Mitigation of Neuroinflammation and Oxidative Stress in Rotenone-Induced Parkinson Mouse Model through Liposomal Coenzyme-Q10 Intervention: A Comprehensive In-vivo Study

Abstract

Parkinson's disease (PD) stands as the sec most prevalent incapacitating neurodegenerative disorder characterized by deterioration of dopamine-producing neurons in the substantia nigra. Coenzyme Q10 (CoQ10) has garnered attention as a potential antioxidant, anti-inflammatory agent and enhancer of mitochondrial complex-I activity. This study aimed to examine and compare the effectiveness of liposomal and non-encapsulated CoQ10 in rotenone induced-PD mouse model over a 21-day treatment duration. 30 mice were divided into 5 equal groups: Group I (mice receiving normal saline), Group II (rotenone was administered to mice), Group III (standard CoQ10 was given to mice), Group IV (mice were treated with non-encapsulated CoQ10) and Group V (mice were treated with CoQ10 Liposomes). Motor performance, the preservation of dopaminergic neurons, levels of neuroinflammation, oxidative stress, neurotransmitter levels, RT-qPCR analysis of PD-linked genes and histopathology were evaluated. The Liposomal CoQ10 group exhibited superior outcomes in behavioral tests such as reduced anxiety in the open field test, enhanced balance and coordination in beam balance test and improved cognitive performance in Y-maze test. Liposomal Coenzyme Q10 displayed pronounced antioxidative effects, evidenced by a significant (p < 0.001) increase in superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) activities. In contrast, the non-encapsulated CoQ10 group showed a delayed response in mitigating the inflammation and oxidative stress. CoQ10 Liposomes demonstrated superior efficacy (p < 0.0001) in restoring dopamine and noradrenaline levels, reducing acetylcholinesterase activity, and downregulating Synuclein Alpha (SNCA) gene expression (0.722-fold change) compared to oral CoQ10, highlighting its potential in suppressing PD symptoms. The results of this study indicated that the liposomal CoQ10 effectively averted motor impairments, memory lapses, oxidative stress, as well as neuroinflammation triggered by rotenone.

Introduction

Parkinson's Disease (PD) is the sec most prevalent neurodegenerative disorder after Alzheimer's. PD is a relentless and debilitating neurodegenerative disorder that afflicts approximately six million people worldwide aged 60 and above. It primarily manifests as motor symptoms due to the degeneration of dopamine-producing neurons in the substantia nigra pars compacta (SNpc), including tremors, bradykinesia (slowness of movement), muscle rigidity, and impaired coordination. PD is also associated with non-motor symptoms such as gastrointestinal issues, cognitive decline and neuropsychiatric changes [1].

While the etiology of PD remains complex and multifaceted, oxidative stress, mitochondrial dysfunction and neuroinflammation are recognized as central players in the pathogenesis of this disorder [2]. The interaction between mitochondrial complex I and α-synuclein is pivotal in PD pathogenesis. α-synuclein fragments are found within mitochondria and phosphorylated α-synuclein is implicated in both sporadic and familial PD. Phosphorylated α-synuclein affects mitochondrial function, leading to cytochrome-c release and altered mitochondrial membrane potential. α-synuclein also interferes with ATP synthesis and can induce mitochondrial lipid peroxidation [3].

Oxidative stress plays a significant role in PD pathogenesis, resulting from an imbalance between oxidants (ROS and RNS) and antioxidative mechanisms. Dopaminergic neuron deterioration is linked to ROS production in mitochondria. Environmental factors like pesticides and neurotoxic substances further increase ROS production. Mitochondrial dysfunction, particularly Complex I deficits, is associated with neural apoptosis in PD [4]. The pesticide rotenone is neurotoxic to dopaminergic neurons in vivo due to its mitochondrial toxicity and complex I inhibition. Rotenone's action increases oxidative stress and α-synuclein toxicity. It also activates microglial cells and induces oxidative stress, prompting research into potential antioxidant therapies like coenzyme Q10 (CoQ10) [5].

In this context, CoQ10, which is biologically active, lipid-soluble quinone that has an isoprenoid side chain attached to the benzoquinone ring was selected [6]. In mitochondrial respiratory chain the CoQ10 is responsible for transferring electrons from complex I and II to complex III [7, 8]. It is a critical co-factor in mitochondrial electron transport and an endogenous antioxidant, has garnered significant attention as a potential therapeutic agent for PD. CoQ10 inherent properties make it a compelling candidate for mitigating the oxidative damage and energy deficits observed in Parkinson's disease [9].

However, it has been observed that CoQ10 can maintain optimal mitochondrial function and alleviate neuronal impairments in individuals with Parkinson's disease. Despite the outstanding results of CoQ10 treatments, the poor bioavailability of this powerful antioxidant limits its clinical application. The significant hydrophobic nature of CoQ10's is due to isoprenoid tail consisted of ten units, the susceptibility to light and sensitivity to temperature constrains its effectiveness when taken orally [10]. Individuals with Parkinson's disease require daily administration of a significantly elevated CoQ10 for an extended duration, often spanning weeks to months. As a result, patients are likely to struggle to stick to the regimen. The effectiveness of therapy can therefore not be easily guaranteed [11].

As per the Unified Parkinson Disease Rating Scale (UPDRS), phase 3 clinical trials did not reveal any favorable impact from increased dosages of CoQ10 taken orally. This lack of benefit might be attributed to its elevated molecular weight and reduced water solubility, both of which restricted its ability to be absorbed effectively in the striatum and impeded its passage through the blood–brain barrier [7].

Different encapsulation techniques have been created to get over these restrictions. One method to address the aforementioned issue is the liposome delivery system. This paper delves into the intricate interplay between CoQ10 and Parkinson's disease, exploring how the formulation of CoQ10 into liposomal structures offers a promising avenue to address these challenges. Liposomes, as versatile nanocarriers, provide a means to enhance CoQ10's solubility, stability, and bioavailability, thereby potentially unlocking its full therapeutic potential [12]. The thin film hydration technique, also known as the Bangham method, was used to prepare stable small unilamellar vesicles (SUVs) containing CoQ10. This approach can improve the delivery of CoQ10 to target areas like the striatum, which is crucial in PD treatment [13].

Our research is focused on investigating the relative effectiveness of CoQ10 and liposomal CoQ10 in a rotenone-induced mouse model of Parkinson’s disease. We aim to meticulously analyze and compare their efficacy in mitigating the symptoms of PD.

Material and Methods

Materials

Cholesterol, Rotenone (≥ 95%) and Coenzyme Q10 (CoQ10; ≥ 98%) were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Stock solutions of rotenone and CoQ10 were prepared by dissolving in normal saline and olive oil respectively [14]. Phospholipids including 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2–dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol)−5000] (DPPE-mPEG5000) were generously donated by Lipoid GmbH (Ludwigshafen, Germany). Phosphate buffered saline (PBS; pH 7.4) was freshly prepared, sterile filtered and stored until further use. All other chemicals and solvents employed in this investigation were of the utmost quality obtainable in the commercial market.

Preparation of CoQ10 Liposomes

The thin film hydration technique was used for the preparation of CoQ10-loaded liposomes. Briefly, stock solutions of lipids were prepared in methanol:chloroform (1:2). Thereafter, calculated amounts of lipids including DPPC:Chol:DPPE-PEG5000 at a molar ratio of (85:13:2) were pipetted into a round bottom flask. The organic solvent was then evaporated under vacuum using a rotary evaporator (Heidolph Instruments, Schwabach, Germany). For CoQ10-loaded liposomes, CoQ10 was added to the lipid mixture in a drug: lipid ratio of 1:20. The formed lipid film was then hydrated with PBS (pH 7.4) and sonicated in a bath-type sonicator for 1 h at 50 °C to get 5 mM final lipid dispersion. The formed liposomal dispersion was then extruded through 200 nm polycarbonate filters (Whatman GmbH, Dassel, Germany) using Avanti mini extruder (Avanti Polar Lipids, Alabama, USA) to get small unilamellar liposomes. The prepared liposomes were transferred to an Eppendorf tube and stored in the fridge until further use [13].

Liposomal Characterizations

Encapsulation Efficiency (EE%)

To determine the encapsulation efficiency of CoQ10-loaded liposomes, 1 mL of the freshly prepared liposomes was centrifuged at 20,784 g for 15 min using benchtop Eppendorf centrifuge 4518 (Eppendorf, Hamburg, Germany) to separate the unentrapped CoQ10. Post centrifugation, 250 µL of the supernatant was diluted with 1.75 mL of ethanol. The pellet was also dissolved in 2 mL of ethanol. 1.75 mL of dissolved pellet was then diluted to 2 mL with 250 µL of PBS (pH 7.4). The absorbance of the supernatant and pellet was then recorded at 275 nm using UV spectrophotometer. The amount of CoQ10 was quantified using the standard curve constructed in the same solvent system with known CoQ10 concentrations. The EE% was calculated using the formula [15].

$$\text{EE}\left(\text{\%}\right)={\text{W}}_{\text{t}}/{\text{W}}_{\text{i}}\times 100$$

whereas, W_i represents the initial amount of CoQ10 added to the formulation and W_t represents the amount of CoQ10 in the supernatant after centrifugation.

Particle Zeta Potential and Size Distribution

To assess the stability of liposomes particle zeta potential and size distribution was measured. Zeta potential serves as an indicator of the electric charge present on the surface of liposomal particles. It offers insights into the electrostatic stability and the propensity of particles to come together in a suspension. The hydrodynamic diameter and zeta potential of the prepared liposomes were determined by dynamic light scattering and laser doppler velocimetry using Nano ZS zetasizer (Malvern Panalytica GmbH, Kassel, Germany). Liposomal dispersion was diluted with Milli-Q water (refractive index:1.33; viscosity:0.88mPaS) to determine the intensity-weighted distribution and polydispersity index. The instrument is equipped with HeNe laser at 633 nm wavelength and a backscattering angle of 173°, with an automatic setting for attenuator and measurement position. Zeta potential measurements relied on scattered light detection at 17° angle. The results obtained are triplicate of three individual measurements [16].

Transmission Electron Microscopy (TEM)

To assess the morphology of liposomes TEM analysis was performed. Moreover, it offers insights into structural integrity and uniformity. For TEM analysis, the liposomal sample was diluted with PBS (pH 7.4) and 20 µL was applied to 300 mesh formvar coated carbon supported copper grids (Plano GmbH, Wetzlar, Germany). After 5 min, excess liquid was removed, and the deposited sample was stained with 2% uranyl acetate for 2 min. The excess stain was bloated away before analysis with a JEM-1400 transmission electron microscope (JEOL Ltd, Tokyo, Japan), operating at 120 kV with a Gatan Inc. high-resolution camera [17].

X-ray Diffraction Analysis (XRD)

The XRD diffractogram of CoQ10 liposomes was obtained by X-ray diffractometer to elucidate crystalline lattice arrangement of substances in order to evaluate structural and functional characteristic of liposomes. The measurements were obtained at a voltage of 40 kV and 25 mA. The diffraction angle 2θ was set at a range of 10 to 80 degrees and the scanned rate was 2 degrees/min [18].

Induction of Parkinson's Disease

Parkinson's disease was induced by intraperitoneal injection (i.p.) of Rotenone at a dose of 2 mg/kg of body weight, every day for 21 days and was administered 1 h before standard drug or treatment [19].

Experimental Animals

Adult albino mice (n = 6), weighing 25–30 g were transported from the University of Veterinary and Animal Sciences, Lahore, and housed in the animal house of the Lahore College for Women University for acclimatization. The mice were housed in individually ventilated cages (6 mice per cage) with a 12:12 h light/dark cycle with free access to food and water. Temperature and humidity levels were maintained at the 23 ± 3 °C and 30–70% ranges, respectively. The study was approved by the Animal Ethical Committee number (ORIC/23–410).

Study Design

The mice were used in the experiment for 21 days. Thirty mice were randomly allocated into five groups (n = 6) as follows:

Group I (Control)	Normal saline with 2% DMSO at a dose of 10 mL/kg i.p. for 3 weeks
Group II (Untreated/Disease Control)	Rotenone (2 mg/kg) i.p. daily for 3 weeks
Group III(Standard)	CoQ10 (20 mg/kg) orally for 3 weeks
Group IV (Rot + CoQ10)	CoQ10 (20 mg/kg) orally 1 h after Rotenone (2 mg/kg), i.p. daily for 3 weeks
Group V (Rot + CoQ10-Liposomes)	Treated with CoQ10 Liposomal formulation (20 mg/kg) orally 1 h after Rotenone (2 mg/kg) i.p. daily for 3 weeks

Before the study, the mice were allowed to acclimatize for a week. The period of the study was 21 days. On days 1,7,14 and 21 behavioral parameters were assessed. Animals were euthanized on day 22 and their brains were collected for biochemical, neuroinflammatory, neurochemical and histopathological investigation (Fig. 1).

Fig. 1.

Summary of Experimental design

Rotenone and CoQ10 Dosing: Preparation and Administration Methods

Rotenone was dissolved in 2%DMSO (2mLDimethyl sulfoxide) and then dilute to q.s normal saline. It was prepared freshly on daily basis. Hence, total volume was 10 mL. Therefore, it was administered to mice at a dose of 2 mg/kg through i.p route for consecutive 21 days [20]. Moreover, 20 mg/kg of CoQ10 was dissolved in olive oil and orally administered to mice by using a gavage needle [14].

Assessment of Behavioral Parameters

Open Field Test (OFT)

To evaluate the locomotion patterns and behaviors associated with anxious tendencies, an open-field experiment was performed. The apparatus utilized for the test is a box which is divided into 16 (4 × 4) squares [21]. The animals were placed individually in the central area of the open field enclosure and allowed to explore the environment freely for three min. Following each test, the enclosure was meticulously cleaned with 70% ethanol. The ToxTrac software was employed to examine various metrics including the overall distance covered (mm), mean velocity (mm/s), mobility average speed (mm/s), frozen events, exploration rate (%) and total number of line crossings (crossing the square boundaries with both forepaws) [22].

Y-Maze Test

The Y-Maze assessment is constructed to evaluate the mice's capacity to explore new environments, as mice generally exhibit a preference for discovering a novel maze arm rather than revisiting a previously explored one. The findings of this investigation mirror spatial cognitive memory, a variant of transient memory. The apparatus utilized for the Y-Maze test consisted of a wooden maze painted black, featuring three arms of equal size arranged in the order of a capital Y, identified as A, B, and C. Each arm measured 12 cm in width, 40 cm in length, and 35 cm in height, positioned at 120° angles to one another. During the examination, every rodent was placed within the central zone of the Y-shaped maze, and their successive entries into the trio of arms were precisely recorded for five min. An entry into an arm was deemed valid once the mouse's hind paws were fully within the arm. Spontaneous alternation behavior indicates memory retention, observed when mice sequentially enter all three arms (e.g. ABC, BCA, or CAB). The calculation of the spontaneous alternation percentage was determined in the following manner:

$$\text{Spontaneous alternation}\left(\text{\%}\right)=\left(_{}^{\text{count of alternation}},,\left./,_{\text{total arm entries}}^{}\right)\right)\times 100$$

Before the arrival of the subsequent mice, the equipment was cleansed using 70% ethanol and then dried using a fresh fabric [23].

Beam Balance Test

The beam balance test is constructed to evaluate motor coordination and balance deficits. In order to assess severity of motor impairment, number of falls are recorded. The capacity of mice to cross a 5 mm square and 100 cm lengthy beam towards a dim target box was gauged through the employment of the beam balance test. Mice were acclimated for three days preceding the evaluation and trained to cross a raised 12 mm square beam to reach a target enclosure. Mice were considered appropriately trained when they were able to cross the complete length of the beam to the "target enclosure". Typically, it would take about four attempts on the beam for mice to attain full proficiency, and this training process would be spread out over two days. Following this, the motor capabilities of the mice under experimentation were subsequently evaluated. The count of instances where mice fell off from the beam was recorded [24].

Preparation of Tissue Homogenate

After completion of treatment period, mice were sacrificed by decapitation under mild anesthesia. The brains of mice from each group were removed immediately and then placed in an ice-cold normal saline in order to remove debris. The brains were homogenized using a homogenizer and then diluted in 0.1 M phosphate buffer with a pH of 7.0 maintaining a 1:10 weight-to-volume ratio. Subsequently, the homogenized sample underwent centrifugation at 600xg for 10 min at 4-degree Celsius. Each sample's supernatants were collected and utilized for analysis of oxidative stress biomarkers. The The substantia nigra, the striatum, the globus pallidus, and the nucleus accumbens areas from hemsphere were used for evaluation of biochemical markers and hippocampus was used for determining the neurotransmitters [25]. For histopathological analysis, the striatum and the substantia nigra part of brain was collected and preserved in 10% formalin solution.

Analysis of Biomarkers for Oxidative Stress

To evaluate the indicators of oxidative stress, a brain tissue homogenate was meticulously prepared. Dissecting the brains of mice from each group yielded tissue homogenate. Using a microplate reader, absorbance from the supernatant of each sample was acquired and applied to estimate indicators of oxidative stress, including enzymatic antioxidants such as superoxide dismutase (SOD), glutathione (GSH), catalase (CAT), lipid peroxidation marker malondialdehyde (MDA) and protein content (PC) and nitric oxide (NO).

Assessment of Superoxide Dismutase (SOD) Activity

SOD plays an essential part in order to protect cells from oxidative injury. Moreover, within the context of PD, oxidative stress leads to diminished SOD activity. Therefore, this test is significant for evaluating potential of CoQ10 in protecting mice from oxidative stress. 2.8 mL of a potassium phosphate buffer solution with a concentration of 0.1 M, adjusted to a pH of 7.4, was combined with 0.1 mL of pyrogallol solution with a concentration of 2.6 mM in 10 mM HCL in a 0.1 mL aliquot of tissue homogenate. The absorbance of the resultant mixture was determined at 325 nm for a duration of 5 min with 30 s interval, employing a UV/VIS spectrophotometer (UV-1280, Shimadzu, Kyoto, Japan) [26]. The results were reported in units per mg of protein(U/mg). The quantification of SOD levels was executed employing a sophisticated mathematical model represented by the regression equation [27].

$$\mathbf{Y}=0.0095\mathbf{x}+0.1939$$

where Y = Absorbance.

Assessment of Catalase (CAT) Activity

Catalase is vitally important antioxidant that facilitates conversion hydrogen peroxide into water and oxygen, thereby inhibiting the accumulation of reactive oxygen species. Furthermore, analysis of CAT activity provides valuable data about antioxidant potential of cells in PD model. A mixture of 1.95 mL of 50 mM PBS (pH 7.4) and 1 mL of 30 mM hydrogen peroxide solution was prepared. This mixture was then added to the 0.05 mL of tissue homogenate. The mixture was analyzed at a wavelength of 240 nm using a UV/VIS spectrophotometer for 30 s with an interval of 15 s for 3 min. Moreover, assay was performed at room temperature with no pre-incubation time [28]. To ascertain CAT levels within each tissue, the subsequent formula was employed [29]. The outcomes were quantified and reported in units of CAT activity/mg of protein (U/mg of protein) [30].

$$\mathbf{C}\mathbf{A}\mathbf{T}=\mathbf{\delta }\mathbf{O}.\mathbf{D}/\mathbf{E}\mathbf{x}\mathbf{v}\mathbf{o}\mathbf{l}\mathbf{u}\mathbf{m}\mathbf{e}\mathbf{o}\mathbf{f}\mathbf{s}\mathbf{a}\mathbf{m}\mathbf{p}\mathbf{l}\mathbf{e}\mathbf{x}\mathbf{m}\mathbf{g}\mathbf{o}\mathbf{f}\mathbf{p}\mathbf{r}\mathbf{o}\mathbf{t}\mathbf{e}\mathbf{i}\mathbf{n}$$

where δ O. D shows change in absorbance, E shows extinction coefficient of hydrogen peroxide (0.071 mmol cm^-1).

Assessment of Glutathione (GSH) and Nitric Oxide (NO) Levels

In PD, lower GSH levels are linked with increased oxidative stress and impaired mitochondrial function. However, evaluating GSH levels provides insights into CoQ10’s role as an antioxidant. To gauge the GSH level, a 1 mL aliquot of tissue homogenate was precipitated with an equivalent volume of a 10% trichloroacetic acid (TCA) solution. Subsequently, 4 mL of PBS and 0.5 mL of 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) reagent were added to the resulting supernatant. The absorbance was then recorded at 412 nm. The outcomes were quantified and reported in nanomoles (nM) of reduced glutathione per mg of protein [31].

To assess the NO concentration, an equal volume of Griss reagent and brain homogenate were mixed and then incubated for 10 min. Afterwards, the absorbance was measured at 546 nm [32]. The amount nitric oxide (μmol/mL) was then estimated using the standard curve.

Assessment of Malondialdehyde (MDA) Concentration

During lipid peroxidation the byproduct which is formed is MDA. However, tracking of MDA levels helps in assessing overall oxidative burden in PD. Briefly, 3 mL of TBA reagent was added to 1 mL of tissue homogenate, followed by vigorous agitation and then for 15 min at room temperature incubate the mixture [29]. After incubation the mixture was then placed in an ice bath. Subsequently, the mixture was centrifuged at 1107 g for 10 min. The supernatant was then collected and the absorbance was then recorded at a wavelength of 532 nm [27]. The subsequent equation was employed to determine the MDA concentration:

$$\mathbf{M}\mathbf{D}\mathbf{A}\mathbf{C}\mathbf{o}\mathbf{n}\mathbf{c}=\mathbf{A}\mathbf{b}\mathbf{s}\mathbf{x}100\mathbf{x}\mathbf{V}\mathbf{t}/(1.56\mathbf{x}{10}^5)\mathbf{x}\mathbf{W}\mathbf{t}.\mathbf{x}\mathbf{V}\mathbf{u}$$

Abs = Absorbance, Vt = Total volume (4 mL), 1.56 × 10⁵ is molar extinction coefficient, Wt = Weight of brain, Vu = Aliquot volume (1 mL).

Measurement of Protein Content

For the determination of protein content, the Lowry method [33], was employed. Briefly, three different solutions were prepared as follows:

• AA solution comprising 2% sodium carbonate (Na₂CO₃) dissolved in 0.1 normal (N) sodium hydroxide (NaOH).
• BA solution containing 1% potassium sodium tartrate (KNaC₄H₄O₆.4H₂O) in distilled water.
• CA solution comprising 0.5% copper sulfate pentahydrate (CuSO₄·5H₂O) in aqueous medium.

Reagent 1 was prepared by mixing 48 mL of solution A, with 1 mL of each of solution B and C.

Reagent 2 was prepared by mixing 2 N Folin-phenol in distilled water (1:1).

Protein content was determined by adding 4.5 mL of reagent 1 in 0.2 mL of tissue homogenate. Afterwards, the mixture was incubated for 10 min. Subsequently, 0.5 mL of reagent 2 was added to the mixture, and incubated for another 30 min. The absorbance of the resulting mixture was recorded at a wavelength of 660 nm. The protein content was then quantified using the standard curve [34].

Evaluation of in vitro Antioxidant Capacity

The in-vitro antioxidant capacity of a CoQ10 was estimated by DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. Briefly, 1 mL of 0.1 mM DPPH solution was added to the varying concentrations (10—100 μg/mL) of CoQ10 solution, followed by the addition of 2 mL of methanol to make up the volume to 4 mL. The reaction mixture was incubated for 30 min at 25 °C in the dark, and the absorbance at 517 nm was measured using a UV-spectrophotometer. Additionally, a standard curve for ascorbic acid was conducted at the comparable concentration. The percentage of inhibition was calculated using the formula [35, 36]:

$$\mathit{I}\mathit{n}\mathit{h}\mathit{i}\mathit{b}\mathit{i}\mathit{t}\mathit{i}\mathit{o}\mathit{n}\left(\text{\%}\right)=\frac{{\mathit{A}\mathit{b}}_{\mathit{c}\mathit{o}\mathit{n}\mathit{t}\mathit{r}\mathit{o}\mathit{l}}-{\mathit{A}\mathit{b}}_{\mathit{s}\mathit{a}\mathit{m}\mathit{p}\mathit{l}\mathit{e}}}{{\mathit{A}\mathit{b}}_{\mathit{c}\mathit{o}\mathit{n}\mathit{t}\mathit{r}\mathit{o}\mathit{l}}}\times 100$$

where Ab_control and Ab_sample represent the absorbance of control and sample respectively.

Quantification of Neurotransmitters

Preparation of Aqueous Phase

For the quantification of the neurotransmitters, brain tissue specimens were weighed and homogenization for 60 s in 5 mL of HCl-butanol solution. Subsequently, the homogenized mixture was centrifuged at 492 g for 10 min. Afterwards, 1 mL of the resulting supernatant was mixed with 2.5 mL of heptane and 0.31 mL of 0.1 M HCl solution. The organic phase was removed to calculate the dopamine and noradrenaline levels, and 0.2 mL of aqueous phase was added.

Dopamine and Noradrenaline Quantification

The quantification of these two neurotransmitters allows a more complete understanding of neurochemical disturbances in PD. Moreover, the quantification of these neurotransmitters helps us to assess the effectiveness of therapies such as conventional versus liposomal CoQ10, in restoring these neurotransmitters and improving PD symptoms. Briefly, 0.2 mL of aqueous phase was mixed with 0.05 mL of 0.4 M HCL and 0.1 mL of ethylenediaminetetraacetic acid (EDTA) solution. 0.1 mL of iodine solution was added for oxidation and incubated for 2 min, followed by the addition of 0.1 mL of Na₂CO₃. Subsequently, acetic acid was added after 90 s, and the solution was heated at 100 °C for 6 min. After cooling to room temperature, the absorbance for dopamine and noradrenaline was measured at wavelengths of 350 nm and 450 nm, respectively. To prepare the blank solutions for dopamine and noradrenaline, the oxidation reagents were added in a reverse order i.e., Na₂CO₃ was added before the addition of iodine solution. Dopamine and noradrenaline concentrations (µg/mg) were determined using standard curves [37].

Assessment of Acetylcholinesterase (AChE) Activity

Acetylcholinesterase activity, responsible for the degradation of acetylcholine, is evaluated to assess potential alterations in cholinergic system. As there is a significant involvement of acetylcholine in regulating motor skills and cognitive functions. However, for determination of impact of CoQ10 treatment on both motor performance and cognitive abilities in mice with PD it is important to assess its levels. To assess AChE activity, the Ellman's method was employed. Briefly, 100 μL of DTNB and 2.6 mL of PBS were added to 0.4 mL of supernatant. The solution was mixed vigorously. Absorbance was recorded at 412 nm using a spectrophotometer. The basal reading was taken when absorbance reached a steady value. Next, 20 μL of acetylthiocholine iodide was added as a substrate, and the changes in absorbance were recorded every 2 min for up to 10 min. The rate of absorbance alteration per min was thus approximated [38].

Estimation of Pro-inflammatory Cytokines

Commercially available sandwich Enzyme-linked immunosorbent assay (ELISA) kits were employed and manufacturer instructions were utilized for the quantification of pro-inflammatory cytokines including IL-6 (Cat NO. PRS-30378), IL-1β (Cat NO. PRS-30369) and TNF-α (Cat NO. PRS-30651) from the brain tissue of rotenone-induced PD mice [39].

RT-PCR Analysis of PD-Linked GENES

Total RNA extraction was performed by HiPure Total RNA Kit (Magen Biotechnology, Guangzhou, China). cDNA synthesis was performed on 3 µg of RNA in a 20 µL sample volume using a cDNA kit (Thermo First, Zokeyo, China) as recommended by the manufacturer. qRT-PCR was performed in a total volume of 15 µL containing 300 ng of cDNA, primers (10 µM each) and 7.5 µL of SYBR™ Select Master Mix. The reactions were conducted in duplicate on the SLAN-96P Real-Time PCR System (Sansure Biotech, Changsha, China). Thermal cycle conditions consisted of 40 cycles of 15 s at 95 °C and 60 s at 60 °C. The SLAN-96P Multi-tasking software interface was used to get the results. The comparative CT method was used for gene expression quantification. The primer’s sequence of SNCA (α-syn), Dopamine transporter (DAT), Tyrosine hydroxylase (TH), Glial fibrillary acidic protein (GFAP) and Caspase 3 are mentioned in Table 1 [40, 41].

Table 1.

List of primer pairs employed in real-time qPCR analysis

Gene	Primer Sequence (Forward)	Primer Sequence (Reverse)
SNCA (α-syn)	5′-GATCCTGGCAGTGAGGCTTA-3′	5′-GCTTCAGGCTCATAGTCTTGG-3′
β-actin (HK gene)	5′-AGAGGGAAATCGTGCGTGAC-3′	5′-CAATAGTGATGACCTGGCCGT-3′
Tyrosine hydroxylase (TH)	5’- GCCCTACCAAGATCAAACCTAC-3’	5’-ATACGAGAGGCATAGTTCCTGA-3’
Dopamine transporter (DAT)	5’-ATGACATCAAGCAGATGACTGG-3’	5’- CACGACCACATACAGAAGGAAG-3’
Glial fibrillary acidic protein (GFAP)	5’- GAGATTCGCACTCAATACGAGG-3’	5’- CTGCAAACTTAGACCGATACCA-3’
18S ribosomal subunit (s18) (HK gene)	5’- GGCGGAAAATAGCCTTCGCT-3’	5’- AGCCCTCTTGGTGAGGTCAA-3’
Caspase 3	5’- GGACAGCAGTTACAAAATGGATTA-3’	5’-CGGCAGGCCTGAATGATGAAG-3’
GAPDH (HK gene)	5’-GGAGTCCCCATCCCAACTCA-3’	5’-GCCCATAACCCCCACAACAC-3’

Histopathological Analysis

The striatum and the substantia nigra part of brain were embedded into the paraffin block and kept at 37 °C. The paraffin blocks were sliced into 4–5 µm thick sections with the help of a rotary microtome (Hunan Kaida Scientific Instruments, Changsha, China). The sections were then stained with hematoxylin–eosin dye and observed under a light microscope [42].

Statistical Analysis

The results are presented as mean ± standard deviation (SD) for three individual experiments. One-way analysis of variance (ANOVA) with Dunnett’s post hoc test (multiple comparison against diseased control) was used to evaluate behavioral tests and to compare the statistical variations between the groups in in-vivo tests to determine the levels of oxidative stress biomarkers, inflammatory markers and neurotransmitters. The DPPH assay was evaluated by using a dose–response inhibition curve using a linear regression equation. GraphPad Prism V 10.2 was used for plotting and data analysis. Significance levels of p < 0.05 were considered for the rejection of null hypothesis and are denoted as * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Results

Liposome Characterization

The CoQ10 was successfully encapsulated in the liposomes with a high encapsulation efficiency of 81.35 ± 4.67%. Hence, the zeta potential 12.5 ± 0.3 mV (Fig. 2B), particle size 213 ± 2.2 nm and PDI of 0.2 (indicating the narrow size distribution) (Fig. 2A) proved the stability of CoQ10 liposomes. The TEM findings shown in (Fig. 2C) revealed spherical-shaped liposomes with distinct inner and outer core. CoQ10 liposomes demonstrated an amorphous or disorganized arrangement in their XRD test outcomes. This implies that the XRD pattern lacks distinct and sharp peaks typically linked with crystalline substances. Instead, it suggests that the CoQ10 molecules contained within the liposomes, most probably within the lipid bilayer, without forming a crystalline lattice, as demonstrated in (Fig. 2D).

Fig. 2.

Physicochemical characterizations of CoQ10 liposomes including: (A) Hydrodynamic diameter (Dh); (B) Zeta potential; (C) TEM micrograph and (D) XRD analysis of liposomes

Assessment for Behavioral Parameters

Effect of CoQ10 and Liposomal CoQ10 on Mice Exploratory and Locomotor Behavior

The comprehensive assessment of locomotor and exploratory activity in mice using the open field test over 21 days illustrates that the mice's average speed exhibited significant variations across treatment groups due to the novelty of the test environment Fig. 3. Notably, the liposomal CoQ10-treated group displayed a gradual response, with a significant increase in average speed observed on day 7 (p < 0.05), which continued to rise significantly on day 14 (p < 0.01) and day 21 (p < 0.0001), reaching 49.01 ± 7.46 mm/s as shown in (Tables 2, 3, 4 and 5). In contrast, the oral CoQ10-treated group initially showed no significant improvement on day 7, but a moderate increase was noted on day 14 (p < 0.05) and day 21 (p < 0.01), reaching 41.8 ± 5.9 mm/s. Furthermore, the total distance travelled by the liposomal CoQ10-treated group significantly increased on day 7 (p < 0.05), day 14 (p < 0.01), and day 21 (p < 0.001) compared to the diseased control group, reaching 5817 ± 621 mm/s. However, the oral CoQ10-treated group did not prevent locomotor deficits on day 7 but exhibited slight improvements on day 14 (p < 0.05) and day 21 (p < 0.01), reaching 5517 ± 365.6 mm/s. In terms of exploration rate, the liposome-treated group exhibited significant increases on day 7 (p < 0.05), day 14 (p < 0.01), and day 21 (p < 0.001), suggesting reduced anxiety and enhanced exploration, whereas the oral CoQ10 group showed limited improvements. Regarding anxiety and exploratory behavior assessment through frozen events, the liposomal CoQ10-treated group exhibited a significant decrease in frozen events (p < 0.05) compared to the diseased control group, with a gradual decline from day 7 to day 21 (p < 0.001). Conversely, the oral CoQ10 group demonstrated a reduction in frozen events on day 14 (p < 0.05) and day 21 (p < 0.01) but displayed less improvement overall. Lastly, in evaluating exploratory behavior using line crossing, the oral liposomal group showed significant increases on day 7 (p < 0.05), day 14 (p < 0.01), and day 21 (p < 0.001), with 57.17 ± 5.11 lines crossed on day 21, indicating reduced anxiety and heightened exploratory behavior. The oral CoQ10 group displayed a significant increase on day 14 (p < 0.05) and day 21 (p < 0.01) compared to the diseased control group. Overall, these findings suggest that liposomal CoQ10 treatment effectively improves locomotor and exploratory activity in mice, whereas oral CoQ10 treatment exhibits more modest benefits.

Fig. 3.

Effect of CoQ10 and Liposomal CoQ10 on the onset of locomotor and exploratory deficits induced by rotenone. To evaluate the locomotor and exploratory behaviors of mice, the open field (OF) test was conducted weekly for a total duration of 21 days. Visual Representation, generated by using the ToxTrac Software, vividly depict the locomotor patterns exhibited by mice during their 3-min exposure to the OF

Table 2.

Effect of CoQ10 and liposomal CoQ10 on OFT parameter’s findings on Day 1

Parameters	Vehicle (Group 1)	Std CoQ10 (Group 2)	Dsd control (Group 3)	Dsd control + CoQ10 (Group 4)	Dsd control + Lipo (Group 5)
Average Speed (mm/s)	54.67 ± 5.05***	49.33 ± 5.13*	40.75 ± 4.45	37.26 ± 6.22	54.33 ± 4.76***
Mob.Average Speed (mm/s)	61.67 ± 5.16***	57.67 ± 4.50*	47.74 ± 5.32	46.29 ± 8.02	60.5 ± 3.45**
Exploration Rate (%)	74.58 ± 4.27***	61.38 ± 5.66	58.22 ± 4.23	67.90 ± 5.42*	61.74 ± 7.58
Total Distance (mm)	6716.67 ± 640.05***	4975 ± 673.61	4860 ± 742.43	5695.33 ± 570.75	5325 ± 608.07
Frozen Events	2.00 ± 0.89*	3.00 ± 1.41	4.00 ± 0.89	2.33 ± 1.51	2.17 ± 0.75*
Line Crossing	67.67 ± 5.39***	61.17 ± 7.22*	51.33 ± 5.99	56.33 ± 6.34	62.17 ± 7.45*

Table 3.

Effect of CoQ10 and liposomal CoQ10 on OFT parameter’s findings on Day

Parameters	Vehicle (Group 1)	Std CoQ10 (Group 2)	Dsd control (Group 3)	Dsd control + CoQ10 (Group 4)	Dsd control + Lipo (Group 5)
Average speed (mm/s)	51.68 ± 6.44***	45.77 ± 7.59**	31.48 ± 5.82	32.22 ± 7.03	44.74 ± 7.77*
Mob. avg speed (mm/s)	60.42 ± 4.07****	53.45 ± 8.16**	39.56 ± 6.37	48.26 ± 3.59	50.13 ± 5.78*
Exploration rate (%)	75.30 ± 2.33****	63.06 ± 5.28**	49.52 ± 7.52	45.33 ± 7.97	61.85 ± 7.43*
Total distance (mm)	6816.67 ± 231.66****	5691.67 ± 560.73**	4383.33 ± 617.79	5036.5 ± 741.98	5478.33 ± 668.14*
Frozen events	1.50 ± 0.55***	2.17 ± 0.75**	4.00 ± 0.89	3.33 ± 1.21	2.50 ± 1.05*
Line crossing	70.50 ± 4.01****	59.33 ± 6.97**	46.50 ± 4.51	50.33 ± 5.50	54.50 ± 4.93*

Table 4.

Effect of CoQ10 and liposomal CoQ10 on OFT parameter’s findings on Day 14

Parameters	Vehicle (Group 1)	Std CoQ10 (Group 2)	Dsd control (Group 3)	Dsd control + CoQ10 (Group 4)	Dsd control + Lipo (Group 5)
Average speed (mm/s)	67.78 ± 5.95****	46.13 ± 9.87***	30.70 ± 2.91	39.86 ± 5.33*	43.75 ± 3.88**
Mob. avg speed (mm/s)	76.06 ± 5.54****	52.87 ± 6.06***	36.87 ± 6.63	47.29 ± 4.38*	50.61 ± 6.35**
Exploration rate (%)	75.67 ± 5.61****	63.83 ± 4.75***	45.17 ± 5.74	56.83 ± 9.06*	60.17 ± 7.33**
Total distance (mm)	7196.67 ± 766.46****	5936.67 ± 435.28***	4300 ± 827.79	5509.83 ± 588.12*	5691.67 ± 567.36**
Frozen events	1.17 ± 0.75****	1.83 ± 0.75***	4.50 ± 1.05	2.67 ± 1.03*	2.17 ± 1.17**
Line crossing	70.83 ± 4.62****	59.67 ± 5.20***	46.50 ± 6.95	55.00 ± 3.34*	58.17 ± 2.48**

Table 5.

Effect of CoQ10 and liposomal CoQ10 on OFT parameter’s findings on Day 21

Parameters	Vehicle (Group 1)	Std CoQ10 (Group 2)	Dsd control (Group 3)	Dsd control + CoQ10 (Group 4)	Dsd control + Lipo (Group 5)
Average speed (mm/s)	70.42 ± 6.88****	47.57 ± 4.82***	30.00 ± 4.33	41.8 ± 5.9**	49.01 ± 7.46****
Mob. avg speed (mm/s)	76.66 ± 3.57****	47 ± 5.69***	34.33 ± 4.67	43.34 ± 7.39*	49.17 ± 3.19***
Exploration rate (%)	76.17 ± 5.64****	59.50 ± 6.16***	42.67 ± 4.76	55.833 ± 5.88**	60.50 ± 7.48***
Total distance (mm)	7200 ± 769.4****	5866 ± 644.9***	4086 ± 629.9	5517 ± 365.6**	5817 ± 621.0***
Frozen events	1.33 ± 0.82****	1.67 ± 1.03***	5.00 ± 1.1	2.50 ± 1.87**	1.50 ± 0.84***
Line crossing	77.17 ± 6.30****	66.33 ± 4.92****	42.50 ± 6.47	54.17 ± 3.76**	57.17 ± 5.11***

Evaluation of Memory and Spontaneous Alternation in Mice

To assess the spatial working memory in animals, the Y-maze test was utilized weekly for 21 days. Figure 4 shows that throughout the therapy, the % alternation was increased in the liposomal treated group from (p < 0.05) on day 7, (p < 0.01) on day 14 and (p < 0.001) on day 21. Comparing the oral CoQ10 treated group to the diseased control group, the % alternation remained statistically non-significant on day 7 but showed a slight increase in % alternation on day 14 (p < 0.05) and (p < 0.01) on day 21. However, the standard CoQ10 group showed significant results relative to the diseased control group.

Fig. 4.

Effect of CoQ10 and liposomal CoQ10 on % behavior alternation of mice in Y-maze test to assess the spatial working memory

Effect on Mice Balance and Coordination

As illustrated in Fig. 5, the liposomal treated group in contrast to disease control group exhibited a reduced number of falls (p < 0.05) (p < 0.01) and (p < 0.001) at days 7,14 and 21 respectively. Whereas there was a detained response towards therapy in the oral CoQ10 treated group as it did not show significant improvements on day 7, but later on, from day 14 to 21 there was a slight reduction in the number of falls (p < 0.05) relative to the diseased control group.

Fig. 5.

The impact of CoQ10 and liposomal CoQ10 on number of falls observed in mice during beam balance test

Effect on Oxidative Stress and Antioxidant Defense

The effect of oral CoQ10 conventional form and liposomal formulation on SOD, CAT, GSH, NO, MDA and protein content in the brain tissue homogenate of mice was assessed. Significantly high SOD activity was presented (p < 0.0001) in the liposome-treated group relative to the diseased control group. The oral CoQ10 treated group raised (p < 0.05) the SOD activity in contrast to the diseased control group. However, the standard treatment raised SOD level (p < 0.0001) in comparison to the diseased control group as shown in Fig. 6A. Both liposome-treated and oral CoQ10-treated groups exhibited a significant increase in CAT and GSH activities, while decreasing MDA and NO levels compared to the diseased control group (p < 0.001 and p < 0.05, respectively). Furthermore, the standard treatment group also demonstrated a significant increase in CAT (Fig. 6B) and GSH (Fig. 6C) concentration (p < 0.0001) and a significant reduction in MDA (Fig. 6D) and NO (Fig. 6E) levels (p < 0.0001) relative to the diseased control group.

Fig. 6.

Effect of CoQ10 and liposomal CoQ10 on rotenone induced changes in: (A) SOD level, (B) CAT activity, (C) GSH levels, (D) MDA levels, and (E) NO levels in brain tissue homogenate

In vitro Antioxidant Capacity of CoQ10

The antioxidant activity of CoQ10 was determined by DPPH assay. According to the findings, CoQ10 showed a dose-dependent increase in antioxidant activity as seen from the minimum % inhibition (68.03%) at 10 µg/mL and the highest % inhibition (87.33%) at 100 µg/mL concentration. The IC50 value of CoQ10 was 85.35 µg/mL whereas the IC50 value of standard ascorbic acid was 69.9 µg/mL. %age inhibition is depicted in Fig. 7.

Fig. 7.

The antioxidant potential of CoQ10

Effect on Neurotransmitter Levels in PD Mice Brain

Dopamine (Fig. 8A) and noradrenaline (Fig. 8B) levels were decreased in the diseased control group. In contrast to the diseased control group, these levels were significantly restored (p < 0.0001) in CoQ10 liposomal group. Furthermore, these levels marginally higher (p < 0.05) in the oral CoQ10-treated group than the diseased control group.

Fig. 8.

Effect of CoQ10 and liposomal CoQ10 on rotenone induced changes in: (A) Dopamine level, (B) Noradrenaline level, and (C) Acetylcholinesterase activity in brain tissue homogenate

Additionally, the activity of acetylcholinesterase (Fig. 8C) increased in the diseased control group but decreased in the liposomal group (p < 0.0001) and oral CoQ10 group (p < 0.05) The standard treatment group also displayed a significant difference (p < 0.0001) compared to the diseased control group.

Effect on Pro-inflammatory Cytokines in PD Mice Brain

The IL-6 (Fig. 9A) and IL-1beta (Fig. 9B), both displayed higher levels in PD mice, which were significantly decreased (p < 0.001) in the CoQ10 liposome-treated group compared to the diseased control group. In contrast, these levels were slightly decreased (p < 0.05) in the oral CoQ10-treated group compared to the diseased control group.

Fig. 9.

The impact of Coenzyme Q10 (CoQ10) and liposomal Coenzyme Q10 on rotenone induced neuroinflammation in brain tissue homogenate. (A) Interleukin-6 levels, (B) Interleukin-1beta, and (C) TNF-alpha levels were examined

However, the level of TNF-α (Fig. 9C) was elevated in the diseased control group while a significant reduction (p < 0.001) was observed in the CoQ10 liposomes group. Meanwhile, in the CoQ10-treated group, TNF-α level was slightly reduced (p < 0.05) compared to the diseased control group. [43]

Gene Expression in Rotenone-induced PD Mice Brain

Upon the administration of CoQ10 liposomes, there was a decline in the expression of SNCA which encodes alpha-synuclein. The levels of SNCA gene expression, however, were significantly upregulated in the diseased control group. Whereas in the oral CoQ10 treatment group, there was an upregulation in gene expression, presented a fold change of 2.144, liposomes showed a fold change of 0.722, as shown in (Fig. 10A). Hence the results suggested the better performance of liposomes as compared to oral CoQ10 in suppressing the PD symptoms. Tyrosine hydroxylase is one of the markers of neurodegeneration in PD as it is necessary for the synthesis of dopamine. The diseased control group significantly downregulated the expression of TH by a fold change of 0.286. In liposomal group the TH expression upregulated by a fold change of 1.812 and in oral CoQ10 treated group there was a decline in fold change by 1.043, demonstrated in (Fig. 10B). The levels of dopamine transporter gene expression significantly downregulated in liposomal group as compared to the oral CoQ10 treated group, presented a fold change 0.743 and 2.704 respectively. However, in diseased control group the DAT expression significantly upregulated with a fold change 3.414, demonstrated in (Fig. 10C). The increased expression of GFAP has been noted in diseased control group, a fold change 3.81. The level of this astroglia activation marker’s expression, however, significantly downregulated, with a fold change of 0.786, in the liposomal treated group and upregulated, a fold change 2.953, in oral supplementation treated group, shown in (Fig. 10D) The expression of this apoptotic marker, Caspase 3 significantly upregulated in the diseased control group, a fold change 4.458 whereas its level in the liposomal treated group was downregulated, a fold change 0.56, but its level increases in oral CoQ10 treated group with a fold change of 3.47 in contrast of diseased control group, as shown in (Fig. 10E).

Fig. 10.

Effects of CoQ10 liposomes on rotenone induced changes in mRNA expression levels of: (A) SNCA, (B) Tyrosine Hydroxylase, (C) Dopamine Transporter; DAT, (D) Glial Fibrillary Acidic Protein; GFAP, and (E) Caspase 3

Assessing the Impact of CoQ10 and Liposomal CoQ10 on PD Mouse Brain Histopathology

Staining of the mid-brain section with hematoxylin and eosin staining exhibited a normal neuronal histological architecture with an obvious vesicular nucleus (Fig. 11A). The midbrain of rotenone-treated mice showed a marked decrease in neuronal cell number and size with obvious neuronal degeneration in the form of irregularly damaged cells, cytoplasmic shrinkage, pyknotic nuclei, and chromatin condensation. The presence of perineuronal vacuolation was also observed (Fig. 11B). The midbrain of the standard CoQ10-treated group showed neuronal structures substantially seem healthy and intact, with minimal neuronal degeneration which could be due to natural biological variability (Fig. 11C). The midbrain of the CoQ10-treated group exhibited few degenerated neurons and neurons with nuclei and cytoplasm (Fig. 11D). Whereas the midbrain of the CoQ10 liposomal treated group showed improvement in histopathological features and neuronal morphology (Fig. 11E).

Fig. 11.

Examining photomicrographs of brain tissue stained with Hematoxylin and Eosin at (100x). The brain of mice treated with: (A) normal Saline; showing the normal architecture of brain. Dopaminergic neurons with vesicular nuclei and basophilic cytoplasm (arrow), (B) rotenone; displaying the neuronal loss, damage, and degeneration. Neurons appear smaller and shrunken (arrows), many neurons illustrating irregular damaged cells, cytoplasmic shrinkage (arrows), perineuronal vacuolations (arrows), Pyknotic darkly stained nuclei (arrow) and cytoplasmic inclusions of Lewy bodies (arrows), (C) standard CoQ10; neuronal structures substantially seem healthy and intact, with minimal neuronal degeneration (arrow), which could be due to natural biological variability. (D) Diseased along with CoQ10 treated mice exhibited few neurons are degenerated (arrow) and neurons with nuclei and cytoplasm (arrow) are present and (E) liposomal CoQ10; there are few degenerated neurons (arrow) along with increase in their size (arrow). The scale bar represents 50 µM scale

Discussion

Parkinson's disease stands as the second most prevalent neurodegenerative disorder, characterized by the gradual and selective degeneration of dopamine-producing neurons within the substantia nigra [44]. Consequently, in the early stages of Parkinson's disease, the predominant therapeutic approach involves dopamine supplementation, often through the prescription of L-dopa or dopamine agonists, aimed at providing temporary relief from the disease's symptoms [45]. However, clinical practice has a shortage of treatments that directly target the fundamental issue in Parkinson's disease, which is the prevention of disease progression [46]. In this context, there has been active research into the neuroprotective compound CoQ10 for its potential capacity to impede the advancement of Parkinson's disease [47]. The present research illustrates that CoQ10 alleviated motor dysfunction and the depletion of dopaminergic neurons induced by rotenone in mice. CoQ10 also improved rotenone-triggered lipid peroxidation and enhanced the cellular antioxidant enzyme activity and proteins responsible for maintaining cellular energy balance. Numerous research investigations have established a connection between oxidative stress and reduced cellular respiration in relation to neurodegenerative disorders [48]. According to research studies, rotenone administration resulted in substantial disruption to the mitochondrial energy equilibrium, potentially heightened the oxidative stress [49, 50]. To minimize the detrimental effects of rotenone, the neuroprotective compound CoQ10 has been extensively studied for its potential capacity to slow down the progression of PD. Apart from the favorable findings of CoQ10, there are some drawbacks that limit its use. However, due to its large molecular weight and hydrophobicity (poor water solubility), the oral administration of CoQ10 exhibits restricted systemic uptake. To overcome these issues, in the present study, we suggested encapsulating CoQ10 in a liposomal formulation to evaluate the pharmacological effects of liposomal CoQ10 against the rotenone-induced mouse model [51].

In our research, we conducted a series of behavioral tests to evaluate the motor symptoms and cognitive function of the mouse model from day 1 to 21. These tests included the open field test (OFT), the Beam Balance test, and the Y-Maze test, each offering unique insights into the mouse's behavior [22]. The OFT assessed various parameters, such as average speed, distance traveled, exploration rate, frozen events, and the number of line crossings, to gauge the anxiety level in the mice. Our findings revealed that mice in the disease control group, exposed to rotenone, exhibited behavioral defects. This was evidenced by a decrease in the average speed, distance travelled, more frozen events, leading to decreased exploration of the field, and fewer line crossings between day 1 and 21. The results indicated that the liposomal group outperformed the oral CoQ10 group in all aspects of the OFT parameters when compared to the rotenone group. The liposomal group exhibited a gradual increase in speed, distance travelled, fewer frozen events, leading to increased exploration of the field, and more line crossings between day 1 and 21. In contrast, the oral CoQ10 group showed only slight improvements during the same period. According to the findings of beam balance test, the number of falls was higher in the disease control group from day 1 to 21. The number of falls was fewer in the liposomal treated group than in the oral CoQ10 treated group, in contrast to the rotenone group from day 1 to 21. The Y-maze test findings suggested that from day 1 to day 21, the percentage of spontaneous alternation decreased in the disease control group. Whereas, the percentage of spontaneous alternation increased in the liposomal group compared to the oral CoQ10 treated group. These outcomes illustrate the successful induction of behavioral anomalies associated with PD in mice by rotenone, consistent with prior research [30, 52, 53] Collectively, the outcomes of behavioral test depict reduced anxiety, enhanced spatial working memory, balance and coordination throughout 21-day experiment. However, the non-encapsulated CoQ10 treated group presented slightly improved results throughout 21-day experimental period. Hence, the findings of behavioral test proved that encapsulated CoQ10 gives beneficial results, whereas non-encapsulated CoQ10 showed a delayed response, which can be attributed to its large molecular weight, limiting its absorption. Effect of CoQ10 on behavioral parameters were evaluated in paraquat induced PD and a remarkable improvement in most of the behavioral tests and decreased protein carbonyl content in the brain was observed [54] but the effect on rotenone induced PD was investigated in the current study. Similarly, in another study CoQ10 supplemented with atorvastatin improved behavioral parameters in 6-hydroxydopamine induced dopaminergic toxicity in rats suggesting the efficacy of CoQ10 in improving PD symptoms [55]

The DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical method was employed to check the antioxidant capacity of CoQ10. Notably, the IC50 value, which exhibit the concentration required to reduce the activity of the DPPH free radical by 50%, was showed to be 85.35 µg/mL, illustrating the antioxidant effect of CoQ10 in this research model. As rotenone triggers the activation of microglial cells and induces oxidative stress. Oxidative stress was one of the contributors in PD. The activity of enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) was decreased upon the exposure of rotenone. In diseased control group the levels of Malondialdehyde (MDA), a lipid peroxidation marker, and NO levels were higher. However, the CoQ10 liposomes decreased the MDA and NO level more significantly than CoQ10 treated group. Effect of CoQ10 on oxidative stress was an established fact. Sangsefidi et al. claimed CoQ10 supplementation increased the levels of TAC and antioxidant enzymes (including SOD, CAT, and GPx) significantly, however MDA levels were decreased supporting the claim of current study [56]. Similar findings were suggested by Akbari et al. [57].

Neuroinflammation results from the release of proinflammatory cytokines in PD [58]. TNF-α, IL-6 and IL-1β levels were found considerably lower in the CoQ10 liposomal group in contrast to disease control group. According to the results, the CoQ10 liposome group exhibited higher anti-inflammatory activity and showed less damage throughout study. Considerably, from day 1 to day 21, beneficial results were seen with CoQ10 supplementation alone. Non-encapsulated CoQ10 showed delayed response by the day 21. Current investigation is supported by the fact that CoQ10 has shown promising results in reducing the levels of TNF-α and proving its influenced role in neuroprotection [11]. CoQ10 was found useful in reducing the levels of IL 6 in arthritic patients supporting the claim of decrease in inflammation in brain [59]. Similarly, the anti-inflammatory effect of CoQ10 in reducing inflammation is already established in chronic inflammatory diseases by reducing different mediators like TNF-α, IL 6 and IL 1β [60, 61]

The primary neurotransmitters implicated in Parkinson's Disease (PD) are dopamine, noradrenaline and acetylcholine. When PD is induced by rotenone, there is a concurrent decrease in both dopamine and noradrenaline levels [52]. In the CoQ10 liposomal-treated group, there was a notable elevation in the concentrations of the brain neurotransmitters dopamine and noradrenaline, both of which play a crucial role in facilitating memory retention. Conversely, acetylcholine, a neurotransmitter crucial for memory function, undergoes rapid degradation when subjected to elevated levels of acetylcholinesterase. In the presence of such conditions, acetylcholine breaks down into acetic acid and choline. Consequently, the levels of acetylcholinesterase in brain are diminished within the liposomal treatment group and elevated in rotenone treated group. The results corroborated the findings of a previous study where CoQ10 supplementation restored AChE activity in rats treated with doxorubicin [43]. The findings justified the previous claims that CoQ10 possess neuroprotective potential [62].

The effect of liposomal CoQ10 treatment on the levels of SNCA, tyrosine hydroxylase (TH), dopamine transporter (DAT), glial fibrillary acidic protein (GFAP) and caspase 3 gene expression was illuminated by real-time PCR amplification by assessing their fold gene expression [41]. SNCA, responsible for encoding alpha-synuclein, stands as a primary hallmark associated with Parkinson's disease (PD). On the other hand, tyrosine hydroxylase serves as a rate-limiting enzyme involved in dopamine synthesis, while dopamine transporter plays a crucial role in the transport of dopamine. GFAP serves as an astrocytic marker, and caspase 3 serves as an apoptotic marker within the study context [63]. Remarkably, the expression levels of all these genes, with the exception of tyrosine hydroxylase (TH) and dopamine transporter (DAT), exhibited a notable increase in the group subjected to rotenone-induced treatment. Conversely, the liposomal CoQ10 treatment group demonstrated a reduction in the expression of SNCA, GFAP, and caspase 3, along with an augmentation in the expression levels of TH and DAT. This transformation can be attributed to the alleviation of oxidative stress, indicating that liposomal CoQ10 possesses the potential to ameliorate the heightened expression of SNCA witnessed in mice exposed to rotenone. Previously it was established from preclinical data in cellular and animal models that CoQ10 inhibits synuclein aggregation in dopaminergic neurons of PD models [64]. Similarly, the CoQ10 intrastriatal treatment was documented to improve the expressions of TH in striatum and substantia nigra supporting the claim of the current study [11]. As far as DAT is concerned it was investigated that CoQ10 provides neuroprotection by inhibiting NF-κB and by augmenting mitochondrial complex 1 in homozygous weaver mutant mice possessing significantly reduced substantia nigra pars compacta (SNpc) CoQ10 and dopamine transporters as judged by reduced 18F-DOPAuptake [65], however not much data is available providing insight into the effect of CoQ10 on DAT and current study explained the gap. Moreover, the CoQ10-treated group exposed to rotenone exhibited a delayed response in terms of gene expression alterations. These findings substantiate the hypothesis that liposomal CoQ10 yields more pronounced therapeutic benefits when compared to non-encapsulated CoQ10 in the context of a rotenone-induced mouse model of Parkinson's disease.

A liposomal formulation of CoQ10 underwent a comprehensive analysis employing various techniques, including Zeta size and potential measurements, Scanning Electron Microscopy (SEM), and X-ray Diffraction (XRD) [66, 67]. The results obtained from these analyses collectively illuminated the properties of this formulation. The SEM examination revealed the presence of stable, spherical liposomes characterized by a robust electrostatic attraction, while the XRD analysis indicated their structural attributes. Notably, the Zeta size and zeta potential measurements played a pivotal role in establishing the stability of these CoQ10-loaded liposomes. These parameters reflect the electrostatic forces acting between the particles in the solution. In the context of CoQ10 liposomes, a significant zeta potential signifies a high degree of electrostatic repulsion, which, in turn, augments their resistance to aggregation. The avoidance of aggregation is a critical determinant for maintaining the quality and efficacy of the formulation. In essence, liposomes with substantial zeta potentials tend to stay dispersed and resist clustering in solution. Consequently, the findings of this analysis affirm the stability and structural integrity of CoQ10 liposomes, substantiating their potential utility in mitigating neuronal deficits associated with Parkinson's Disease (PD). This stability is a crucial factor, as it ensures the liposomal CoQ10 formulation's capacity to remain intact and efficacious, thereby facilitating its therapeutic application. Furthermore, histological assessments provided compelling evidence of the effectiveness of CoQ10 liposomes. These assessments revealed a notable reduction in the pathological alterations induced by rotenone. This finding underscores the therapeutic potential of the liposomal CoQ10 formulation in ameliorating the neuropathological aspects of PD.

Therefore phase 3 clinical trials stipulated that high dose oral supplementation of CoQ10 did not produce significant advantages, potentially due to obstacles related to its bioavailibity, which limited its absorption in striatum and hinders its transport across the blood brain barrier [7]. Different encapsulation techniques have been created to get over these restrictions. One method to address the aforementioned issue is the liposome delivery system. The continuous release of a medicine from a liposomal encapsulation might be the reason to improve the pharmacological impact of the CoQ10. Liposome CoQ10 pharmacological superiority over CoQ10 might be attributed to extended circulating components, as it has already proven that liposomal delivery is the most effective methods for enhancing CoQ10 stability, lengthening circulation times, and increasing bioavailability [12]. CoQ10 liposomes may have a much higher therapeutic effect in lower doses than conventional dosage forms. In this study, we investigate the effectiveness of formulated Liposomal CoQ10 as compared to conventional CoQ10 through behavioral testing, biochemical estimation and histopathological investigation. The possible mechanism for enhanced pharmacological response of liposomal CoQ10 across blood brain barrier can be phospholipid bilayer of liposome, facilitating the permeation of the drug across the biological membrane and release of entrapped CoQ10 at the target site via passive diffusion [68–70].

Experimental Limitations

Therefore, this study outlines therapeutic potential of CoQ10 liposomal formulation in PD disease model. Apart from several advantages of CoQ10 liposomal formulation there might exist some significant challenges and limitations which should be acknowledged. First, main objective of study was to focus on efficacy of liposomal formulation in contrast to conventional CoQ10, without extensive analysis of significant adverse effects linked with long-term liposomal delivery. Hence, future studies could explore any progressive toxicity or side effects related with prolonged administration of CoQ10 liposomes. Furthermore, due to limited resources, we did not include any empty liposome control group, which could offer further apprehension into any baseline effects linked with liposomal carrier alone. Moreover, in this study we performed neurochemical analysis, histopathological analysis and gene expression analysis on entire brain instead of targeting specific brain region such as substantia nigra and striatum. However, this approach offers detailed overview of brain response to our treatment groups but it may have confined our capacity to detect more specific changes in specific brain regions. Therefore, future studies should perform specific brain-region analysis in order to enhance the understanding of these mechanistic changes.

Conclusion

In conclusion, our research substantiates the proposition that encapsulated Coenzyme Q10 (CoQ10) possesses the capability to act as both an antioxidant and an anti-inflammatory agent, thereby serving as a potential therapeutic strategy for ameliorating the deleterious effects of Parkinson's disease (PD). Our study revealed noteworthy effectiveness in the utilization of CoQ10 in averting behavioral impairments induced by rotenone. In addition to addressing behavioral deficits, CoQ10 liposomal formulations also exhibited a commendable capacity to mitigate inflammation and oxidative stress, as evidenced by the reduction in levels of pro-inflammatory cytokines and the augmentation of enzymatic antioxidant activity. These findings collectively emphasize the promising potential of CoQ10 in the management of PD, offering a multi-faceted approach to mitigate its pathophysiological consequences.

Abbreviations

PD

Parkinson Disease

CoQ10

Coenzyme-Q10

UPDRS

Unified Parkinson Disease Rating Scale

ROS

Reactive oxygen species

RNS

Reactive nitrogen species

SUV

Small unilamellar vesicles

TEM

Transmission electron microscopy

XRD

X-ray diffraction

OFT

Open field test

SOD

Superoxide dismutase

CAT

Catalase

MDA

Malondialdehyde

GSH

Glutathione

PC

Protein content

NO

Nitric oxide

DPPH

2,2-Diphenyl-1-picrylhydrazyl

AChE

Acetylcholinesterase

TH

Tyrosine hydroxylase

DAT

Dopamine transporter

GFAP

Glial fibrillary acidic protein

Author Contributions

H.U performed investigation and data curation, A.S was involved in Conceptualization, Data curation, project administration, supervision, writing – review and editing, S.M.M.A helped in methodology, H.M.K did conceptualization and helped in project administration, B.A was involved in Software application and data curation, S.A helped in methodology and editing, M.A.H and M.A helped in Writing, review & editing. All authors read and approved the final manuscript.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Ethics Approval

The study was conducted followed by the approval of Institutional Animal Ethics Committee. Ethics no: ORIC / 23–410 in accordance with the NC3Rs ARRIVE Guidelines, adhere to ethical guidelines of The Basel Declaration, the International Council for Laboratory Animal Science (ICLAS) ethical guidelines, and Directive 2010/63/EU.

Consent for Publication

Not applicable.

Competing Interests

The authors declare no competing interests.