Isotopically enriched ⁶⁴ZN-aspartate attenuates systemic inflammation and gut dysbiosis in an LPS-induced rat model of Parkinson’s disease

Abstract

A growing body of research indicates that systemic inflammation contributes substantially to the progression of Parkinson’s disease (PD). Foundational studies propose that targeting inflammatory pathways may offer therapeutic benefits for PD and other neurodegenerative conditions. Our previous work demonstrated that a novel zinc aspartate compound enriched with the light isotope ⁶⁴Zn (⁶⁴Zn-asp) can counteract inflammatory and cognitive impairments triggered by intra-hippocampal Aβ_1-40 in rats, and can also mitigate neuroinflammation while promoting neuronal survival in a PD model. In the present study, we investigated the impact of this isotopically modified zinc compound on systemic inflammatory responses and gut microbiota composition in a rat model of PD induced by a single stereotactic intranigral injection of lipopolysaccharide (LPS). LPS-lesioned rats exhibited impaired locomotion, heightened anxiety-like behavior, and progressive dopaminergic dysfunction. ⁶⁴Zn-asp administration attenuated behavioral deficits and reduced apomorphine-induced rotations. Treatment normalized CRP levels, reversed LPS-induced increases in granulocytes and platelets, and corrected elevations in systemic inflammatory indices (including NLR, PLR, SII, and SIRI). ⁶⁴Zn-asp shifted circulating and peritoneal phagocytes toward an anti-inflammatory phenotype and partially restored thymus structure and cellularity. In the gut, LPS-induced PD resulted in marked reductions in Bifidobacterium and Lactobacillus spp. and an expansion of opportunistic Enterobacteriaceae and Staphylococcus spp. ⁶⁴Zn-asp largely preserved beneficial anaerobes and suppressed opportunistic taxa in both luminal and mucosa-associated compartments. These findings demonstrate that ⁶⁴Zn-aspartate exerts anti-inflammatory, immunomodulatory, and microbiota-stabilizing effects, suggesting potential therapeutic value as a disease-modifying strategy targeting neuroimmune-gut axis dysfunction in PD.

Introduction

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, affecting approximately 11.77 million people worldwide as of 2021^1,2. PD has seen the fastest growth in prevalence and associated disability among neurological disorders, emerging as a major global contributor to neurological impairment³. PD, once viewed primarily as a motoric disorder, is now understood to be a complex, multifactorial systemic condition. Its pathogenesis involves a range of interconnected mechanisms, including neuroinflammation, α-synuclein aggregation, dysfunction of the lysosomal-autophagy system, mitochondrial impairment with oxidative stress, and vesicular transport defects. An expanding body of research highlights the pivotal role of immune activation and inflammatory processes as key drivers in both the onset and progression of the disease. Persistently activated microglia release high levels of pro-inflammatory mediators that not only damage neurons but also perpetuate their own activation, establishing a self-amplifying cycle of neuroinflammation and neurodegeneration⁴. Activated microglia release diverse inflammatory mediators (matrix metalloproteinases, reactive oxygen and nitrogen species, chemokines, cytokines, etc.) and can phagocytose astrocytic end-feet, thereby compromising blood–brain barrier (BBB) integrity and increasing permeability^5–7. In these circumstances, pro-inflammatory mediators released by chronically activated microglia may escape into the systemic circulation, initiating peripheral low-grade pro-inflammatory immune responses. In turn, chronic low-grade peripheral inflammation, especially under conditions of increased BBB permeability, can exacerbate neuroinflammation by enabling the circulating immune cells and inflammatory mediators to access the central nervous system. This bidirectional communication forms a vicious, self-perpetuating inflammatory loop that accelerates PD progression^8,9.

Current evidence-based treatment for PD focuses on symptomatic management with levodopa (L-DOPA) and does not specifically target pathological disease progression. However, long-term use frequently leads to complications such as dyskinesia, limiting its efficacy. Moreover, treatment usually begins in advanced stages, after substantial and irreversible neural damage has occurred. This underscores the urgent need for safer, more effective, and truly disease-modifying therapies¹⁰. One of the central therapeutic targets in the development of disease-modifying strategies for PD is the neuroimmune-inflammatory response. Inflammation plays a pivotal role in amplifying oxidative stress and creating a microenvironment conducive to neuronal injury. By modulating inflammatory pathways, it may be possible to attenuate oxidative modifications and disrupt the cascade of α-synuclein misfolding, aggregation, and intercellular propagation, thereby slowing or halting disease progression¹¹. A range of immuno-inflammatory pathways is being explored as therapeutic targets in PD. Agents under investigation include the NLRP3 inflammasome inhibitor selnoflast¹², GLP-1 receptor agonists such as exenatide¹³, the leukotriene receptor antagonist montelukast¹⁴, PPARγ agonists¹⁵, azathioprine¹⁶, and selected traditional Chinese medicines¹⁷, alongside trials of NSAIDs¹⁸. While some have shown modest benefits, most are limited by narrow target profiles and immunosuppressive side effects¹⁹. The gut-brain axis (GBA) has also emerged as a promising focus, with gut microbiota shown to modulate systemic immunity and microglial activity. Probiotics may relieve constipation, and certain strains exhibit neuroprotective potential, but robust clinical evidence for disease modification in PD is still lacking²⁰. Collectively, these findings underscore the urgent need for effective anti-inflammatory therapies that can modify PD progression.

In our previous work, we showed that intravenous administration of a novel zinc preparation – the isotopically-modified zinc aspartate enriched with 99.2% of the light zinc isotope ⁶⁴Zn (coded KLS-1) – reversed both inflammatory responses and cognitive impairments induced by intra-hippocampal Aβ_1-40 in rats²¹, as well as alleviated neuroinflammation and motor dysfunction in rats with LPS-induced PD²². A meta-analysis revealed that serum zinc levels are significantly lower in PD patients than in healthy controls. Zinc, abundant in the hippocampus and cerebral cortex, is essential for behavior, learning, memory, and emotional regulation, and its deficiency may contribute to neurodegenerative disorders such as PD, Alzheimer’s disease, and amyotrophic lateral sclerosis²³. In animal models of neurodegenerative diseases, zinc supplementation has shown neuroprotective and disease-modifying effects^24,25. Zinc supplementation in elderly individuals significantly improves health outcomes by reducing the incidence of infectious diseases and mitigating inflammaging-associated pathologies, such as age-related macular degeneration, cardiovascular diseases, and others^26,27. Zinc supplementation lowers serum inflammatory and oxidative stress markers in adults, suggesting a modulatory effect on systemic inflammation and oxidative pathways^28,29. In a case report by Quiroga et al. (2014), resolution of movement disorder symptoms in a PD patient was reported following treatment with zinc sulfate in combination with vitamin C³⁰. All these studies have used zinc with its natural isotopic composition, which is primarily an aggregate of heavy zinc isotopes. The brain normally favors the lighter ⁶⁴Zn isotope (the ratio of ⁶⁶Zn/⁶⁴Zn δ⁶⁶Zn < 1) due to its isotope bonding preference with sulfur-containing amino acids like cysteine in metallothioneins^31,32. With age and in neurodegenerative diseases, δ⁶⁶Zn increases as heavier isotopes accumulate, likely due to altered binding preferences^33,34. In Alzheimer’s disease, for example, heavy zinc isotopes preferentially bind to histidine residues in amyloid-β plaques³⁵. Similar mechanisms may influence α-synuclein aggregation in PD, where histidine-50 is a key binding site for heavy zinc^36–38.

This study aimed to assess the effects of intravenous ⁶⁴Zn-asp (KLS-1) administration on systemic inflammation and gut microbiota in a rat model of PD induced by LPS.

Materials and methods

Test agent

The therapeutic agent used in this study was isotopically modified ⁶⁴Zn di-aspartate (⁶⁴Zn-asp), an investigational zinc aspartate complex. The molecule consisted of one atom of ⁶⁴Zn chelated with two L-aspartic acid molecules (NeoFroxx, Einhausen, Germany), with the molecular formula C₈H₁₂O₈N₂⁶⁴Zn and a molar mass of 328 g/mol. Zinc accounted for 17.98% of the compound by weight, with the zinc component enriched to 99.2% ⁶⁴Zn. The compound, designated KLS-1, was synthesized by Pharmaceutical Factory Biopharma LLC (Bila Tserkva, Ukraine).

Animals, experimental design, and LPS-induced Parkinson’s disease model

The study was conducted using adult male Wistar rats (8 weeks old, weighing 220–250 g) obtained from the vivarium of the Educational and Scientific Centre “Institute of Biology and Medicine” at Taras Shevchenko National University of Kyiv, Ukraine. Animals were housed under standard laboratory conditions with unrestricted access to food and water (standard chow). All animal experiments in this study were performed in accordance with the ARRIVE guidelines.

A total of 51 rats were randomly assigned into four experimental groups using the “RAND” function in Microsoft Excel (Fig. 1):

Fig. 1.

Animal groups and study design.

Group I—intact (non-operated) controls (n = 12), did not underwent any manipulations;

Group II—sham-operated controls (n = 12), intra-nigral sterile saline injection;

Group III—LPS-induced PD model (n = 12), intra-nigral LPS injection (10 μg);

Group IV—LPS-induced PD model treated with ⁶⁴Zn-asp (n = 15), intra-nigral LPS injection (10 μg). ⁶⁴Zn-asp was administered i.v. at 1.5 mg/kg, daily for 10 days.

To induce unilateral lesions of the nigrostriatal pathway in groups III and IV, stereotaxic injections of 10 μg lipopolysaccharide (LPS, Escherichia coli O111:B4, Sigma) dissolved in 2 μL sterile saline (JSC “Infusion”, Ukraine) were administered directly into the substantia nigra as described previously³⁹. Animals in group II received equivalent injections of sterile saline only. Prior to surgery, rats in groups II–IV were anesthetized with a combination of ketamine (75 mg/kg, Sigma, USA) and 2% xylazine (400 μL/kg, Alfasan International BV, Netherlands), and positioned in a stereotaxic apparatus (SEJ-4, Ukraine). Coordinates for injection were based on Hoban et al. (2013): AP –5.3 mm, ML ± 2.0 mm from bregma, and DV – 7.2 mm below the dura⁴⁰. The injection rate was 1 μL/min, with the needle left in place for 5 min post-injection to facilitate diffusion and prevent backflow. Postoperative survival was 100%.

Intranigral LPS administration induces irreversible degeneration of dopaminergic neurons in the substantia nigra pars compacta within one week⁴¹. Therefore, an 8-day period post-injection was allowed for disease development. Disease development was confirmed using the apomorphine-induced rotation test, which assesses motor dysfunction associated with dopaminergic neuron loss⁴². Starting on Day 9, rats in group IV received daily intravenous injections of ⁶⁴Zn-asp (1.5 mg/kg) via the lateral tail vein for 10 consecutive days. Parkinsonian pathology was confirmed through behavioral assessments and post-mortem analysis of the nigrostriatal system using semi-quantitative tyrosine hydroxylase (TH) immunohistochemistry (data are not presented). On day 28, the rats were sacrificed using carbon dioxide inhalation and subsequent cervical dislocation⁴³, after which biological samples—including brain tissue, thymus, peritoneal lavage fluid, sections of the gastrointestinal tract, and blood—were obtained for analysis.

Ethics statement

All experimental procedures were approved by Ethics Committee of the Taras Shevchenko National University of Kyiv (protocol No. 4, 10.10.2021) and complied with the Animal Welfare Act, as well as national (Kyiv National Bioethics Congress, 2001–2007) and international (EU Directive 86/609/EEC) guidelines on the ethical treatment of laboratory animals.

Behavioral assessments

Behavioral testing was conducted to evaluate locomotion, anxiety-like behavior, and dopaminergic dysfunction associated with the LPS-induced Parkinson’s disease model.

Open field test

On Day 24 post-surgery, spontaneous locomotor activity and anxiety-related behavior were assessed using the Open Field test, following the methodology adapted from⁴⁴. The test arena consisted of a square enclosure (100 × 100 cm) with 30 cm high walls, illuminated by two overhead 60W LED lamps positioned 2 m above the floor. A 6 × 6 grid (36 squares) was marked on the floor to aid spatial tracking. Each rat was individually placed in the center of the arena and allowed to explore for 5 min. Movements were recorded from above using a digital camera (Casio EX-Z850, China) mounted 1 m above the arena. Video data were later analyzed using MATLAB (MATLAB online, free: https://matlab.mathworks.com/). Measured parameters included total distance traveled, time spent in the central zone (inner perimeter), thigmotaxis (time spent near walls), number of rearings and grooming episodes, and frequency of defecation^45,46. The arena was cleaned and dried between trials to eliminate olfactory cues.

Apomorphine-induced rotation test

This test was used to evaluate the extent of dopaminergic neuron loss. It was conducted on Days 8 and 21 following stereotaxic surgery. Apomorphine hydrochloride (Sigma, USA) was administered intraperitoneally at a dose of 0.5 mg/kg. Five minutes after injection, each rat was placed in a cylindrical observation chamber (40 cm diameter), and contralateral (counterclockwise) rotations were recorded manually for 30 min using a stopwatch. Rats displaying more than 6 full-body turns per minute (rpm) were considered to have severe dopaminergic neuron (DN) loss (approximately 86.6%), while animals with ≤ 2 rpm were classified as having moderate DN loss (~ 44%)⁴⁷.

Elevated plus maze (EPM) test

Anxiety-like behavior was further assessed using the Elevated Plus Maze on Day 24 post-surgery, in accordance with Walf and Frye (2007)⁴⁸. The maze consisted of a cross-shaped platform elevated 50 cm above the floor, featuring two open arms (50 × 10 cm) and two enclosed arms (50 × 10 × 30 cm), all connected by a central square (10 × 10 cm). Rats were placed in the center square facing an open arm at the start of the 5-min test session. The setup was illuminated using two ceiling-mounted 60W LED lamps. Behavioral activity was recorded via IP camera and analyzed using MATLAB (MATLAB online, free: https://matlab.mathworks.com/). Key parameters included total distance traveled, frequency of transitions between arms (open to closed and vice versa), time spent in open vs. closed arms, number of risk assessment behaviors (stretched attend postures), and total number of arm entries. All animals were given a short habituation period to minimize stress before testing.

CRP level measurement

C-reactive protein (CRP) levels in blood plasma, collected using the anticoagulant EDTA, were measured by enzyme-linked immunosorbent assay (ELISA) with the ELISA-CRP test system (Labcare Diagnostics India Pvt Ltd), following the manufacturer’s instructions.

Lymphoid organ assessment

Thymus and spleen tissues were carefully excised, weighed, and prepared for a single-cell suspension. Each thymus (spleen) was placed onto a 200-mesh sieve and gently homogenized using a tissue grinder in cold phosphate-buffered saline (PBS, pH 7.3) containing 2% heat-inactivated fetal calf serum (FCS; Gibco, Grand Island, NY, USA) until no visible clumps remained. The resulting single-cell suspensions were then counted using a standard hemocytometer. Cell viability, assessed via Trypan blue exclusion, consistently exceeded 95%. The measured parameters included the relative weights of the thymus and spleen, as well as the relative numbers of thymocytes and splenocytes. These values were calculated as follows:

Hematological analysis

Whole blood samples anticoagulated with EDTA were analyzed to assess hematological parameters. A fully automated hematology analyzer (Particle Counter Model PCE 210, ERMA, Japan), calibrated for rodent blood profiling, was used to evaluate blood cell indices in rats. Derived immune-inflammatory ratios were calculated using standard formulas based on absolute cell counts:

These indices were used to evaluate systemic inflammatory status and immune response patterns in the experimental groups.

Peritoneal macrophage isolation

Peritoneal macrophages (PMs) were harvested from the abdominal cavity of non-sensitized rats using a standard rapid peritoneal lavage technique, as previously described⁴⁹. The peritoneal cavity was flushed with phosphate-buffered saline (PBS) supplemented with 100 U/mL heparin and 3% fetal bovine serum (FBS) to prevent clotting and preserve cell viability. The collected lavage fluid was centrifuged at 300 × g for 5 min at 4 °C. The resulting cell pellet was washed twice with Hanks’ Balanced Salt Solution (HBSS) to remove residual serum and debris.

Assessment of phagocyte metabolic profile

The functional status of phagocytes was evaluated by analyzing their phagocytic capacity, oxidative metabolism, and expression of surface phenotypic markers using flow cytometry. Phagocytosis was assessed based on a previously established method⁴⁹. For this purpose, FITC-conjugated, heat-inactivated Staphylococcus aureus Cowan I (sourced from the microbiological collection of the Department of Microbiology and Immunology, ESC "Institute of Biology and Medicine", Taras Shevchenko National University of Kyiv) served as the phagocytic target. A suspension containing 2 × 10⁵ microglia or macrophages was incubated with 5 µL of the bacterial stock (1 × 10⁷ cells/mL) at 37 °C for 30 min. Phagocytic activity was halted by adding a quenching solution composed of PBS with 0.02% EDTA and 0.04% paraformaldehyde.

Phagocytic efficiency was quantified in two ways: the phagocytosis index (PhI), representing the average fluorescence intensity per phagocytic cell (reflecting the number of bacteria ingested), and the phagocytosis percentage (PP), denoting the proportion of fluorescently positive cells.

Reactive oxygen species (ROS) generation, indicative of oxidative metabolic activity, was assessed using the fluorescent probe 2’,7'-dichlorodihydrofluorescein diacetate (H2DCFDA, Invitrogen) as previously described⁴⁹.

To determine the phenotypic characteristics of phagocytes, cells were stained with FITC-conjugated anti-CD86, PE-conjugated anti-CD80, and Alexa Fluor 647-conjugated anti-CD206 antibodies (BD Pharmingen, USA). All samples were analyzed using a FACSCalibur flow cytometer, and data were processed with CellQuest Pro 5.2.1 (BD Biosciences, USA).

Fecal water content assessment

Fecal water content was determined using a modified method of Zhu et al.⁵⁰. Each rat was placed individually in a clean cage lined with filter paper. Freshly expelled fecal pellets were collected immediately and placed in sealed tubes. The total sample was weighed to obtain the wet weight (WW), then dried at 60 °C for 24 h and reweighed to determine the dry weight (DW). Stool water content (%) was calculated using the formula:

Analysis of Luminal and Mucosa-associated gut microbiota

Microbiota analysis was performed using conventional culture-based techniques as described earlier⁵¹. To evaluate both luminal and wall-adherent microbial populations, distinct sections of the gastrointestinal tract were sampled. For mucosal microbiota assessment, colon segments measuring 1 cm² (located 2 cm from the anal verge) and sections of the small intestine (located 2 cm from the ileocecal valve) were excised, washed three times with chyme in saline, and homogenized using a Potter homogenizer. For luminal microbiota analysis, fecal samples were weighed and homogenized in 9 mL of sterile 0.5% sodium chloride solution to obtain a 10^–1 dilution. Serial tenfold dilutions (ranging from 10^–2 to 10^–11) were prepared using the same procedure.

Aliquots (10 μL) from each dilution were aseptically inoculated onto selective and differential culture media (HiMedia Laboratories Pvt. Ltd., India), including Bifidobacterium Agar, MRS Agar, Endo Agar, Mannitol Salt Agar, Iron Sulphite Agar, Simmons Citrate Agar, and Blood Agar Base (supplemented with 5% sterile defibrinated sheep blood). Cultures were incubated at 37 °C for 24–48 h.

Microbial identification was conducted following the taxonomic keys provided in Bergey’s Manual of Determinative Bacteriology. Colony morphology, Gram staining, and a battery of biochemical tests were used for classification, including plasma coagulation, DNAse activity, lysozyme and phosphatase production, oxidase activity, carbohydrate fermentation profiles, Voges-Proskauer reaction, motility assessment, and novobiocin susceptibility (to differentiate S. aureus, S. epidermidis, and S. saprophyticus). Lactose-negative E. coli strains were distinguished from other opportunistic Enterobacteriaceae by their ability to produce hydrogen sulfide.

Results are expressed as the mean ± SD in logarithmic colony-forming units per gram of feces (lg CFU/g) and per square centimeter of intestinal tissue (lg CFU/cm²).

Statistical analysis

Statistical analysis was performed using Statistica 12 (StatSoft Inc., Tulsa, OK, USA). The Shapiro–Wilk test was applied to assess the normality of data distribution, following the approach described by Mishra et al.⁵². Variables with a normal distribution were expressed as mean ± standard deviation (SD), while those not normally distributed were reported as medians with interquartile ranges (IQR). For group comparisons, normally distributed data were analyzed using one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons. Non-normally distributed data were evaluated using the Mann–Whitney U test for pairwise comparisons and the Kruskal–Wallis test for comparisons across multiple groups, as outlined by Chan and Walmsley⁵³. The Fisher’s Exact Test was employed to compare the proportions of rats showing an increase or decrease in rotation rate between the first and second apomorphine-induced rotation tests. A p value of ≤ 0.05 was considered statistically significant.

Results

Administration of ⁶⁴Zn-aspartate attenuated dopaminergic system damage and improved behavioral outcomes in rats with LPS-induced Parkinson’s disease

The apomorphine-induced rotation test is widely recognized as a standard method for evaluating dopaminergic system impairment and behavioral deficits in rat models of PD, including LPS-induced models^54,55. On Day 8 post-surgery (prior to the initiation of treatment), rats administered LPS exhibited an average contralateral turning rate of 2.2 rpm, which corresponds to an estimated 44–60% loss of dopaminergic neurons (Table 1). By Day 21, 67% of LPS-lesioned rats demonstrated a 23% increase in rotation rate, suggesting continued neurodegeneration. The remaining 33% of animals in this group showed either stable or slightly reduced turning behavior. In contrast, among the rats receiving ⁶⁴Zn-asp, 86% showed a 13% decrease in rotation rate by Day 21, indicating potential neuroprotection or partial recovery of dopaminergic function. Only 14% of the treated animals displayed stable or slightly increased rotation rates, suggestive of ongoing neuronal loss.

Table 1.

Behavioral characteristics of rats with LPS-induced Parkinson’s disease treated with ⁶⁴Zn-asp.

	Intact animals, n = 12	Sham-operated animals, n = 12	LPS-induced PD, n = 12	LPS-induced PD + 64Zn-asp, n = 15
Open field test
Total distance traveled, sm	3088.6 [2823.7; 3473.3]	2959.7 [2702.4; 4759.5]	2097.3 [1232.4; 2498.9]	2679.6 [1521.3; 3518.7]
Time spent exploring the inner perimeter, sec	21.5 [9.3; 29.5]	14.0 [7.0; 37.0]	5.0 [0.5; 12.0]a	11.5 [7.3; 22.3]
Time spent in squares surrounded by two walls, min	0.91 [0.90; 0.97]	0.95 [0.87; 0.96]	1.0 [0.98; 1.1]ab	0.98 [0.95; 1.0]
Number of rearings	18.8 [16.5; 25.1]	21.3 [16.0; 28.3]	15.8 [10.3; 21.9]	16.2 [11.9; 23.3]
Rearing duration, s	15.0 [12.5; 18.5]	11.8 [8.9; 18.8]	14.0 [13.9; 14.3]	14.8 [11.9; 16.8]
Number of grooming episodes	4.0 [2.8; 6.3]	6.4 [4.4; 9.3]	8.8 [6.5; 9.3]a	6.2 [4.0; 7.3]
Number of defecations	3.7 [1.8; 6.4]	4.5 [1.9; 6.7]	3.8 [1.8; 7.2]	5.2 [2.3; 8.4]
Elevated plus maze (EPM) test
Total distance traveled, sm	1214.1 [1112.7; 1282.7]	1216.7 [957.6; 1256.0]	481.04 [401.3; 635.0]ab	999.7 [728.3; 1200.3]c
Number of transitions	14.8 [10.8; 18.9]	16.6 [9.4; 21.6]	10.1 [6.2; 12.4]	13.6 [10.2; 19.9]
Time in closed arms/time in open arms	5.1 [2.8; 6.1]	9.9 [5.8; 10.1]	15.2 [10.8; 18.3]ab	11.9 [7.8; 13.1]a
Time spent in a stretched attend posture (risk assessment), s	200.3 [187.3; 326.1]	283.6 [211.2; 324.5]	61.6 [57.2; 115.1]ab	185.6 [135.9; 234.9]c
Number of rearings	18.0 [17.0; 28.0]	25.0 [17.5; 30.5]	13.5 [9.3; 17.3]b	16.7 [10.3; 22.5]
Apomorphine test
Contralateral rotation rate, rpm
Day 8 post lesion			1.23 [0.98; 1.65]	1.53 [1.23; 1.79]
Day 21 post lesion			1.90 [1.28; 2.38]	0.85 [0.46; 1.23]c
Percentage of rats exhibiting an increase/ decrease in rotation rate between the first and second apomorphine-induced rotation tests			67/33	14/86c

Data are presented as median and IQR or %.

Data from different animal groups were compared using Kruskal–Wallis’s test or Fisher Exact Test for % correspondingly. ^ap < 0.05 as compared to intact animals;

^bp < 0.05 as compared to sham-operated animals; ^cp < 0.05 as compared to untreated animals with LPS-induced PD.

Italic and bold in the cells indicates statistically significant differences.

Motor function was assessed using the open field and EPM tests. LPS-induced dopaminergic damage was associated with impaired locomotion (Table 1). In the open field test, LPS-lesioned rats exhibited a 37% reduction in the median distance traveled compared to controls (p = 0.06). Similarly, in the EPM test, the distance traveled was reduced by approximately 2.4-fold relative to control animals (p ≤ 0.05). Treatment with ⁶⁴Zn-asp ameliorated these deficits: rats in the LPS-PD + ⁶⁴Zn-asp group demonstrated higher locomotor activity in both tests compared to untreated LPS-lesioned animals, with performance levels comparable to those of control groups.

By the study’s end, LPS-lesioned rats exhibited moderate anxiety-like behavior. In the open field test, these animals spent about 2.5 times less time in the central area compared to controls (p ≤ 0.05), indicating increased anxiety (Table 1). The number of transitions between open and closed arms in the EPM was moderately reduced in LPS-lesioned rats. Conversely, LPS-lesioned rats treated with ⁶⁴Zn-asp tended to show increased transitions, suggesting reduced anxiety. Additionally, the ratio of time spent in closed arms to time in open arms was three times higher in LPS-lesioned rats than in controls, a pattern that was reversed with ⁶⁴Zn-asp treatment.

Thigmotactic behavior, reflected by the time spent in corner zones (squares bordered by two walls), was slightly elevated in LPS-lesioned rats. Furthermore, these rats displayed reduced risk assessment behavior, as evidenced by a more than twofold reduction in the time spent in a stretched-attend posture compared to controls. Treatment with ⁶⁴Zn-asp normalized these parameters, bringing them in line with those observed in healthy rats.

Rearing frequency, another indicator of exploratory behavior, was also reduced in LPS-lesioned animals. A tendency toward recovery of this behavior was observed in the treated group. Excessive grooming—commonly associated with anxiety⁵⁶—was prominent in the LPS-PD group but normalized following ⁶⁴Zn-asp administration.

Collectively, these findings suggest that ⁶⁴Zn-aspartate treatment not only protects against dopaminergic neurodegeneration but also alleviates motor impairments and mitigates anxiety-like behavior in the LPS-induced PD rat model.

⁶⁴Zn-aspartate treatment improves hematological inflammatory profiles in LPS-induced parkinsonian rats

The serum level of C-reactive protein (CRP), a widely recognized and reliable marker of systemic inflammation, exhibited substantial individual variability across all experimental groups (Fig. 2). Despite this variability, the median CRP level in the LPS-PD group was approximately 35% higher than that in control animals. In contrast, the median CRP level in the LPS-PD + ⁶⁴Zn-asp group was comparable to that of the control groups.

Fig. 2.

Plasma CRP levels in rats with LPS-induced Parkinson’s disease treated with ⁶⁴Zn-asp. Data are presented as medians and IQR. Data from non-operated, sham-operated, and LPS-lesioned animals were compared using Kruskal–Wallis’s test.

White blood cell (WBC) counts and their differential components—neutrophils, lymphocytes, monocytes, and platelets—are well-established markers of systemic inflammation. In our study, LPS-lesioned rats exhibited a significant increase in both absolute and relative granulocyte (Gr) counts, a decrease in the relative lymphocyte (Ly) count, and a 1.7-fold increase in platelet (PLT) count (Table 2). These changes are indicative of a systemic inflammatory response. In contrast, LPS-lesioned animals treated with ⁶⁴Zn-aspartate showed no significant differences in these parameters compared to non-operated and sham-operated control groups, suggesting an anti-inflammatory effect of the treatment.

Table 2.

Hematological parameters in rats with LPS-induced Parkinson’s disease treated with ⁶⁴Zn-asp.

	Intact animals, n = 12	Sham-operated animals, n = 12	LPS-induced PD, n = 12	LPS-induced PD + 64Zn-asp, n = 15
WBC count with differentials
WBC, × 10^3/μl	5.2 ± 1.6	5.9 ± 1.2	5.2 ± 1.2	7.0 ± 2.3
Ly, × 10^3/μl	3.9 ± 1.1	4.0 ± 0.7	3.3 ± 1.0	5.0 ± 1.0 c
Mo, × 10^3/μl	0.4 ± 0.1	0.7 ± 0.2a	0.4 ± 0.1b	0.5 ± 0.2
Gr, × 10^3/μl	1.0 ± 0.2	1.2 ± 0.2	1.6 ± 0.2ab	1.4 ± 0.2c
PLT, × 10^3/μl	140.3 ± 29.4	173.5 ± 60.0	240.6 ± 24.1ab	160.6 ± 55.8c
Ly, %	73.9 ± 5.0	68.0 ± 4.4	60.5 ± 4.5ab	70.2 ± 5.9c
Mo, %	8.0 ± 1.8	12.1 ± 1.7a	7.7 ± 1.2b	7.3 ± 1.9b
Gr, %	18.2 ± 4.6	20.0 ± 3.6	36.3 ± 5.4ab	23.5 ± 5.0c
WBC-based indices of systemic inflammation
NLR	0.25 [0.19; 0.29]	0.26 [0.25; 0.30]a	0.54 [0.41; 0.62]ab	0.34 [0.27; 0.39]abc
LMR	9.9 ± 2.8	5.9 ± 1.1a	8.3 ± 2.2b	10.3 ± 3.0b
PLR	37.7 ± 8.9	45.8 ± 11.9	51.8 ± 6.7a	37.9 ± 11.3
PMR	347.8 ± 16.3	253.7 ± 34.8	614.5 ± 98.7ab	534.3 ± 94.7ab
PNR	159.8 ± 47.3	158.6 ± 45.8	152.5 ± 67.3	116.4 ± 41.6
NMR	2.4 ± 0.7	1.8 ± 0.3	4.1 ± 0.8ab	3.3 ± 0.7bc
SII	38.0 [18.3; 51.6]	39.1 [33.4; 48.6]	76.6 [75.9; 78.7]ab	49.0 [41.9; 57.9]abc
SIRI	0.11 [0.06; 0.15]	0.15 [0.13; 0.19]	0.22 [0.19; 0.23]ab	0.10 [0.07; 0.12]c
NPLHbR	2.58 [1.97; 3.15]	3.46 [3.03; 4.10]	5.73 [5.29; 8.80]ab	3.24 [2.87; 3.53]c

Data are presented as median and IQR or as mean ± SD.

Data from different animal groups were compared using Kruskal–Wallis’s test or ANOVA with Tukey post-hoc test, respectively.

^ap < 0.05 as compared to intact animals; ^bp < 0.05 as compared to sham-operated animals; ^cp < 0.05 as compared to untreated animals with LPS-induced PD.

Ly lymphocytes, Mo monocytes, Gr granulocytes, PLT platelets.

WBC-based indices are now recognized as valuable markers for assessing the severity of systemic inflammation in various conditions, including neurodegenerative diseases. Our study demonstrated a marked increase in nearly all calculated WBC-based inflammatory indices in LPS-PD rats (Table 2). In LPS-lesioned rats, NLR was increased 2.2-fold as compared to controls. The median PLR value was elevated by 27.2%. Compared to other inflammatory conditions, PMR and NMR are less commonly used in neurodegenerative disease research, yet they are considered informative markers of systemic inflammation. In our study, both PMR and NMR median values were approximately twofold elevated in LPS-lesioned rats. The median SII value was approximately twofold higher in the LPS-PD group compared to control animals, while another complex WBC-based index – SIRI – was 1.7 times higher than in controls. The median value of a newly proposed WBC-based inflammatory marker, NPLHbR, in the LPS-PD group was approximately 1.9 times higher than that in the control rats. Treatment with ⁶⁴Zn-asp effectively prevented LPS-induced alterations in WBC-based inflammatory indices, maintaining values at levels comparable to those of non-operated and sham-operated controls, unlike in untreated LPS-PD rats.

⁶⁴Zn-asp modulates polarization of peripheral blood phagocytes in LPS-induced PD rats

The functional state of circulating phagocytes was assessed using parameters commonly applied to characterize their polarized activation profile: phagocytic activity (PI), oxidative metabolism (ROS generation), and the expression of phenotypic markers CD80/86 and CD206. In the LPS-PD group, the median monocyte PI value was twice lower than that of control animals (Fig. 3A). Treatment of LPS-lesioned rats with ⁶⁴Zn-asp increased the monocyte PI median by 31% compared to untreated parkinsonian rats, with values comparable to those in the sham-operated control group. The median neutrophil PI value in the LPS-PD group did not differ significantly from that of control rats, and treatment with ⁶⁴Zn-asp had no effect on this parameter (Fig. 3B). Notably, neutrophil phagocytic activity was elevated in sham-operated animals, likely reflecting an N2 polarization shift associated with their participation in reparative processes following surgical intervention⁵⁷. Monocyte ROS generation was markedly elevated in LPS-lesioned rats, indicating a pro-inflammatory shift characteristic of systemic inflammation (Fig. 3C). Treatment with ⁶⁴Zn-asp substantially reduced oxidative metabolism: the median ROS generation in the LPS-PD + ⁶⁴Zn-asp group was sevenfold lower than in the LPS-PD group and approximately fourfold lower than in controls. Neutrophil ROS generation in LPS-PD rats was significantly higher than in controls. In contrast, LPS-lesioned animals treated with ⁶⁴Zn-asp showed ROS levels comparable to sham-operated rats (Fig. 3D). Notably, sham-operated rats exhibited reduced ROS generation compared to non-operated animals, further supporting the notion of an anti-inflammatory, tissue-repairing metabolic profile. Neither the proportion of CD80/86⁺ cells (Fig. 3E) nor the CD80/86 expression level (Fig. 3F) in circulating phagocytes from LPS-lesioned rats differed significantly from controls. In the LPS-PD + ⁶⁴Zn-asp group, the median proportion of CD80/86⁺ cells was reduced by half compared with untreated animals, while the expression level was approximately threefold higher. The proportion of CD206⁺ circulating phagocytes was slightly elevated in LPS-lesioned rats, whereas treatment with ⁶⁴Zn-asp restored this value to control levels (Fig. 3G). CD206 expression level in the LPS-PD group was similar to non-operated animals but 6.5-fold lower than in sham-operated rats (Fig. 3H). Administration of ⁶⁴Zn-asp modestly increased CD206 expression. Since elevated CD206 is linked to an anti-inflammatory phagocyte phenotype, the higher values in sham-operated rats likely reflect post-surgical reparative processes, while the increase in the LPS-PD + ⁶⁴Zn-asp group may result from the drug’s anti-inflammatory action.

Fig. 3.

Metabolic characteristics of peripheral blood phagocytes in rats with LPS-induced Parkinson’s disease treated with ⁶⁴Zn-asp. (A) monocyte phagocytosis index; (B) neutrophil phagocytosis index; (C) monocyte ROS generation; (D) neutrophil ROS generation; (E) fraction of CD80/86-positive cells; (F) CD80/86 expression level; (G) fraction of CD206-positive cells; (H) CD206 expression level. Data are presented as medians and IQR. a - p ≤ 0.05 as compared to non-operated animals, b - p ≤ 0.05 as compared to sham-operated animals; c - p ≤ 0.05 as compared to LPS-PD group (Kruskal–Wallis’s test).

Influence of ⁶⁴Zn-asp on lymphoid organ weight and cellularity

PD-related inflammation was reflected in alterations of the lymphoid organs. LPS-PD rats showed significantly reduced thymus weight and increased thymus cellularity compared with controls (Table 3).

Table 3.

Lymphoid organ parameters in rats with LPS-induced Parkinson’s disease treated with ⁶⁴Zn-asp.

	Intact animals, n = 12	Sham-operated animals, n = 12	LPS-induced PD, n = 12	LPS-induced PD + 64Zn-asp, n = 15
Relative weight of thymus	1.32 [1.27; 1.36]	1.26 [1.18; 1.33]	1.07 [0.08; 1.08]a,b	1.16 [1.07; 1.29]c
Relative number of thymocytes	1.75 [1.49; 2.01]	3.77 [1.72; 5.82]a	7.86 [6.13; 9.86]a,b	6.26 [4.24; 8.31]a,b
Relative weight of spleen	2.97 [2.62; 3.33]	3.31 [3.07; 3.56]	2.55 [2.48; 2.59]b	3.19 [2.32; 3.45]c
Relative number of splenocytes	3.68 [3.26; 4.10]	5.58 [5.53; 5.63]a	6.45 [5.69; 8.62] a,b	2.46 [2.44; 4.27]c

Treatment with ⁶⁴Zn-asp restored thymus weight to control levels and partially normalized cellularity. Spleen weight was unchanged in LPS-lesioned rats compared to non-operated controls, but was marginally lower than in sham-operated animals; cellularity showed only a slight increase. In the LPS-PD + ⁶⁴Zn-asp group, both spleen weight and splenocyte counts were indistinguishable from controls.

⁶⁴Zn-asp alters polarization profiles of peritoneal macrophages in LPS-induced PD rats

Peritoneal macrophage PI was comparable across all experimental groups (Fig. 4A). In LPS-PD rats, median ROS generation was approximately twice that of non-operated and sham-operated controls (Fig. 4B), indicating heightened oxidative activity. Administration of ⁶⁴Zn-asp normalized ROS production to control levels. Neither the percentage of CD80/86⁺ cells (Fig. 4C) nor CD80/86 expression levels (Fig. 4D) in LPS-lesioned rats differed significantly from controls, and ⁶⁴Zn-asp treatment had no effect on these parameters.

Fig. 4.

Metabolic characteristics of peritoneal macrophages in rats with LPS-induced Parkinson’s disease treated with ⁶⁴Zn-asp. (A) phagocytosis index; (B) ROS generation; (C) fraction of CD80/86-positive cells; (D) CD80/86 expression level; (E) fraction of CD206-positive cells; (F) CD206 expression level. Data are presented as medians and IQR. a - p ≤ 0.05 as compared to non-operated animals, b - p ≤ 0.05 as compared to sham-operated animals; c - p ≤ 0.05 as compared to LPS-PD group (Kruskal–Wallis’s test).

The proportion of CD206⁺ cells (representing large resident peritoneal macrophages) and CD206 expression levels in LPS-lesioned rats were similar to those in non-operated animals (Fig. 4E, F). Treatment with ⁶⁴Zn-asp reduced the CD206⁺ cell fraction but increased CD206 expression.

⁶⁴Zn-asp mitigates inflammation-linked gut dysbiosis in a rat with LPS-induced PD

We analyzed the composition of the culturable gut microbiota in conjunction with measurements of fecal water content. At the end of the experiment, stool water content did not differ significantly among the experimental groups (Table 4). Nevertheless, rats in the LPS-PD group produced approximately 1.5-fold more feces (wet weight: 0.553 ± 0.11 g; dry weight: 0.268 ± 0.02 g) than controls (sham: 0.387 ± 0.18 g wet; 0.183 ± 0.05 g dry; p < 0.05), indicating impaired colonic motility, with stool accumulating in the colon rather than being efficiently propelled and expelled. In the LPS-PD + ⁶⁴Zn-asp group, fecal output was slightly lower than in untreated LPS-PD rats but remained higher than in controls.

Table 4.

Fecal wet weight, dry weight, and stool water content in rats with LPS-induced Parkinson’s disease treated with ⁶⁴Zn-asp.

	Intact animals, n = 12	Sham-operated animals, n = 12	LPS-induced PD, n = 12	LPS-induced PD + 64Zn-asp, n = 15
Fecal wet weight, g	0.356 ± 0.07	0.387 ± 0.18	0.554 ± 0.11a,b	0.521 ± 0.08a,b
Fecal dry weight, g	0.191 ± 0.02	0.183 ± 0.05	0.268 ± 0.02a,b	0.233 ± 0.02
Stool water content, %	54.1 ± 3.15	48.5 ± 7.47	48.9 ± 8.24	47.9 ± 6.57

Data are presented as mean ± SD.

Data from different animal groups were compared using ANOVA with a Tukey post-hoc test.

a - p ≤ 0.05 as compared to intact animals; b - p ≤ 0.05 as compared to sham-operated animals.

In gut microbiota assessment, particular attention was focused on quantitative analysis of anaerobic saccharolytic genera Bifidobacterium and Lactobacillus, which synthesize γ-aminobutyric acid (GABA) involved in gastrointestinal motility and the gut-brain axis. In LPS-PD rats, counts of these bacteria were moderately reduced in the small-intestinal and large-intestinal wall-adherent microbiota (Fig. 5A) and markedly decreased in the luminal microbiota of the large intestine (Fig. 5B). Specifically, the number of Bifidobacterium species decreased by one order of magnitude—from lg 8.20 ± 0.6 CFU/g in sham-operated animals to lg 6.90 ± 0.20 CFU/g in the LPS-PD group. The number of Lactobacillus species decreased by two orders of magnitude compared to the control group—from lg 7.00 ± 0.40 CFU/g in controls to lg 5.50 ± 0.70 CFU/g in the LPS-lesioned rats. Treatment with ⁶⁴Zn-asp preserved bacterial abundance at control levels.

Fig. 5.

Culturable wall-adherent and luminal gut microbiota in rats with LPS-induced Parkinson’s disease treated with ⁶⁴Zn-asp. (A) wall-adherent Bifidobacterium and Lactobacillus; (B) luminal Bifidobacterium and Lactobacillus; (C) wall-adherent enterobacteria; (D) luminal enterobacteria; (E) wall-adherent staphylococci; (F) luminal staphylococci. Data are presented as mean ± SD. Data from different animal groups were compared using ANOVA with a Tukey post-hoc test. a - p ≤ 0.05 as compared to non-operated animals; b - p ≤ 0.05 as compared to sham-operated animals; c - p ≤ 0.05 as compared to untreated animals with LPS-induced PD.

Under normophysiological conditions, neither lactose-positive nor lactose-negative E. coli were detected in the mucosa-associated biotope (Fig. 5C). In the LPS-PD group, lactose-positive E. coli reached 10³ CFU/cm², while lactose-negative strains increased to 10⁵ CFU/cm² in the mucosa-associated microbiota of the small intestine, with trace amounts also detected in colonic tissue. LPS-PD was additionally associated with a pronounced increase in opportunistic enterobacteria, reaching lg 2.90 ± 1.00 CFU/cm².

In fecal microbiota from LPS-PD animals, total E. coli abundance did not differ significantly from sham-operated controls (Fig. 5D). However, opportunistic enterobacteria counts increased by approximately two orders of magnitude, from lg 2.46 ± 0.73 CFU/g in controls to lg 4.30 ± 0.52 CFU/g in the LPS-PD group.

Treatment with ⁶⁴Zn-asp resulted in a notable restoration of microbiota composition. Lactose-fermenting E. coli levels decreased, and lactose-negative strains were completely eliminated from the mucosa-associated microbiota. Moreover, ⁶⁴Zn-asp administration significantly reduced opportunistic enterobacteria abundance in both mucosa-associated and luminal compartments.

Staphylococcus spp., including both mannitol-fermenting (S. aureus) and mannitol-negative strains, were also detected⁵⁸. No Staphylococci were present in the mucosa-associated biotopes of control animals. In LPS-PD animals, Staphylococcus spp. were identified in the wall-adherent microbiota of the large intestine, suggesting increased aerobiosis (Fig. 5E). Treatment with ⁶⁴Zn-asp slightly decreased the abundance of staphylococci in the mucosa-associated biotope. Quantitative characteristics of staphylococci in the luminal microbiota did not differ significantly between groups (Fig. 5F).

Discussion

LPS-induced PD models are among the most commonly used inflammatory models of PD, as they effectively reproduce key pathological features, including motor impairment, neuroinflammation, and systemic inflammation^39,59. Furthermore, these models provide valuable tools for investigating anti-inflammatory and neuroprotective agents⁶⁰. Given the growing recognition of systemic inflammation as a contributor to PD development and progression, as well as a potential therapeutic target, we employed this model to evaluate the effects of intravenous ⁶⁴Zn-asp administration on systemic immune-inflammatory responses in PD rats. Findings from in vitro and in vivo studies suggest that zinc may exert disease-modifying effects in PD through several mechanisms. Zinc appears to influence autophagy and lysosomal function, which may help limit α-synuclein accumulation, and it can reduce aggregation by enhancing albumin chaperone activity^61,62. It has also been reported to modulate inflammatory pathways, including NF-κB signaling⁶³, the NLRP3 inflammasome⁶⁴, and STAT3 activation⁶⁵, while preserving immune competence³¹. In addition, zinc contributes to redox homeostasis by inducing metallothioneins and glutathione, thereby supporting antioxidant defenses⁶⁶.

In our study, intravenous administration of ⁶⁴Zn-asp exerted neuroprotective effects by reducing dopaminergic neurodegeneration, ameliorating motor dysfunction, and attenuating anxiety-like behavior in Parkinsonian rats. These outcomes were associated with the pronounced anti-inflammatory activity of the drug.

Systemic inflammation in LPS-induced PD rats was confirmed by established markers, including elevated serum CRP levels, increased granulocyte and platelet counts, and a concomitant reduction in lymphocyte count^67,68. Beyond these parameters, indices derived from complete blood counts provide a more nuanced assessment of immune-inflammatory status by capturing the relative proportions of different leukocyte populations. Among them, the neutrophil-to-lymphocyte ratio, coupled with relative lymphopenia, has been linked to alterations in neurodegeneration-associated proteins, particularly within the α-synuclein and amyloid-β pathways, and correlates with greater clinical burden in PD patients^69,70. The PLR represents another established indicator of peripheral immune dysregulation and systemic inflammation in PD⁷¹. Similarly, an elevated neutrophil-to-monocyte ratio suggests an intensified inflammatory response⁷². Platelets, in addition to their roles in hemostasis and thrombosis, are increasingly recognized as active mediators of inflammation, supporting the recruitment of lymphocytes, neutrophils, and monocytes to inflamed tissues, thereby amplifying immune responses. In PD, chronic inflammation promotes platelet hyperreactivity, which may further exacerbate neuroinflammation⁷³. This highlights the relevance of the PMR as an informative marker of systemic inflammatory activity. Composite indices have also been proposed to better capture the complexity of immune-inflammatory dynamics. The SII, which incorporates neutrophil, platelet, and lymphocyte counts, provides an integrated measure of the immune-inflammatory balance. Elevated SII is strongly associated with increased PD risk, particularly in females⁷⁴. Likewise, the SIRI, which combines neutrophil, monocyte, and lymphocyte counts, reflects the interplay between immune activation and suppression and may indicate states of immunodeficiency or immune exhaustion⁷⁵. More recently, the NPLHbR has been introduced as a reliable WBC-based marker of systemic inflammation. Unlike other indices, NPLHbR also incorporates hemoglobin, a parameter frequently reduced in chronic inflammatory states, including age-related “inflammaging,” which is associated with anemia and sustained immune activation⁷⁶. In our study, the median values of all WBC-based inflammatory indices were significantly (~ 2 times) elevated in LPS-lesioned rats, reflecting pronounced and persistent systemic inflammation accompanied by features of immune exhaustion. ⁶⁴Zn-aspartate prevented LPS-induced changes in WBC-based inflammatory indices, preserving values comparable to controls. In addition to the well-established anti-inflammatory properties of zinc mediated through modulation of key pro-inflammatory signaling pathways such as NF-κB and IL-6, promotion of anti-inflammatory protein expression, and regulation of nitric oxide production⁶⁶, one potential mechanism underlying the observed effects of ⁶⁴Zn-asp may involve its impact on thymic function. In the context of systemic inflammation, acute thymic atrophy, as observed in LPS-lesioned rats in our study and in the MPTP model of PD in mice⁷⁷, may occur, leading to extensive loss of developing T cells during thymic selection, along with infiltration of immune cells that can further disrupt thymic architecture⁷⁸. Zinc plays a pivotal role in normal T-cell development and in recovery following acute injury and inflammation, as it promotes thymic regeneration by inducing endothelial cell production of bone morphogenetic protein 4 (BMP4)⁷⁹. In addition, zinc can promote differentiation of regulatory T-cells⁸⁰, which interfere with inflammatory signaling pathways controlling systemic inflammation⁸¹.

In addition to restoring immune cell counts in LPS-PD animals, intravenous administration of ⁶⁴Zn-asp abrogated the pro-inflammatory metabolic shift of phagocytic cells – key mediators of inflammatory responses—in both peripheral blood and the peritoneal cavity. In PD, systemic inflammation is driven in part by monocytes and neutrophils. Clinical studies consistently report elevated circulating levels of these cells along with increased concentrations of inflammatory markers⁸². Among monocytes, the classical pro-inflammatory (M1) subset is particularly implicated, as these cells release cytokines and chemokines that exacerbate neuroinflammation and contribute to neuronal damage. Neutrophils, the most abundant immune cells in circulation, act as early responders to inflammatory stimuli. In PD, pro-inflammatory N1 neutrophils are believed to play a role in the initial immune response to potential triggers, including pathogens or cellular damage. Although their role in PD has been less extensively investigated than that of monocytes and microglia, they are increasingly recognized as important contributors to early inflammatory events⁸³. In our study, LPS-lesioned rats exhibited an increased proportion of circulating CD206⁺ phagocytic cells. Although CD206 is commonly regarded as a marker of anti-inflammatory M2 phagocytes, emerging evidence indicates that CD206 expression does not invariably reflect an anti-inflammatory phenotype. Notably, Trombetta et al. described a population of circulating CD206⁺ phagocytes with a pro-inflammatory metabolic bias that co-expresses both M1 (pro-inflammatory) and M2 (anti-inflammatory) markers⁸⁴. These cells are less mature than classical M2 phagocytes and lack CD204 expression. According to Trombetta et al., this CD206⁺ pro-inflammatory subset contributes to the pathogenesis of inflammatory complications in systemic sclerosis. In our experiments, the expanded CD206⁺ cell fraction was associated with reduced phagocytic activity in monocytes and markedly increased reactive oxygen species production in both monocytes and neutrophils. Together, these functional alterations support the interpretation that the elevated CD206⁺ population observed in LPS-lesioned rats represents a mixed M1/M2 phenotype with a pro-inflammatory metabolic shift, contributing to the maintenance of systemic inflammation^85,86. Treatment with ⁶⁴Zn-asp returned the CD206 + phagocyte cell fraction to the norm, marginally increased monocyte phagocytic activity, and substantially decreased the oxidative metabolism of both phagocytic cell populations, indicating an anti-inflammatory metabolic shift. In treated Parkinsonian rats, we observed a reduced fraction of CD80/86⁺ cells accompanied by increased CD80/86 expression levels. While the functional significance of this seemingly paradoxical pattern cannot be conclusively established within the scope of the present study, it may tentatively suggest a shift toward the differentiation of CD80/86⁺ monocyte-derived myeloid suppressor cells, potentially associated with the anti-inflammatory action of the treatment. Costimulatory molecules of the B7.1 family, CD80 and CD86, are known to play a dual role in phagocyte biology. On the one hand, increased CD80/CD86 expression reflects acquisition of antigen-presenting capacity by circulating phagocytes and is commonly associated with a pro-inflammatory metabolic profile⁸⁷. On the other hand, the majority of ex vivo–generated myeloid-derived suppressor cells (MDSCs) exhibit high CD80/CD86 expression, and in vivo monocytic MDSCs are characterized by pronounced CD86 positivity^88,89.

The metabolic profile of peritoneal macrophages in LPS-lesioned rats was moderately altered compared with controls, with increased ROS production indicating a pro-inflammatory metabolic shift⁹⁰. Treatment with ⁶⁴Zn-aspartate reduced ROS generation and enhanced CD206 expression, a reliable marker of an anti-inflammatory phenotype in tissue-resident macrophages⁹¹.

The hypothesis that PD may originate in the periphery and involve environmental risk factors has brought increasing attention to the role of the gut microbiota. Intestinal dysbiosis, which can precede the clinical diagnosis of PD by several years, has been proposed as a potential trigger of the disease through the disruption of microbial and mucosal immune homeostasis, thereby initiating an inflammatory cascade that extends to the brain and contributes to the pathophysiology of PD⁹². Nevertheless, the prevailing view is that dysbiosis is more likely a consequence of PD rather than its primary cause. Although it may arise years before clinical onset and contribute to disease progression, it is not generally considered the initiating event⁹³. Instead, inflammation inherent to PD appears to be a major driver of gut dysbiosis. Under inflammatory conditions, the relative abundance of obligate anaerobes belonging to the Bacteroidetes and Firmicutes phyla declines, while Proteobacteria – particularly Gammaproteobacteria such as Enterobacteriaceae – expand and become dominant within the gut microbiota⁹⁴. In our study, systemic inflammation in parkinsonian rats was closely associated with pronounced intestinal dysbiosis, suggesting a bidirectional interplay between peripheral immune activation and gut microbial imbalance. Representatives of the anaerobic saccharolytic genera Bifidobacterium and Lactobacillus exhibited a marked reduction in both mucosa-associated and luminal microbiota. In parallel, members of the family Enterobacteriaceae demonstrated a significant expansion. Specifically, both lactose-fermenting and non-fermenting Escherichia coli, as well as opportunistic enterobacteria, were detected in the wall-adherent microbiota of LPS-lesioned animals, whereas these taxa were absent in control rats. A comparable increase in opportunistic enterobacteria was also observed in the colonic luminal microbiota. Both systemic and intestinal inflammation are known to alter the physiological oxygen gradient, leading to localized increases in oxygen availability. This shift generates conditions that are more aerobic than those observed in the healthy gut. Within this altered niche, members of the Enterobacteriaceae family gain a selective advantage, while commensal symbiotic bacteria are negatively affected by inflammation-induced environmental changes⁹⁵. Among the pathobionts characteristic of the gut microbiota in patients with PD, adherent-invasive E. coli (AIEC) has been identified. AIEC is thought to contribute to PD through the production of substances such as curli, which promote the aggregation of α-synuclein, a key pathogenic process in the disease⁹⁶. Inflammation in the peritoneal cavity, reflected by the pro-inflammatory metabolic profile of peritoneal macrophages and accompanied by pronounced dysbiosis, suggests concurrent inflammation within the intestinal mucosa-associated lymphoid tissue, which may promote the growth and virulence of AIEC. Notably, the emergence of Staphylococcus, atypical for mucosa-associated intestinal microbiota, likely reflects inflammation-driven aerobization of the biotope, further highlighting microbial community disruption. In LPS-lesioned rats, gut microbiota imbalance was associated with increased fecal output without a corresponding rise in water content, indicating gastrointestinal dysfunction – one of the most common non-motor symptoms in PD^97,98. This dysbiosis may also contribute to the thymic atrophy observed in these animals⁹⁹. Treatment of LPS-PD rats with ⁶⁴Zn-asp promoted the rebalancing of gut microbial communities, evidenced by an increase in bifidobacteria and lactobacilli in the luminal microbiota and a concomitant reduction or disappearance of enterobacteria in the wall-adherent compartment, suggesting a microbiota-stabilizing effect. This effect may result from the anti-inflammatory action of the compound, although a direct impact of the preparation on the microorganisms cannot be excluded. Zinc has been reported to exert bidirectional effects on Lactobacillus species, promoting the growth of some while inhibiting others¹⁰⁰. In addition, zinc can suppress the growth and virulence of AIEC by reducing bacterial adherence, biofilm formation, and the expression of key virulence factors¹⁰¹.

This study is subject to several limitations. First, it did not directly investigate how ⁶⁴Zn-asp influences zinc homeostasis or neuronal function, leaving important aspects of its underlying mechanism unresolved and emphasizing the need for further exploration. Second, although clearly pathologically relevant, the impact of ⁶⁴Zn-asp on cytoplasmic α-synuclein accumulation in nigral tyrosine hydroxylase–positive neurons was not evaluated and warrants investigation in future studies. Third, a more detailed characterization of phagocyte metabolism would require additional phenotypic markers to better distinguish myeloid-derived suppressor cells, while cytokine profiling in plasma and colonic tissue would further refine the assessment of systemic inflammation and the effects of ⁶⁴Zn-asp. Finally, molecular approaches are necessary to gain deeper insight into the impact of ⁶⁴Zn-asp on the intestinal microbiota.

Conclusion

Systemic inflammation is a recognized contributor to the onset and progression of PD, making it an important therapeutic target. Peripheral inflammatory events can intensify neuroinflammation and accelerate neurodegeneration; therefore, anti-inflammatory interventions are being actively explored. In this study, we demonstrate that intravenous administration of ⁶⁴Zn-aspartate markedly attenuates systemic inflammation in an LPS-induced rat model of PD. Treatment with ⁶⁴Zn-asp reduced dopaminergic neurodegeneration, improved motor performance and anxiety-like behavior, normalized systemic immune-inflammatory indices, and reversed pro-inflammatory metabolic shifts in circulating and tissue-resident phagocytic cells. These effects indicate a robust peripheral anti-inflammatory action of the compound.

Importantly, suppression of systemic inflammation was accompanied by partial restoration of gut microbial homeostasis. ⁶⁴Zn-asp increased the abundance of beneficial anaerobic commensals while limiting the expansion of inflammation-associated opportunistic bacteria, supporting a functional link between peripheral immune modulation and gut dysbiosis in PD. Together, these findings highlight the relevance of targeting systemic immune dysregulation and the gut–immune–brain axis to mitigate neurodegenerative processes.

Although additional studies are required to elucidate the molecular mechanisms underlying these effects—particularly those related to zinc homeostasis, α-synuclein pathology, and host–microbiota interactions—our data identify ⁶⁴Zn-asp as a promising multifunctional anti-inflammatory intervention with potential relevance for PD.

Author contributions

MT: supervision and review. MR: methodology, investigation, formal analysis. AB: methodology, conceptualization, and project administration. SG: conceptualization, writing, review, and editing. TD: methodology, investigation. RB: methodology, conceptualization. ND: methodology, investigation. RD: methodology, investigation. TS: methodology, investigation, and writing original draft. GT: supervision and writing—review and editing. LS: methodology, conceptualization, project administration, and writing the original draft. All authors read the final version of the manuscript.

Funding

The study was supported by a TSNUK project N 18DP036-10.

Data availability

The data that support the findings of this study are available from the corresponding author, upon reasonable request.

Declarations

Competing Interests

The authors declare no competing interests.