a strong pro-fibrotic activation following E. multilocularis infection.

To investigate whether E. multilocularis-induced hepatic fibrosis is associated with lactate accumulation and histone lactylation, we measured lactate levels in liver tissues and serum of E. multilocularis-infected mice. Lactate concentrations in liver tissue and serum were substantially elevated in infected mice, reaching 0.908 mmol/g protein and 1.318 mmol/g protein, respectively, compared with 0.355 mmol/g protein and 0.244 mmol/g protein in control samples (Fig 1C). These values correspond to an average increase of 155.77% in liver lactate content and 440.16% in serum lactate levels.

To further validate whether parasite-derived factors contribute to lactate accumulation, AML-12 hepatocytes were exposed to culture supernatants from E. multilocularis PSCs for 72 h. Lactate content in AML-12 cells began to increase by 48 h, increasing by 16.86% (from 3.557 mmol/g protein to 4.157 mmol/g protein) (Fig 1C). These findings indicate that E. multilocularis infection disrupts host glucose metabolism, leading to increased hepatic and systemic lactate levels, which may contribute to the progression of fibrosis.

HK3 upregulation is associated with hepatic lactate accumulation and elevated H3K14 and H3K18 lactylation in AE mice

To investigate the factors contributing to lactate accumulation in the liver tissues of mice infected with E. multilocularis, we examined the expression of HKs, which catalyze the conversion of glucose into glucose-6-phosphate and initiate glycolysis. As a key rate-limiting enzyme in glycolysis, HK activity may directly affect lactate production in host tissues. We evaluated the expression levels of all four HK isoforms, HK1, HK2, HK3, and HK4. A significant increase in HK3 expression was detected in hepatic tissues of mice with HAE, whereas HK1 (P = 0.1075) and HK2 (P = 0.1537) and HK4 (P = 0.0062) mRNA levels showed no significant differences compared with the control group (S2A Fig). Western blot and RT-qPCR analyzes further confirmed a significant elevation of HK3 protein and mRNA expression in the livers of HAE mice relative to controls (Fig 2A). Immunofluorescence staining also demonstrated a significant expansion of HK3-positive regions within HAE mouse livers, accompanied by spatial colocalization with TGF-β expression (Fig 2B and 2C). These findings suggest that HK3 upregulation may be associated with lactate accumulation in the livers of AE mice and may be involved in the development of liver fibrosis.

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Fig 2. Upregulated expression of HK3 and increased histone lactylation in the liver of HAE mice.

A: Quantification of HK3 protein and mRNA expression levels in hepatic alveolar echinococcosis (HAE) and control (NC) groups by WB and RT-qPCR (n = 3; ****P < 0.0001; two-tailed unpaired t-test). B: Pearson correlation analysis between HK3 and TGF-β1 expression levels in liver tissues from HAE and NC groups. C: Immunofluorescence staining of TGF-β1 (green) and HK3 (red) in liver sections from NC and HAE mice (100×). Nuclei were counterstained with DAPI (4,’6-diamidino-2-phenylindole). D: WB analysis using a pan-lactylation antibody to assess global histone lactylation in liver tissues of NC and HAE mice. E: WB analysis of H3K18la and H3K14la expression levels in liver tissues from NC and HAE mice (n = 3; **P < 0.01; ****P < 0.0001; two-tailed unpaired t-test).

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Although E. multilocularis infection leads to HK3 upregulation and aberrant lactate accumulation in the mouse liver, it remains unclear whether this accumulation promotes histone lactylation. To address this question, WB analysis using a pan-lactylation antibody was performed on liver tissues from AE mice. Lactylation levels were significantly increased in AE samples compared with controls (Fig 2D). To identify specific histone residues undergoing lactylation in AE liver, WB analysis was conducted to assess lactylation at ten lysine sites previously reported to undergo lactyl modifications (S1C Fig). The results demonstrated a substantial elevation of lactylation in HAE mice, with H3K14la and H3K18la increasing by 216.4% and 183.7%, respectively, compared with the control group (Fig 2E). These data indicate that E. multilocularis infection induces pronounced histone lactylation, particularly at H3K14 and H3K18. These findings support a model in which E. multilocularis infection triggers HK3 upregulation in hepatic tissue, resulting in elevated lactate production. The increased lactate then increases histone lactylation, especially at H3K14 and H3K18, which may ultimately contribute to the progression of liver fibrosis.

CUT&Tag analysis shows H3K18la lactylation in HAE mice affects TGFBR

To further elucidate the functional relevance of H3K18la, particularly its role in liver fibrosis, a genome-wide CUT&Tag assay was performed on liver tissues from HAE mice using an anti-H3K18la antibody. The results showed that the overall alignment rate of sequencing reads reached approximately 90%, with fragment lengths ranging from 1 to 689 bp (Fig 3A). Using Deeptools, we mapped the distribution of H3K18la-associated reads across peak regions. Clear differences emerged between the control and HAE groups, with control samples showing a more concentrated signal around peak centers (Fig 3B).

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Fig 3. CUT&Tag analysis of liver tissues from HAE mice.

A. Length distribution of enzymatically digested fragments in liver tissue from the control group (NC) and mice with hepatic alveolar echinococcosis (HAE). B: Distribution of sequencing reads around peak regions in liver samples from control (NC) and hepatic alveolar echinococcosis (HAE) mice. C: Functional annotation of peak-associated genomic regions. During annotation, regions were assigned in the following default priority order: promoter > 5′UTR > 3′UTR > exon > intron > downstream > intergenic, with no duplicate annotations. D: Summary of the number of differentially enriched H3K18la peaks between NC and HAE groups. E: Integrative Genomics Viewer (IGV) tracks showing H3K18la enrichment at the Tgfbr and Smad3 loci in liver tissues from NC and HAE mice.

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Functional annotation of enrichment peaks using the ChIPseeker R package quantified their genomic distribution across functional regions. Compared with the control group (21.17%), the HAE group exhibited a higher proportion of peaks within promoter regions (Promoter≤1 kb, 23.18%) and fewer peaks within first intron regions (Fig 3C). Differential peak analysis identified a total of 6,948 peaks that differed between groups, including 5,507 peaks elevated and 1,441 reduced in the HAE group relative to controls (Fig 3D). Among these peaks, five regions were associated with regulation of TGF-β receptors (TGFBRs), covering all three family members, Tgfbr1, Tgfbr2, and Tgfbr3, along with two regions associated with Smad3, a downstream effector of Tgfb1 signaling (Fig 3E). These observations indicate that H3K18la may contribute to liver fibrosis in HAE through its differently enrichment at TGFBR-related regulatory regions, facilitating activation of the TGF-β1-Smad3 axis.

E. multilocularis supernatant upregulates HK3 and promotes histone lactylation and fibrosis-related protein expression in hepatocytes

To examine the relationship between HK3 and histone lactylation, lysates from induced AML-12 cells were subjected to co-immunoprecipitation using an anti-HK3 antibody, and the immunocomplexes were probed by Western blot. H3K18la co-precipitated with HK3 (Fig 4A), indicating that HK3 is associated with H3K18-lactylated histones.

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Fig 4. E. multilocularis stimulation induces histone lactylation in hepatocytes by upregulating HK3 expression.

A: Co-immunoprecipitation (Co-IP) analysis of HK3 and H3K18la in E. multilocularis-stimulated AML-12 cell lysates. B: Western blot (WB) analysis of fibrosis-related proteins (TGF-β1 and α-SMA) and HK3, with corresponding mRNA quantification and statistical comparison of expression levels in the control (NC) and AML-12 cells stimulated with of E. multilocularis (Em) culture supernantant for 72 h (n = 3; *P < 0.05, ****P < 0.0001; two-way ANOVA). C: WB analysis using a pan- lactylation antibody in NC and AML-12 cells stimulated with E. multilocularisculture supernatant for 72 h. D: WB analysis of H3K18la and H3K14la expression in NC and stimulated AML-12 cells(72 h) (n = 3; ***P < 0.001, ****P < 0.0001; two-tailed unpaired t-test). E: Immunofluorescence staining of HK3 (green) in NC and stimulated AML-12 (72 h) (magnification, 100 × ; nuclei stained with DAPI). F: WB analysis using a pan-lactylation antibody in NC and AML-12 cells treated with corosolic acid following E. multilocularis stimulation for 72 h (CorA + Em). G: WB analysis of TGF-β1, α-SMA, and HK3, with corresponding mRNA quantification and statistical comparison in NC and AML-12 cells treated with corosolic acid following E. multilocularis stimulation for 72 h (n = 3; ns = not significant; two-way ANOVA). H: WB analysis of H3K18la and H3K14la, with corresponding mRNA quantification and statistical comparison in NC and AML-12 cells treated with corosolic acid following E. multilocularis stimulation for 72 h (n = 3; ns = Non-significant; two-tailed unpaired t-test).

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Quantitative analysis showed that, relative to controls, induced AML-12 cells exhibited significant upregulation of HK3 (by 63.36%), α-SMA (by 181.76%), and TGF-β1 (by 137.89%) (Figs 4B and S2B). Consistently, pan-lactylation signals were elevated in E. multilocularis-induced AML-12 cells (Fig 4C). Both H3K18la and H3K14la were significantly increased, with H3K18la showing a greater increase (by 71.27%) than H3K14la (Fig 4D). Immunofluorescence staining further confirmed increased HK3 expression in induced AML-12 cells (Fig 4E).These findings indicate that secreted factors of E. multilocularis drive HK3 upregulation in hepatocytes, promote histone lactylation predominantly at H3K18, and induce the expression of fibrosis-related proteins.

To assess whether corosolic acid (CorA) could suppress the E. multilocularis-induced fibrogenic response, AML-12 cells were pretreated with 10 μM corosolic acid for one hour before undergoing a 72-h induction with E. multilocularis supernatant. Pan-lactylation signals in corosolic acid-pretreated cells did not differ significantly from control levels (Fig 4F). HK3, α-SMA, and TGF-β1 levels in inhibitor-treated cells did not increase after induction and remained comparable to control levels (Fig 4G). Similarly, H3K18la levels did not differ significantly from those in the control group (Fig 4H). These findings show that CorA suppresses the E. multilocularis-induced increases in HK3, histone lactylatio, and fibrosis-related proteins.

HK3 knockdown and lactate rescue reveal that HK3-derived lactate promotes H3K18 lactylation and fibrogenesis

To further validate the role of HK3 in lactylation-associated liver fibrosis during AE, we knocked down HK3 expression in AML-12 cells using RNAi, and the knockdown was confirmed at the protein level by WB. Following HK3 knockdown, HK3 expression remained low and was not restored by stimulation with E. multilocularis protoscolex culture supernatant, exogenous lactate supplementation, or corosolic acid treatment (Fig 5A), indicating that HK3 upregulation is an upstream event triggered by E. multilocularis stimulation rather than a secondary consequence of lactate accumulation or downstream signaling.

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Fig 5. HK3-derived lactate mediates E. multilocularis-induced histone lactylation and fibrosis-related protein expression in AML-12 cells.

A: Western blot analysis and quantification of HK3. Left panel: NC, HK3-knockdown cells (HK3^kd), HK3^kd cells stimulated with E. multilocularis culture supernatant for 72 h (HK3^kd+Em), and HK3^kd cells treated with 10 mM lactic acid for 72 h (HK3^kd+LA) (n = 3; ****P < 0.0001; two-way ANOVA). Right panel: NC, HK3^kd, HK3^kd+LA, and HK3^kd cells treated with 10 mM lactic acid plus corosolic acid for 72 h (HK3kd+LA + CorA) (n = 3; ****P < 0.0001; two-way ANOVA). B: Western blot analysis and quantification of α-SMA, TGF-β1, H3K18la in NC, HK3^kd, HK3^kd+Em, and HK3^kd+LA cells (n = 3; ****P < 0.0001, ns = not significant; two-way ANOVA). C: Western blot analysis and quantification of α-SMA, TGF-β1, and H3K18la in NC, HK3^kd, HK3^kd+LA, and HK3^kd+LA + CorA cells (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant; two-way ANOVA).

https://doi.org/10.1371/journal.pntd.0014582.g005

Following HK3 knockdown, protoscolex culture supernatant no longer induced a significant increase in H3K18la, and the upregulation of α-SMA and TGF-β1 was significantly suppressed(Fig 5B), indicating that HK3 is required for E. multilocularis -induced histone lactylation and fibrogenic signaling. To determine whether lactate is the key intermediate mediating these effects, we supplemented HK3-knockdown cells with 10 mM exogenous sodium lactate in the absence of protoscolex stimulation. Exogenous lactate significantly increased the levels of H3K18la, α-SMA and TGF-β1 despite HK3 knockdown (Fig 5B), demonstrating that lactate functions as the critical intermediate linking HK3 activity to histone lactylation and fibrogenesis.

We next examined whether corosolic acid could reverse this lactate-driven fibrogenic response. When corosolic acid was added to HK3-knockdown cells supplemented with exogenous lactate, it partially reduced H3K18la levels but did not suppress the lactate-induced upregulation of α-SMA and TGF-β1 (Fig 5C). Notably, since HK3 was already silenced and lactate was supplied exogenously, the residual reduction of H3K18la by corosolic acid suggests that its effect on histone lactylation is not entirely dependent on HK3-mediated lactate production.

Corosolic acid attenuates hepatic fibrosis in E. multilocularis-infected mice

To explore whether the HK3–lactylation–TGF-β1 axis can be targeted during hepatic fibrosis in vivo, E. multilocularis-infected mice were treated with corosolic acid (10 mg/kg, intraperitoneally, every 3 days for a total of 5 doses). Although corosolic acid did not significantly change maximum cyst diameter (Fig 6A), histopathology showed a clear reduction in hepatic fibrosis area in the drug-treated group compared with controls (Fig 6B). Molecular analysis revealed that HK3 levels in corosolic acid-treated mice showed a significantly reduced after E. multilocularis infection, rather than the increase observed in untreated AE mice (Figs 6C and S2C). TGF-β1 expression remained stable without the increase typically seen during infection, and α-SMA expression showed a significant reduction in the liver tissues of corosolic acid-treated AE mice (Figs 6C and S2C). Furthermore, H3K18la levels were significantly decreased in corosolic acid-treated mice, indicating that corosolic acid prevented the E. multilocularis infection-induced increase in histone lactylation (Fig 6C). These findings suggest corosolic acid alleviates E. multilocularis–induced hepatic fibrosis in association with reduced HK3 expression and histone lactylation, particularly during early infection. The results also suggest that targeting HK3 may offer a potential strategy for slowing fibrosis progression in AE.

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Fig 6. Corosolic acid treatment attenuates liver fibrosis in E. multilocularis-infected mice.

A: Gross morphology of livers and statistical analysis of maximum cyst diameter in control mice (NC), hepatic alveolar echinococcosis (HAE) mice, and HAE mice treated with corosolic acid (HAE + CorA) (n = 3; ns = not significant; two-tailed unpaired t-test). B: Histopathological staining of liver tissues using hematoxylin–eosin (HE; top), Masson’s trichrome (middle), and Sirius Red (bottom) from NC, HAE, and HAE + CorA groups, with comparative quantification of fibrotic area ratios (n = 3; ***P < 0.001, ****P < 0.0001; two-way ANOVA). C: Western blot analysis and quantitative comparison of α-SMA, TGF-β1, HK3, H3K18la, and H3K14la in liver tissues from NC, HAE, and HAE + CorA groups (n = 3; *P < 0.05, **P < 0.01, ****P < 0.0001, ns = not significant; two-way ANOVA).

https://doi.org/10.1371/journal.pntd.0014582.g006