Purinergic system dysregulation and depression: The neuroinflammatory impact of silica nanoparticles

Abstract

Major depressive disorder (MDD) is a neuropsychiatric condition linked to neurotransmitter imbalances, neuroinflammation, and purinergic signaling dysregulation. Emerging evidence suggests that environmental pollutants, such as silica nanoparticles (SiNPs), contribute to neuroinflammatory responses and depressive-like behaviors, though the mechanisms remain unclear. This study investigates the effects of repeated SiNP exposure on depressive-like behaviors and purinergic signaling in the hippocampus of adult male rats. Thirty-six Wistar rats were divided into three groups (control, low-dose SiNP, high-dose SiNP) and received intraperitoneal injections for 28 days. Depressive-like behaviors were assessed using the Forced Swimming Test (FST) and Tail Suspension Test (TST), while the enzymatic activities of ectonucleotidases (E-NTPDase, E-NPP, and ecto-5′-nucleotidase) and ATPase function were measured in hippocampal tissue. Gene expression of purinergic receptors (A2A, P2X7, P2Y2) and ectonucleotidases (CD73, NTPDase 1–3) was analyzed via RT-qPCR, with immunohistochemistry and immunofluorescence assessing CD73 and CD90 protein levels. SiNP exposure significantly increased immobility time in both behavioral tests, indicating depressive-like behavior. It also upregulated ectonucleotidase activity, purinergic receptors (A2A, P2X7, P2Y2), and CD73/CD90 expression, while disrupting ATPase function by decreasing both Na⁺/K⁺-ATPase and Ca²⁺-ATPase activities. These findings suggest that SiNPs induce depressive-like behavior through purinergic pathway dysregulation, promoting neuroinflammation and neurotransmission alterations. Further studies are needed to explore purinergic signaling as a potential therapeutic target in depression.

Introduction

Depression is a mood disorder characterized by symptoms such as hopelessness, irritability, anxiety, feelings of guilt, and loss of motivation [1]. Recent advances in the understanding of major depressive disorder (MDD) have highlighted the role of inflammatory processes and immune responses in its development [2-4]. The World Health Organization (WHO) predicted that depression would become the second most common illness globally by 2020 [1].

Depression is commonly accompanied by biochemical disturbances in neurotransmission [5]. Evidence suggests that individuals suffering from depression have an increased risk of developing neurodegenerative diseases such as Parkinson's and Alzheimer's [6]. Additionally, untreated depression has been associated with various disorders, including diabetes, stroke, obesity, and inflammatory diseases [7-9].

ATP and adenosine are known to influence a broad spectrum of physiological functions, particularly neurotransmission and neuromodulation [10]. Adenosinergic signaling plays a critical role in neurodevelopmental and pathophysiological processes, including mood disorders, inflammation, differentiation, and cell proliferation [11]. The purinergic system is composed of two receptor families: P1 receptors, which are activated by adenosine, and P2 receptors, which are activated by ATP [12]. Extracellular levels of adenosine and ATP are regulated by the balance between their release from cells and the enzymatic activity of ectonucleotidases. Ectonucleoside triphosphate diphosphohydrolase-1 (CD39) catalyzes the conversion of ATP and adenosine diphosphate (ADP) to adenosine monophosphate (AMP), while ecto-5’-nucleotidase (CD73) further converts AMP to adenosine [13]. Adenosine can also be released into the extracellular space via the equilibrative nucleoside transporter (ENT) [14].

Dysfunctions in purinergic signaling at genetic, biochemical, or functional levels can lead to altered behaviors and mood disorders [15]. The purinergic system is particularly implicated in the pathophysiology of major depressive disorder by influencing neurotransmitter systems and hormonal pathways of the hypothalamic-pituitary-adrenal (HPA) axis [16].

Environmental pollutants such as nanoparticles, xenobiotics, and heavy metals have been linked to various health disorders [17]. These pollutants are responsible for approximately one-quarter to one-third of diseases in developing countries and contribute to 2–5% of global mortality [18]. Silica nanoparticles (SiNPs) have been identified in several toxicity studies as potential triggers of neurological disturbances. Although many questions remain, the purinergic system represents a promising area of research for understanding the molecular basis of depression and identifying potential therapeutic targets [19, 20]. Silica nanoparticles (SiNPs) are increasingly integrated into industrial, environmental, and biomedical contexts, raising concern about unintended neurological consequences. Beyond their widespread use, accumulating evidence suggests that SiNPs can traverse or disrupt the blood–brain barrier, triggering neuroinflammatory cascades and altering purinergic signaling pathways central to mood regulation. Against this backdrop, we set out to determine whether repeated systemic exposure to SiNPs is sufficient to provoke depression-like behaviors in rats, and whether these behavioral outcomes are paralleled by hippocampal alterations in ectonucleotidase activity, purinergic receptor expression, and ATPase function.

Therefore, this study aims to investigate the relationship between the purinergic pathway and depressive states by examining the activation of this pathway following repeated exposure to SiNPs in adult male rats.

Materials and Methods

Animal model and experimental design

Thirty-six adult male Wistar rats (200–250 g, 8–10 weeks old) were obtained from a certified animal facility (SIPHAT, Tunisia). Animals were housed under controlled temperature (22 ± 2°C) and humidity (50–60%) conditions with a 12-hour light/dark cycle. Standard chow and water were provided ad libitum. Animals were housed 3–4 per cage and allowed a 7-day acclimatization period prior to experimental procedures. Rats were randomly divided into three experimental groups (n = 12 per group). The control group received daily intraperitoneal injections of an equal volume of vehicle (deionized water) without nanoparticles. Animals underwent daily health checks for posture, piloerection, ocular/nasal discharge, and grooming. Body weight was recorded weekly; no animal experienced ≥10% weight loss, and all animals completed the study without humane endpoints. The selected doses of 25 and 100 mg/kg (i.p.) were chosen based on previous in vivo studies, including our own earlier work, which demonstrated that this range induces measurable CNS and hippocampal responses without overt systemic toxicity, thereby allowing assessment of dose-graded response effects.

Ethical considerations

All procedures were approved by the Faculty of Sfax Committee on Research Ethics (Project No. 20/PRD-10). Approval for animal care and all experimental protocols was obtained from the local Ethics Committee of the Sciences Faculty-Sfax prior to the experiments, in accordance with the guidelines of the National Institutes of Health.

Chemicals and reagents

Amorphous silica nanoparticles (SiO₂; CAS No. 7631-86-9; Sigma-Aldrich, Deissenhofen, Germany; Cat. No. 637238), specified by the manufacturer as 10–20 nm (BET), and characterized in our previous work as 11 ± 3 nm, were used in this study [21]. XRD analysis confirmed an amorphous structure with a broad halo at 2θ ≈ 22.9°, while TEM and SEM imaging revealed spherical morphology with an average particle size of 11 ± 3 nm. EDX analysis confirmed high purity, detecting only Si and O, with Pd/Pt arising from conductive coating and C from the sample holder. In physiological saline, DLS showed a hydrodynamic diameter of ~39 nm, and electrophoretic light scattering indicated a ζ-potential of −32.13 mV, confirming favorable dispersibility and stability [21].

Due to their tendency to aggregate, the nanoparticles were sonicated in deionized water for 15 minutes before administration. Silica nanoparticles were administered via daily intraperitoneal injection at doses of 25 and 100 mg/kg, freshly suspended in deionized water. The injection volume was adjusted to approximately 1 mL/kg body weight to ensure accurate dosing. All reagents and chemicals were of analytical grade. Trizol reagent (ThermoFisher Scientific, Cat. No. 15596026); SYBR Green Master Mix (Takara, Cat. No. RR420A); p-Nph-5’-TMP (Sigma-Aldrich, Cat. No. T4510); Bradford reagent (Bio-Rad, Cat. No. 5000006).

Behavioral assessments

Forced Swim Test (FST)

The FST was conducted following established protocols to evaluate depression-like behavior [22]. Rats were placed individually in transparent cylindrical containers (20 cm height, 10 cm diameter) filled with water (23 ± 1°C) to a depth of 10 cm. Behavior was recorded for 6 minutes, with immobility time assessed during the last 4 minutes. Immobility was defined as minimal movement necessary to stay afloat without active swimming or struggling. Observations were conducted by two independent evaluators blinded to group allocation.

Tail Suspension Test (TST)

For the TST, rats were suspended by the tail using adhesive tape, placed approximately 1 cm from the tip, and hung from a metal rod 50 cm above the ground [23]. The test duration was 6 minutes, with immobility recorded during the final 4 minutes. Immobility was characterized by the absence of escape-oriented movements.

Enzymatic activity assays

E-NTPDase and Ecto-5'-Nucleotidase (CD73) Activity

Hippocampal tissues were homogenized in an ice-cold buffer and centrifuged to obtain the supernatant. The samples were incubated in reaction buffers containing essential cofactors and divalent cations (Mg²⁺ or Ca²⁺) to facilitate ATP, ADP, and AMP hydrolysis. The enzymatic activity was quantified by measuring the release of inorganic phosphate (Pi) using a molybdate-based colorimetric assay. Absorbance was recorded at 650 nm with a microplate reader, and results were expressed as nmol Pi/min/mg protein.

Ecto-Nucleotide Pyrophosphatase/Phosphodiesterase (E-NPP) Activity

Phosphodiesterase activity was determined using p-nitrophenyl-5'-thymidine monophosphate (p-Nph-5'-TMP) as a substrate [24]. Tissue extracts were incubated at 37°C in 50 mM Tris-HCl (pH 8.0) containing 5 mM MgCl₂ or CaCl₂, with 0.5 mM p-nitrophenyl-5'-thymidine monophosphate (p-Nph-5'-TMP) as the substrate. After 30 min, the reaction was stopped with 1 M NaOH, and the release of p-nitrophenol was measured at 400 nm using a microplate reader. Enzymatic activity was expressed as nmol p-nitrophenol released per minute per milligram of protein [24].

ATPase Activity (Na⁺/K⁺-ATPase, Mg²⁺-ATPase, and Ca²⁺-ATPase)

ATPase activity was assessed using a molybdate-based colorimetric assay, where free inorganic phosphate was quantified at 690 nm [25]. Specific inhibitors were used to differentiate Na⁺/K⁺-ATPase from total ATPase activity. Results were reported as mol Pi/hour/mg protein.

Gene expression analysis via RT-qPCR

Total RNA was extracted from hippocampal tissue using Trizol reagent [26]. The RNA integrity and concentration were determined via A260/A280 absorbance ratios. Complementary DNA (cDNA) synthesis was performed using reverse transcription kits (ThermoFisher Scientific), and quantitative PCR (qPCR) was conducted using SYBR Green Master Mix in a Bio-Rad CFX96 Real-Time PCR System (Bio-Rad, USA) with SYBR detection (excitation 497 nm, emission 520 nm). Cycling conditions included an initial denaturation step at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Gene expression levels of A2A, P2X7, P2Y2, NTPDase1, NTPDase2, and NTPDase3 were normalized to GAPDH using the 2^−ΔΔ CT method [27] . Negative controls lacking RNA templates were included to confirm specificity (Table 1). All analyses were performed in triplicate.

Table 1.

Primer sequences used for quantitative real-time PCR (RT-qPCR) analysis of target genes and GAPDH as the reference gene.

Gene	Primer : Forward/Reverse	Sequences
GAPDH	Forward	5′ -CCTCTCTCTTGCTCTCAGTAT-3′
	Reverse	5′ -GTATCCGTTGTGGATCTGACA-3′
A2A	Forward	5′ -GTTTTGTCCTGGTCCTCACG-3′
	Reverse	5′ -CAAGCCATTGTACCGGAGTG-3′
P2X7	Forward	5′ -CTGCAGCTGGAACGATGTCT-3′
	Reverse	5′ -GACGGTCATGTGCAAGATCC-3′
P2Y2	Forward	5′ -GGGGACGAACTGGGTTACA -3′
	Reverse	5′ -ATGTAGAGGGCCACGACGT-3′
NTPDase1	Forward	5′ -TCAAGGACCCGTGCTTTTAC-3′
	Reverse	5′ -TCTGGTGGCACTGTTCGTAG-3′
NTPDase2	Forward	5′ -TGCTTCGACACAGATCACCT -3′
	Reverse	5′ -GATGAACAGCCCTGTGATGA-3′
NTPDase3	Forward	5′CGGGATCCTTGCTGTGCGTGGC-3′
	Reverse	5′ -TCTAGAGGTGCTCTGGCAGGAATCAGT-3′

Immunohistochemistry and immunofluorescence

Brain sections (3 μm thick) were prepared from paraffin-embedded hippocampal tissue for immunohistochemical and immunofluorescence analysis. Samples were incubated with primary antibodies against CD73 (1:200, Life Technologies) and CD90 (1:100, Bioss), followed by incubation with appropriate secondary antibodies. Diaminobenzidine (DAB) staining and hematoxylin counterstaining were performed for immunohistochemistry, and fluorescence signals were visualized using a Leica TCS SP5 confocal microscope (Leica Microsystems, Germany) with a 63× oil immersion objective, laser excitation at 405 nm (DAPI) and 488/568 nm (fluorophores). Nuclei were counterstained with DAPI [28]. Quantification of immunofluorescence and immunohistochemistry images (Figures 5 and 6) was performed using ImageJ software (NIH, USA). Regions of interest (ROIs) were manually defined, background was subtracted, and mean fluorescence intensity or positive cell counts were measured. Thresholding parameters were kept constant across all groups.

Figure 5.

Immunofluorescence staining of CD90 in the hippocampus of control, SiNPs-25 mg/kg, and SiNPs-100 mg/kg groups. Representative images and bar graph quantification of CD90-positive cells are shown. Arrows indicate positively stained cells. Scale bar = 20 μm. Data are mean ± SEM (n ≥ 3 animals/group); *p < 0.05, **p < 0.01, ***p < 0.001 vs. control.

Figure 6.

Immunohistochemical staining of CD73 in the hippocampus of control, SiNPs-25 mg/kg, and SiNPs-100 mg/kg groups. Representative images and bar graph quantification of CD73-positive cells are shown. Arrows indicate positively stained cells. Scale bar = 20 μm. Data are mean ± SEM (n ≥ 3 animals/group); *p < 0.05, **p < 0.01, ***p < 0.001 vs. control.

Protein quantification

Total protein concentrations in hippocampal lysates were determined using the Bradford assay (Cat. No. 5000006; Bradford, 1976) [29], with bovine serum albumin as the standard.

Statistical analysis

All statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, USA). Data are presented as mean ± standard error of the mean (SEM). Comparisons between groups were conducted using one-way ANOVA followed by Tukey’s post hoc test. Statistical significance was set at P < 0.05.

Results

Effects of SiO2-NP exposure on depressive-like behaviors

The forced swimming test (FST) and tail suspension test (TST) were used to assess depressive-like behaviors in rats. Increased immobility time is indicative of behavioral despair. A high significant increase (P < 0.001 for 25 and 100 mg/kg of SiNPs) in immobility time was observed in the treated groups compared to the control group during both the FST (Figure 1A) and the TST (Figure 1B), suggesting that SiNP exposure induces depressive-like behavior in rats.

Figure 1.

Behavioral assessment in the Forced Swim Test (FST) and Tail Suspension Test (TST) in control, SiNPs-25 mg/kg, and SiNPs-100 mg/kg groups. Immobility time is shown. Data are mean ± SEM (n = 12); *p < 0.05, **p < 0.01, ***p < 0.001 vs. control..

E-NTPDase and E-5′-ectonucleotidase activities in the hippocampus

The activities of ecto-nucleotidases (E-NTPDase and E-5′-ectonucleotidase) in the hippocampus following 28 days of intraperitoneal SiNP injections are shown in Figure 2. The activities of E-NTPDase (ATP and ADP substrate) were significantly elevated (P < 0.001 for 100 mg/kg of SiNPs) in the treated groups compared to the control respectively (Figure 2A and B ). Similarly, E-5′-ectonucleotidase activity (Figure 2C) responsible for ATP and ADP hydrolysis, was significantly higher (P < 0.001 for 25 and 100 mg/kg of SiNPs) in the treated groups compared to the control group.

Figure 2.

Activities of E-NTPDase (ATPase and ADPase) (A, B), E-5′-Ectonucleotidase (AMPase) (C), and E-NPP (D, E) in the hippocampus of control, SiNPs-25 mg/kg, and SiNPs-100 mg/kg groups. These enzymes are involved in extracellular nucleotide hydrolysis within purinergic signaling pathways. Enzyme activities were measured by hydrolysis of specific substrates (ATP, ADP, AMP, and p-Nph-5′-TMP). Data are mean ± SEM (n = 12); *p < 0.05, **p < 0.01, *p < 0.001 vs. control.

E-NPP activities

Figure 2 (D) illustrates the effect of repeated intraperitoneal injections of 25 and 100 mg/kg SiNPs on E-NPP activity in the hippocampus. Hydrolysis of the substrate p-Nph-5′-TMP, used as a marker of E-NPP activity, was significantly increased (P < 0.001 ) in the hippocampus of treated groups (100 mg/kg-SiNPs) compared to controls.

Effects of SiNPs on Na+/K+-ATPase, Ca2+-ATPase, and Mg2+-ATPase activity in the hippocampus

Figure 3 presents the activity of membrane-bound ATPases in hippocampal tissue. SiNP treatment significantly (P < 0.01 ) decreased Na+/K+-ATPase activity compared to controls. Then, Ca2+-ATPase activity was significantly decreased in the treated group. Mg2+-ATPase activity was highly decreased (P < 0.001), comparable to control levels.

Figure 3.

Na⁺/K⁺-ATPase, Ca²⁺-ATPase, and Mg²⁺-ATPase activities in the hippocampus of control, SiNPs-25 mg/kg, and SiNPs-100 mg/kg groups. These ATPases maintain ion gradients essential for neuronal excitability, indirectly linked to purinergic signaling. Data are mean ± SEM (n = 12); *p < 0.05, **p < 0.01, ***p < 0.001 vs. control..

PCR analysis of nucleoside triphospate phosphohydrolase (E-NTPDase 1, 2, and 3) and purinergic receptors (A2A, P2X7, P2Y2)

The mRNA expression levels of E-NTPDase 1, 2, and 3 in hippocampal tissues following subacute exposure to SiNPs are shown in Figure 4. The treated group exhibited a significant (P < 0.001) increase for the high dose of SiNPs (100 mg/kg) in E-NTPDase 1, 2, and 3 mRNA levels compared to the control. Similarly, the expression of purinergic receptors A2A, P2X7, and P2Y2 was significantly (P < 0.001) upregulated in the hippocampus of SiNP-exposed rats (100 mg/kg) compared to controls.

Figure 4.

qPCR analysis of purinergic receptors (P2X7, P2Y2, A2A) and NTPDase enzymes in the hippocampus of control, SiNPs-25 mg/kg, and SiNPs-100 mg/kg groups. Relative mRNA expression levels are shown. Upregulation of these genes reflects activation of purinergic signaling pathways. Data are mean ± SEM (n = 12); *p < 0.05, **p < 0.01, ***p < 0.001 vs. control.

Effects of SiNPs on CD90 expression in the hippocampus

Immunofluorescence analysis revealed that CD90 expression was significantly higher in Hippocampus of rats exposed to a high dose (100 mg/kg) of SiNPs compared to those receiving a low dose (25 mg/kg) and controls (Fig.5). The red fluorescence represents CD90-positive cells, while the blue fluorescence (DAPI staining) marks the nuclei. The intensity and number of CD90-positive cells appear to increase with higher SiNPs doses. CD90 expression was minimal in the low-dose group and nearly absent in normal brains. These findings suggest that CD90 expression may indicate the severity of nanoparticle exposure.

Effects of SiNPs on CD73 expression in the Hippocampus

The Figure 6 presents histological and quantitative data illustrating the immunolabeling of CD73 in brain tissue across three experimental conditions: Control, SiNPs-25 mg/kg, and SiNPs-100 mg/kg. The histological images demonstrate an increase in CD73-positive cells (brown staining) with increasing doses of SiNPs, suggesting a dose- graded response. In the control group, CD73 expression appears minimal, whereas in the SiNPs-25 mg/kg and SiNPs-100 mg/kg groups, there is a progressive increase in the number and intensity of CD73-positive cells.

The bar graph quantifies the percentage of CD73-positive cells relative to the control. The SiNPs-25 mg/kg group shows a significant increase (p < 0.01) while the SiNPs-100 mg/kg group exhibits a highly significant increase (p < 0.001). The data suggest that exposure to SiNPs induces an upregulation of CD73 expression, potentially influencing purinergic signaling and immunomodulation in the hippocampus.

Discussion

Depression is associated with altered neuronal plasticity and cellular resilience, leading to cognitive and behavioral impairments. The purinergic system plays a key role in this pathology, with A2A and P2X7 receptors emerging as potential therapeutic targets [20]. Purinergic enzyme dysregulation, particularly ATP hydrolysis by E-NTPDase and ecto-5'-nucleotidase, has been implicated in various diseases, affecting neurotransmission and inflammation [30, 31].

The hippocampus, critical for mood regulation and memory, is highly vulnerable to neuroinflammatory insults. Its role in HPA axis regulation makes it central to depression pathology, with neuroimaging studies showing significant volume reduction in affected individuals [32].

Environmental factors, including nanoparticles, have raised concerns due to their neurotoxic potential. Their ability to cross the blood-brain barrier and activate neuroinflammatory pathways may disrupt purinergic signaling, contributing to depression [33]. Recent studies further support these findings. For example, [34-36] demonstrated that silica nanoparticles can cross the blood–brain barrier and accumulate in brain tissue after systemic exposure. Regarding neurobehavioral impacts, studies in rodents [37-39] and (zebrafish behavior) [40] have reported significant changes in hippocampal function, oxidative stress, and depressive-like behavior. Consistent with these findings, our previous study also showed dose-related increases in brain silicon concentrations after intraperitoneal administration, supporting direct hippocampal exposure. Given their widespread use in industry and medicine, understanding their long-term effects on brain health is essential to addressing emerging public health risks [41].This study aimed to assess the effects of SiNP exposure on depressive behavior and purinergic signaling in the hippocampus [42]. The silica nanoparticles used in this study were characterized as described in our previous work [21]. Whether hippocampal changes arise from direct exposure or peripheral signaling has important implications. Multiple in vivo studies report that silica nanoparticles traverse the blood–brain barrier and accumulate in brain tissue after systemic dosing. Consistent with this, our previous work quantified brain Si content and found 3.3- and 7.7-fold increases after 25 and 100 mg/kg i.p. exposure, respectively, confirming brain entry and supporting a direct effect on hippocampus [21]. Alternatively, peripheral routes e.g., systemic cytokines, endothelial activation, and microglial priming can secondarily drive hippocampal responses; these pathways likely act in parallel.

Behavioral tests, including the forced swim test (FST) and the tail suspension test (TST), demonstrated a significant increase in immobility time in SiNP-exposed rats (25 mg/kg and 100 mg/kg BW), indicating depressive-like behavior. These findings align with previous studies demonstrating that silica nanoparticles induce depression-like states, possibly through serotonin depletion [43]. Synaptic dysfunction involving serotonin, dopamine, and norepinephrine has been observed in depression, with serotonin release in the hippocampus being downregulated by A1 receptor activation and upregulated by A2A receptor activation [44, 45].

Consistent with these findings, Xiang Li et al. 2017 reported that exposure to SiNPs (⌀19 nm) increased depressive and anxiety-like behaviors [43]. Our study further suggests that SiNPs affect neuronal function through purinergic signaling. We observed increased activities of E-NTPDase, E-NPP, and ecto-5'-nucleotidase in the hippocampus of SiNP-exposed rats, along with overexpression of E-NTPDase1, E-NTPDase2, and E-NTPDase3. These changes indicate a shift in extracellular adenine nucleotide metabolism, which may disrupt neurotransmission and contribute to depressive behavior. A limitation of this study is the absence of a pharmacological positive control, such as an antidepressant, to reinforce the specificity of the observed depression-like effects. Our experimental design intentionally focused on isolating SiNP-induced outcomes without confounding by drug treatment. Nevertheless, previous reports have shown that antidepressant administration can reverse nanoparticle-induced behavioral and molecular alterations [46, 47] .In addition, our earlier work on cobalt oxide nanoparticles demonstrated that subacute exposure can trigger behavioral, biochemical, molecular, and histopathological changes in aged rats [48], further supporting the concept that engineered nanoparticles can drive neurobehavioral disturbances. Future studies will incorporate pharmacological comparators to strengthen construct and predictive validity in SiNP-induced models of depression-like behavior.

Our findings offer novel insights into how SiNPs drive purinergic system dysregulation, positioning these nanoparticles as previously unrecognized environmental neurotoxicants. Immunofluorescence and immunohistochemical analysis confirmed increased protein expression of CD73 and CD90 in the hippocampus of SiNP-treated rats. CD90, known for its role in neurite outgrowth inhibition and stem cell differentiation [49] suggests that SiNP exposure triggers neuroinflammatory remodeling. Meanwhile, the overexpression of P2Y2, P2X7, and A2A receptors, key mediators of neuroinflammation and synaptic plasticity indicates that prolonged SiNP exposure disrupts extracellular ATP metabolism, amplifying pro-inflammatory responses and impairing neuronal resilience.

Our findings extend beyond the laboratory, offering potential insight into real-world exposure scenarios where workers or populations may encounter SiNPs through manufacturing, environmental release, or medical applications. Although the doses and routes of administration in experimental models do not fully mirror human exposure, the convergence of depressive-like behaviors with hippocampal molecular disruptions highlights a plausible neurobehavioral risk. These observations underscore the need for further investigation into SiNP-related brain effects and the development of strategies to mitigate potential occupational and environmental hazards.

Beyond neurotransmitter imbalances, our study highlights the intersection between SiNP exposure and inflammatory pathways implicated in depression. The observed upregulation of P2X7 receptors, coupled with increased ecto-5'-nucleotidase activity, suggests that SiNPs exacerbate neuroinflammation by promoting ATP-induced cytokine release. This aligns with clinical evidence linking elevated IL-1β, IL-6, and TNF-α to major depressive disorder, reinforcing the idea that SiNPs may act as environmental stressors capable of triggering depressive pathology. Notably, our findings go beyond previous studies by identifying a distinct molecular network involving ATP hydrolysis, P2 receptor activation, and A2A-mediated immune modulation—mechanisms that could serve as potential therapeutic targets.

These insights shift the paradigm of depression research by introducing nanoparticle exposure as a critical, yet underexplored, environmental risk factor. With the increasing ubiquity of SiNPs in medicine, industry, and consumer products, their potential to silently alter brain function warrants urgent investigation. Our findings underscore the necessity of developing strategies to counteract purinergic dysregulation, particularly through selective modulation of A2A and P2X7 receptors, to mitigate depression linked to environmental neurotoxins.

This study provides strong evidence that SiNP exposure induces depressive-like behavior via purinergic pathway dysregulation, promoting neuroinflammation and altering neurotransmission. By linking nanoparticle exposure to purinergic receptor overactivation, ATP metabolism shifts, and inflammatory cascades, our findings not only expand the understanding of depression's molecular basis but also highlight an urgent public health concern. Future research should explore targeted therapeutic interventions that restore purinergic homeostasis, mitigating the impact of environmental neurotoxicants on mental health.

Conclusions

Our study demonstrates that silica nanoparticle (SiNP) exposure induces depressive-like behavior in rats, accompanied by significant disturbances in hippocampal purinergic signaling. SiNPs increased the activity and expression of key purinergic enzymes and receptors, promoting neuroinflammation and altering neurotransmission. These findings identify SiNPs as emerging environmental neurotoxicants capable of disrupting brain function through ATP metabolism imbalance and receptor overactivation. Given the widespread use of SiNPs, understanding their impact on mental health is essential. Future research should focus on therapeutic strategies aimed at restoring purinergic homeostasis to mitigate the neurobehavioral risks associated with nanoparticle exposure.