Inhibition of AMPK activation in Echinococcus granulosus sensu stricto limits the parasite’s glucose metabolism and survival

ABSTRACT

Cystic echinococcosis (CE) is a zoonotic parasitic disease caused by larvae of the Echinococcus granulosus sensu lato (s.l.) cluster. There is an urgent need to develop new drug targets and drug molecules to treat CE. Adenosine monophosphate (AMP)-activated protein kinase (AMPK), a serine/threonine protein kinase consisting of α, β, and γ subunits, plays a key role in the regulation of energy metabolism. However, the role of AMPK in regulating glucose metabolism in E. granulosus s.l. and its effects on parasite viability is unknown. In this study, we found that targeted knockdown of EgAMPKα or a small-molecule AMPK inhibitor inhibited the viability of E. granulosus sensu stricto (s.s.) and disrupted the ultrastructure. The results of in vivo experiments showed that the AMPK inhibitor had a significant therapeutic effect on E. granulosus s.s.-infected mice and resulted in the loss of cellular structures of the germinal layer. In addition, the inhibition of the EgAMPK/EgGLUT1 pathway limited glucose uptake and glucose metabolism functions in E. granulosus s.s.. Overall, our results suggest that EgAMPK can be a potential drug target for CE and that inhibition of EgAMPK activation is an effective strategy for the treatment of disease.

INTRODUCTION

Cystic echinococcosis (CE) is a chronic and neglected parasitic disease caused by the larvae of Echinococcus granulosus sensu lato (s.l.) cluster. CE is mainly distributed in western China, Central Asia, South America, Mediterranean countries, and East Africa (1). In 2020, the World Health Organization reported that more than one million people were affected by echinococcosis at any time, and the annual costs associated with CE were estimated to be US$ 3 billion for treating cases and losses to the livestock industry (2). Currently, treatments for CE include percutaneous treatment, surgery, and anti-infective drug treatment. Surgery is the only therapy that can completely cure CE, but it is not always possible, and those affected may be prone to secondary infections caused by fluid leakage. Anti-infective drugs are still the most widely used treatment method and are used before and after surgery to prevent recurrence (3). The WHO has recommended benzimidazoles, such as albendazole (ABZ) and mebendazole (MBZ), as the only two drugs used to treat CE (4). However, these drugs need to be taken for the long term and in high doses and thus are prone to the development of drug resistance and side effects (5). Unfortunately, there is still a lack of effective alternative drugs; thus, there is an urgent need to develop new drug targets and drug molecules.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a Ser/Thr protein kinase composed of α, β, and γ subunits and plays a key role in the regulation of energy metabolism (6). When the body is under energy stress, AMPK is activated and participates in various metabolic pathways to restore the balance of energy metabolism, which can be broadly divided into two pathways: inhibition of anabolic metabolism to reduce ATP consumption (protein and cholesterol synthesis, etc.) and promotion of catabolic metabolism to stimulate ATP production (glucose uptake and fatty acid oxidation, etc.) (7, 8). AMPK may be actively involved in the regulation of energy metabolism throughout the life cycles of several eukaryotic parasites, including Toxoplasma gondii (9), Trypanosoma cruzi (10), Trypanosoma brucei (11), Schistosoma mansoni (12), and Echinococcus multilocularis (13). Due to its lack of anabolic capacity, E. granulosus s.l. needs to obtain exogenous sugars from the host environment as an energy metabolic reserve and use aerobic and anaerobic carbohydrate metabolism to produce sufficient ATP for growth and development. Therefore, the regulation of energy metabolism plays a key role in the parasitic infection of E. granulosus s.l. in the host, with glucose metabolism representing an important energy metabolic pathway (14, 15).

AMPK regulates a variety of complex energy metabolic pathways but there has been controversy regarding the role of AMPK as a therapeutic target (16). Considering the highly conserved nature of AMPK in eukaryotes, the controversy regarding AMPK may also extend to parasitic infections. Previous studies demonstrated that the AMPK activator metformin is therapeutically effective against E. granulosus s.l. infections both in vitro and in vitro (17, 18). However, the regulatory mechanism of EgAMPK in E. granulosus s.l. infection is not fully understood. In this study, we explored the value of EgAMPK as a therapeutic drug target for CE with the help of siRNA and the small-molecule AMPK inhibitor Compound C, (also known as dorsomorphin), and investigated the effects of inhibiting EgAMPK activation on glucose metabolism in the E. granulosus sensu stricto (s.s.).

RESULTS

Knockdown of EgAMPK reduces the viability of E. granulosus s.s. protoscoleces

To test the effects of EgAMPK on the viability of E. granulosus s.s., protoscoleces (PSCs) cultured in vitro were transfected with siRNA. First, the transfection efficiency of siRNA needed to be determined, and quantitative real-time PCR (qRT-PCR) was used to detect the effect of siRNA on the level of EgAMPKα mRNA. qRT-PCR results showed that the transcript level of EgAMPKα in PSCs treated with siRNA-EgAMPKα was significantly reduced on day 3 compared to that in the siRNA-NC group (P < 0.01) (Fig. 1A). To clarify the effect of siRNA knockdown of EgAMPKα gene expression on the viability of PSCs, the viability of PSCs was examined on days 0, 3, 6, 10, and 15 after transfection by 1% eosin staining. The results showed that the viability of PSCs in the siRNA-EgAMPKα group gradually decreased over time compared to that in the siRNA-NC group. Fifteen days after transfection, the viability of PSCs transfected with 5 µM siRNA-EgAMPKα was only 30.66% ± 3.82%, while the viability of PSCs in the siRNA-NC group was 97.84% ± 1.30% (P < 0.01). There was no statistically significant difference in the viability of PSCs between the untreated and siRNA-NC groups during the experiment (Fig. 1B).

Fig 1.

Effects of siRNA transfection on the expression of AMPKα and viability of E. granulosus s.s. PSCs. (A) Expression of EgAMPKα mRNA in PSCs after siRNA-EgAMPKα transfection. (B) Effects of siRNA-EgAMPKα on the viability of PSCs after 0, 3, 6, 10, and 15 days of transfection. Each point represents the average percentage of viable PSCs in three different experiments. Untreated, PSCs were not treated with siRNA transfection. siRNA-NC (negative control, independent interfering sequence transfection) was used as a control. ^**P < 0.01 indicates statistically significant differences between the siRNA-EgAMPKα and siRNA-NC groups.

AMPK inhibitor exhibits parasiticidal activity against E. granulosus s.s. protoscoleces and metacestodes in vitro

To further explore the effects of EgAMPK inhibition on the viability of E. granulosus s.s., we used the small-molecule inhibitor Compound C. The viabilities of PSCs in the untreated and dimethyl sulfoxide (DMSO) control groups were both >90% throughout the experiment. By contrast, the PSCs were completely dead after 6 days of incubation when treated with 50 and 100 µM Compound C. At the same time points, the viabilities of PSCs in medium containing 12.5 and 25 µM Compound C were 19.6% ± 5.3% and 11.92% ± 2.2%, respectively. The viability of the PSCs incubated with 15 µM albendazole sulfoxide (ABZSO) was 53.5% ± 2.5% (Fig. 2A). In addition, ultrastructural damage to PSCs was observed by scanning electron microscopy (SEM) after incubation for 6 days with different concentrations of Compound C. As shown in Fig. 2B, the ultrastructure of PSCs cultured with Compound C was disrupted. It is noteworthy that the ultrastructures of PSCs cultured with 50 and 100 µM Compound C were more severely disrupted, exhibiting roughly wrinkled bodies and collapsed suckers. By contrast, the PSCs of the DMSO control group exhibited an intact rostellar region, clear hooks, and normal sucker morphology.

Fig 2.

Effects of the AMPK inhibitor on PSC viability in vitro. (A) PSCs were incubated with different concentrations (12.5–100 µM) of Compound C for 6 days and the viability of PSCs was assessed by 1% eosin. Each point represents the average percentage of viable PSCs from three different experiments. Untreated, PSCs were not treated with solvents or inhibitors. DMSO, PSCs were supplemented with 1% DMSO as a control. ^**P < 0.01 indicates statistically significant differences between the Compound C groups at different concentrations and the 1% DMSO group. (B) SEM of E. granulosus s.s. PSCs incubated with Compound C in vitro. rr, rostellar region; su, suckers; bo, body.

To further investigate the in vitro killing effect of the AMPK inhibitor on E. granulosus s.s., metacestodes (MTCs) were incubated with Compound C for 5 days. The viability of MTCs is shown in Fig. 3A. After 5 days of in vitro incubation, MTC viabilities were significantly lower in the 15 µM ABZSO (42.96% ± 5.00%) and 12.5, 25, 50, and 100 µM Compound C groups (38.43% ± 4.58%, 31.81% ± 7.28%, 20.87% ± 2.43%, and 0) than in the DMSO control group (94.31% ± 0.41%) (P < 0.01). The SEM results revealed that there were many different types of morphologically intact cells on the germinal layer in the DMSO control group, while the structures of germinal cells on the germinal layer were disrupted in the Compound C groups (Fig. 3B). The results of transmission electron microscopy (TEM) revealed that in the DMSO control group, the microtriches were well arranged, the nuclei were dense and the nuclear membranes were clear, while in the Compound C groups, the ultrastructures of the MTCs were severely damaged, the structure of the germinal layers was sparse, the microtriches had disappeared, the nuclear membranes were missing, and the matrices were dissolved (Fig. 3C).

Fig 3.

Effects of the AMPK inhibitor on MTC viability in vitro. (A) MTCs were incubated with different concentrations (12.5–100 µM) of Compound C for 5 days. Each point represents the average percentage of viable MTCs from three different experiments. Each point represents the average percentage of viable MTCs from three different experiments. Untreated, MTCs were not treated with solvents or inhibitors. DMSO, MTCs were supplemented with 1% DMSO as a control. ^**P < 0.01 indicates statistically significant differences between the Compound C groups at different concentrations and the 1% DMSO group. (B and C) SEM (B) and TEM (C) of E. granulosus s.s. MTCs incubated with Compound C in vitro. GL, germinal layer; mt, microtriches; nu, nucleus.

Therapeutic effects of the AMPK inhibitor in vivo in mice infected with E. granulosus s.s

To investigate the in vivo therapeutic effects of the AMPK inhibitor, C57BL/6 mice were infected with E. granulosus s.s. PSCs by intraperitoneal injection. After 6 months of infection, ABZ (50 mg/kg/d) and Compound C (10 mg/kg/d) were injected intraperitoneally. The treatment effects were observed after 28 days of continuous administration. The results showed significant reductions in the weights of cysts in the ABZ (4.61 ± 2.10 g, P < 0.01) and Compound C (7.66 ± 4.05 g, P < 0.05) groups compared to the control group (15.98 ± 5.96 g) (Fig. 4A). The in vivo treatment effects of ABZ and Compound C were further examined by TEM. The germinal layer of cysts removed from control group mice had a typical structure. By contrast, microtriches in the germinal layers of cysts from ABZ- and Compound C-treated mice were irregularly arranged, the nuclear membranes of the cell nuclei were absent, and the chromatin was unevenly dense (Fig. 4B).

Fig 4.

In vivo treatment of E. granulosus s.s.-infected mice with AMPK inhibitor. (A) Box plots showing the weights of cysts in mice after 28 days of in vivo treatment with 50 mg/kg ABZ and 10 mg/kg Compound C. ^*P < 0.05 and ^**P < 0.01 indicate statistically significant differences between the treatment and control groups. Mice in the control group were given only the same volume of 0.5% CMC-Na. (B) Representative TEM images of cysts from 50 mg/kg ABZ- and 10 mg/kg Compound C-treated E. granulosus s.s.-infected mice. GL, germinal layer; mt, microtriches; nu, nucleus.

In addition, a preliminary assessment of the effects of Compound C on the host was performed. The changes in the body weights of mice in the ABZ and Compound C treatment groups remained essentially the same, while the body weights of mice in the control group increased gradually with the duration of infection (Fig. S1A). Liver pathology assessments indicated that the livers of mice in the ABZ group showed a large number of hepatocytes with focal necrosis and a large number of inflammatory cells infiltrating the hepatic blood sinusoids and the portal duct areas (Fig. S1B), and the livers of mice in the Compound C group showed occasional inflammatory cell infiltration in the hepatic blood sinusoids. There were no histopathological changes or damage in the kidneys of mice in the ABZ and Compound C groups (Fig. S1C). The blood glucose results in mice revealed no differences in blood glucose levels between mice in the ABZ and Compound C groups and the control group throughout the experimental period (Fig. S1D).

Inhibition of the EgAMPK/EgGLUT1 pathway disrupts glucose uptake and glucose metabolism in E. granulosus s.s

Docking simulation technology is a convenient and effective method to detect small-molecule-target interactions. EgAMPK is a heterotrimeric complex consisting of a catalytic α-subunit and noncatalytic, regulatory β and γ-subunits. To explore the potential interaction site between the small molecule AMPK inhibitor and EgAMPK, docking between the small molecule and the subunit was simulated using bioinformatics techniques. The total energies between Compound C and EgAMPKα, EgAMPKβ, and EgAMPKα were −8.5, –7.5, and −7.7 kcal/mol, respectively, suggesting that Compound C had a strong binding activity with all three subunits of EgAMPK and binds via hydrophobic interactions (Fig. S2).

To analyze the effects of the AMPK inhibitor Compound C on the EgAMPK activity of E. granulosus s.s., PSCs were incubated in the presence of 50 µM Compound C for 2 days, and then the parasites were collected. As shown in Fig. 5A, the distribution of EgAMPKα was investigated by immunofluorescence, and the expression levels of total and phosphorylated EgAMPKα were detected in the tegument, posterior bladder, and surrounding calcareous corpuscles. Protein extracts from the PSCs were subjected to immunoblotting (Fig. 5B), which revealed that the AMPK inhibitor Compound C caused downregulation of basal levels of phosphorylated EgAMPKα in PSCs, similar to the immunofluorescence results, while the expression levels of total EgAMPKα and β-actin in the parasite were unaffected.

Fig 5.

Expression levels of total and phosphorylated EgAMPKα in the E. granulosus s.s. PSCs. (A) Expression levels of EgAMPKα and phosphorylated EgAMPKα in PSCs incubated with 50 µM Compound C for 48 h were detected by immunofluorescence. rr, rostellar region; tg, tegument; pb, posterior bladder. (B) Expression levels of EgAMPKα and phosphorylated EgAMPKα in PSCs after incubation with 50 µM Compound C for 48 h were detected by immunoblotting. Bar graphs show the ratios of phosphorylated EgAMPKα to EgAMPKα. ^*P < 0.05 indicates statistically significant differences between the 50 µM Compound C and 1% DMSO groups.

AMPK is a key regulator of energy metabolism and is involved in glucose metabolism (19). To assess the effects of inhibiting EgAMPK activation on the glucose metabolism function of E. granulosus s.s., we examined the level of glucose uptake in PSCs (Fig. 6A), and the results showed that glucose uptake was inhibited by Compound C (P < 0.05). EgGLUT1 belongs to a class of glucose transport proteins responsible for glucose transport across membranes (20, 21). Immunofluorescence analysis revealed that glucose transporter protein 1 of E. granulosus s.s. (EgGLUT1) was expressed in the nucleus, cytoplasm, rostellar region, sucker, tegument, the calcareous corpuscle, and posterior bladder of PSCs. Compared with the DMSO control group, Compound C suppressed the overall expression levels of EgGLUT1 (Fig. 6B). Next, we performed glucose, lactate, and ATP content assays. The results showed that Compound C had no effect on lactate levels in PSCs (Fig. 6D) (P = 0.449) but reduced glucose (Fig. 6C) and ATP (Fig. 6E) levels in E. granulosus s.s. PSC compared with the DMSO control group (P < 0.01). In addition, to investigate the effect of EgAMPK inhibition on apoptosis in PSCs, Caspase-3 activity analysis was performed (Fig. S3). The results showed that treatment with siRNA-EgAMPKα (P = 0.549) and 50 µM Compound C (P = 0.709) did not lead to an increase in Caspase-3 activity treated in PSCs compared to controls.

Fig 6.

Effects of an AMPK inhibitor on glucose metabolism and glucose uptake. (A) Glucose uptake levels of PSCs after 150 min of 50 µM Compound C incubation in vitro. (B) EgGLUT1 expression in PSCs after incubation with 50 µM Compound C for 48 h was detected by immunofluorescence. rr, rostellar region; tg, tegument; su, suckers; pb, posterior bladder. (C) Glucose levels of PSCs after 48 h of incubation with 50 µM Compound C. (D) Lactate content of PSCs after 48 h of incubation with 50 µM Compound C. (E) ATP levels of PSCs after 48 h of incubation with 50 µM Compound C. “ns” indicates that the difference between the 50 µM Compound C group and the 1% DMSO group was not statistically significant (P = 0.449); ^*P < 0.05 and ^**P < 0.01 indicate statistically significant differences between the 50 µM Compound C and 1% DMSO groups.

DISCUSSION

For parasites, dynamic interactions of energy between host and parasite are necessary for the effective coordination of the different developmental stages of the parasite, and the balance between these mechanisms is essential for parasite survival (22). Glucose metabolism is the main pathway of energy metabolism in E. granulosus s.s (14), and E. granulosus s.s. requires glucose-based energy metabolism substrates from the host environment to produce ATP for growth and development (23). AMPK is a major coordinator of metabolic and growth pathways and can be involved in the regulation of glucose metabolism processes when it is activated (24–26); however, the effects of EgAMPK on the activity and glucose metabolism function of E. granulosus s.s. are unknown. In this study, we demonstrated that inhibition of EgAMPK activation disrupted the glucose uptake and glucose metabolism functions of E. granulosus s.s., resulting in the blockage of ATP synthesis and parasite death. We clarified that EgAMPK is an important drug target for CE and that inhibition of EgAMPK activation is an effective strategy for the pharmacological treatment of CE.

Hunter et al. (12) found that AMPKα of Schistosoma mansoni was expressed in all stages of parasite growth and development but was not actively expressed in the adult stage. Saldivia et al. (11) reported that TbAMPKα1 regulates the transition of Trypanosoma brucei from the proliferative slender form to the quiescent stumpy form, playing a critical role. Loos J et al. (17) cloned AMPK in E. granulosus and found that AMPK was highly expressed during the growth and development of E. granulosus. These studies suggest that AMPK activation plays important roles in regulating parasite survival and infection but these biological roles are not identical. To clarify the effects of EgAMPK on E. granulosus s.s. viability, we inhibited EgAMPK using siRNA and chemical inhibition. When EgAMPKα expression was knocked down by siRNA, PSC viability was inhibited in a time-dependent manner. Compound C is a potent AMPK inhibitor widely used to study AMPK signaling (27). In in vitro experiments, Compound C showed significant killing effects on both PSCs and MTCs and E. granulosus s.s. larvae were more sensitive to Compound C than to ABZSO. Interestingly, a previous study reported that metformin activates EgAMPK and has a killing effect on E. granulosus s.s (17). In the present study, we propose a new strategy in which inhibition of EgAMPK activation still has a significant killing effect on E. granulosus s.s., and this information has important implications for the potential treatment of CE. Electron microscopy results revealed that Compound C disrupted the ultrastructures of both PSCs and MTCs. The SEM results showed that Compound C caused the parasite bodies to crumple and their suckers to collapse, which we hypothesized led to the restriction of the mobility of the PSCs, which, in turn, prevented them from accessing nutrients in the medium and directly or indirectly led to their death (28). SEM of the MTCs revealed that Compound C caused significant disruption of the structure of the germinal cells, which are mainly responsible for the parasite development (29). The destruction of the germinal cells by Compound C was ultimately manifested by the collapse of the germinal layer and reduced MTC viability. In addition, the TEM results revealed that the nuclear structure on the germinal layer was disrupted, which further confirmed the killing effect of Compound C on MTCs.

To further investigate the efficacy of Compound C in the treatment of CE, we constructed an animal model of secondary infection, which is now widely used in laboratories to assess the therapeutic effects of drugs on CE (30, 31). After intraperitoneal administration of 10 mg/kg Compound C to E. granulosus s.s.-infected mice for 28 days, the weight of cysts was significantly reduced, and the ultrastructure of cysts was disrupted in the Compound C group compared with the control group, suggesting that Compound C can play a therapeutic role in CE in vivo. Although the in vivo therapeutic effect of each dose in the Compound C group was not superior to that in the ABZ group, considering that patients need to take ABZ for a long period of time and in large doses, which can cause the development of drug tolerance (32), it is suggested that AMPK inhibitors have the value of serving as a potential alternative drug to ABZ.

Due to the importance of AMPK in the regulation of glucose uptake and glucose metabolism (33), we hypothesized that the inhibition of EgAMPK could lead to the disruption of glucose uptake and glycolytic function in E. granulosus s.s., which, in turn, induced parasite death. Molecular docking analysis revealed that Compound C binds to all three subunits of EgAMPK through hydrophobic interactions and has the highest binding affinity to EgAMPKα. Immunoblotting and immunofluorescence results confirmed that Compound C inhibited the phosphorylation level of EgAMPKα. In this study, we found that the glucose and ATP levels of E. granulosus s.s. decreased after Compound C treatment in vitro but the lactate content was not affected, suggesting that the inhibition of EgAMPK did not change the glycolytic metabolism of E. granulosus s.s.. The regulation of glycolysis by AMPK is not always consistent; AMPK activation in monocytes promotes glycolysis (34), while in T lymphocytes AMPK inhibits glycolysis (35). Hunter et al. (36) reported that inhibition of schistosome AMPK resulted in increased parasite glycolysis. Our study suggests that the inhibition of ATP levels by the chemical inhibitor Compound C does not appear to occur by affecting glycolysis. It has been previously reported that cancer cells satisfy their infiltrative and unrestricted proliferation by taking up large amounts of glucose (37). When glucose levels are not sufficient for cancer cell proliferation, AMPK is activated, which increases glucose transport by GLUTs (38). Glucose, as an important substrate for energy synthesis, is extremely important for the growth and development of E. granulosus s.s.; however, as E. granulosus s.s. is unable to synthesize glucose and needs to uptake it from the host environment, and EgGLUTs are responsible for the transmembrane transport of glucose in E. granulosus s.s (20, 21). In our previous studies, we successfully cloned the open reading frame gene sequences of the EgGLUT1 gene from E. granulosus s.s (20), so we examined the effects of the EgAMPK/EgGLUT1 signaling pathway and its glucose uptake function in E. granulosus s.s. The results revealed that the EgAMPK/EgGLUT1 signaling pathway was inhibited after Compound C intervention in E. granulosus s.s., and the glucose uptake ability of E. granulosus s.s. was also suppressed. Notably, although EgGLUT1 is widely distributed, it cannot be excluded that other GLUT family proteins may also play a role in glucose uptake in E. granulosus s.s (21).

Despite efforts to characterize AMPK homologs in parasitic organisms, their roles in parasitic infections remain elusive. The main reason is that AMPK is at the center of the energy metabolism regulatory network and is involved in the regulation of complex downstream signaling pathways. When AMPK is activated, it can not only positively regulate glucose uptake and glycolysis but also negatively regulate ATP-consuming biosynthesis pathways, including gluconeogenesis and protein and lipid synthesis (6). It has been found that in Echinococcus multilocularis infection, metformin can activate EmAMPK and inhibit EmTOR phosphorylation to induce autophagy (13). Previous studies have demonstrated that metformin restricts mitochondrial function to activate AMPK, which exerts therapeutic effects on echinococcosis through the autophagy pathway (13, 17); however, we note the role of AMPK in the regulation of glucose uptake. Unlike the therapeutic strategy of activating AMPK, inhibition of AMPK aims at limiting the downstream glucose uptake function to achieve disruption of energy metabolism in E. granulosus s.s. Overall, AMPK serves as an important sensor for regulating energy metabolism homeostasis, and either activation or inhibition of AMPK may disrupt energy metabolism homeostasis, thereby affecting disease development, suggesting the importance of AMPK as a drug target. In this study, although we found that inhibition of EgAMPK can directly or indirectly lead to the blockage of glucose uptake in E. granulosus s.s., the inhibition of EgAMPK activation may be accompanied by alterations in other pathways of metabolism and compensatory mechanisms. At present, we cannot fully explain the regulation of energy metabolism by EgAMPK in E. granulosus s.s.; this limitation will be further elucidated in future work. Most of the potential drug candidates with therapeutic effects in preclinical studies have not been able to enter clinical studies due to problems with side effects (39). In this study, it was reported that Compound C showed significant therapeutic effects in vivo but we cannot ignore the potential adverse effects of Compound C on the host. Compound C, although it has been widely used as a pharmacological tool, has not yet been applied in clinical studies. Unfortunately, most of the existing AMPK-targeting drugs are activators, and the development of small-molecule inhibitors is extremely limited, thus greatly restricting the study of AMPK as a drug target in the clinic. With the gradual maturation of computer-aided drug design technology, an increasing number of drug molecules with better targeting and lower toxicity have been introduced (40, 41); thus, the development of EgAMPK-targeted inhibitors is promising.

In summary, we demonstrated that inhibition of EgAMPK activation leads to the downregulation of EgGLUT1 expression, with reduced levels of glucose uptake and ATP synthesis, in turn leading to parasite death. Our results also demonstrated that EgAMPK can be a candidate drug target for the treatment of CE.

MATERIALS AND METHODS

Chemicals

Compound C and ABZSO were obtained from MedChem Express (Monmouth Junction, NJ, USA), and DMSO and ABZ were obtained from Sigma-Aldrich (Monmouth Junction, NJ, USA).

In vitro culture of E. granulosus s.s. protoscoleces, metacestodes, and drug treatment

E. granulosus s.s. PSCs were obtained from infected hydatid cysts in the liver of sheep slaughtered in an abattoir, in Urumqi, Xinjiang, China. These sheep were initially planned for routine slaughter. Otherwise, E. granulosus s.s. MTCs were obtained from the peritoneums of C57BL/6 female mice infected with 2,000 PSCs (PSCs were derived from hydatid cysts of infected sheep slaughtered in an abattoir) for 6 months. PSCs and MTCs were cultured separately in vitro as we described in detail previously (42). Before the start of the experiment, the viabilities of the PSCs and MTCs needed to be >90%. PSCs with complete morphology were spread on 96-well plates (n = 200/0.32 cm² growth area per well). MTCs (approximately 2–3 mm in diameter) with swelling and no collapsed germinal layers were randomly assigned to 6-well plates (n = 10–20/9.6 cm²/well growth area). PSCs and MTCs were cultured at 37°C and 5% CO₂, and the complete medium was changed every 2 days. Compound C and ABZSO were prepared as stock solutions in DMSO at concentrations of 10 and 1.5 mM. In vitro PSC and MTC treatments were assayed with concentrations of 12.5, 25, 50, and 100 µM Compound C and ABZSO alone at a concentration of 15 µM (17) for 6 and 5 days. 1% DMSO was used as a control. The viability of PSCs was assessed by 1% eosin staining (PSCs that were not stained with eosin were considered viable, while dead PSCs were stained red) (20), and the viability of MTCs was assessed by inverted light microscopy to observe the loss of turgidity and collapse of the germinal layer (43). Each experiment was carried out in triplicate and repeated three times. The viabilities of PSCs and MTCs were assessed every 24 h. Randomly selected PSCs and MTCs were analyzed by SEM (ZEISS, LEO1430VP, Germany) and TEM (JEOL, JEM-1230, Japan) for ultrastructural studies, as described previously (44). For molecular and biochemical assays, PSCs were collected for 48 h after 1% DMSO and 50 µM Compound C (45) intervention and stored at −80°C until further use.

siRNA interference

The siRNA specific for EgAMPKα and the siRNA used as a negative control were designed and synthesized by Guangzhou Ruibo Biotechnology Co., Ltd. The siRNA-EgAMPKα (three siRNA-EgAMPKα constructs were combined as a mixture) sequences were as follows: siRNA-EgAMPKα−1: 5′-CAAGCGTACGTGATCTCTT-3′; siRNA-EgAMPKα−2: 5′-TCAGCTCTATCAGGTGGAT-3′; and siRNA-EgAMPKα−3: 5′-CATCGATGAGGCTGTCTAT-3′.

The experiments were divided into three groups, the siRNA-EgAMPKα-treated group, the siRNA-negative control-treated group, and the untreated group. siRNA transfection was performed by electroporation as described previously with some modifications (46). Briefly, 200 µL of electroporation buffer containing approximately 4,000 PSCs was placed in a 4 mm cuvette and siRNA was added to obtain a final concentration of 5 µM. Square wave protocol of 125 V for 20 ms. After incubation at 37°C and 5% CO₂ for 10 min, PSCs were transferred to 1 mL of medium and then incubated in 24-well plates at 37°C and 5% CO₂. After the PSCs had been incubated for 72 h, the PSCs were collected, and qRT-PCR was used to determine the effects of siRNA on the level of EgAMPKα mRNA. Meanwhile, PSCs were transferred to 96-well plates (200 PSCs/well), and the effect of siRNA on PSC viability was assessed using 1% eosin staining on days 0, 3, 6, 10, and 15. Each treatment was performed in triplicate and the experiment was repeated three times using samples of PSCs collected at different times.

Quantitative real-time PCR analysis of EgAMPKα gene expression

To clarify the effects of siRNA on EgAMPKα mRNA levels, total RNA was extracted from PSCs using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA was reverse transcribed into cDNA using a Prime Script RT kit (TAKARA, Tokyo, Japan) according to the manufacturer’s instructions. qRT-PCRs were carried out using the QuantStudio 6 Real-Time Fluorescence PCR System (Thermo Fisher Scientific, Waltham, MA, USA) in a final volume of 20 µL. The reaction system was constructed with 10 µL TB Green Premix Ex Taq II (TAKARA, Tokyo, Japan), 0.8 µL of each of the primers, 2 µL cDNA product, 6.4 µL ddH₂O. The sense and antisense primers for EgAMPKα were 5′-GGTGCAAGCGTACGTGATCT-3′, and 5′-TGAGGAAACAGATGCGGTGG-3′. Primers for β-actin as an internal control were 5′-ATGGTTGGTATGGGACAAAAGG-3′ and 5′-TTCGTCACAATACCGTGCTC-3′. The reaction conditions were as follows: 95°C for 30 s; 40 cycles at 95°C for 5 s, and 58°C for 34 s. To determine the optimal amount of template, a fivefold dilution of cDNA was performed and qRT-PCR amplification occurred within the linear range. The data were analyzed according to the 2^−ΔΔCt method. All samples were analyzed in triplicate.

In vivo efficacy of AMPK inhibitor treatment in mice experimentally infected with E. granulosus s.s

For in vivo experiments, ABZ and Compound C were suspended in 0.5% carboxymethylcellulose sodium (CMC-Na). Female C57BL/6 mice (aged 6–8 weeks, weighing 18–20 g) were injected intraperitoneally with 200 µL of saline containing 2,000 PSCs (from the same source as the PSCs described above), as previously described (20). At 6 months post-infection, all mice were randomly divided into three groups: the control group (0.5% CMC-Na, n = 8), ABZ (50 mg/kg/day, n = 10) group, and Compound C (10 mg/kg/day, n = 10) group. All drugs were administered by intraperitoneal injection (dose: 0.1 mL/10 g per mouse) every day for 28 days, and then the mice were euthanized by cervical dislocation under isoflurane anesthesia. The body weights of the mice were recorded, the livers and kidneys were collected, and the cysts were weighed for each animal. During the administration period, blood glucose and body weight were monitored on days 0, 7, 14, 21, and 28 for each group of mice.

Histopathologic and ultrastructural observations

PSCs and MTCs from in vitro interventions and cysts isolated in vivo from mice involved in efficacy studies were fixed in 2.5% glutaraldehyde for SEM and TEM, as previously described (44). Meanwhile, cysts, livers, and kidneys of mice were collected, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for histopathological examination.

Molecular docking analysis

Homology modeling of EgAMPKα, EgAMPKβ, and EgAMPKγ was performed using the SWISS-MODEL (https://www.swissmodel.expasy.org) (47); the Compound C 3D structure was obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/compound/11524144) (48) and energy was minimized under the MMFF94 force field using Chem3D (Version 20). In this study, AutoDock Vina 1.1.2 software was used for molecular docking work (49), and all crystal ligands were hydrogenated using PyMol 2.5 before docking began. Next, the PyMOL plugin Centerof_mass.Py was used to calculate the crystal ligand centroid, define the center of the docking box, and ensure that the docking box was positioned around the crystal ligands with box sides of 90 angstroms in length. The detailed degree of the global search was set to 20, and the other parameters were left at their default settings. The docking results were visualized and analyzed using PyMol 2.5.

Immunoblot and immunofluorescence analyses

Proteins from PSCs were extracted using a Total Protein Extraction Kit (Solarbio, Beijing, China), and protein concentrations were measured using a BCA protein assay (Thermo Fisher Scientific, Waltham, MA, USA). Proteins were separated by SDS-PAGE on 10% polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. PVDF membranes were blocked for 2 h using 5% skim milk powder and incubated at 4°C overnight with the primary antibodies anti-AMPKα (Abcam, ab32047, 1:1,000), anti-phospho-AMPKα (Cell Signaling Technology, 2537, 1:1,000), anti-beta actin (Abcam, ab8226, 1:1,000). Secondary antibodies were rabbit IgG-HRP (Bioss, bs-80295G-HRP, 1:5,000) and mouse IgG-HRP (Bioss, bs-0296G-HRP, 1:5,000). Signals were detected using ECL reagents and analyzed using ImageJ software (NIH, Bethesda, MD, USA).

For immunofluorescence, PSCs were treated as described previously (50). Briefly, PSCs were washed three times with precooled PBS and then fixed for 4 h at 4°C. Next, PSCs were washed with PBS (containing vol/vol 0.3% Triton X-100, wt/vol 0.5% BSA) for 24 h, and primary antibody (1:50 dilution) was added and incubated for 5 days at 4°C. A polyclonal antibody specific for EgGLUT1 (the peptide sequence CDVQEEFVRMASGGG) was generated and affinity-purified by GenScript (Piscataway, NJ, USA). PSCs were washed three times with PBS and incubated at 4°C for 24 h. After incubation for 24 h, goat anti-rabbit IgG conjugated with Alexa 594 (1:500 dilution) was added and incubated for 24 h at 4°C. Two μg/mL DAPI was added and incubated for 30 min in the dark, washed three times with PBS for 5 min each time, and observed under a laser confocal microscope (Leica, TCS SP8, Germany).

ATP, lactate, and glucose assays

ATP and glucose assays of PSCs and cysts were performed as described previously with some modifications (51). Briefly, PSCs and cysts were lysed using precooled 20 mM Tris-HCL. Next, the samples were boiled for 5 min and centrifuged at 4°C for 30 min at 12,000 r/min to obtain the supernatant. The ATP and glucose contents of the parasites were measured using an ATP detection assay kit (Cayman Chemical, Ann Arbor, MI, USA) and a glucose colorimetric kit (Cayman Chemical, Ann Arbor, MI, USA), respectively, according to the manufacturer’s instructions. ATP and glucose contents were generated based on the standard curve calculation. The lactate content assay was performed as described previously (52) using a lactate assay kit (Solarbio, Beijing, China). Three replicate wells were measured for each experiment, and the experiment was repeated three times.

Glucose uptake assay

Glucose uptake was measured as described previously with some modifications (20). PSCs were incubated overnight in 96-well plates (200 PSCs/well) containing sugar-free DMEM. The next day, each well was washed twice with PBS and incubated with 50 µM Compound C at 37°C, 5% CO₂ for 150 min. 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]−2-deoxy-d-glucose (2-NBDG, a glucose analog, final concentration of 100 µM) (Invitrogen, Carlsbad, CA, USA) was added to each well, and the supernatant was removed after 15 min. The wells were washed twice with ice-cold PBS and then the fluorescence intensity was detected directly using a microplate reader from Varioskan Flash (Thermo Fisher Scientific, Waltham, MA, USA) at an excitation/emission wavelength of 467 nm/542 nm.

Caspase-3 activity assay

To detect caspase-3 activity, a caspase-3 activity kit was purchased from Solarbio (Beijing, China). PSCs were treated with siRNA-EgAMPKα and 50 µM Compound C for 72 h and 48 h, respectively. According to the manufacturer’s instructions, PSC proteins were mixed into a reaction system containing a caspase-specific substrate (Ac-DEVD-ρNA for caspase-3) and buffer. After incubation in 96-well plates at 37°C for 2 h, the OD value of the mixture was measured at 405 nm.

Statistical analysis

IBM SPSS Statistics 20 software was used for all analyses. The results are presented as the mean ± standard deviation. The chi-square test was used to analyze the viability of PSCs and MTCs in vitro. Student’s t-test was applied when the data complied with normality and homogeneity of variance; otherwise, the Kruskal-Wallis test was executed and followed by Dunn’s multiple comparison test. The P values are indicated as ^*P < 0.05 and ^**P < 0.01.***

ETHICS APPROVAL

The animal experimental protocols involved in this study were approved by the Animal Welfare Committee of the First Affiliated Hospital of Xinjiang Medical University (IACUC-20180213–07). Surgery was performed under isoflurane anesthesia to reduce the pain experienced by the animals.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/aac.01202-23.

Fig. S1 to S3. aac.01202-23-s0001.docx.

Figure S1 (Preliminary assessment of host effects after AMPK inhibitor Compound C treatment), Figure S2 (Molecular docking studies of AMPK inhibitor Compound C with EgAMPKα [A], EgAMPKβ [B], and EgAMPKγ [C[), and Figure S3 (The impact of siRNA-EgAMPKα [A] and 50 μΜ Compound C [B] on Caspase-3 levels in PSCs of E. granulosus s.s.).

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