Introduction
Leishmaniasis remains one of the most challenging neglected parasitic diseases worldwide (Gradoni et al., Reference Gradoni, López-vélez and Mokni2017; World Health Organization, 2026). Although the disease primarily affects the skin, mucosal tissues or visceral organs, neurological involvement has also been documented in selected clinical and experimental settings. Parasites, parasite-derived material, anti-Leishmania antibodies and inflammatory alterations have been detected in central nervous system (CNS)-associated compartments, supporting the biological relevance of cerebral leishmaniasis. Nevertheless, the mechanisms underlying CNS involvement remain incompletely understood (Melo and Machado, Reference Melo and Machado2011; Melo et al., Reference Melo, Seraguci, Schweigert, Silva, Grano, Peiró, Lima and Machado2013, Reference Melo, Goyard, Fiette, Boissonnas, Combadiere, Machado, Minoprio and Lang2017; Islek et al., Reference Islek, Ucisik and Sahin2024). Although macrophages are the classical host cells of Leishmania, the potential contribution of glial cells to parasite-associated neuroinflammation and local host responses has received limited attention.
Within the CNS, astrocytes are increasingly recognized as immune-responsive cells that contribute to redox homeostasis, cytokine production, antigen presentation and the regulation of local inflammatory responses. In our previous infected bone marrow-derived macrophage–astrocyte co-culture model, the gp63-positive L. infantum population progressively decreased over 72 h, accompanied by increased IFN-γ and IL-6 levels, suggesting that astrocytes may contribute to both parasite regulation and neuroinflammatory signalling (Islek et al., Reference Islek, Ucisik and Sahin2024). Nevertheless, the direct response of astrocytes to L. infantum exposure and the extent to which this response can be pharmacologically modulated remain unclear.
Current therapeutic strategies for leishmaniasis rely primarily on a limited number of chemotherapeutic agents, including pentavalent antimonials, pentamidine, amphotericin B (AmpB) and its lipid-based formulations, miltefosine and paromomycin (Sundar et al., Reference Sundar, Makharia, More, Agrawal, Voss, Fischer, Bachmann and Murray2000, Reference Sundar, Pai, Sahu, Kumar and Murray2002, Reference Sundar, Jha, Thakur, Mishra, Singh and Buffels2003, Reference Sundar, Mehta, Suresh, Singh, Rai and Murray2004; Sundar and Chatterjee, Reference Sundar and Chatterjee2006; Tiuman et al., Reference Tiuman, Santos, Ueda-Nakamura, Filho and Nakamura2011; Sundar and Chakravarty, Reference Sundar and Chakravarty2013). While these treatments remain the cornerstone of clinical management, their effectiveness is increasingly compromised by several critical limitations (Kumari et al., Reference Kumari, Kumar, Samant, Singh and Dube2008; Freitas-Junior et al., Reference Freitas-Junior, Chatelain, Kim and Siqueira-Neto2012; De Menezes et al., Reference De Menezes, Guedes, De Oliveira Almeida Petersen, Fraga and Veras2015). These include significant systemic toxicity due to a narrow therapeutic index, the requirement for prolonged or intravenous administration and the growing emergence of drug-resistant strains.
Collectively, these challenges underscore a pressing need for alternative therapeutic approaches that are not only more effective but also safer and capable of overcoming resistance mechanisms. In this context, the development of novel drug candidates and host-directed or immunomodulatory strategies has emerged as a promising direction in antileishmanial therapy (Kumari et al., Reference Kumari, Kumar, Samant, Singh and Dube2008; Freitas-Junior et al., Reference Freitas-Junior, Chatelain, Kim and Siqueira-Neto2012; De Menezes et al., Reference De Menezes, Guedes, De Oliveira Almeida Petersen, Fraga and Veras2015).
Boron-based compounds have attracted increasing attention because of their antimicrobial, redox-modulating and immunoregulatory properties. Among these compounds, sodium perborate tetrahydrate (SPT) was previously investigated by our group for its antileishmanial activity against L. infantum promastigotes and its biological effects in infected macrophages. SPT inhibited promastigote proliferation and induced mitochondrial dysfunction, cell-cycle arrest and apoptosis-like death in the parasites (Islek et al., Reference Islek, Uraz, Ozeken, Reis, Ucisik, Taskan and Sahin2026). In L. infantum-infected RAW264.7 macrophages, SPT also modulated inflammatory gene expression by increasing IFN-γ expression and downregulating selected anti-inflammatory mediators, including IL-10 and TGF-β1, while preserving the viability of non-infected macrophages. These findings provided evidence for both the direct parasite-targeting and host immunomodulatory activities of SPT. However, its effects on parasite infection, oxidative homeostasis and inflammatory responses in CNS-relevant host cells have not previously been investigated.
The immunomodulatory activity of boron compounds appears to be context-dependent and may involve both pro- and anti-inflammatory responses. Boron has been reported to promote lymphocyte proliferation and increase the expression of TNF-α, IL-6, IL-1β and iNOS in lipopolysaccharide (LPS)-stimulated macrophages, particularly under M1-polarizing conditions (Routray and Ali, Reference Routray and Ali2016). Conversely, other boron derivatives have been shown to suppress nitric oxide (NO) production and inflammatory mediators. This dual redox and immunological activity provides a rationale for investigating SPT in L. infantum-infected astrocytes.
This study aimed to evaluate whether SPT could reduce L. infantum-associated astrocyte infection and regulate inflammatory gene expression in a murine astrocyte-based in vitro model while preserving astrocyte viability. To our knowledge, this is the first study to investigate the antileishmanial and immunomodulatory effects of SPT in L. infantum-infected astrocytes. By integrating dual-fluorescence flow cytometry with reactive oxygen species (ROS), glutathione (GSH) and inflammatory gene-expression analyses, the study examines SPT as a potential modulator of both parasite infection and astrocyte-associated neuroinflammatory responses in a CNS-relevant cellular context.
Materials and methods
Cell lines
Leishmania infantum promastigotes (MHOM/MA/67/ITMAP-263) were kindly provided by Dr Ana Tomás (IBMC, University of Porto, Portugal). The murine astrocyte cell line C8-S (Astrocyte type II clone) was obtained from the American Type Culture Collection (Rockville, MD). L. infantum promastigotes were cultured in Roswell Park Memorial Institute medium (RPMI, 11875093, Invitrogen, Gibco, UK) while murine astrocytes were cultured in Dulbecco’s Modified Eagle Medium (DMEM, 41966-029, Invitrogen, Gibco, UK).
Cell culture studies
Parasite culture
The promastigotes were cultured in RPMI 1640 Glutamax (Gibco) supplemented with 10% (v/v) inactivated fetal bovine serum (iFBS), 50 U mL–1 penicillin, 50 μg mL–1 streptomycin and 20 mM HEPES sodium salt, pH 7.4 (Sigma-Aldrich, Germany) at 25 °C. To ensure the infectivity of parasites in culture, parasites were limited to a maximum of 7 passages in culture flasks. Infection experiments were performed with recently thawed aliquots of virulent parasites. Infective metacyclic promastigotes were obtained from stationary-phase cultures of L. infantum. The promastigotes were collected from the pellet after centrifugation at 1560 × g for 10 min (Islek et al., Reference Islek, Ucisik, Keskin, Sucu, Gomes‐Alves, Tomás, Guzel and Sahin2022a, Reference Islek, Ucisik and Sahin2024).
Astrocyte cell culture
The murine astrocyte cell line C8-S (Astrocyte type II clone) was cultured as a monolayer at 37 °C in a humid environment with 5% CO2. The cell line was grown in DMEM, supplemented with 10% iFBS, 100 U mL–1 of penicillin, 100 μg mL–1 of streptomycin and 2 mM l-glutamine. Cells were maintained in culture by subpassaging every 3 days.
Cell viability assay on astrocytes
The C8-S murine astrocytes were subjected to incubation in the presence of SPT, and cell viability was assessed through the MTS assay. The cells were seeded in 96-well plates at a density of 8000 cells per well and incubated for 24 h. Subsequently, the astrocytes were treated with SPT at various concentrations (25–150 µM) for 24, 48 and 72 h. After the incubation period, the cells were exposed to a solution of 10% (v/v) MTS with glucose and phosphate-buffered saline (PBS) for 1 h, followed by the detection of absorbance at 490 nm using a microplate spectrophotometer (xMark-Biorad, Canada). Cell viability was calculated as the percentage compared to the negative control (NC) culture. Data were analysed with GraphPad Prism 8.01 software.
Quantification of astrocytes infected with L. infantum promastigotes
The infection rate of C8-S astrocytes was quantified by adapting the dual-fluorescence flow-cytometric method previously developed and validated by our group for L. infantum-infected macrophages (Islek et al., Reference Islek, Ucisik and Sahin2022b, Reference Islek, Ucisik and Sahin2024). The same PKH-based staining principle and quadrant-analysis approach were applied, with C8-S astrocytes used as the host-cell population instead of macrophages. Astrocytes and L. infantum promastigotes were labelled with PKH26 and PKH67, respectively, according to the manufacturer’s instructions. Before infection, successful and selective labelling of both cell populations was verified separately by flow cytometry.
PKH26-labelled C8-S astrocytes were seeded in 12-well plates and allowed to adhere for 24 h. PKH67-labelled stationary-phase L. infantum promastigotes were then added at an astrocyte-to-promastigote ratio of 1:10. Following 3 h of co-incubation at 37 °C, non-internalized promastigotes were reduced by successive washing with PBS. Cultures were maintained for an additional 24 h before treatment with SPT at concentrations ranging from 0 to 400 µM. Infection rates were evaluated after 24, 48 and 72 h of SPT treatment.
For flow cytometric analysis, the astrocyte population was first identified according to its FSC-A/SSC-A characteristics to exclude debris and events outside the astrocyte gate. Fluorescence thresholds and quadrant boundaries were established using unstained astrocytes, PKH26-labelled astrocytes and PKH67-labelled promastigotes. PKH67-associated green fluorescence and PKH26-associated red fluorescence were detected using the FITC and PE channels, respectively. Within the gated astrocyte population, PKH26-positive/PKH67-negative events in the lower-right quadrant represented non-infected astrocytes, whereas PKH26-positive/PKH67-positive double-positive events were classified as parasite-associated astrocytes for the flow-cytometric determination of the infection rate. PKH26-negative/PKH67-positive events in the upper-left quadrant were considered residual-free or non-associated promastigote events and were excluded from the infection-rate analysis. The astrocyte infection rate was expressed as the percentage of PKH26-positive/PKH67-positive double-positive events within the gated astrocyte population. At least 20 000 events were acquired for each sample using a CytoFLEX S flow cytometer (Beckman Coulter, USA). Representative fluorescence images were acquired to support the detection of parasite-associated fluorescence in astrocytes using a fluorescence microscope.
Measurement of ROS
Intracellular ROS levels were measured using a 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA)-based assay as previously described (Islek et al., Reference Islek, Uraz, Ozeken, Reis, Ucisik, Taskan and Sahin2026). Briefly, astrocytes were infected with L. infantum promastigotes (1:10 ratio), followed by treatment with SPT (0–200 µM). After incubation, cells were stained with DCFDA, and fluorescence intensity was measured using flow cytometry. Tert-butyl hydroperoxide (TBHP) was used as a positive control (PC). Data were obtained from at least 3 independent experiments.
Determination of oxidative stress by the measurement of GSH level in Leishmania-infected astrocytes
GSH levels were quantified using a DTNB (Ellman’s reagent – 5,5′-dithiobis (2-nitrobenzoic acid))-based colorimetric assay as previously reported (Reis et al., Reference Reis, Orak, Yilmaz, Cimen and Sipahi2021; Islek et al., Reference Islek, Uraz, Ozeken, Reis, Ucisik, Taskan and Sahin2026). Briefly, cell lysates were incubated with DTNB in EDTA buffer, and absorbance was measured at 412 nm. Results were expressed as µmol g–1 protein and performed in triplicate.
Determination of gene-expression level in Leishmania-infected astrocytes
The relative expression levels of the inflammatory, immune-, oxidative stress- and tissue-remodelling-related genes, namely TNF-α, IFN-γ, IL-1β, IL-12B, IL-10, NF-κB, TGF-β1, iNOS, STAT1 and MMP2, were quantified by qPCR in Leishmania-infected astrocytes following SPT treatment.
Briefly, C8-S astrocytes were seeded in 12-well plates (90 000 cells/well) and infected with stationary-phase L. infantum promastigotes at a 1:10 ratio. After 3 h of incubation, non-internalized parasites were removed by washing with PBS. Cells were then incubated for 24 h and treated with SPT (0–200 µM) for an additional 24 h. Total RNA was isolated using TRIzol reagent, and cDNA synthesis was performed using the QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer’s instructions. Gene-specific primers (Supplementary Table 1) were designed using the Primer-BLAST online tool from the National Center for Biotechnology and were synthesized by Sentebiolab (Ankara, Türkiye). qPCR was performed using a SYBR Green PCR kit (Qiagen) on a Bio-Rad real-time PCR system. Relative gene-expression levels were calculated after normalization to housekeeping genes (ATP5 or β-actin) using standard Bio-Rad CFX Manager software.
Statistical analysis
GraphPad Prism Software (version 8.0.1) was used to perform statistical analysis. Error bars represent the standard error of the mean. The data sets were compared using ordinary one-way ANOVA and two-way ANOVA, followed by Tukey’s multiple comparison test. Differences were considered statistically significant at (*) P ≤ 0·05, (**) P ≤ 0·01, (***) P ≤ 0·001 and (****) P ≤ 0·0001. All data were obtained from at least 3 independent experiments.
Results
Determination of cytotoxicity of SPT in astrocytes
Based on the concentration range previously shown to be effective against L. infantum promastigotes (Islek et al., Reference Islek, Uraz, Ozeken, Reis, Ucisik, Taskan and Sahin2026), the cytotoxicity of SPT was first evaluated in non-infected C8-S astrocytes. SPT treatment at concentrations ranging from 25 to 150 µM maintained the viability of non-infected C8-S astrocytes above approximately 90% after 24, 48 and 72 h of incubation. At 72 h, astrocyte viability following treatment with 100 µM SPT was approximately 100% relative to the untreated control. These findings indicated that the SPT concentrations tested for cytotoxicity did not cause significant cytotoxicity in non-infected astrocytes (Figure 1A).
Cytotoxicity of SPT and flow-cytometric determination of L. infantum infection rates in C8-S astrocytes. (A) Cytotoxicity of SPT at concentrations ranging from 25 to 150 μM in C8-S astrocytes after 24, 48 and 72 h of incubation compared with untreated negative control cells (NC). (B–E) Flow-cytometric evaluation of PKH labelling efficiency. Representative plots show (B) unstained and (C) PKH67-labelled L. infantum promastigotes, as well as (D) unstained and (E) PKH26-labelled C8-S astrocytes. (F) Infection rates of C8-S astrocytes infected with L. infantum promastigotes following treatment with SPT at concentrations ranging from 50 to 400 μM for 24, 48 and 72 h, as determined by flow cytometry. Quantitative data represent the results of 3 independent experiments. Statistical significance was determined using two-way ANOVA with Tukey’s multiple-comparison test. *P ≤ 0·05, **P ≤ 0·01, ***P ≤ 0·001 and ****P ≤ 0·0001.
Figure 1 Long description
A bar chart titled, C8-S murine astrocytes. The y-axis label is, Cell viability percent. The x-axis label is, Time h. The x-axis tick labels are 24, 48 and 72. A legend lists: Negative Control, 25 micro M, 50 micro M, 75 micro M, 100 micro M, 125 micro M, 150 micro M and DMSO 20 percent. Each time point contains a cluster of bars for the listed groups. Cell viability remains approximately 90 percent or higher in the SPT-treated groups, whereas markedly lower viability is observed in the DMSO 20 percent group. Significant differences are indicated by asterisks. The image B shows flow cytometry plots with unstained L. infantum promastigotes. Scatter plots show point clouds with gating outlines and histograms display a single peak. The image C shows flow cytometry plots with PKH67-labelled L. infantum promastigotes. Scatter plots show point clouds with gating outlines and histograms display a single peak. The image D shows flow cytometry plots with unstained C8-S astrocytes. Scatter plots show point clouds with gating outlines and histograms display a single peak. The image E shows flow cytometry plots with PKH26-labelled C8-S astrocytes. Scatter plots show point clouds with gating outlines and histograms display a single peak. The image F shows a line graph of infection rates. The y-axis label is, Infection rate of astrocytes percent. The x-axis label is, Concentration micro M. The x-axis categories are NC, 50 micro M SPT, 75 micro M SPT, 100 micro M SPT, 150 micro M SPT, 200 micro M SPT and 400 micro M SPT. A legend lists three series: 24h, 48h and 72h. The 24h series starts at approximately 23 percent in the NC group, remains close to 24 percent at 50 micro M SPT, and then decreases progressively, reaching its lowest value at approximately 200 micro M SPT before increasing slightly at 400 micro M SPT. The 48h series remains low across all groups. The 72h series decreases from approximately 13 percent in the NC group to its lowest value at 75 micro M SPT and remains below the NC level at higher SPT concentrations. Multiple asterisk annotations indicate statistical significance.
Antileishmanial effect of SPT on astrocytes infected by L. infantum promastigotes by flow cytometry
Prior to infection, C8-S astrocytes and L. infantum promastigotes were selectively labelled with PKH26 and PKH67, respectively, according to the previously established dual-fluorescence flow-cytometric method (Islek et al., Reference Islek, Ucisik and Sahin2022b). The labelling efficiency of each cell population was evaluated separately before infection using unstained and single-labelled controls. Flow-cytometric analysis confirmed that 91·78% of the promastigote population was successfully labelled with PKH67, whereas 99·67% of the astrocyte population was labelled with PKH26. The corresponding unstained controls showed negligible fluorescence, confirming the selective labelling of both populations (Figure 1B-E).
The astrocyte infection rate was subsequently quantified by adapting the same dual-fluorescence approach previously developed for L. infantum-infected macrophages, with C8-S astrocytes used as the host-cell population instead of macrophages (Islek et al., Reference Islek, Ucisik and Sahin2022b). In the original method, host cells and parasites were selectively labelled with PKH26 and PKH67, respectively, and the double-positive population was used to identify infected host cells. The method was validated using unstained and single-labelled controls, quadrant-based discrimination of infected and non-infected host cells, successive washing to remove non-internalized parasites and parallel fluorescence microscopy analysis. In the present study, the same staining and analytical principle was adapted to C8-S astrocytes.
As illustrated in Figure 2, astrocyte-associated events were first selected according to their FSC-A/SSC-A characteristics, thereby excluding debris and events outside the astrocyte gate. PKH67-associated green fluorescence and PKH26-associated red fluorescence were then evaluated using the FITC and PE channels, respectively. In the final FITC/PE quadrant analysis, PKH26-positive/PKH67-positive double-positive events in the upper-right quadrant were classified as parasite-associated astrocytes for the flow-cytometric determination of the infection rate. PKH26-positive/PKH67-negative events in the lower-right quadrant represented non-infected astrocytes, whereas PKH26-negative/PKH67-positive events in the upper-left quadrant were considered residual free or non-associated promastigote events and were excluded from the infection-rate analysis. Representative flow-cytometric plots illustrating the gating strategy and identification of the double-positive infected astrocyte population are shown in Figure 2, Supplementary Figures 1 and 2. Quantitative infection-rate data presented in Figure 1F were calculated from 3 independent experiments.
Representative dual-fluorescence flow-cytometric analysis of L. infantum-infected C8-S astrocytes following SPT treatment. C8-S astrocytes and L. infantum promastigotes were labelled with PKH26 and PKH67, respectively. Representative plots are shown for (A) unstained astrocytes, (B) PKH26-labelled non-infected astrocytes, (C) untreated L. infantum-infected astrocytes, and infected astrocytes treated with (D) 100 µM, (E) 200 µM or (F) 400 µM SPT.
Figure 2 Long description
Six sets of scatter plots labeled A to F, each representing flow cytometry analysis of C8-S astrocytes and L. infantum promastigotes. Each set includes four plots: FSC-A vs SSC-A, FSC-A vs PE-A, FSC-A vs FITC-A and FITC-A vs PE-A. The x axis for FSC-A and SSC-A ranges from 0 to 250K, while PE-A and FITC-A also range from 0 to 250K. In each set, the first plot shows the gating of astrocyte-associated events based on FSC-A and SSC-A characteristics. The second and third plots display the gating of PKH26 and PKH67 fluorescence using PE-A and FITC-A channels, respectively. The fourth plot presents quadrant analysis of FITC-A vs PE-A, indicating PKH26-positive/PKH67-positive double-positive events classified as parasite-associated astrocytes. Key values for quadrant percentages are: A) 1.12, B) 0.78, C) 11.46, D) 7.99, E) 7.48, F) 7.27. The plots show variations in infection rates across different conditions: A) unstained astrocytes, B) PKH26-labelled non-infected astrocytes, C) untreated infected astrocytes, D) 100 microM SPT treatment, E) 200 microM SPT treatment, F) 400 microM SPT treatment. The colored gates and points represent different populations and events, highlighting changes in infection rates and treatment effects.
Antileishmanial activity of SPT was investigated on Leishmania-infected astrocytes for concentrations of 50, 75, 100, 150, 200 and 400 µM (Figures 1F and 2). At 24 h, the mean infection rate in the untreated infected astrocyte group was approximately 23%. Treatment with 75, 100, 150, 200 and 400 µM SPT significantly reduced the infection rate to 20·00 ± 3·33% (P ≤ 0·05), 17·00 ± 2·86% (P ≤ 0·0001), 11·89 ± 3·56% (P ≤ 0·0001), 7·97 ± 1·51% (P ≤ 0·0001) and 12·63 ± 1·78% (P ≤ 0·0001), respectively (Figure 1F and Supplementary Figure 1). The greatest reduction at this time point was observed following treatment with 200 µM SPT.
At 48 h, the infection rate in the untreated infected group decreased to 2·91% and remained close to 3% across the SPT-treated groups, irrespective of further increases in SPT concentration (Figure 1F and Supplementary Figure 2). At 72 h, the infection rate was approximately 13% in the untreated infected group. Treatment with 75 µM SPT significantly reduced the infection rate to 3·75% (P ≤ 0·0001), whereas infection rates of approximately 7% were observed following treatment with 100, 150, 200 and 400 µM SPT (Figure 1F). Overall, the results obtained in cell culture analysis demonstrated that SPT concentrations ≥75 µM exerted a significant antileishmanial effect on Leishmania-infected astrocytes, while concentrations within the cytotoxicity-tested range (25–150 µM) did not cause significant toxicity in non-infected astrocytes.
Morphological analysis of astrocytes infected with L. infantum promastigotes phase-contrast microscopy analysis
Phase-contrast microscopy was used to visualize the morphological changes of Leishmania-infected astrocytes (infection ratio: 1:10) mediated by SPT treatment, as shown in Figure 3. The number of Leishmania-infected C8-S astrocytes was observed to decrease after SPT treatment, during which the surface morphology and attachment of cells changed dramatically. The star-shaped structure of astrocytes, formed by tendrils that extend from the cell, has transformed into a round, spherical character, and cells departed from each other because of this transformation in morphology (Figure 3).
Morphological evaluation of L. infantum-infected C8-S astrocytes following SPT treatment. Representative phase-contrast microscopy images of (A) untreated L. infantum-infected astrocytes and infected astrocytes treated with (B) 75 µM, (C) 100 µM and (D) 200 µM SPT after 24, 48 and 72 h of incubation. Scale bars represent 50 μm.
Fluorescence microscopy analysis of Leishmania-infected astrocytes treated with SPT
In parallel with the flow-cytometric analysis, PKH26-labelled C8-S astrocytes and PKH67-labelled L. infantum promastigotes were examined by fluorescence microscopy using the red, green and merged fluorescence channels (Figure 4). Untreated parasite-exposed cultures showed PKH26-positive astrocytes, PKH67-positive promastigote-associated signals and areas of overlapping red and green fluorescence. Isolated green fluorescent signals were also observed and were interpreted as free or non-associated promastigotes.
Representative fluorescence microscopy images of PKH-labelled C8-S astrocytes exposed to L. infantum promastigotes following SPT treatment. C8-S astrocytes were labelled with PKH26 (red), whereas L. infantum promastigotes were labelled with PKH67 (green). Representative images are shown for (A) untreated infected astrocytes and infected astrocytes treated with 100 µM SPT for (B) 24 h and (C) 48 h (magnification: 20×). Scale bars correspond to 50 μm.
Figure 4A shows representative images of untreated parasite-exposed astrocyte cultures, whereas Figure 4B and C shows infected astrocytes treated with 100 µM SPT for 24 and 48 h, respectively. Following SPT treatment, the frequency and intensity of PKH67-associated green fluorescent signals appeared lower at 48 h than at 24 h. These representative images qualitatively support the flow-cytometric findings indicating a reduction in parasite-associated fluorescence after SPT treatment.
ROS measurement in Leishmania-infected astrocytes after treatment with SPT
All tested SPT concentrations, with the exception of 100 µM, resulted in a significant reduction in intracellular ROS levels in astrocytes at 24 h, as indicated by decreased DCF fluorescence intensity measured by flow cytometry. In all treated groups, ROS levels were reduced to below 30% relative to the NC (Figure 5). The treatment of Leishmania-infected astrocytes with 50, 75, 150 and 200 µM SPT for 24 h resulted in a significant decrease close to 28% (i.e., 28·24%) (P ≤ 0·01), 22% (i.e., 21·83%) (P ≤ 0·001), 29% (i.e., 29·22%) (P ≤ 0·01) and 18% (i.e., 17·85%) (P ≤ 0·0001), respectively. In parallel, exposure to the PC for 1 h markedly increased intracellular ROS levels, reaching approximately 84%, as demonstrated by flow cytometry histograms and the corresponding relative ROS (%) graph (P ≤ 0·0001) (Figure 5A–C).
Intracellular ROS level measurement in Leishmania-infected astrocytes after 24 h of SPT at different concentrations (50–200 µM). (A) Representative histograms of ROS accumulation (%) in SPT treatment groups. (B) Bar graph showing the mean relative ROS (%) of 3 independent experiments. (C) Overlay histogram analysis of untreated infected astrocytes without DCFDA (green), untreated infected astrocytes with DCFDA (NC) (dark blue), positive control (PC) TBHP (100 µM) (black), and SPT treatment groups including 50 µM (red), 75 µM (yellow), 100 µM (light blue), 150 µM (pink) and 200 µM (dark yellow) at 24 h of exposure. Quantitative data represent the results of 3 independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s multiple-comparison test. *P ≤ 0·05, **P ≤ 0·01, ***P ≤ 0·001 and ****P ≤ 0·0001.
Figure 5 Long description
Eight histogram plots titled, NC (w/o DCF), NC, PC, 50 micro-M SPT, 75 micro-M SPT, 100 micro-M SPT, 150 micro-M SPT and 200 micro-M SPT. Each histogram uses the horizontal axis label, FL1-Height, with tick labels 10 to the power of 0, 10 to the power of 1, 10 to the power of 2, 10 to the power of 3 and 10 to the power of 4. Each histogram shows a single filled distribution with a main peak near the lower end of the FL1-Height scale and a tail extending toward higher FL1-Height values. The PC histogram shows the distribution shifted toward higher FL1-Height values compared with NC. The SPT histograms show distributions generally closer to the lower FL1-Height range than PC, with visible differences in peak position and tail length across 50 micro-M, 75 micro-M, 100 micro-M, 150 micro-M and 200 micro-M SPT. The image B showing a bar graph with the vertical axis label, Relative ROS level percent, with values ranging from 0 to 100. The horizontal axis label reads, Time (hour), and the bars represent measurements obtained after 24 h of exposure. A legend lists seven groups: NC, PC, 50 micro-M, 75 micro-M, 100 micro-M, 150 micro-M and 200 micro-M. The PC bar is the tallest among the groups. Relative ROS levels are lower in the 50, 75, 150 and 200 micro-M SPT groups compared with NC, whereas the 100 micro-M SPT group does not show a reduction relative to NC. Brackets and asterisk markers indicate statistically significant differences between groups. The image C showing an overlaid histogram plot with the horizontal axis label, FL1-Height and tick labels 10 to the power of 0, 10 to the power of 1, 10 to the power of 2, 10 to the power of 3 and 10 to the power of 4. Multiple line traces are overlaid, corresponding to untreated infected astrocytes without DCFDA, untreated infected astrocytes with DCFDA (NC), positive control (PC) TBHP (100 micro-M) and SPT treatment groups 50 micro-M, 75 micro-M, 100 micro-M, 150 micro-M and 200 micro-M. The overlaid traces show the PC shifted toward higher FL1-Height values compared with the other groups, whereas the SPT-treated groups generally show distributions closer to the lower FL1-Height range. The bar graph in image B summarizes the relative ROS differences represented by the histogram distributions in image A and the overlaid comparison in image C.
Assessment of oxidative stress via GSH level measurement in SPT-treated Leishmania-infected astrocytes
The antioxidant capacity of SPT was investigated in Leishmania-infected astrocytes by the measurement of GSH levels in the untreated and treated cells. According to the results, 50, 100, 150 and 200 µM SPT led to a significant decrease in oxidative stress by increasing GSH levels compared with NC (Figure 6). However, 75 µM SPT did not cause a significant increase in GSH levels.
Effect of SPT treatment on intracellular GSH levels in L. infantum-infected C8-S astrocytes. Intracellular GSH levels were quantified to evaluate the oxidative-stress response in infected astrocytes following 24 h of SPT treatment at the indicated concentrations. Data represent the results of 3 independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s multiple-comparison test. *P ≤ 0·05, **P ≤ 0·01, ***P ≤ 0·001 and ****P ≤ 0·0001.
Gene-expression level of Leishmania-infected astrocytes after SPT treatment
Following treatment of Leishmania-infected astrocytes with SPT at concentrations of 50, 75, 100, 150 and 200 µM, the relative expression levels of TNF-α, IFN-γ, IL-1β, IL-12B, MMP2, IL-10, NF-κB, TGF-β1, iNOS and STAT1 (normalized to β-actin or ATP5) were evaluated by real-time PCR after 24 h (Supplementary Table 1).
As shown in Figure 7, qPCR analysis indicated that IFN-γ levels were significantly overexpressed (1.33-fold) in only 100 µM SPT treatment on infected astrocytes (P ≤ 0·05) (Figure 7A), when compared to the NC group. On the other hand, 200 µM SPT treatment significantly increased TNF-α gene-expression level (1.34-fold) in the infected astrocytes (P ≤ 0·001), whereas 50, 75, 100 and 150 µM SPT treatment led to a significant decrease compared to the NC group (Figure 7B). Contrary to TNF-α gene expression, the gene-expression level of IL-12B was significantly upregulated in infected astrocytes treated with 100 µM (3·45-fold increase) and 150 µM SPT (3·08-fold increase) (P ≤ 0·0001) (Figure 7C), while treatment of 50, 75 and 200 µM SPT caused a significant decrease in IL-12B gene expression. In contrast, NF-κB gene expression did not differ significantly between the SPT-treated groups and the NC group in infected astrocytes (Figure 7D). A significant 3·81-fold increase in IL-1β expression was observed only in infected astrocytes treated with 150 µM SPT (P ≤ 0·01) (Figure 7E). Besides, the gene-expression levels of IL-10 were downregulated upon treatment of 50, 100, 150 and 200 µM SPT in infected astrocytes compared to NC (Figure 7F). STAT1 gene expression increased (not significantly) after SPT treatment, when compared to NC (Figure 7G); however, iNOS, MMP2 and TGF-β1 gene expressions did not show any significant alterations after SPT treatment in infected astrocytes (Figure 7H–J).
Effect of SPT treatment on immune-, inflammatory-, and oxidative stress-related gene expression in L. infantum-infected C8-S astrocytes. Relative gene-expression levels of (A) IFN-γ, (B) TNF-α, (C) IL-12B, (D) NF-κB, (E) IL-1β, (F) IL-10, (G) STAT1, (H) iNOS, (I) MMP2, and (J) TGF-β1 were determined in infected C8-S astrocytes following treatment with 0-200 µM SPT for 24 h. Gene-expression levels were calculated relative to the untreated infected control group. Data represent the results of three independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s multiple-comparison test. *P ≤ 0·05, **P ≤ 0·01, ***P ≤ 0·001 and ****P ≤ 0·0001.
Figure 7 Long description
Ten bar graphs depicting relative gene-expression levels in L. infantum-infected C8-S astrocytes after 24 hours of SPT treatment. Each graph represents a different gene: IFN gamma, TNF alpha, IL 12B, NF kappa B, IL 1 beta, IL 10, STAT1, iNOS, MMP2 and TGF beta 1. The x axis for all graphs is labeled, 24H, and the y axis represents relative gene-expression levels. Panels A to G include Control, 50 micro M SPT, 75 micro M SPT, 100 micro M SPT, 150 micro M SPT and 200 micro M SPT groups. Panels H, I and J include Control, 50 micro M SPT, 75 micro M SPT, 100 micro M SPT and 150 micro M SPT groups. Error bars and significance markers are shown where applicable. IFN gamma shows increased expression at 100 micro M SPT. TNF alpha shows increased expression at 200 micro M SPT and lower expression at 50, 75, 100 and 150 micro M SPT compared with Control. IL 12B shows increased expression at 100 and 150 micro M SPT and lower expression at 50, 75 and 200 micro M SPT. NF kappa B shows little variation across treatment groups. IL 1 beta shows increased expression at 150 micro M SPT. IL 10 shows lower expression in several SPT-treated groups compared with Control. STAT1 shows a slight increase following SPT treatment. iNOS, MMP2 and TGF beta 1 show relatively small changes across the concentrations presented.
Discussion
Previous clinical and experimental studies have indicated that Leishmania species may reach CNS-associated compartments and contribute to neurological and inflammatory manifestations. Anti-Leishmania antibodies, parasites and inflammatory alterations have been reported in the CNS or cerebrospinal fluid in canine and experimental models, supporting the biological relevance of cerebral involvement during leishmaniasis (Abreu-Silva et al., Reference Abreu-Silva, Calabrese, Tedesco, Mortara and Gonçalves Da Costa2003; Font et al., Reference Font, Mascort, Altimira, Closa and Vilafranca2004; Ikeda et al., Reference Ikeda, Laurenti, Corbett, Feitosa, Machado and Perri2007). In previous studies, the presence of anti-Leishmania antibodies was detected in the CSF of Leishmania-positive dogs, which could lead to alterations in the nervous tissue and may result in a disease known as ‘cerebral leishmaniasis’ (Melo et al., Reference Melo, Seraguci, Schweigert, Silva, Grano, Peiró, Lima and Machado2013, Reference Melo, Goyard, Fiette, Boissonnas, Combadiere, Machado, Minoprio and Lang2017; Islek et al., Reference Islek, Ucisik and Sahin2024). Moreover, Melo et al. (Reference Melo, Goyard, Fiette, Boissonnas, Combadiere, Machado, Minoprio and Lang2017) reported the presence of Leishmania donovani parasites in the brain and brain inflammation in experimental visceral leishmaniasis, which demonstrated a remarkable process of inflammatory response, including cytokine and chemokine release and inflammatory cells (e.g., macrophages) in the CNS (Melo et al., Reference Melo, Goyard, Fiette, Boissonnas, Combadiere, Machado, Minoprio and Lang2017). Also, it was revealed that the parasite burden in the brain of L. donovani-infected mice was estimated at between 900 and 12 000, especially in the choroid plexus, meninges and cerebrospinal fluid in humans and dogs, considering the immune-privileged blood–brain barrier and immune barrier of the nervous system (Melo et al., Reference Melo, Goyard, Fiette, Boissonnas, Combadiere, Machado, Minoprio and Lang2017).
Astrocytes are abundant glial cells in the CNS and play important roles in tissue homeostasis, redox regulation, phagocytic activity and cytokine production. In response to infection, injury or neurodegenerative stimuli, astrocytes may acquire a reactive phenotype accompanied by morphological and transcriptional changes. Activated astrocytes can express inflammatory mediators, including IL-1β, IL-6, TNF-α, CCL2, COX-2 and iNOS, thereby contributing to local immune and neuroinflammatory responses (Lu et al., Reference Lu, He, Zhang, Xu, Zhang, He, Chen, Liu, Pi, Yu and Zhou2014). These properties make astrocytes a relevant cellular model for investigating host responses during CNS-associated Leishmania infection.
The present study provides evidence that SPT significantly reduces the flow-cytometrically determined L. infantum infection rate in astrocytes and modulates inflammatory responses. The dual-PKH approach used here was previously developed and validated by our group in L. infantum-infected macrophages and published in Parasitology (Islek et al., Reference Islek, Ucisik and Sahin2022b). In addition, parasite-specific anti-gp63 intracellular staining in our previously published astrocyte-infected macrophage co-culture study demonstrated time-dependent changes in the Leishmania population at 24, 48 and 72 h (Islek et al., Reference Islek, Ucisik and Sahin2024). These studies provide complementary methodological and biological support for the present analytical strategy, although the gp63 experiment was performed in a related but distinct co-culture model and is not presented as direct proof of internalization in the current C8-S astrocyte model.
To our knowledge, this is the first study to investigate the antileishmanial and immunomodulatory effects of SPT in L. infantum-infected astrocytes. The findings provide in vitro evidence that SPT reduces the flow-cytometrically determined infection rate while modulating oxidative and inflammatory responses. The biological relevance of boron-containing compounds in cellular stress regulation has also been supported by previous studies in an in vitro amyloid-β toxicity model (Ozansoy et al., Reference Ozansoy, Altintaş, Ozansoy, Günay, Kiliç and Kiliç2020).
Antileishmanial activity of SPT against L. infantum promastigotes and its cytotoxicity profile in astrocytes
SPT was previously shown to exert significant antileishmanial activity against L. infantum promastigotes while preserving the viability of non-infected macrophages (Islek et al., Reference Islek, Uraz, Ozeken, Reis, Ucisik, Taskan and Sahin2026). In that study, the most pronounced inhibition of promastigote proliferation was observed within the 75–150 µM concentration range. The biological effects of SPT were also characterized in detail, including its effects on parasite viability, mitochondrial membrane potential, cell-cycle progression, apoptosis-like death, oxidative status and inflammatory responses in infected macrophages. These findings suggest that, as a peroxide-releasing compound, SPT may exert its antileishmanial activity through a combined mechanism involving direct effects on the parasite and modulation of host-cell redox and inflammatory responses. Consistent with its previously observed safety profile in macrophages, the present study showed that SPT preserved the viability of non-infected C8-S astrocytes across the cytotoxicity-tested concentration range, supporting its subsequent evaluation in the infected astrocyte model.
Antileishmanial activity of SPT in L. infantum promastigote-infected astrocytes
Flow cytometric analysis demonstrated that SPT treatment significantly reduced the proportion of PKH26-positive/PKH67-positive astrocytes classified as parasite-associated astrocytes, particularly at 24 h. Although the response was not strictly concentration-dependent, SPT produced a significant antileishmanial effect at concentrations of 75 µM and above at 24 h. These results support the antileishmanial effect of SPT, as evidenced by the significant reduction in PKH26-positive/PKH67-positive astrocytes determined by flow cytometry.
The relatively low infection rate observed in C8-S astrocytes may be related to their limited phagocytic capacity and comparatively lower expression of phagocytosis-associated surface receptors. It may also reflect the immune-responsive and potentially parasite-restrictive properties of astrocytes, which is consistent with our previous study (Islek et al., Reference Islek, Ucisik and Sahin2024). In the previous astrocyte-infected macrophage co-culture study, a time-dependent decrease in the gp63-FITC-positive L. infantum population was accompanied by increased IFN-γ and IL-6 secretion (Islek et al., Reference Islek, Ucisik and Sahin2024). These findings provide relevant biological context suggesting that astrocytes are not merely passive cellular components and may contribute to regulation of the parasite population. Moreover, in this study, the significant reduction observed following SPT treatment compared with the untreated infected group clearly supports the antileishmanial activity of SPT in this model.
The absence of a proportional improvement at the highest concentrations may indicate that the maximum effective response was achieved within the intermediate concentration range. Importantly, SPT significantly decreased the proportion of parasite-associated astrocytes while preserving the viability of non-infected astrocytes within the cytotoxicity-tested concentration range.
Overall, these findings demonstrate that SPT exerts a pronounced antileishmanial effect in L. infantum-infected astrocytes and may represent a promising candidate for limiting parasite infection without causing marked host-cell toxicity. Further studies using differential extracellular/intracellular immunostaining or confocal microscopy are required to distinguish internalized parasites from surface-associated parasites and to clarify whether SPT acts through direct parasiticidal effects, inhibition of parasite replication or modulation of host-cell responses.
ROS levels and oxidative stress in Leishmania-infected astrocytes following SPT treatment
All SPT concentrations, except 100 µM, resulted in a significant reduction in intracellular ROS levels in astrocytes at 24 h, as determined by DCF fluorescence intensity using flow cytometry. In all treated groups, ROS levels were reduced to below 30% compared to the NC. In a previous study, reduced mitochondrial ROS production was associated with a decrease in intracellular Trypanosoma cruzi infection and inhibition of parasite multiplication in astrocytes primed with extracellular vesicles derived from T. cruzi trypomastigotes (Urquiza et al., Reference Urquiza, Cevallos, Elizalde, Delpino and Quarleri2020). T. cruzi expresses antioxidant enzymes that protect the parasite against oxidative damage, thereby facilitating its survival and persistence under oxidative stress within host cells. Similarly, although macrophage-derived ROS plays an important role in reducing the intracellular Leishmania burden, this antimicrobial response is closely associated with T cell-mediated immune activation. In particular, cytokines released by activated T cells, such as IFN-γ, can activate macrophages and enhance ROS-dependent antimicrobial activity. However, Leishmania parasites are continuously exposed to oxidative stress during the intracellular stage of their life cycle and possess antioxidant defence mechanisms that enable them to tolerate host-derived ROS. Under certain conditions, ROS may therefore contribute not only to parasite elimination but also to parasite adaptation and persistence (Urquiza et al., Reference Urquiza, Cevallos, Elizalde, Delpino and Quarleri2020).
On the other hand, another study reported that the induction of ROS production controls lesion sizes at early stages of infection rather than parasite killing; they control the inflammatory reaction, possibly by regulating neutrophil numbers in lesions (Roma et al., Reference Roma, Macedo, Goes, Gonçalves, Castro, Cisalpino and Vieira2016).
In parallel with the determination of ROS levels, the antioxidant capacity of SPT was investigated in Leishmania-infected astrocytes by the measurement of GSH levels in untreated and treated cells. According to the results, 50, 100, 150 and 200 µM SPT led to a significant decrease in oxidative stress by increasing GSH levels significantly when compared to NC. Therefore, Leishmania infection-induced oxidative stress was remarkably reduced in infected astrocytes by SPT treatment through the induction of elevated GSH levels.
The simultaneous reduction in intracellular ROS levels and astrocyte infection rate suggests that the antileishmanial activity of SPT may not primarily depend on ROS-mediated parasite killing. Instead, SPT may exert a direct effect on the parasite while simultaneously restoring redox homeostasis in infected astrocytes. This interpretation is supported by our previous findings showing that SPT impaired L. infantum proliferation, mitochondrial function and cell-cycle progression. Therefore, the reduction in ROS observed in the present study may reflect attenuation of infection-associated oxidative stress rather than suppression of an essential antiparasitic defence mechanism. The concomitant increase in intracellular GSH levels further supports the redox-regulating effect of SPT.
qPCR analysis of Leishmania-infected astrocytes after SPT treatment
qPCR analysis indicated that both IFN-γ and TNF-α genes were significantly overexpressed in 100 and 200 µM SPT treatment on infected astrocytes, respectively, when compared to the NC group. In the previous study, Chung and Benveniste (Reference Chung and Benveniste1990) reported that astrocytes have the capacity to release TNF-α in response to biologic stimuli such as infection, especially cytokines IL-1 and IFN-γ, which play a critical role in the CNS during neurodegeneration associated with inflammation (Chung and Benveniste, Reference Chung and Benveniste1990). Similarly, in another study using an in vitro model of acute neuroinflammation, cerebellar granule neurons were cultured in IFN-γ-containing medium in the absence of astrocytes, resulting in the induction of quick neuronal apoptosis characterized by the expression of cleaved caspase-3, and intercellular Ca2+ influx. In contrast, the presence of astrocytes exerted a protective effect through IFN-γ-induced astrocytic IL-6 secretion (Sun et al., Reference Sun, Li, Jia, Wang, Li, Hu, Tian, Yang, Xing, Zhang, Wang, Xu, Wang, Zhang and Ren2017).
On the other hand, the gene-expression level of IL-12B was significantly upregulated in infected astrocytes treated with 100 µM (3·45-fold increase) and 150 µM SPT (3·08-fold increase). Interestingly, treatment with 200 µM SPT caused a significant decrease in IL-12B gene expression. This could be explained by the fact that SPT modulated IL-12B transcript levels on Leishmania-infected macrophages, resulting in the production of an immune response and the development of Th1-type immune responses. For instance, after infection, astrocytes have been indicated to secrete cytokines, such as IFN-γ, TNF-α and IL-12B, that alter microglia and macrophages to a more pro-inflammatory phenotype (Soung and Klein, Reference Soung and Klein2020). However, under similar circumstances, astrocytes have also been found to significantly generate cytokines, including TGF-β and IL-10, which can cause monocytes to reduce their inflammation (Soung and Klein, Reference Soung and Klein2020). In line with the literature, as expected, the gene-expression levels of IL-10 were downregulated upon treatment of 50, 100, 150 and 200 µM SPT in infected astrocytes compared to the NC group, whereas TGF-β1 gene expression did not show any significant alterations after SPT treatment in infected astrocytes.
The pro-inflammatory cytokine IL-1β gene was overexpressed (3·81-fold increase) in infected astrocytes treated with only 150 µM SPT (P ≤ 0·01), and all treatment groups except 150 µM SPT did not lead to significant alterations in IL-1β gene expression in infected astrocytes, when compared to the NC group. IL-1β gene is a crucial regulator of inflammation and neuronal death in acute CNS traumas, including stroke and brain trauma (Chen et al., Reference Chen, Hu and Ju2020; Adamu et al., Reference Adamu, Li, Gao and Xue2024), and has been linked to neurodegenerative diseases like Alzheimer’s disease (Clayton et al., Reference Clayton, Van Enoo and Ikezu2017; Adamu et al., Reference Adamu, Li, Gao and Xue2024). It has been proposed that factors secreted from IL-1β-activated astrocytes cause primary human neurons to undergo apoptosis and undergo significant alterations in their metabolic activity (Deshpande et al., Reference Deshpande, Zheng, Borgmann, Persidsky, Wu, Schellpeper and Ghorpade2005; Lawrence et al., Reference Lawrence, Schardien, Wigdahl and Nonnemacher2023). Besides, Scorei and his co-workers have reported that a boron derivative, calcium fructoborate treatment, has decreased IL-1β and IL-6 protein expression and NO levels in the culture supernatant (Scorei et al., Reference Scorei, Ciubar, Ciofrangeanu, Mitran, Cimpean and Iordachescu2008). Therefore, no significant alteration in IL-1β gene expression could be explained by the effect of SPT treatment in the case of astrocyte infection by Leishmania parasites. Similarly, STAT1, iNOS and MMP2 expressions did not show any significant alterations after SPT treatment in infected astrocytes, when compared to the NC group, which could be linked to the SPT-mediated dual role in both inflammatory and anti-inflammatory responses, together. The previous study indicated that both boric acid and sodium borate suppress the critical inflammatory mediator genes, iNOS and COX-2, and reduce NO production levels in a dose-dependent manner (Demirci et al., Reference Demirci, Doğan, Aydın, Dülger and Şahin2016). On the other hand, boron (borax orally) could increase lymphocyte proliferation and further induce TNF-α, IL-6, IL-1β secretion and iNOS expression by the LPS-primed macrophages, more specifically the M1 macrophages, in a dose-dependent manner (Routray and Ali, Reference Routray and Ali2016).
In light of our previous findings (Islek et al., Reference Islek, Uraz, Ozeken, Reis, Ucisik, Taskan and Sahin2026), the present results further support the antileishmanial and immunomodulatory activity of SPT in Leishmania-infected astrocytes while preserving the viability of non-infected astrocytes. The observed regulation of both pro- and anti-inflammatory mediators suggests that SPT may contribute to a coordinated host-cell response associated with the reduction in astrocyte infection rate.
Although the qPCR findings provide evidence of SPT-associated regulation of inflammatory mediators, the present study was limited to transcriptional analysis. Therefore, the observed changes in IL-12B, TNF-α and IL-10 expression should be interpreted at the mRNA level. Further validation of these mediators at the protein level will be important to confirm the inflammatory response associated with SPT treatment.
Conclusion
This study demonstrated that SPT significantly reduced the flow-cytometrically determined L. infantum infection rate in astrocytes while preserving the viability of non-infected C8-S cells within the cytotoxicity-tested concentration range. SPT treatment was also associated with reduced intracellular ROS levels, increased GSH levels and modulation of inflammatory gene expression, suggesting combined antileishmanial, redox-regulating and immunomodulatory effects. To our knowledge, this is the first study to demonstrate the antileishmanial activity of SPT in L. infantum-infected astrocytes together with its modulatory effects on astrocyte-associated inflammatory and redox responses. The promising neuroinflammatory-based antileishmanial efficacy of the SPT in vitro Leishmania-infection model highlights the potential of antileishmanial therapy in future in vivo studies and clinical studies.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0031182026102868.
Data availability statement
The authors do not have permission to share data.
Acknowledgements
We would like to thank Burçin Asutay for her support in flow cytometry studies. During the preparation of this manuscript, the authors used ChatGPT (OpenAI; accessed 18 August 2026) solely for language editing and improvement of readability. The authors subsequently reviewed and edited the content and take full responsibility for the final version of the manuscript.
Author contributions
F.S. constructed the study, provided research esources, and supervised the study. Z.I. and M.H.U. designed the framework of cell culture studies. Z.I. performed and coordinated the cell culture studies and analysed and interpreted the resulting data. H.I.O. and H.U. contributed to the cell viability and infection studies. E.T. contributed to the qPCR studies. Z.I. and R.R. carried out the glutathione analysis. Z.I. was actively involved in writing the manuscript. All authors were involved in revising the manuscript and approved its final version.
Financial support
This study was supported by the Department of Genetics and Bioengineering at Yeditepe University.
Competing interests
Based on the findings of this study, a patent application was filed with the Turkish Patent and Trademark Office (TR2023001878A1) and subsequently published as an international patent application (WO2024177602A1) and a European patent application (EP4633643A1). Apart from this, the authors declare no other competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Ethical standards
Not applicable.
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