WZB117 and albendazole sulfoxide (ABZSO) were obtained from MedChem Express (USA), and dimethyl sulfoxide (DMSO) and ABZ from Sigma (USA).

Sterile PSCs were obtained aseptically from the cysts of sheep infected with E. granulosus sensu stricto (s.s.) in the Hualing slaughter market of Urumqi in Xinjiang, PR China. All experiments were conducted on this species. Viable and morphologically intact PSCs were cultured using RPMI-1640 medium (Gibco, USA) (10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin), and maintained at 37°C under a humidified atmosphere containing 5% CO₂ (Walker et al., 2004; Wang et al., 2018). For in vitro experiments on E. granulosus s.s. metacestode, in vitro sterile cultures were maintained for 4 months to obtain vesicles with a diameter of approximately 2 mm.

Healthy female Kunming mice (20 ± 2 g of 8 weeks old) were adaptively reared by the Experimental Animal Center of Xinjiang Medical University for one week under controlled laboratory conditions (temperature 20 ± 2°C and 50 ± 5% humidity) (Loos and Cumino, 2015). The mice were inoculated with 2,000 PSCs in 0.2 mL normal saline by intraperitoneal injection. After 6 months, the mice were examined by B-scan ultrasonography; when the diameter of the lesion was more than 0.5 cm, this indicated that the infection was successful.

For a first set of experiments, after PSCs were cultured in vitro for one week, their viability was tested by 1% eosin staining (the dead PSCs were stained red with 1% eosin dye, while living PSCs were unstained); to be used in the study, the required percentage of viability required was higher than 90% (Loos et al., 2017). In vitro PSCs treatments were performed with 3.125, 6.25, 12.5, 25, 50 and 100 μmol/L WZB117 (dissolved in DMSO). For a second set of experiments, the PSC-derived vesicles were cultured in vitro for four months and then cultured in a 6-well plate (25 vesicles/well). In vitro vesicle treatments were performed with 3.125, 6.25, 12.5, 25, 50 and 100 μmol/L WZB117 and 15 μmol/L ABZSO. For both control PSCs and vesicles, the culture medium with an identical amount of DMSO (without inhibitor; final concentration, 1%) was used, and the PSCs and vesicles were cultured in an incubator (5% CO₂, 37°C) for 4 days, and each viability test was repeated three times. The collapse of vesicles, loss of swelling and contraction of germinal layer were used as criteria for evaluating vesicle viability (Elissondo et al., 2007). Viability of PSCs and vesicles was observed under a microscope every 24 h (Fabbri et al., 2016). Each experiment was performed in triplicate and repeated three times.

Glucose uptake levels were measured using the 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) (final concentration, 100 μmol/L) assay. Briefly, the PSCs with siRNA sequence or the WZB117-treated PSCs were incubated at 37°C for 48 h. Then, the PSCs were incubated in the darkness with 2-NBDG for 180 min and 60 min at 37°C in 5% CO₂ humidified atmosphere. The fluorescence intensity was detected at the excitation wavelength of 466 nm and emission wavelength of 540 nm by the fluorescence marker.

For in vivo experiments, ABZ and WZB117 was dissolved in PBS/DMSO (1:1, v/v) (Liu et al., 2012). All E. granulosus s.s.-infected mice (n = 45) judged appropriate at the 6^th month after infection (see above) were randomly divided into five experimental groups (9 mice per group): control group (receiving injections of the PBS/DMSO solvent, 1:1, v/v), ABZ (50 mg/kg/day) group (Wen et al., 1996) and WZB117 (5, 10, 20 mg/kg/day) groups (Wei et al., 2018). After 28 days of continuous intraperitoneal injection (dosage: 0.1 mL/10 g per mouse), animals were sacrificed by cervical dislocation (Loos et al., 2017). At necropsy, the peritoneal cavity was opened, the number of cysts was counted, cyst weights and diameters were measured. The efficacy of treatments was calculated as follows: 100 × {(average cyst weight in the control group) - (average cyst weight in the treatment group)}/(average cyst weight in the control group) (Loos et al., 2017).

After in vitro and in vivo drug treatment, the PSCs, vesicles, or cysts were fixed with 4% glutaraldehyde for 24 h (Xin et al., 2019). The samples were processed for SEM using a LEO1430VP (LEO company, Germany) microscopy and TEM using a JEM1230 (JEOL company, Japan) microscope, as previously described.

To determine the glucose and ATP content of cysts in treated mice, the cysts were washed three times with precooled PBS and added with 100 μL of 20 mM Tris buffer (Thermo, USA). The cyst wall was homogenized in a homogenizer for 2~4 minutes and boiled in a water bath for 5 minutes before centrifugation (at 15,800 g at 4°C for 30 min) (You et al., 2009). The cyst fluid was processed by gradient dilution according to the instructions in the kit. Glucose Colorimetric Assay Kit (Cayman, USA) was used to determine glucose contents. The ATP content was detected by the ATP Detection Assay Kit (Cayman, USA). Experimental groups include at least 5 mice. Each experiment was repeated three times.

Total RNA was extracted from E. granulosus s.s. PSCs by Mini BEST Universal RNA Extraction Kit (Takara, Japan). Me Script II 1^st Strand cDNA Synthesis Kit (Takara, Japan) was reverse transcriptionally synthesized for 1^st cDNA. The reaction conditions were: incubation at 42°C for 60 min, heating at 95°C for 5 min, termination of the reaction, and storage at -20°C (Cumino et al., 2010). We used the 1^st cDNA synthesized by reverse transcription as a template and used the Premix Ex Taq™ Hot Start Version (Takara, Japan) to amplify the full-length sequence of the EgGLUT1-ss gene; primers for the target genes included: EgGLUT1-ss (forward primer 5’- ATGGTTAACTTTCACTACGT-3’ and reverse primer 5’-CTAAAATCTGACCTTATCG-3’). The PCR amplification conditions were: pre-denaturation at 94°C for 5 min; denaturation at 98°C for 30 s; annealing at 45°C for 30 s; extension at 72°C for 90 s, for a total of 34 cycles. The reaction was terminated after 10 min extension at 72°C; 1% agarose gel was used to determine whether the gene band location was found where expected. PCR products of EgGLUT1-ss gene were recovered from Agarose Gel with Agarose Gel DNA Extraction Kit (Takara, Japan), and the amplified fragments were cloned into pMD19-T vector with Mighty ta-cloning Reagent Set for Prime STAR (Takara, Japan), and verified by sequencing. The gene was named as EgGLUT1-ss (GenBank accession number: QTT61014.1).

The amino acid sequences of the homologous genes of GLUT1 in E. multilocularis (GenBank ID: CDS42031.1), Hymenolepis microstoma (GenBank ID: CDS25463.1), Caenorhabditis elegans (GenBank ID: NP_493981.1), Drosophila melanogaster (GenBank ID: NP_001097467.1), Labrus bergylta (GenBank ID: XP_020502389.1), Astyanax mexicanus (GenBank ID: XP_007258287.2), Xiphophorus maculatus (GenBank ID: XP_023187106.1), Aplysia californica (GenBank ID: XP_012944940.1), Biomphalaria glabrata (GenBank ID: XP_013087453.1), Crassostrea gigas (GenBank ID: XP_019925400.1), Danio rerio (GenBank ID: NP_001034897.1), Mus musculus (GenBank ID: XP_006502971.1) and Homo sapiens (GenBank ID: NP_006507.2) were obtained from GenBank. The post-translational modification sites were predicted using the MotifScan software (Rai et al., 2014). The transmembrane region was predicted using TMHMM (Gulyaeva et al., 2020). The conserved domain was predicted using GenBank tools. The multiple sequence alignment was analyzed by DNAMAN (Version 7.0.2.176) (Kaukonen et al., 2000). The phylogenetic tree was constructed by MEGA (Version 10.0.5) (Hall, 2013).

According to the cloned EgGLUT1-ss Gene sequence (GenBank accession number: QTT61014.1), siRNA interference sequences of three EgGLUT1-ss genes were designed using Gene Pharma siRNA Designer 3.0 software. Experimental groups were: siRNA-386-treated group, siRNA-578-treated group, siRNA-723-treated group, negative control group (NC, transfection independent interference sequence) and untreated group. The three siRNA sequences were transfected into in vitro cultured PSCs separately. The targeting sequence of each siRNA in EgGLUT1-ss cDNA is summarized in Table 1 . The EgGLUT1-ss-siRNA interference assay was carried out as previously described (Mizukami et al., 2010). Briefly, electroporation was performed at 125 V using a pulse. For electroporation, 200 μL electroporation buffer (5 mM magnesium chloride, 200 mM glucose, 20 mM Tris, 2 mM 2-Hydroxy-1-ethanethiol, pH adjusted to 7.4 with HCl) containing approximately 4,000 PSCs was placed in a 4-mm cuvette, and the final concentration of siRNA interference sequence was 5 μM. After electroporation, the shock cup was placed in a 37°C incubator for 30 min and then transferred to a 6-well plate with 2 mL mixed medium for 48 h. After washing with PBS, PSCs in each group were stained with 1% eosin for 5 min (the dead PSCs were stained red with 1% eosin dye, while living PSCs were unstained), respectively. The PSCs viability was evaluated by counting living and dead PSCs using a microscope.

Total RNA was extracted from PSCs and vesicles by Mini BEST Universal RNA Extraction Kit (Takara, Japan), as previously described (Li et al., 2004). RNA was converted to cDNA with the PrimeScript™ RT reagent Kit Prime Script TMRT reagent Kit (Takara, Japan), and the cDNA was analyzed by qRT-PCR using TB Green^® Premix Ex Taq™ II (Takara, Japan). The primer sequences are shown in Table 2 . All samples were run in triplicate using the following cycle parameters: 95°C for 30 s; 40 cycles at 95°C for 5 s and 55°C for 30 s. All data were used for standard curve analysis.

Through the Pubchem website (https://pubchem.ncbi.nlm.nih.gov/) to retrieve the 3D structure of WZB117, we adopted the Autodock software (version 4.2.6) to hydrogenation processing, calculating charge EgGLUT1-ss protein, and setting the receptor proteins docking lattice parameter. We used AutoDock Vina (version 1.1.2) to dock EgGLUT1-ss and WZB117 and obtained 20 conformations. EgGLUT1-ss and WZB117 were used to chart the binding sites and amino acid residues and PyMOL (version 2.4.0) was used as a 3D diagram to show the interaction between receptor proteins and ligand small molecules.

Primary hepatocytes were isolated from 10 - 12 weeks old female C57BL/6 mice and the isolation was performed as previously described (Liu et al., 2021). Isolated primary mouse hepatocytes were inoculated at a density of 1 × 10⁴ cells/0.32 cm² growth area per well in rat tail collagen type I-coated 96-well plates for 4 h at 37°C, 5% CO₂, and incubated in a growth medium [William’s E (Gibco, USA), 10% fetal bovine serum, 0.01% dexamethasone, 0.25% insulin and 1% of 100 U/ml penicillin/100 μg/ml streptomycin] for further experiments. Cell Counting Kit-8 (CCK-8) (Bioss, China) assay is used to detect the viability of primary mouse hepatocytes in each group. Briefly, primary mouse hepatocytes were cultured at 37°C and 5% CO₂ for 4 h. The medium was replaced with medium (without serum) and 1% DMSO was used as the control group, while WZB117 was added to the medium at final concentrations of 3.125, 6.25, 12.5, 25, 50 and 100 μmol/L to continue the culture for 48 h. Then 10 μL of CCK-8 solution was added to each mixed well, and after 2 h of continuous incubation, zeroing was completed using blank control wells, and the optical density of each well was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, USA).

All analyses were performed using IBM SPSS Statistics 20 software. The chi-square test was used at each time point to assess the effect of E. granulosus s.s. PSCs and vesicles viability in the treatment and control groups. The one-way analysis of variance (ANOVA) was used to analyze the effect of siRNA interference and WZB117 on the glucose metabolism of E. granulosus s.s. PSCs. The one-way ANOVA and nonparametric test of Kruskal-Wallis test were used to assess the efficacy of WZB117 treatment in E. granulosus s.s.-infected female Kunming mice. All data were expressed as (mean ± S.D.), and P values are indicated in each assay (*P < 0.05, **P < 0.01). For all analyses P < 0.05 was considered statistically significant.

The terminology proposed by the World Association of Echinococcosis was followed all along the text of the publication. Especially, we followed the distinction between ‘vesicles’, obtained in vitro, and ‘cysts’ obtained from in vivo experiments, and between E. granulosus sensu lato for the cluster of species previously known as ‘E. granulosus’ and ‘E. granulosus sensu stricto’ for the species used in our experiments (Vuitton et al., 2020).

To assess whether GLUT1 gene was present in E. granulosus s.s, we successfully cloned a conserved GLUT1 homologous gene from E. granulosus s.s., named EgGLUT1-ss (GenBank accession number: QTT61014.1) ( Supplementary Figure 1 ), which encodes 500 amino acids ( Figure 1A ). Bioinformatics analysis showed that EgGLUT1-ss had one major facilitator superfamily (MFS) domain and 12 transmembrane regions, which are a typical feature of the GLUT gene ( Figure 1A ). This special structure plays an important role in the transmembrane transport of glucose (Hruz and Mueckler, 2001). In addition, we also found that EgGLUT1-ss contained two amidation sites, one glycosylation site, one cAMP- and cGMP-dependent protein kinase phosphorylation site, two casein kinase II phosphorylation sites, five myristoylation sites, and five protein kinase C phosphorylation sites ( Figure 1A ). Whole sequence alignment analysis showed that the highest overall homologies on the amino acid sequence level could be detected between EgGLUT1-ss and the GLUT1 of E. multilocularis (96.81% identical). When EgGLUT1-ss sequence was compared with the sequences of GLUT1 in other species, percentages of homology were between 32.77% to 72.33% ( Figure 1A ). Phylogenetic analysis revealed that the amino acid sequence of EgGLUT1-ss was generally closely related to parasite species GLUT, especially helminths, and was more distantly related to mammalian species such as human and mouse GLUT ( Figure 1B ).

To investigate EgGLUT1 function in E. granulosus s.l., we examined the EgGLUT1-ss expression in PSCs and vesicles of E. granulosus s.s. We found EgGLUT1-ss was expressed in both PSCs and vesicles, and that EgGLUT1-ss expression level in PSCs was 3.5 times higher than that of vesicles (P < 0.01) ( Figure 2A ).

To determine the functional relevance of EgGLUT1-ss in PSCs, we designed three siRNA sequences (siRNA-386, siRNA-578 and siRNA-723) targeting EgGLUT1-ss. The three siRNA compositions treatment led to a significant reduction in EgGLUT1-ss-mRNA expression in PSCs compared with the NC group (siRNA-386, P < 0.01; siRNA-578, P < 0.01; siRNA-723, P < 0.01) ( Figure 2B ). To detect the viability of PSCs, the number of PSCs was calculated after 1% eosin staining (the dead PSCs were stained red with 1% eosin dye, while living PSCs were unstained), and the results showed that the viability of PSCs was significantly reduced in all three siRNA intervention groups compared with the NC group (siRNA-386, P < 0.01; siRNA-578, P < 0.01; siRNA-723, P < 0.01) ( Figure 2C ). To examine the influence of EgGLUT1-ss knockdown on the glucose uptake of PSCs, three siRNA compositions were transfected into PSCs, and 2 days after transfection, compared with the NC group, 2-NBDG uptake by PSCs decreased significantly (siRNA-386, P < 0.01; siRNA-578, P < 0.01; siRNA-723, P < 0.01) ( Figure 2D ).

By docking predictions, we found that WZB117 had the potential ability to combine with EgGLUT1-ss. The binding of WZB117 to EgGLUT1-ss involved four hydrogen bonds, with Arg120, Asn29, Asn285 and Gln280. Amino acid residues Phe66, Thr25, Val63, Phe289, Ala286, and Trp410 interacted with the WZB117 molecule through hydrophobic bonds ( Figure 3A ). After 60 min of incubation with WZB117 in vitro, the glucose uptake level of PSCs was significantly decreased (P < 0.01) ( Figure 3B ). After 48 h incubation with 25 (P < 0.05), 50 (P < 0.01), and 100 (P < 0.01) μmol/L WZB117 in vitro, the glucose content of PSCs was significantly decreased compared with the DMSO group ( Figure 3C ). In parallel, compared with the NC group, the ATP content was decreased ( Figure 3D ) when the PSCs were treated with 50 (P < 0.01) and 100 (P < 0.01) μmol/L WZB117.

To investigate the effect of WZB117 on the viability of E. granulosus s.s. larval stages in vitro, we analyzed the survival rate of PSCs exposed to various concentrations of WZB117. As shown in Figure 3E , compared to the DMSO group, 4 days-WZB117 exposure led to a dose-dependent decrease in the viability of PSCs in cultures (P < 0.01). After exposure to 100 μmol/L WZB117 in vitro for one day, all the PSCs died. Scanning electronic microscopy (SEM) showed that, compared to the DMSO group, WZB117-treated PSCs exhibited incomplete rostellum structure, absence of hooks, collapsed suckers and various damages to the other PSC structures, as shown in Figure 3F . Transmission electronic microscopy (TEM) showed that there were significant changes in the ultrastructure of WZB117-treated PSCs: the microtriches disappeared, the nuclei were ruptured, associated with disappearance of the nucleoli, and vacuoles appeared in the cytoplasm. ( Figure 3G ).

Cysts are the lesions produced by E. granulosus s.l. in the intermediate host and their development is related to the pathogenicity of this helminth (Wen et al., 2019). Therefore, we further explored the effect of WZB117 on in vitro-cultured vesicles exposed to increasing concentrations of WZB117. Compared with the DMSO group, the viability of the vesicles was significantly decreased when they were exposed to various concentrations of WZB117 for 4 days (P < 0.01); it was decreased by 100% on day 3 when exposed to 100 μmol/L WZB117 ( Figure 4A ). These data and morphological changes indicate that WZB117 had a significant effect on the reduction of the viability of in vitro-developed E. granulosus s.s. vesicles. Compared with the DMSO group, the SEM features of the vesicles in the WZB117 group changed significantly: the germinal layer was condensed, and the vesicles collapsed. The degree of collapse was positively correlated with the dose of WZB1117 ( Figure 4B ). TEM images obtained from the WZB117-treated vesicles showed that the structures of germinal layer and laminated layer were damaged, lipid droplet appeared, the microtriches were reduced, nucleoli disappeared, the heterochromatin edges were clustered, and the cortical matrix was fuzzy ( Figure 4C ).

We measured the glucose and ATP content of the cysts in vivo. Compared with the PBS/DMSO group, there was a marked reduction in glucose (5 mg/kg WZB117-treated group, P < 0.01; 10 mg/kg WZB117-treated group, P < 0.01; 20 mg/kg WZB117-treated group, P < 0.01) and ATP (5 mg/kg WZB117-treated group, P < 0.05; 10 mg/kg WZB117-treated group, P < 0.01; 20 mg/kg WZB117-treated group, P < 0.01) levels in the cyst tissue after WZB117 treatment ( Figures 5A, C ). A significant reduction of glucose (P < 0.05) and ATP (P < 0.01) levels in the cyst fluid was observed for the 20 mg/kg dose of WZB117 ( Figures 5B, D ).

After 6 months of WZB117 treatment, the weight of cysts recovered from 5mg/kg and 10 mg/kg WZB117-treated mice was significantly lower than that recovered from the PBS/DMSO group (P < 0.05) ( Figure 5E ). Additionally, the number (P < 0.01) and diameter (P < 0.05) of cysts in WZB117-treated mice were lower than those of the PBS/DMSO group ( Figures 5F, G ). Overall, the ‘therapeutic’ effect of 10 mg/kg WZB117 was comparable to that of ABZ ( Figure 5H ).

In the PBS/DMSO group ( Figure 5I ), the cysts had no significant structural abnormalities when observed with TEM. However, the structure of cysts removed from the treated groups was damaged at different degrees ( Figure 5I ). Compared with the PBS/DMSO group, cysts from WZB117-treated mice had loose germinal layers, the microtriches were shortened and reduced dramatically in length, the tegument was thinned, the structure of the laminated layer was destroyed, and the nuclei were condensed.

For a preliminary assessment of the safety of WZB117 in the treatment of CE in experimental hosts, we first evaluated the toxicity of WZB117 on primary mouse hepatocytes by the CCK-8 assay. cells were treated with WZB117 at concentration series (3.125, 6.25, 12.5, 25, 50 and 100 μmol/L) for 48 h ( Supplementary Figure 2 ). The results showed that the primary mouse hepatocyte activity in the 100 μmol/L WZB117 group was 95.98 ± 10.38% compared to the 1% DMSO group (P > 0.05), and the primary mouse hepatocyte viability in the other treatment groups was also not significantly different from the 1% DMSO group (P > 0.05), indicating that WZB117 was not significantly toxic to primary mouse hepatocytes at the effective dose. In the in vivo experiments, we measured the usual indices of adverse effects. There were no differences in body weight between treated and control mice. The aspartate transaminase (AST) level was significantly lower in the 5 mg/kg WZB117-treated group than in the PBS/DMSO group (P < 0.05) ( Supplementary Figure 3A ) and there were no significant differences in alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels, the most accurate markers of liver damage ( Supplementary Figures 3B, C ). The 5 mg/kg WZB117-treated group had lower blood urea levels than the PBS/DMSO group (P < 0.05) ( Supplementary Figure 3D ) but all observed values remained in the physiological range and creatinine levels were similar in all WZB117-treated groups and in the PBS/DMSO group ( Supplementary Figure 3E ). We measured blood glucose in mice on days 0, 7, 14, 21 and 28 prior to administration and showed no difference in blood glucose levels between WZB117-treated mice and PBS/DMSO control mice ( Supplementary Figure 4 ). Overall, WZB117, at the dosages used in our study did not cause significant toxicity in mice.