https://orcid.org/0000-0002-9783-3775
Figures
Abstract
Background
Angiostrongylus cantonensis is a zoonotic nematode that causes eosinophilic meningitis and central nervous system injury in humans; 3-hydroxybenzaldehyde (3-HBA) is a benzaldehyde compound that exhibits antioxidant and anti-inflammatory activities. Brain injury promotes Ca²⁺ influx and mitochondrial Ca²⁺ loading via voltage-dependent anion channel 1 (VDAC1) and the mitochondrial Ca²⁺ uniporter (MCU), leading to mitochondrial dysfunction and cytochrome c-mediated apoptosis.
Methodology/Principal Findings
This study aimed to evaluate the therapeutic effects of 3-HBA combined with albendazole on brain injury and the expression of mitochondria-related molecules in A. cantonensis-infected mice. In BALB/c mice infected with A. cantonensis, the infection significantly increased glial fibrillary acidic protein expression in five regions: the cerebral cortex, hippocampus, subcortical areas, cerebellum, and brainstem and elevated the expression of MCU and cytochrome c in the cerebral cortex and hippocampus. Hematoxylin and eosin staining revealed pathological changes, such as meningitis, hemorrhage, and vascular congestion. However, combined treatment with 3-HBA and albendazole reduced these pathological changes and the expression of mitochondria-related molecules, including glial fibrillary acidic protein, VDAC1, MCU, and cytochrome c. In cultured mouse astrocytes, soluble antigens from fifth-stage larval-activated astrocytes induced mitochondria-related molecule expression, but 3-HBA suppressed these effects.
Conclusions/Significance
These results suggest that the combination of 3-HBA and albendazole downregulates astrocyte activation and VDAC1/MCU-associated mitochondrial pathways following A. cantonensis infection. These findings support the use of 3-HBA as a promising adjuvant to albendazole in the treatment of angiostrongyliasis.
Author summary
Angiostrongylus cantonensis (rat lungworm) is a major food-borne zoonotic parasite that causes severe central nervous system pathologies in humans, including eosinophilic meningitis and meningoencephalitis. Therapeutic strategies for cerebral angiostrongyliasis remain controversial; 3-hydroxybenzaldehyde (3-HBA) is a benzaldehyde derivative. Our previous studies demonstrated that treatment with 3-HBA combined with an anthelmintic exerted therapeutic effect by modulating oxidative stress and apoptosis in A. cantonensis-infected mouse brains. Therefore, this study aimed to elucidate the mitochondrial mechanisms underlying these effects. We investigated the mechanism by which 3-HBA regulates mitochondria-associated pathways during A. cantonensis infection. These findings enhance our understanding of 3-HBA-mediated neuroprotection and may inform the development of adjunctive therapeutic strategies for human angiostrongyliasis.
Citation: Huang Y-H, Chang S-H, Cheng C-J, Lin Y-H, Chou Y-C, Chen C-L, et al. (2025) Combination therapy with 3-Hydroxybenzaldehyde and Albendazole modulates mitochondrial protein expression in astrocytes after Angiostrongylus cantonensis infection. PLoS Negl Trop Dis 19(11): e0013773. https://doi.org/10.1371/journal.pntd.0013773
Editor: Robert Adamu SHEY, University of Buea, CAMEROON
Received: September 10, 2025; Accepted: November 19, 2025; Published: November 24, 2025
Copyright: © 2025 Huang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All raw data underlying the results are presented in figures.
Funding: This work was supported in part by grants from the National Science and Technology Council, ROC (112-2320-B-182 -052-MY3 to KYC), the Chang Gung Memorial Hospital Research Grant (CMRPD1N0081-2 to KYC and CLC, CMRPD1P0241 to KYC and CLC, and BMRPL30 to KYC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Angiostrongylus cantonensis, commonly known as the rat lungworm, is a parasitic nematode that causes angiostrongyliasis in humans. This zoonotic disease can lead to conditions, such as eosinophilic meningitis and meningoencephalitis, which may result in severe or even fatal outcomes. The optimal treatment for cerebral angiostrongyliasis remains a topic of ongoing debate [1]. The life cycle of A. cantonensis includes rats as definitive hosts and mollusks as intermediate hosts. Adult worms reside and reproduce in the pulmonary arteries and right ventricles of rats. Female worms lay eggs that hatch into first-stage larvae within the blood capillaries of the lungs. These larvae are eventually expelled in rat feces. Mollusks become infected either by ingesting larvae or through skin penetration. Humans, as accidental hosts, become infected when they consume an intermediate or paratenic host containing infective third-stage (L3) larvae. The larvae penetrate the intestinal wall, enter the bloodstream, migrate to the central nervous system (CNS), and develop into fifth-stage (L5) larvae [2].
Infection with L5 larvae can cause severe inflammatory responses, mechanical injury, and cell death, particularly in humans. This process involves the recruitment of eosinophils and secretion of cytokines and chemokines within the CNS [3–8]. In our study of A. cantonensis, we found that oxidative stress, apoptosis, and inflammation were induced in the brains of infected mice [5,9].
Astrocytes are the most abundant glial cells in the CNS and interact with various brain cell types to perform different functions, such as maintaining neurotransmitter homeostasis, supporting synapse formation and plasticity, clearing excess neurotransmitters, and contributing to the formation of the blood–brain barrier [10,11]. Astrocytes play a critical role in CNS disorders and neurodegenerative diseases, including autoimmune inflammation, multiple sclerosis, and Alzheimer’s disease [12].
Furthermore, mitochondrial dysfunction in astrocytes is a key contributor to the pathology of neurodegenerative diseases [13,14]. In Alzheimer’s disease, mitochondrial dysfunction has been observed, including reduced mitochondrial metabolic capacity, decreased mtDNA synthesis, increased reactive oxygen species (ROS) production, and impaired lactate generation [15].
3-Hydroxybenzaldehyde (3-HBA) is an oxidized benzaldehyde with a hydroxyl group in its structural unit and possesses antioxidant, anti-inflammatory, and antimicrobial properties [1,16,17]. 3-HBA has vasoprotective potential, which suggests that it could be used in the treatment of atherosclerosis [16]. Additionally, 3-HBA has been evaluated as an experimental compound for new cancer treatments [18]. Our previous research showed that combined treatment with albendazole and 3-HBA in A. cantonensis-infected mice enhanced antioxidant activity, reduced the expression of apoptosis-related molecules in the brain, and increased cell viability [1,19]. Moreover, 3-HBA inhibits the production of ROS, thereby reducing endoplasmic reticulum stress [20].
The infection with A. cantonensis in mice induces oxidative stress in the CNS, ultimately leading to astrocyte apoptosis and brain injury. By contrast, mitochondrial dysfunction is recognized as a key mechanism through which astrocytes contribute to the pathogenesis of neurodegenerative diseases. Therefore, in this study, we investigated the therapeutic effects of 3-HBA combined with albendazole against A. cantonensis-induced pathogenesis, focusing on mitochondrial function. The results of this study elucidate the therapeutic capacity of 3-HBA as an adjuvant therapeutic agent and provide novel insights into potential treatment strategies for CNS injury caused by parasitic infections.
Materials and methods
Ethical approval
All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Chang Gung University, Taiwan (IACUC approval CGU111–167 and CGU113–082), and conducted in accordance with the Guidelines for Laboratory Animal Facilities and Care issued by the Council of Agriculture, Executive Yuan (ROC). Rats and mice were housed in plastic cages with ad libitum access to food and water. Animals were euthanized under inhalational isoflurane anesthesia (1 mL/min).
Establishment and maintenance of A. cantonensis life cycle
The A. cantonensis strain used in this study was originally isolated from Achatina fulica in Neihu, Taipei, in 1985. The laboratory life cycle was established by infecting Sprague–Dawley rats with L3 larvae [2]. First-stage larvae were collected from rat feces using a modified Baermann funnel method [21] and used to infect Biomphalaria glabrata. After 21 d, L3 larvae were harvested from the infected snails by tissue homogenization (Cole-Parmer Instrument Co., USA), followed by enzymatic digestion with artificial gastric juice (0.6% w/v pepsin, pH 2–3). L3 larvae were then used to infect rats by oral inoculation.
Experimental animals
Sprague–Dawley rats and BALB/c mice were purchased from the National Laboratory Animal Center, Taipei, Taiwan, for the maintenance of the A. cantonensis life cycle. Animals were housed individually in plastic cages under controlled conditions with free access to standard chow and drinking water. All procedures involving animal care and handling were conducted at the Chang Gung University Animal Center and reviewed and approved by the Institutional Animal Care and Use Committee of Chang Gung University.
Drug treatment
In this study, BALB/c mice were infected orally with 25 L3 larvae of A. cantonensis. Body weight was monitored daily throughout the experimental period. After 7 d, the mice received albendazole (10 mg/kg) and 3-HBA (100 mg/kg) once daily until they were sacrificed at 21 dpi. At 21 dpi, the mice were euthanized, and blood and brain tissues were collected. The brains were dissected into five regions: the cerebral cortex, hippocampus, subcortical areas, cerebellum, and brainstem.
Immunohistochemistry staining
Brain tissues were processed for paraffin embedding. Samples were fixed in 10% formalin and dehydrated in a graded ethanol series. The dehydrated tissues were cleared in xylene for 1 h to ensure transparency and compatibility with paraffin infiltration. Subsequently, the brain sections were washed with PBS. H&E staining was performed to evaluate the pathological feature. Finally, the slides were dehydrated in xylene and permanently mounted using NeoMount (Cat# 1.09016; Merck, Germany). Images were acquired using a light microscope.
Hematoxylin and eosin (H&E) staining and histopathological assessment
H&E staining was performed to evaluate four pathological features: meningitis, congestion, hemorrhage, and the presence of larvae. The evaluation criteria were adapted from previously established methods [22,23].
Western blot analysis
Protein samples were separated on 12% sodium dodecyl sulfate–polyacrylamide gels and transferred onto nitrocellulose membranes using a wet-transfer system. Membranes were blocked in blocking buffer before incubation with primary antibodies overnight at 4 °C. The following primary antibodies were used: GFAP (Proteintech, USA), VDAC1 (ABclonal, USA), MCU (ABclonal, USA), cytochrome c (ABclonal, USA), and β-actin (Sigma-Aldrich, USA). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. The blots were visualized using enhanced chemiluminescence with equal volumes of stable peroxide and luminol enhancer solutions.
Cell culture
Mouse astrocytes (CRL-2535) were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were maintained in Dulbecco’s modified Eagle’s medium (Corning, NY, USA) supplemented with 10% fetal bovine serum and 100 U/ml penicillin–streptomycin. Cells were seeded at a density of 0.25 × 106 cells/cm2 onto poly-L-lysine-coated culture plates and incubated at 37 °C in 5% CO₂ until they reached confluence (1–2 × 104 cells/cm2). Immunostaining for GFAP confirmed that >95% of the cultured cells were astrocytes.
Immunofluorescence staining
Cells were fixed with 3.7% paraformaldehyde and treated with 0.1% Triton X-100. After blocking with 1% BSA, the cells were incubated with primary antibodies (GFAP, Proteintech, USA; VDAC1, ABclonal, USA; MCU, ABclonal, USA; cytochrome c, ABclonal, USA), followed by incubation with a fluorescent secondary antibody. Nuclei were counterstained with DAPI using the mounting medium.
Results
Evaluation of body weight changes in BALB/c mice infected with A. cantonensis and therapeutic treatment
To evaluate the therapeutic effect of 3-HBA in A. cantonensis-infected mice, we monitored body weight following L3 larvae infection and treatment. The animals were infected with 25 L3 larvae and assigned to four groups (n = 13/group): normal (uninfected), infected, infected + albendazole, and infected + albendazole + 3-HBA. Body weight was recorded daily, and treatments were initiated from 7 to 21 dpi.
As shown in Fig 1A, the body weight of the infected group significantly decreased by day 21. Moreover, on day 21, the infected + albendazole and infected + albendazole + 3-HBA groups exhibited significantly higher body weights than those of the infected group (Fig 1B). These data indicate that 3-HBA combined with albendazole is an effective therapeutic strategy against A. cantonensis infection.
Mice were assigned to four groups (n = 11/group): Normal (N), Infected (Infect), Infected + albendazole (Infect + Alb), and Infected + albendazole + 3-HBA (Infect + Alb + 3-HBA). Body weight was recorded daily for 21 days. (A) The changes in body weight for N and Infect groups at 0, 7, 14, and 21 days post-infection (dpi). (B) The changes in body weight for Infect, Infect + Alb, and Infect + Alb + 3-HBA groups at 0, 7, 14, and 21 dpi. The data are obtained from independent experiments; ***P < 0.001.
Evaluation of pathological changes in BALB/c mice infected with A. cantonensis and therapeutic treatment
To evaluate the therapeutic effect of 3-HBA on pathological changes in the CNS after A. cantonensis infection, histopathological examination was performed using H&E staining. We detected four features: eosinophilic meningitis, larval findings, hemorrhage, and vascular congestion. As shown in Figs 2–5, these pathological features were significantly increased in the infected group. However, the combination of albendazole and 3-HBA significantly reduced pathological damage. Collectively, these findings indicate that 3-HBA attenuates severe neuropathological changes in the brains of mice following A. cantonensis infection.
H&E stained brain tissues from (A) Normal (N), (B) Infected (Infect), (C) Infected + albendazole (Infect + Alb), and (D) Infected + albendazole + 3-HBA (Infect + Alb + 3-HBA) (E) Quantification of eosinophilic meningitis thickness. The data are obtained from independent experiments (n = 3); **P < 0.01, ****P < 0.0001.
H&E stained brain tissues from (A) Normal (N), (B) nfected (Infect), (C) Infected + albendazole (Infect + Alb), and (D) Infected + albendazole + 3-HBA (Infect + Alb + 3-HBA) (E) Quantification of larval presence. The data are obtained from independent experiments (n = 3); ***P < 0.001.
H&E stained brain tissues from (A) Normal (N), (B) nfected (Infect), (C) Infected + albendazole (Infect + Alb), and (D) Infected + albendazole + 3-HBA (Infect + Alb + 3-HBA) (E) Quantification of hemorrhage. The data are obtained from independent experiments (n = 3); ****P < 0.0001.
H&E stained brain tissues from (A) Normal (N), (B) nfected (Infect), (C) Infected + albendazole (Infect + Alb), and (D) Infected + albendazole + 3-HBA (Infect + Alb + 3-HBA) (E) Quantification of vascular congestion. The data are obtained from independent experiments (n = 3); ****P < 0.0001.
Effect of astrocyte activation following treatment with 3-HBA in BALB/c mice infected with A. cantonensis
First, we determined whether 3-HBA could reduce astrocyte activation in A. cantonensis-infected BALB/c mice. Western blotting was used to detect the protein expression of GFAP in five brain regions: the cerebral cortex, hippocampus, subcortex, cerebellum, and brainstem. As shown in Fig 6, GFAP expression was significantly elevated in the cerebral cortex, hippocampus, subcortex, and cerebellum after A. cantonensis infection. Albendazole combined with 3-HBA significantly downregulated GFAP expression in the hippocampus. These findings demonstrate that 3-HBA attenuates astrocyte activation in BALB/c mice following A. cantonensis infection, with particularly pronounced effects in the hippocampus.
The western blotting was employed to detect the protein expression of GFAP in each treatment group in vivo. (A) cerebral cortex, (B) hippocampus, (C) subcortical areas, (D) cerebellum, and (E) brain stem. N: Normal, Inf: Infection, Inf + Alb: infection+albendazole, Inf + Alb + 3-HBA: Infection+albendazole+3-HBA. The data are expressed as the means ± SEM from independent experiments (n = 3); *P < 0.05, **P < 0.01, ***P < 0.001.
Evaluation of VDAC1 expression following treatment with 3-HBA in BALB/c mice infected with A. cantonensis
During the initiation of mitochondria-mediated intrinsic apoptosis, VDAC1 expression increases, promoting the release of cytochrome c into the cytosol and triggering downstream apoptotic cascades [24]. As shown in Fig 7, VDAC1 expression was significantly elevated in the cerebral cortex and hippocampus after A. cantonensis infection. In addition, albendazole combined with 3-HBA significantly downregulated VDAC1 expression in the cerebral cortex, hippocampus, and subcortex. These findings demonstrate that 3-HBA downregulates VDAC1 expression in brain regions following A. cantonensis infection.
The western blotting was employed to detect the protein expression of VDAC1 in each treatment group in vivo. (A) cerebral cortex, (B) hippocampus, (C) subcortical areas, (D) cerebellum, and (E) brain stem. N: Normal, Inf: Infection, Inf + Alb: infection+albendazole, Inf + Alb + 3-HBA: Infection+albendazole+3-HBA. The data are expressed as the means ± SEM from independent experiments (n = 3); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Evaluation of MCU expression following treatment with 3-HBA in BALB/c mice infected with A. cantonensis
Under cellular injury conditions, Ca2+ is transported into the mitochondrial matrix through the MCU. Excessive mitochondrial Ca2+ loading increases ROS levels, disrupts mitochondrial function, and stimulates the intrinsic apoptotic pathway [25]. As shown in Fig 8, MCU expression was significantly elevated in the hippocampus after A. cantonensis infection. In addition, albendazole combined with 3-HBA significantly downregulated MCU expression in the cerebral cortex, subcortex, and cerebellum. These findings demonstrate that 3-HBA downregulates MCU expression in various brain regions following A. cantonensis infection.
The western blotting was employed to detect the protein expression of MCU in each treatment group in vivo. (A) cerebral cortex, (B) hippocampus, (C) subcortical areas, (D) cerebellum, and (E) brain stem. N: Normal, Inf: Infection, Inf + Alb: infection+albendazole, Inf + Alb + 3-HBA: Infection+albendazole+3-HBA. The data are expressed as the means ± SEM from independent experiments (n = 3); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Evaluation of cytochrome c expression following treatment with 3-HBA in BALB/c mice infected with A. cantonensis
Mitochondrial damage alters membrane permeability, leading to the release of cytochrome c into the cytosol and initiation of apoptosis. As shown in Fig 9, cytochrome c expression was significantly elevated in the cerebral cortex and hippocampus after A. cantonensis infection. Additionally, albendazole combined with 3-HBA significantly downregulated cytochrome c expression in these two regions. These findings demonstrate that 3-HBA downregulates cytochrome c expression in BALB/c mice following A. cantonensis infection, particularly in the cerebral cortex and hippocampus.
The western blotting was employed to detect the protein expression of cytochrome c in each treatment group in vivo. (A) cerebral cortex, (B) hippocampus, (C) subcortical areas, (D) cerebellum, and (E) brain stem. N: Normal, Inf: Infection, Inf + Alb: infection+albendazole, Inf + Alb + 3-HBA: Infection+albendazole+3-HBA. The data are expressed as the means ± SEM from independent experiments (n = 3); *P < 0.05, **P < 0.01, ***P < 0.001.
Expression of GFAP and mitochondria-related proteins in astrocytes following exposure to A. cantonensis L5 soluble antigens (SA)
To evaluate the effects of A. cantonensis on the expression of mitochondria-related proteins (VDAC1, MCU, and cytochrome c) in astrocytes, astrocytes were stimulated with SA from A. cantonensis L5. As shown in Fig 10, the expression of these proteins was significantly elevated after SA treatment compared with that in the untreated group. These data indicate that A. cantonensis L5 SA stimulates astrocyte activation and induces mitochondria-related protein expression.
Mouse astrocytes were treated with A. cantonensis L5 soluble antigen (SA) for 0 (N), 6, 12, or 24 h. The western blotting was employed to detect the protein expression in each treatment group in vitro. (A) GFAP, (B) VDAC1, (C) MCU, and (D) cytochrome c. The data are expressed as the means ± SEM from independent experiments (n = 3); *P < 0.05, **P < 0.01, ***P < 0.001.
3-HBA downregulates mitochondria-related protein expression in astrocytes after A. cantonensis L5 SA treatment
We confirmed the effects of 3-HBA on the expression of mitochondria-related proteins (GFAP, VDAC1, MCU, and cytochrome c) in astrocytes. As shown in Fig 11, A. cantonensis L5 SA induced the expression of these proteins in astrocytes. Moreover, the expression of GFAP and cytochrome c was significantly decreased after 0.1 or 0.5 mM 3-HBA treatment compared with observations in SA alone. Immunofluorescence staining was then used to detect mitochondria-related proteins (GFAP, VDAC1, MCU, and cytochrome c) in astrocytes (Fig 12). The data showed that the expression of mitochondria-related proteins significantly increased after SA treatment but significantly decreased after 3-HBA treatment compared with that after SA alone. Collectively, these data demonstrate that 3-HBA attenuates astrocyte activation and reduces mitochondria-associated damage.
Astrocytes were stimulated with L5 A. cantonensis L5 soluble antigen (SA) or 3-HBA (0.1 or 0.5 mM) for 24 h. The western blotting was employed to detect the protein expression in each treatment group in vitro. (A) GFAP, (B) VDAC1, (C) MCU, and (D) cytochrome c. The data are expressed as the means ± SEM from independent experiments (n = 3); *P < 0.05, ***P < 0.001, ****P < 0.0001.
Astrocytes were stimulated with L5 A. cantonensis L5 soluble antigen (SA) or 3-HBA (0.1 or 0.5 mM) for 24 h. The immunofluorescence staining was employed to detect the protein expression in each treatment group in vitro. (A) GFAP, (B) VDAC1, (C) MCU, and (D) cytochrome c. The data are expressed as the means ± SEM from independent experiments (n = 5); *P < 0.05, **P < 0.01, ***P < 0.001.
Discussion
In this study, we evaluated the therapeutic potential of 3-HBA in angiostrongyliasis. We characterized CNS injury during A. cantonensis infection and assessed the protective efficacy of 3-HBA in combination with albendazole. In addition, we evaluated the therapeutic effects of 3-HBA on astrocytic responses after stimulation with SA derived from A. cantonensis L5 larvae. A. cantonensis infection induces mitochondrial injury in the CNS [26]. Therefore, we primarily focused on investigating mitochondria-related pathways in astrocytes.
3-HBA is a relatively small molecular weight, lipophilic phenolic compound (a phenolic aldehyde contains one hydroxyl group and one aldehyde group), and a few polar substituents. It may have the capacity to facilitate passive diffusion across the blood-brain barrier (BBB). Moreover, previous studies on A. cantonensis infection have demonstrated that the BBB can be disrupted or become leaky to some degree, leading to clinical signs such as neuroinflammation or hemorrhage. This BBB breakdown could facilitate the enhanced passage of compounds that would otherwise be restricted. Our previous investigation also clarified the protective efficacy of 3-HBA in the mouse brain after A. cantonensis infection. We showed that 3-HBA exhibits BBB protective efficacy in the CNS [1]. In conclusion, 3-HBA (alone or in combination with albendazole) could reach the CNS under infection conditions. However, this characteristic requires further experimental confirmation, such as using a radiolabeled tracer. Investigating the therapeutic efficacy of 3-HBA in the CNS will help us further understand its potential for the treatment of neuroangiostrongyliasis.
Our previous studies demonstrated the therapeutic potential of 3-HBA in the A. cantonensis-infected mouse brain [1,19,20]. The data revealed that 3-HBA exhibits potent antioxidant and anti-inflammatory properties, which are closely linked to its ability to inhibit cell apoptosis in astrocytes and prevent breakdown of the BBB. After A. cantonensis infection, the neuroinflammation and cell apoptosis can stimulate CNS pathology changes and neuronal injury. 3-HBA treatment was shown to reduce oxidative stress and astrocytic hyperactivation, thereby improving BBB function. However, treatment with 3-HBA alone did not demonstrate direct parasiticidal activity against A. cantonensis L5. This compound role is predominantly host-directed, aiming to counteract infection-induced oxidative and inflammatory damage in the CNS. In combination therapy, 3-HBA combined with albendazole showed synergistic benefits (albendazole exerted an anthelmintic effect, whereas 3-HBA provided neuroprotective and anti-inflammatory actions). Therefore, while 3-HBA does not directly kill the A. cantonensis L5, it acts as a neuroprotective adjunct that enhances CNS recovery and reduces neuroinflammation after A. cantonensis infection. These data revealed that 3-HBA is a potential therapeutic candidate for use alongside conventional anthelmintics.
The MCU is the principal Ca2+ uptake channel in the inner mitochondrial membrane. LPS activates the IP3R–GRP75–VDAC1–MCU Ca2+ transfer axis, thereby inducing Ca2+ entry into the mitochondrial matrix [27]. Moreover, the MCU contributes to neurotransmission and redox homeostasis in the CNS [28]. The MCU has also been implicated in hippocampal neuroinflammation in Parkinson’s disease [29] and in synaptic loss following brain injury [30]. In this study, we observed that astrocyte activation (GFAP expression) and MCU expression were significantly elevated in the hippocampus after A. cantonensis infection in vivo and after L5 larvae SA treatment in vitro. These findings support the hypothesis that A. cantonensis infection enhances mitochondrial Ca2+ uptake by activated astrocytes. Our previous work showed that 3-HBA has antioxidant capacity in vitro [20]. In the present study, 3-HBA combined with albendazole reduced MCU expression in the cerebral cortex, subcortex, and cerebellum in vivo.
Cytochrome c is a component of the electron transport chain that plays a major role in redox regulation and apoptosis. In CNS injury, excessive Ca2+ influx causes mitochondrial dysfunction, oxidative stress, and cell death [31]. In cerebral ischemia, mitochondrial Ca2+ accumulation and cytochrome c release trigger caspase-dependent apoptosis [32]. Our previous data confirmed that A. cantonensis infection or ESP stimulation elevated intracellular Ca2+ in the hippocampus [33]. In the present study, we found that cytochrome c expression significantly increased in astrocytes. These data suggest that A. cantonensis infection stimulates mitochondrial Ca2+ overload and ROS production, thereby promoting cytochrome c release and apoptosis. However, 3-HBA attenuates this mechanism [1,19].
Regional variation in mitochondrial protein expression likely reflects the distinct metabolic demands and vulnerability of different brain regions during A. cantonensis infection. In our observations, the cortex exhibited a more pronounced decrease in mitochondrial proteins, such as VDAC1 and MCU, accompanied by reduced retention of cytochrome C. In contrast, the cerebellum maintained relatively stable mitochondrial protein expression and lower ROS accumulation, suggesting an intrinsic resistance or delayed response to infection-induced stress.
3-hydroxybenzaldehyde (3-HBA) combined with albendazole treatment can restore mitochondrial function through several complementary mechanisms. Our previous studies determined that 3-HBA can enhance antioxidant capacity via reducing reactive oxygen species and improving antioxidant activity, thereby supporting mitochondrial integrity and energy metabolism in astrocytes. Concurrently, albendazole can eliminate A. cantonensis L5, alleviating ongoing inflammatory stress that suppresses mitochondrial biogenesis. Although this investigation has not yet directly quantified markers of mitochondrial biogenesis, such as TFAM, the observed astrocytic mitochondrial protein expression suggests that 3-HBA combined with albendazole therapy may promote mitochondrial recovery at both functional and structural levels. This possibility should be discussed as a future direction, emphasizing the potential of host-targeted metabolic restoration as an adjunct strategy for antiparasitic therapy in neuroangiostrongyliasis.
Subsequently, we examined the neuropathology of A. cantonensis-infected mice. Mitochondrial repair reduces oxidative stress, apoptosis, neuroinflammation, and astrocyte reactivity in traumatic brain injury [31]. Mitochondrial damage promotes inflammation by releasing mtDNA and other DAMPs that activate PRRs and inflammasomes [34]. Moreover, our investigations showed that A. cantonensis induces meningitis, hemorrhage, and vascular congestion in vivo and upregulates IL-1β and IL-6 secretion following ESP stimulation in astrocytes in vitro [1,5]. In the present study, H&E staining confirmed that meningitis, hemorrhage, and congestion increased after infection and were markedly ameliorated by combined 3-HBA and albendazole therapy.
In summary, we determined that A. cantonensis triggers mitochondrial dysfunction in astrocytes both in vitro and in vivo. However, 3-HBA attenuates this injury by reducing astrocyte activation and the expression of mitochondria-related molecules. Based on our findings, a combination of 3-HBA and albendazole may be a promising adjunct therapy for human angiostrongyliasis.
Acknowledgments
We would like to express our appreciation to the Instrumentation Center, Chang Gung University, Taoyuan, Taiwan for supplying invaluable technical assistance.
References
- 1. Chen K-Y, Cheng C-J, Chen Y-J, Chiu C-H, Wang L-C. Protective effect of benzaldehyde combined with albendazole against brain injury induced by Angiostrongylus cantonensis infection in mice. Int J Antimicrob Agents. 2023;62(5):106963. pmid:37666435
- 2. Wang LC, Chao D, Chen ER. Acquired immunity in rats against Angiostrongylus cantonensis infection. Int J Parasitol. 1989;19(6):617–20. pmid:2807717
- 3. Wei J, Wu F, He A, Zeng X, Ouyang L, Liu M, et al. Microglia activation: one of the checkpoints in the CNS inflammation caused by Angiostrongylus cantonensis infection in rodent model. Parasitol Res. 2015;114(9):3247–54. pmid:26002828
- 4. Yu L, Wu X, Wei J, Liao Q, Xu L, Luo S, et al. Preliminary expression profile of cytokines in brain tissue of BALB/c mice with Angiostrongylus cantonensis infection. Parasit Vectors. 2015;8:328. pmid:26070790
- 5. Chen K-Y, Wang L-C. Stimulation of IL-1β and IL-6 through NF-κB and sonic hedgehog-dependent pathways in mouse astrocytes by excretory/secretory products of fifth-stage larval Angiostrongylus cantonensis. Parasit Vectors. 2017;10(1):445. pmid:28950910
- 6. Zhang R, Miao T, Qin M, Zhao C, Wang W, Zhang C, et al. CX3CL1 Recruits NK Cells Into the Central Nervous System and Aggravates Brain Injury of Mice Caused by Angiostrongylus cantonensis Infection. Front Cell Infect Microbiol. 2021;11:672720. pmid:34017692
- 7. Feng Y, Zhou Z, Liu Z, Zheng C, Feng F, Xie F, et al. Interleukin 17A Derived from γδ T Cell Induces Demyelination of the Brain in Angiostrongylus cantonensis Infection. Mol Neurobiol. 2021;58(8):3968–82. pmid:33904019
- 8. Zhou H, Zhou M, Hu Y, Limpanon Y, Ma Y, Huang P, et al. TNF-α Triggers RIP1/FADD/Caspase-8-Mediated Apoptosis of Astrocytes and RIP3/MLKL-Mediated Necroptosis of Neurons Induced by Angiostrongylus cantonensis Infection. Cell Mol Neurobiol. 2022;42(6):1841–57. pmid:33683530
- 9. Chen K-Y, Chiu C-H, Wang L-C. Anti-apoptotic effects of Sonic hedgehog signalling through oxidative stress reduction in astrocytes co-cultured with excretory-secretory products of larval Angiostrongylus cantonensis. Sci Rep. 2017;7:41574. pmid:28169282
- 10. Endo F, Kasai A, Soto JS, Yu X, Qu Z, Hashimoto H, et al. Molecular basis of astrocyte diversity and morphology across the CNS in health and disease. Science. 2022;378(6619):eadc9020. pmid:36378959
- 11. Giovannoni F, Quintana FJ. The Role of Astrocytes in CNS Inflammation. Trends Immunol. 2020;41(9):805–19. pmid:32800705
- 12. Linnerbauer M, Rothhammer V. Protective Functions of Reactive Astrocytes Following Central Nervous System Insult. Front Immunol. 2020;11:573256. pmid:33117368
- 13.
Lampinen R, Belaya I, Boccuni I, Malm T, Kanninen KM. Mitochondrial Function in Alzheimer’s Disease: Focus on Astrocytes. Astrocyte - Physiology and Pathology. InTech. 2018. https://doi.org/10.5772/intechopen.71825
- 14. Liang KX, Chen A, Kianian A, Kristiansen CK, Yangzom T, Furriol J, et al. Activation of Neurotoxic Astrocytes Due to Mitochondrial Dysfunction Triggered by POLG Mutation. Int J Biol Sci. 2024;20(8):2860–80. pmid:38904024
- 15. Angelova PR, Abramov AY. Role of mitochondrial ROS in the brain: from physiology to neurodegeneration. FEBS Lett. 2018;592(5):692–702. pmid:29292494
- 16. Kong BS, Im SJ, Lee YJ, Cho YH, Do YR, Byun JW, et al. Vasculoprotective Effects of 3-Hydroxybenzaldehyde against VSMCs Proliferation and ECs Inflammation. PLoS One. 2016;11(3):e0149394. pmid:27002821
- 17. Esmaeili A, Kakavand S. Antioxidant and antibacterial activity evaluation of 3‐hydroxybenzaldehyde: the product of thymol oxidation by a new magnetic nanocatalyst. IET Nanobiotechnology. 2017;11(6):630–6.
- 18. Stamboliyska BA, Velcheva EA, Binev IG. IR spectral and structural changes caused by the conversion of 3-hydroxybenzaldehyde into the oxyanion. Spectrochim Acta A Mol Biomol Spectrosc. 2003;59(7):1541–8. pmid:12714077
- 19. Chen K-Y, Chen Y-J, Cheng C-J, Jhan K-Y, Chiu C-H, Wang L-C. 3-Hydroxybenzaldehyde and 4-Hydroxybenzaldehyde enhance survival of mouse astrocytes treated with Angiostrongylus cantonensis young adults excretory/secretory products. Biomed J. 2021;44(6 Suppl 2):S258–66. pmid:35300947
- 20. Chen K-Y, Chen Y-J, Cheng C-J, Jhan K-Y, Wang L-C. Benzaldehyde Attenuates the Fifth Stage Larval Excretory-Secretory Product of Angiostrongylus cantonensis-Induced Injury in Mouse Astrocytes via Regulation of Endoplasmic Reticulum Stress and Oxidative Stress. Biomolecules. 2022;12(2):177. pmid:35204678
- 21. Wang LC, Chao D, Chen ER. Experimental infection routes of Angiostrongylus cantonensis in mice. J Helminthol. 1991;65(4):296–300. pmid:1795091
- 22. Jhan K-Y, Cheng C-J, Jung S-M, Lai Y-J, Chen K-Y, Wang L-C. Co-Therapy of Albendazole and Dexamethasone Reduces Pathological Changes in the Cerebral Parenchyma of Th-1 and Th-2 Dominant Mice Heavily Infected with Angiostrongylus cantonensis: Histopathological and RNA-seq Analyses. Biomolecules. 2021;11(4):536. pmid:33917604
- 23. Wang L-C, Jung S-M, Chen K-Y, Wang T-Y, Li C-H. Temporal-spatial pathological changes in the brains of permissive and non-permissive hosts experimentally infected with Angiostrongylus cantonensis. Exp Parasitol. 2015;157:177–84. pmid:26299243
- 24. Keinan N, Tyomkin D, Shoshan-Barmatz V. Oligomerization of the mitochondrial protein voltage-dependent anion channel is coupled to the induction of apoptosis. Mol Cell Biol. 2010;30(24):5698–709. pmid:20937774
- 25. Patron M, Raffaello A, Granatiero V, Tosatto A, Merli G, De Stefani D, et al. The mitochondrial calcium uniporter (MCU): molecular identity and physiological roles. J Biol Chem. 2013;288(15):10750–8. pmid:23400777
- 26. Lam HYP, Chen C-C, Chen TT-W, Chang K-C, Wu W-J, Yang T-H, et al. Mitochondrial dynamics in Angiostrongylus cantonensis-infected mouse brain. Parasitol Int. 2021;80:102231. pmid:33147498
- 27. Meng M, Jiang Y, Wang Y, Huo R, Ma N, Shen X, et al. β-carotene targets IP3R/GRP75/VDAC1-MCU axis to renovate LPS-induced mitochondrial oxidative damage by regulating STIM1. Free Radic Biol Med. 2023;205:25–46. pmid:37270031
- 28. Rose J, Brian C, Pappa A, Panayiotidis MI, Franco R. Mitochondrial Metabolism in Astrocytes Regulates Brain Bioenergetics, Neurotransmission and Redox Balance. Front Neurosci. 2020;14:536682. pmid:33224019
- 29. Cai H, Qiao J, Chen S, Yang J, Hölscher C, Wang Z, et al. MCU knockdown in hippocampal neurons improves memory performance of an Alzheimer’s disease mouse model. Acta Biochim Biophys Sin (Shanghai). 2022;54(10):1528–39. pmid:36239352
- 30. Pannoni KE, Fischer QS, Tarannum R, Cawley ML, Alsalman MM, Acosta N, et al. MCU expression in hippocampal CA2 neurons modulates dendritic mitochondrial morphology and synaptic plasticity. Sci Rep. 2025;15(1):4540. pmid:39915602
- 31. Chakraborty R, Tabassum H, Parvez S. Dantrolene alleviates mitochondrial dysfunction and neuroinflammation in traumatic brain injury by modulating the NF-ĸβ/Akt pathway. Biochem Pharmacol. 2024;224:116244. pmid:38685280
- 32. Andreyev A, Tamrakar P, Rosenthal RE, Fiskum G. Calcium uptake and cytochrome c release from normal and ischemic brain mitochondria. Neurochem Int. 2018;117:15–22. pmid:29042253
- 33. Chen K-Y, Chen Y-J, Cheng C-J, Jhan K-Y, Wang L-C. Excretory/secretory products of Angiostrongylus cantonensis fifth-stage larvae induce endoplasmic reticulum stress via the Sonic hedgehog pathway in mouse astrocytes. Parasit Vectors. 2020;13(1):317. pmid:32552877
- 34. Vezzani B, Carinci M, Patergnani S, Pasquin MP, Guarino A, Aziz N, et al. The Dichotomous Role of Inflammation in the CNS: A Mitochondrial Point of View. Biomolecules. 2020;10(10):1437. pmid:33066071