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RESEARCH ARTICLE

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The Neuroprotective Effect of Micronized Purified Flavonoid Fraction in an Experimental Parkinson’s Disease Model  

Omar M.E. Abdel-Salam1, Eman R. Youness2, Enayat A. Omara3, and Amany A. Sleem4  

1Department of Toxicology and Narcotics, National Research Centre, Cairo, Egypt; 2Department of Medical Biochemistry, National Research Centre, Cairo, Egypt; 3Department of Pathology, National Research Centre, Cairo, Egypt; 4Department of Pharmacology, National Research Centre, Cairo, Egypt  

Correspondence: omasalam@hotmail.com (O.M.A-S.)  

Abdel-Salam OM et al. Reactive Oxygen Species 10(29):xxx–xxx, 2020; ©2020 Cell Med Press 

http://dx.doi.org/10.20455/ros.2020.835 

(Received: June 8, 2020; Revised: July 14, 2020; Accepted: July 16, 2020)  

ABSTRACT | Micronized purified flavonoid fraction (MPFF; Daflon®) containing 90% diosmin and 10% hesperidin is a vasoactive drug used for the treatment of chronic venous insufficiency and hemorrhoids. The aim of this study was to examine the effect of MPFF on the development of brain oxidative stress and neuronal damage induced in the rat by systemic rotenone injection. Rats received subcutaneous injections of rotenone (1.5 mg/kg) every other day for two weeks and were orally treated with MPFF (9 or 18 mg/kg). Results indicated that rotenone caused a significantly elevated oxidative stress in the cerebral cortex, striatum, and the rest of the brain tissue. Malondialdehyde (MDA) and nitric oxide concentrations were increased. In contrast, the level of reduced glutathione (GSH) and the activity of paraoxonase-1 (PON-1) were decreased in the above brain regions. Moreover, the concentration of the antiapoptotic protein B cell/lymphoma-2 (Bcl-2) in the striatum decreased after rotenone injection. Rotenone caused neuronal vacuolation and apoptotic neurons in the striatum, cortex, and hippocampus. Treatment with MPFF reduced, in a dose-dependent manner, the rotenone-induced increase in brain lipid peroxidation and nitric oxide and restored GSH level and PON-1 activity to vehicle control values. Moreover, MPFF was found to attenuate the decrease in Bcl-2 and the histopathological changes in the brain of rotenone-treated animals. These results suggest that MPFF could be a potential therapeutic “add on” for the pharmacological treatment of Parkinson’s disease.  

KEYWORDS | Micronized purified flavonoid fraction; Neuroprotection; Oxidative stress; Parkinson’s disease; Rotenone  

ABBREVIATIONS | GSH, reduced glutathione; iNOS, inducible nitric oxide synthase; MDA, malondialdehyde; MPFF, micronizedpurified flavonoid fraction; nNOS, neuronal nitric oxide synthase; PD, Parkinson’s disease; PON-1, paraoxonase-1; ROS, reactive oxygen species; SNPc, substantia nigra pars compacta 

CONTENTS  

  1. Introduction
  2. Materials and Methods

2.1. Animals 

2.2. Drugs and Chemicals 

2.3. Experimental Design 

2.4. Determination of Lipid Peroxidation 

2.5. Determination of Nitric Oxide 

2.6. Determination of GSH 

2.7. Determination of Paraoxonase-1 (PON-1) Activity 

2.8. Determination of B Cell/Lymphoma-2 (Bcl-2) 

2.9. Histopathological Studies 

2.10. Quantitative Image Analysis 

2.11. Statistical Analysis 

  1. Results

3.1. Biochemical Results 

3.1.1. MDA 

3.1.2. Nitric Oxide 

3.1.3. GSH 

3.1.4. PON-1 

3.1.5. Bcl-2 

3.2. Histopathological Results 

3.2.1. Striatum and Cerebral Cortex 

3.2.2. Hippocampus 

3.2.3. Quantitative Neuronal Damage 

  1. Discussion

1. INTRODUCTION  

Parkinson’s disease (PD) is a common neurodegenerative disorder affecting ~1% of elderly people above the age of 65 years [1]. It is caused by a preferential and continued death of pigmented dopaminergic neurons in the substantia nigra pars compacta (SNPc) [2]. This results in loss of dopamine in SNPc and striatum, disturbed basal ganglia functioning, and the emergence of the motor symptoms of the disease including the slowing of movements or bradykinesia, muscular rigidity, postural instability, and a resting tremor of the hands [3]. PD is essentially sporadic in ~95% of cases (idiopathic PD) for which there is no specific cause [4]. It is largely thought, however, that exposure to environmental toxins besides genetic susceptibility is the trigger for initiating the process of dopaminergic cell death via pathways involving oxidative stress and neuroinflammation [5, 6]. In this context, epidemiological studies suggested a role for pesticides such as rotenone in increasing the risk for developing PD [7, 8]. Rotenone, a pesticide of plant origin, has been shown to reproduce the biochemical, motor, and pathological changes of idiopathic PD when injected into rodents [9, 10].  

Reactive oxygen species (ROS) are produced in the cell as byproducts of normal cellular metabolism. The important source for ROS is the mitochondrial respiratory chain where leakage of electrons onto O2 results in the formation of superoxide (O2˙) which can be converted to hydrogen peroxide (H2O2) by superoxide dismutase or react with nitric oxide forming peroxynitrite (ONOO). Moreover, the hydroxyl radical (HO˙) can be produced from the reaction of H2O2 with the reduced forms of the transition metal ions (e.g., Fe2+, Cu+) [11, 12]. Mitochondrial complex I damage that occurs in the brain of PD patients [13] can lead to increased production of O2˙ [14]. Other sources of ROS are activated phagocytes, that release O2˙, H2O2, and hypochlorous acid (HOCl), and activated lipoxygenase and cyclooxygenase [11]. In face of ROS, there is a number of antioxidant defenses including the enzymes superoxide dismutase and glutathione peroxidase and the low molecular weight antioxidants such as reduced glutathione (GSH), ascorbate, and a-tocopherol [15]. Oxidative stress occurs when the formation of ROS is increased or the cell’s antioxidants are insufficient or depleted. The result is oxidative damage to the cell biomolecules such as membrane lipid peroxidation, enzyme inactivation, or DNA damage causing cellular metabolic dysfunction or death [15, 16]. In this context, evidence of oxidative stress, such as increased lipid peroxidation [17], protein carbonyls [18], and oxidative DNA damage [19] was found in the brain of PD patients.  

Currently, the pharmacological management of PD is based on replacing the biochemical deficit by the administration of the dopamine precursor L-3,4-dihyroxyphenylalanine (L-DOPA) which provides great improvement in motor function but is associated with motor fluctuations (off phenomenon) and peak-dose dyskinesias [20]. Other dopaminergic therapies used to increase synaptic dopamine levels include monoamine oxidase B inhibitors and catechol-O-methyltransferase inhibitors. These agents, however, become less effective as the disease advances because of the continued death of SNPc dopaminergic neurons. It has become clear that none of the existing therapies is able to lessen the progressive death of dopaminergic cells [21, 22]. This necessitates the search for new drugs with the hope of improving SNPc dopaminergic cell survival.  

Micronized purified flavonoid fraction (MPFF; Daflon®) is a widely used drug for the treatment of venous circulatory disorders such as varicose veins and hemorrhoids. The drug improves the symptoms of pain, heaviness, and edema in patients with venous reflux and accelerates healing of venous ulcers [23, 24]. It contains diosmin as the major constituent (90%) and 10% hesperidin. The latter is a flavanone glycoside found in large amounts in citrus fruits such as lemon and orange and possesses antioxidant and anti-inflammatory activities [25]. Diosmin is a bioactive flavonoid naturally occurring and can be derived from hesperidin [26]. Both are safe with no or minor side effects [25, 27]. The vascular beneficial effects of MPFF have been ascribed to decreased granulocyte and macrophage infiltration and decreased leukocyte adhesion to the vascular endothelium which result in reduced levels of proteolytic enzymes, and decreased microvascular permeability and edema [28–30]. Moreover, diosmin was shown to decrease the elevated isoprostane levels, an indicator of lipid peroxidation in patients with chronic venous insufficiency [31]. Hesperidin alone or combined with diosmin showed free radical-scavenging and anti-inflammatory activities in models of ischemia/reperfusion (I/R) injury [32, 33]. Other studies reported antioxidant and anti-inflammatory effects for hesperidin in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD in mice [34] and in 6-hydroxydopamine (6-OHDA)-neurotoxicity in vitro [35].  

In this study, we aimed to investigate the effect of MPFF on the oxidative stress and neurodegeneration in the rotenone-induced PD in the rat. 

2. MATERIALS AND METHODS  

2.1. Animals  

Male Sprague-Dawley rats (180–200 g) from the National Research Centre colony (Cairo, Egypt) were used in the study. Rats were housed on a 12-h light/dark cycle and standard laboratory food and tap water freely given. Animal procedures were done following the recommendations of the Institution Ethics Committee and that of the U.S. National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985).  

2.2. Drugs and Chemicals  

Rotenone was purchased from Sigma-Aldrich (St Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO). MPFF (Daflon®, Servier, Paris, France) consisting of 90% diosmin and 10% hesperidin was used and dissolved in saline solution immediately before use. The doses of the drug were selected based on previous studies [36, 37]. Other chemicals and reagents were of analytical grade and purchased from Sigma.  

2.3. Experimental Design  

Rats were randomly divided into four equal groups, with six rats in each group. Group 1 received the vehicle (DMSO) via the subcutaneous (s.c.) route. Group 2 received injection of rotenone at the dose of 1.5 mg/kg, s.c., once every other day. Groups 3 and 4 received rotenone at the dose of 1.5 mg/kg, s.c., once every other day along with MPFF orally at doses of 9 or 18 mg/kg at time of rotenone administration. Treatments were continued for 2 weeks and at the end of the experiment, rats were euthanized by decapitation for tissue collection; their brains were rapidly removed out on an ice-cold plate, dissected into different areas (the cerebral cortex, striatum, and the rest of the brain) washed with ice-cold phosphate-buffered saline (pH 7.4), weighed, and stored at −80 °C until the biochemical analysis. The brain tissues were homogenized with 0.1 M phosphate-buffered saline at pH 7.4 to give a final concentration of 10% for the biochemical assays.  

2.4. Determination of Lipid Peroxidation  

Lipid peroxidation was determined by measuring the amount of the lipid peroxidation end product malondialdehyde (MDA). MDA reacts with thiobarbituric acid (TBA) to form a red colored TBA-MDA adduct, which can be measured spectrophotometrically at 532 nm [38].  

2.5. Determination of Nitric Oxide  

The generation of nitric oxide was determined by measuring accumulation of nitrite using the Greiss reagent as described previously[39]. The concentration of nitrite was calculated using a standard curve for sodium nitrite.  

2.6. Determination of GSH  

The Ellman assay was used to determine GSH. Briefly, 5,5-dithiobis(2-nitrobenzoic acid) (DTNB, also known as the Ellman’s reagent) is reduced by the free sulfhydryl group of GSH molecule to generate 5-thio-2-nitrobenzoic acid which can be determined spectrophotometrically at 412 nm [40].  

2.7. Determination of Paraoxonase-1 (PON-1) Activity  

PON-1 activity was estimated in the brain tissue using phenyl acetate as a substrate. In this assay, phenyl acetate is hydrolyzed by PON-1 to produce phenol which is measured spectrophotometrically at 270 nm. One unit of arylesterase activity is considered equivalent to 1 μmol of phenol formed per min. Enzyme activity is expressed in kilo international unit/liter (kU/L) [41].  

2.8. Determination of B Cell/Lymphoma-2 (Bcl-2)  

Bcl-2 was measured in striatal homogenates using a commercially available human Bcl-2 enzyme-linked immunosorbent assay kit from Glory Science (Del Rio, TX, USA). The manufacturer’s instruction was followed for carrying out the assay.  

2.9. Histopathological Studies  

The brain tissues were immediately fixed in 10% neutral-buffered formalin, dehydrated in gradual ethanol (50%–100%), cleared in xylene, and embedded in paraffin. Sections of 5-μm thickness were prepared and stained with hematoxylin and eosin (H&E) dye for photomicroscopic examination.  

2.10. Quantitative Image Analysis  

Quantitative analysis of the degenerating neurons in the in cortex, striatum, and hippocampus was performed to determine the labeling index, which denotes the percentage of degenerating cells. Briefly, labeling index = [(number of degenerating neurons in 5 high-power fields) ÷ (number of all neurons in these fields)] × 100 [42]. The degenerating cells in 5 random high-power fields were counted on the screen using Leica Qwin 500 Image Analyzer (LEICA, Cambridge, England,) in the Pathology Department at the National Research Centre (Cairo, Egypt).  

2.11. Statistical Analysis  

Results are expressed as mean ± SE. Data were statistically analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test for multiple group comparison. Statistical significance was considered at a probability value of less than 0.05. GraphPad Prism 6 for Windows (GraphPad Prism Software Inc., San Diego, CA, USA) was used. 

3. RESULTS  

3.1. Biochemical Results  

3.1.1. MDA  

Rotenone caused significant elevations in the level of the lipid peroxidation end product MDA in the striatum (42.8% increase: 23.27 ± 0.57 vs. 16.29 ± 1.0 nmol/g tissue), cerebral cortex (55.5% increase: 26.22±1.33vs.16.86±1.1nmol/gtissue),andthe rest of brain tissue (58.1% increase: 27.1 ± 1.20 vs. 17.14 ± 0.83 nmol/g tissue) as compared with the vehicle group. MPFF given to rotenone-treated rats at 18 mg/kg resulted in significant decreases in MDA levels in these brain regions by 25.4% (17.35 ± 0.86 vs. 23.27 ± 0.57 nmol/g tissue), 26.6% (19.23 ± 0.37 vs. 26.22 ± 1.33 nmol/g tissue), and 25.7% (20.14 ± 0.81 vs. 27.1 ± 1.20 nmol/g tissue), respectively (Figure 1).

 

FIGURE 1. Box plots of the effect of MPFF on the levels of MDA in the brain of rotenone-treated rats. Results are mean ± SEM (n = 6). *, p < 0.05 versus saline; +, p < 0.05 versus rotenone only group; #, p < 0.05 versus MPFF 9 mg/kg.  

3.1.2. Nitric Oxide  

Rotenone caused significant increments in brain nitric oxide levels in the striatum (67.4% increase: 31.20 ± 0.19 vs. 18.64 ± 1.31 mmol/g tissue), cerebral cortex (60.1% increase 28.70 ± 1.50 vs. 17.93 ± 0.45 mmol/g tissue), and the rest of the brain tissue (73.9% increase: 31.61± 1.0 vs. 18.17 ± 0.69 mmol/g tissue) as compared with the vehicle control values. The administration of MPFF at 18 mg/kg resulted in significant decreases in nitric oxide levels in these brain regions by 40.5% (18.56 ± 0.94 vs. 31.20 ± 0.19 mmol/g tissue), 34.1% (18.90 ± 0.71 vs. 28.70 ± 1.50 mmol/g tissue), and 41.8% (18.40 ± 0.51 vs. 31.61± 1.0 mmol/g tissue), respectively (Figure 2). 

 

FIGURE 2. Box plots of the effect of MPFF on the levels of nitric oxide in the brain of rotenone-treated rats. Results are mean ± SEM (n = 6). *, p < 0.05 versus saline; +, p < 0.05 versus rotenone only group; #, p < 0.05 versus MPFF 9 mg/kg.  

3.1.3. GSH  

In rotenone-treated rats, significant decreases in GSH content were observed in the striatum (40.3% decrease: 1.79 ± 0.08 vs. 3.0 ± 0.07 mmol/g tissue), cerebral cortex (41.4% decrease 1.78 ± 0.08 vs. 3.04 ± 0.14 mmol/g tissue), and the rest of the brain tissue (33.1% decrease: 2.16± 0.01 vs. 3.23 ± 0.10 mmol/g tissue) as compared with the vehicle control group. The administration of MPFF at 18 mg/kg resulted in restoration of GSH content to their vehicle control values (Figure 3). 

 

FIGURE 3. Box plots of the effect of MPFF on GSH content in the brain of rotenone-treated rats. Results are mean ± SEM (n = 6). *, p < 0.05 versus saline; +, p < 0.05 versus rotenone only group; #, p < 0.05 versus MPFF 9 mg/kg.  

3.1.4. PON-1  

PON-1 activity was significantly decreased in different brain regions following rotenone injection. It decreased by 45.1%, 53.4%, and 53.7% in striatum (7.12 ± 0.36 vs. 12.98 ± 0.54 kU/l), cerebral cortex (6.15±0.82vs.13.19±0.69kU/l),andrestofthe brain tissue (6.37 ± 0.52 vs. 13.75 ± 0.65 kU/l), respectively. MPFF given at 9 or 18 mg/kg resulted in significant increments in PON-1 activity in the striatum (by 73.2% and 96.6%), cerebral cortex (by 62.6% and 142.9%), and the rest of the brain tissue (by 43.0% and 114.6%) compared with the rotenone only group (Figure 4). 

 

FIGURE 4. Box plots of the effect of MPFF on PON-1 activity in the brain of rotenone-treated rats. Results are mean ± SEM (n = 6). *, p < 0.05 versus saline; +, p < 0.05 versus rotenone only group; #, p < 0.05 versus MPFF 9 mg/kg.  

3.1.5. Bcl-2 

Rotenone caused a significant decrease in striatal Bcl-2 levels by 36.4% as compared to the vehicle treated group (2.1 ± 0.06 vs. 3.3 ± 0.05 ng/ml). Striatal Bcl-2 increased significantly by 26.2% and 42.9% (2.65 ± 0.05 and 3.0 ± 0.04 vs. 2.1 ± 0.06 ng/ml) in rats treated with rotenone and MPFF at 9 or 18 mg/kg compared to the rotenone only group (Figure 5). 

 

FIGURE 5. Box plots of the effect of MPFF on striatal Bcl-2 in rotenone-treated rats. *, p < 0.05 versus vehicle-treated group; +, p < 0.05 versus rotenone group. #, p < 0.05 versus MPFF 9 mg/kg.  

3.2. Histopathological Results  

3.2.1. Striatum and Cerebral Cortex  

Sections of the striatum and cerebral cortex from the vehicle-treated group showed neurons arranged in neat rows with abundant cytoplasm and round basophilicnuclei(Figures 6A and 7A).Rotenoneonly-treated rats showed distorted architecture, vacuolated cells, necrosis of neurons, and congestion of blood vessels. Also, some neuronal cells appeared darkly stained, irregular (pyknotic), and apoptotic (Figures 6B and 7B). These changes were attenuated after treatment with MPFF at 9 mg/kg. However, moderate disarrangement, vacuolated neurons, some pyknoyic and apoptotic neurons, and congestion of blood vessels were also seen (Figures 6C and 7C). Rats treated with rotenone and MPFF at 18 mg/kg showed neuronal cells nearly normal as in the vehicle-treated animals, while very few cells exhibited vacuolation, pyknosis, and apoptosis (Figures 6D and 7D). 

 

FIGURE 6. Representative photomicrographs of H&E-stained sections of rat striatum. (A) Vehicle control group showing normal architecture and neurons (N). (B) Rotenone group showing distorted architecture, vacuolation (long arrow), congestion of blood vessel (arrow), pyknotic (thick arrow), and apoptotic cells (arrowhead). (C) Rotenone + MPFF 9 mg/kg group showing less degeneration changes, vacuolated (long arrow), pyknotic (thick arrow), and apoptotic cells (arrowhead). (D) Rotenone + MPFF 18 mg/kg group showing nearly normal neurons, with few pyknotic (thick arrow) and apoptotic cells (arrowhead) (×400). 

 

FIGURE 7. Representative photomicrographs of H&E-stained sections of rat cerebral cortex.(A) Vehicle control group showing normal architecture and neurons (N). (B) Rotenone group showing distorted architecture, vacuolation (long arrow), congestion of blood vessel (arrow), pyknotic (thick arrow), and apoptotic cells (arrowhead), in site pyknotic (1) and apoptotic cells (2). (C) Rotenone + MPFF 9 mg/kg group showing less degeneration changes, vacuolated cells (long arrow),congestion of blood vessel (arrow) pyknotic ( thick arrow) and apoptotic cells (arrowhead). (D) Rotenone + MPFF 18 mg/kg group showing that neurons cells were nearly normal, with few pyknotic (thick arrow) and apoptotic cells (arrowhead) (×400).  

3.2.2. Hippocampus  

The hippocampus from the vehicle group showed pyramidal cells of normal appearance (Figure 8A).Rotenone treated rats showed neuronal shrinkage, deeply stained and irregular nuclei, vacuolated cells, apoptotic cells, and decreased thickness of the hippocampal layer due to degeneration of pyramidal cells, when compared to vehicle group (Figure 8B). The group treated with rotenone and MPFF showed a thick pyramidal layer of near normal appearance, and obvious improvement in most of the tissue, in a dose-dependent manner. However, few cells were vacuolated, pyknotic, and some apoptotic (Figure 8C and 8D). 

 

FIGURE 8. Representative photomicrographs of H&E-stained sections of rat hippocampus. (A) Vehicle control group showing normal pyramidal cells. (B) Rotenone group showing neuronal shrinkage, pyknotic (thick arrow), apoptotic cells (arrowhead), vacuolated (long arrow), irregular in shape, and decreased thickness of hippocampal layer. (C) Rotenone + MPFF 9 mg/kg group showing thick pyramidal layer of near normal appearance and moderate improvement with moderate vacuolated (long arrow), pyknotic (thick arrow), and apoptotic cells (arrowhead). (D) Rotenone + MPFF 18 mg/kg group showing thick pyramidal layer of near normal appearance and obvious improvement with few vacuolated (long arrow), pyknotic (thick arrow), and apoptotic cells (arrowhead) (×400).  

3.2.3. Quantitative Neuronal Damage  

In the group treated with rotenone alone, the percentage of vacuolated cells, apoptotic, and pyknotic cells in cortex, striatum, and hippocampus were higher than that in the vehicle group. This effect of rotenone decreased following treatment with MPFF in a dose-depended manner (Tables 1–3).  

TABLE 1. Percentage of degenerating cells in the striatum in different experimental groups
Group Vacuolated cells Apoptotic cells Pyknotic cells
Vehicle 0.0 % 0.0 % 0.0 %
Rotenone 52% 69% 78%
Rotenone + MPFF 9 mg/kg 26% 39% 47%
Rotenone + MPFF 18 mg/kg 3% 20% 25%

 

TABLE 2. Percentage of degenerating cells in cortex in different experimental groups
Group Vacuolated cells Apoptotic cells Pyknotic cells
Vehicle 0.0 % 0.0 % 0.0 %
Rotenone 68% 76% 89%
Rotenone + MPFF 9 mg/kg 34% 42% 49%
Rotenone + MPFF 18 mg/kg 4% 24% 28%

 

TABLE 3. Percentage of degenerating cells in hippocampus in different experimental groups
Group Vacuolated cells Apoptotic cells Pyknotic cells
Vehicle 0.0 % 0.0 % 0.0 %
Rotenone 56% 66% 71%
Rotenone + MPFF 9 mg/kg 22% 35% 39%
Rotenone + MPFF 18 mg/kg 6% 22% 23%

4. DISCUSSION 

Rotenone, a pesticide and a mitochondrial complex I inhibitor, is widely used to model human PD by virtue of its ability to cause oxidative stress and nigrostriatal dopaminergic neurodegeneration [43]. In this study, MPFF (Daflon®) was evaluated for its ability to reduce the rotenone-induced neurotoxicity. Results of this study show that rotenone exposure induced oxidative stress in the striatum, cerebral cortex, and the rest of the brain tissue as evidenced by an increase in the lipid peroxidation end product MDA and a decrease in the antioxidant and radical scavenger GSH, indicative of an increase in ROS and consequent attack on membrane lipids [11, 15]. There were also decreased striatal levels of the anti-apoptotic protein Bcl-2 [44] and histological evidence of neuronal necrosis and apoptosis in the striatum, cerebral cortex, and hippocampus of rotenone-treated rats. MPFF given at the dose of 18 mg/kg prevented the increase in brain lipid peroxidation and nitric oxide levels and restored GSH and PON-1 to their normal values and protected against the rotenone-induced neuronal damage. 

The increased generation of ROS and consequent oxidative damage to cellular biomolecules are considered major mechanisms underlying the rotenone-inducedneurodegeneration[45].The pesticidehasbeen shown to be a selective complex I inhibitor [46], resulting in increased formation of superoxide and hydrogen peroxide from both complex I and complex III [47]. Rotenone can also activate microglia to release superoxide [48] or hypochlorous acid [49] via the activation of NADPH oxidase and myeloperoxidase, respectively. The superoxide can reduce cytochrome c or transition metals or react with nitric oxide to form the strong oxidant peroxynitrite (ONOOˉ) [50]. This results in damage to the mitochondrial complexes I and III [47] and activation of the apoptotic cell death pathway [51]. Our results show that rotenone caused increased brain lipid peroxidation and depletion of GSH. These data are consistent with previous studies that indicated increased lipid peroxidation products [52–54] along with depletion of the antioxidant GSH and decreased activity of the antioxidant enzymes superoxide dismutase [55] and catalase [56] following rotenone exposure. The role of oxidative stress in rotenone neurotoxicity is further supported by the finding that treatment with antioxidants such as glutathione, N-acetylcysteine, and vitamin C protected against rotenone-induced dopaminergic cell apoptosis and nigrostriatal neurodegeneration [51, 57, 58]. 

In this study, increased nitric oxide was observed in the brain of rotenone-exposed rats which is in accordance with previously published data [52, 53, 59]. Excessive nitric oxide production may occur from neuronal (nNOS) or inducible nitric oxide synthase (iNOS) following its induction by activated microglia and inflammatory cells in response to proinflammatorycytokinesorbacterialendotoxin[60]. High levels of nitric oxide can be neurotoxic through the formation of peroxynitrite and other reactive nitrogen species, resulting in oxidation of proteins, lipids, and DNA, nitrosylation of thiol residues and nitration of tyrosine residues in proteins [61, 62]. Neuronal death occurs via inactivation of mitochondrial electron transport complexes, inhibition of mitochondrial respiration, and energy failure [60]. Nitric oxide may contribute to dopaminergic neurodegeneration in PD since increased nitric oxide production and protein tyrosine nitrations as well as increased nNOS mRNA expression were shown in neutrophils isolated from PD patients [63]. Nitric oxide production by nNOS or iNOS also contributes to dopaminergic neurotoxicity caused by rotenone, MPTP, or lipopolysaccharide endotoxin [53, 59, 64, 65] and inhibition of iNOS or nNOS activity was shown to afford protection against rotenone or MPTP neurotoxicity [59, 66, 67]. Here we showed that MPFF was able to prevent the increase in brain nitric oxide induced by rotenone, suggesting that inhibition of nitric oxide is involved in the neuroprotection by the drug. Other studies demonstrated an inhibitory effect for MPFF on hepatic iNOS overexpression following bacterial endotoxin injection in the rat [36]. 

This study also demonstrate marked inhibition of brain PON-1 following rotenone injection; a finding which is consistent with previous observations [54, 58]. Recent evidence supports an important role for PON-1 in neurodegeneration that occurs in PD. This is because the enzyme is important in the detoxification of a number of organophosphorus insecticides such as parathion, diazinon, and chlorpyrifos [68] and exposure to these chemicals, other insecticides, or rotenone has been shown to increase the risk for developing PD in exposed individuals [7, 8, 69]. The catalytic efficiency of PON-1 and hence its ability to hydrolyze organophosphates largely determines the susceptibility to these compounds [68] and also the propensity for developing sporadic PD [70]. In this context, studies showed that PON1 L55M genetic variation is associated with faster progression of motor and depressive symptoms in PD [71]. The peroxidative and anti-inflammatory activities of PON-1 and the decrease in its activity in a number of neurologic disorders suggest a neuroprotective role for the enzyme [72]. PON-1 is inactivated by oxidative stress [73] and this might explain the decline in enzyme activity in rotenone-treated rats. Taken together, the above data suggest that increasing or maintaining PON-1 activity should pursue a neuroprotective role. In the present study, there was restoration of PON-1 activity by treatment with MPFF. Studies in rats with endotoxemia and systemic inflammation caused by bacterial endotoxin showed that the decrease in serum PON-1 activity was prevented with MPFF at 9 and 18 mg/kg [36]. 

The mitochondrial apoptotic pathway is controlled by the B cell lymphoma-2 protein (Bcl2) family which includes both pro- and anti-apoptotic members. Bcl-2 is an anti-apoptotic protein whose function is to maintain the integrity of the outer mitochondrial membrane. This prevents the release of mitochondrial cytochrome c into the cytosol and consequent activation of caspase proteins that execute the process of apoptosis [44]. The presence of apoptosis has been detected in substantia nigra melanized neurons in PD patients [74] and evidence has also been provided for the occurrence of apoptosis in MPTP and rotenone models of PD [75–77]. In our study, we found decreased Bcl2 protein after rotenone administration which suggests that apoptosis is a mechanism by which rotenone induces neuronal death. Similar data were provided by other researchers indicating decreased expression of Bcl-2 in human dopaminergic cells in vitro [78] and decreased Bcl2 protein levels in the brain of rotenone-treated rats [79]. In this study, treatment with MPFF at 18 mg/kg resulted in the restoration of Bcl-2 protein levels in the striata of rotenone-treated rats, suggesting an anti-apoptotic effect for MPFF. 

The antioxidant and anti-inflammatory actions of hesperidin, diosmin, or their combination (MPFF) have been reported by several studies. Hesperidin prevented the decline in superoxide dismutase and catalase activities in tissues of exercise-exhausted rats [80]. It displayed neuroprotective effects against memory disruption by scopolamine in the rat, decreasing hippocampal tumor necrosis factor-a levels [81]. In vitro, hesperidin and, to a lesser extent, diosmin attenuated the increase in intracellular ROS, following exposure of human SH-SY5Y neuroblastoma cells to 6-OHDA [35]. In I/R injury of rat retina, diosmin was shown to decrease lipid peroxidation and increase the activities of the antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase [33]. It also decreased tissue MDA and myeloperoxidase activity in intestinal I/R injury in the rat [32]. In an in vivo model of systemic inflammatory response induced by bacterial endotoxin in rats, MPFF inhibited brain and liver oxidative stress, and markedly attenuated hepatic expression of iNOS and cleaved caspase-3 [36]. 

In conclusion, the antioxidant and anti-inflammatory properties of natural compounds such as hesperidin and diosmin provides an attractive adjunctive therapy in PD with the hope of improving dopaminergic cell survival. Our results demonstrate that MPFF was able to prevent rotenone neurodegeneration by a mechanism that involves decreased oxidative and nitrosative stress in addition to an anti-apoptotic action. Taking into consideration the proven safety of MPFF and its widespread use, we suggest that MPFF could be a useful “add on” treatment in PD. 

ACKNOWLEDGMENTS 

This works was not supported by research grants. 

CONFLICTS OF INTEREST STATEMENT 

The authors declare no conflict of interest. 

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