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CNS & Neurological Disorders - Drug Targets, 2020, 19, 000-000 1

RESEARCH ARTICLE

Neuroprotection of Rotenone-Induced Parkinsonism by Ursolic Acid in PD

Mouse Model

Walia Zahra1, Sachchida Nand Rai2, Hareram Birla1, Saumitra Sen Singh1, Aaina Singh Rathore1,
Hagera Dilnashin1, Richa Singh1, Chetan Keswani1, Rakesh K. Singh1 and Surya Pratap Singh1,*

1
Department of Biochemistry, Institute of Science, Banaras Hindu University, Varanasi-221005, India; 2Centre of Bio-
technology, University of Allahabad, Prayagraj-211002, India

ARTICLE HISTORY
Received: April 24, 2020
Revised: June 29, 2020
Accepted: July 01, 2020
DOI:
10.2174/1871527319666200812224457
Abstract: Background: Parkinson’s disease (PD) is characterized by both motor and non-motor
symptoms. The presynaptic neuronal protein, α-Synuclein, plays a pivotal role in PD pathogenesis and
is associated with both genetic and sporadic origin of the disease. Ursolic Acid (UA) is a well-known
bioactive compound found in various medicinal plants, widely studied for its anti-inflammatory and
antioxidant activities.
Objective: In this research article, the neuroprotective potential of UA has been further explored in the
Rotenone-induced mouse model of PD.
Methods: To investigate our hypothesis, we have divided mice into 4 different groups, control, drug
only control, Rotenone-intoxicated group, and Rotenone-intoxicated mice treated with UA. After the
completion of dosing, behavioral parameters were estimated. Then mice from each group were sacri-
ficed and the brains were isolated. Further, the biochemical tests were assayed to check the balance
between the oxidative stress and endogenous anti-oxidants; and TH (Tyrosine Hydroxylase),
α-Synuclein, Akt (Serine-threonine protein kinase), ERK (Extracellular signal-regulated kinase) and
inflammatory parameters like Nuclear factor-κB (NF-κB) and Tumor Necrosis Factor- α (TNF-α) were
assessed using Immunohistochemistry (IHC). Western blotting was also done to check the expressions
of TH and α-Synuclein. Moreover, the expression levels of PD related genes like α-Synuclein,
β-Synuclein, Interleukin-1β (IL-1β), and Interleukin-10 (IL-10) were assessed by using Real-time
PCR.
Results: The results obtained in our study suggested that UA significantly reduced the overexpression
of α-Synuclein and regulated the phosphorylation of survival-related kinases (Akt and ERK) apart
from alleviating the behavioral abnormalities and protecting the dopaminergic neurons from oxidative
stress and neuroinflammation.
Conclusion: Thus, our study shows the neuroprotective potential of UA, which can further be ex-
plored for possible clinical intervention.

Keywords: Parkinson’s disease, Rotenone, α-Synuclein, neuroinflammation, oxidative stress, ursolic acid.

1. INTRODUCTION are found in the form of intracellular inclusions known as

PD is the second most common neurodegenerative disor-
der, clinically detected by several motor symptoms such as
bradykinesia, rigidity, resting tremors, and postural instabil-
ity that might be originated sporadically or have a familial
origin. Different genetic and environmental factors are sup-
posed to be responsible for the pathobiogenesis of PD [1]. In
Substantia Nigra pars compacta (SNpc) of basal ganglia,
primarily insoluble aggregates of ubiquitin and α-Synuclein
*Address correspondence to this author at the Department of Biochemistry,
Institute of Science, Banaras Hindu University, Varanasi-221005, India;
Tel: 0542-6701543; E-mail: suryasinghbhu16@gmail.com
Lewy Bodies (LBs) and Lewy Neurites (LNs), which are
also predominantly considered as the neuropathological
hallmarks of PD. The fact that elevated levels of LBs and
LNs in the basal ganglia concomitantly lead to increased
degeneration of Dopaminergic (DA) neurons in-vivo strong-
ly puts forward that the overexpression of α-Synuclein is one
of the main pathological hallmarks of PD [2-4]. Oxidative
stress and neuroinflammation play a crucial role in the pro-
gression of PD. In response to neuroinflammation, microglia
get activated, and - in turn - produce and release neurotoxic
factors such as Tumor Necrosis Factor-α (TNF-α) and Inter-
leukin-1β (IL-1β). Some experimental studies have reported

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2 CNS & Neurological Disorders - Drug Targets, 2020, Vol. 19, No. 0
that suppression of pro-inflammatory mediators and free
radical generation, along with the inhibition of α-Synuclein
aggregation, could be achieved by exploiting the naturally
occurring compounds from medicinal herbs [5, 6]. Also,
evidences from several studies suggest that abnormality in
the function of different protein kinases; such as the kinases
of the Phosphoinositide 3-Kinase (PI3K) and Mitogen-
Activated Protein Kinase (MAPK) contribute to PD’s etiolo-
gy [7, 8]. ERK and Akt are the respective modulators of
MAPK and PI3K pathways [8-11]. Overexpression of α-
Synuclein is also linked with suppression in the phosphoryla-
tion and of these molecules in different studies [12-14]. There-
fore, by understanding how the activities of these enzymes get
changed during the disease pathogenesis would allow us to
modulate these signaling pathways for PD therapy.
Rotenone is a widely used neurotoxin for generating an-
imal models of chronic PD and is being frequently used to
screen potential compounds that might be used therapeutical-
ly to improve the symptoms of PD [15, 16]. Medicinal plants
are being used as an alternative drug therapy because of hav-
ing minimal side-effects [17-23]. Ursolic Acid (UA) is found
as a component of medicinal herbs and ornamental species.
It is also found in fruits like prunes, apples, blueberries, and
cranberries [24, 25], with their amounts ranging between
0.15 to 1.8 g/100 g dry matter [26]. Moreover, UA has been
reported as a potent anti-oxidative agent in 1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mice
model of PD [27]. The compound is capable of crossing the
blood-brain barrier [28] and various reports have demon-
strated the neuroprotective effects of UA through its anti-
oxidative and anti-inflammatory properties in different ani-
mal models [27, 29-31]. Therefore, the keystone of our study
was to explore further the neuroprotective potential of UA in
Rotenone-induced Parkinsonian mice model.
Regardless of various studies already being conducted
based on the anti-oxidative and neuroprotective properties of
UA, the present study demonstrates the potential of UA
against nigrostriatal degeneration of DA neurons by the inhi-
bition of α-Synuclein-mediated neurotoxicity in Rotenone-
induced mouse model of PD.
2. MATERIALS AND METHODS
2.1. Reagents and Antibodies
Normal goat serum (NGS), UA, and Rotenone were pro-
cured from Sigma-Aldrich (St. Louis, MO, United States).
Bovine serum albumin (BSA), ammonium chloride, potassium
chloride, acetic acid, reduced nicotinamide adenine dinucleo-
tide phosphate (NADPH), disodium hydrogen phosphate, and
sodium dihydrogen phosphate were purchased from Sisco
Research Laboratories (SRL, Mumbai, India). The protein
estimation kit by Bradford GeNeiTM, potassium dichromate
and hydrogen peroxide (H2O2) were bought from Merck
(Darmstadt, Germany), and thiobarbituric acid (TBA), 1,4-
diazabicyclo[2.2.2]octane (DABCO), sodium dodecyl sul-
phate (SDS), and Griess reagent were obtained from HiMe-
dia (Mumbai, India). Paraformaldehyde and sodium nitrite
were bought from Lobachemie, India. Protease inhibitor and
RNase inhibitor were procured from Sigma Aldrich and Ge-
nei, respectively. Primary antibodies for β-actin (SC-47778)
and TH (SC-25269) were purchased from Santa Cruz, Bio-
Zahra et al.
technology (Santa Cruz, CA, United States), and the primary
antibodies for α-Synuclein (ab191692), TNF-α (ab1793),
Akt (ab81283), ERK (ab76165) and NF-κB (ab16502) were
procured from Abcam Life Science, Biogenuix Medsystems
Pvt. Ltd. (New Delhi, India). Secondary antibodies for
western blot were mouse anti-rabbit IgG-HRP (Horseradish
peroxidase) (sc-2357) and Goat anti-mouse (GeNei-
62114068001A), and secondary fluorescent-tagged antibod-
ies for IHC (such as Cy2 conjugated and Cy3 conjugated for
immunohistochemistry) were purchased from Merck Milli-
pore and Chemicon, respectively.
2.2. Experimental Animals
Adult Swiss albino male mice weighing 25 ± 5g were
provided by an animal research facility of the Institute of
Medical Sciences, Banaras Hindu University, Varanasi, In-
dia, to conduct the experiment. Mice were housed in clean
polypropylene cages in a regulated air-conditioned environ-
ment on 12 h light-dark cycles with access to standard mice
pellet diet and water. All efforts were made to minimize the
suffering of the animals, and all the animal experiments were
performed in accordance with the experimental practices and
standards approved by the Animal Ethics Committee of Ba-
naras Hindu University, Varanasi, India.
2.3. Experimental Design
After the mice were acclimatized to the laboratory condi-
tions, they were divided into 4 groups with 8 animals in each
group. Group-I was used as a control group. Group-II was
drug only control: mice were orally administered with UA
(25mg/kg bwt/day) for 42 days. Group-III was given Rote-
none (2 mg/kg bwt/day) subcutaneously for 35 days. Lastly,
Group-IV was first orally-administered with UA for a week,
then simultaneous administration of UA and Rotenone-
intoxication was done for the next 35 days, similar to that of
Group-III. Dosing of UA was done according to Rai et al.,
2016, with slight modifications [27].
2.4. Behavioral Assessment
After the completion of the administration of the dosages,
behavioral parameters were performed at room temperature
without any outside interference and their motor discrepan-
cies were studied. Various tests were performed for the as-
sessment, including rotarod, narrow beam walking, and
hanging tests.
2.4.1. Rotarod Test
The mice were trained for three consecutive days at a
fixed speed (15 rpm) and then subjected to analysis. The
performance time was recorded for a maximum of 5 min,
while mice ran on the rod. Each mouse was subjected to four
trials, and the average time was recorded [32]. Similarly,
after the completion of the experiments, the rotarod test was
again performed and the mean of their falling latency was
calculated.
2.4.2. Narrow Beam Walking Test
The purpose of performing this test was to assess the
mouse’s motor coordination, where the mouse needed to stay
Neuroprotection of Rotenone-Induced Parkinsonism by Ursolic Acid
upright and walk across an elevated narrow beam to a safe
platform. Primarily, the mice were unaware/untrained for this
test. Hence, they were trained to walk on a stationary narrow
wooden beam placed 100 cm above the floor (L100 cm × W1
cm). The experiment was repeated thrice and the time taken
by the mice to cross over the beam was recorded [33].
2.4.3. Hanging Test
On a horizontal grid, each mouse was placed and allowed
to hold it firmly before inverting it so that the mouse hung
upside down until it lost its grip and fell. The time of hang-
ing was recorded, and the experiment was repeated thrice
[34].
2.5. Biochemical Parameters
The mice were sacrificed by cervical dislocation, fol-
lowed by decapitation with minimum pain. The brains were
dissected out quickly on dry ice and were stored at -80oC
until further use. The SNpc was procured [35] and further
homogenized in KCl buffer (Tris–HCl 10 mM, NaCl 140
mM, KCl 300 mM, Ethylenediaminetetraacetic acid 1 mM,
Triton-X 100 0.5%) at pH 8.0, supplemented with protease
and phosphatase inhibitors. The acquired tissue homogenates
were then subjected to centrifugation at 12,000 × g for 20
min at 4oC to obtain a supernatant. The specific antioxidant
activities of the enzyme were measured for Superoxide Dis-
mutase (SOD) and catalase, along with determining the re-
duced Glutathione content, levels of lipid peroxidation, and
nitrite from the supernatant.
2.5.1. Estimation of Lipid Peroxidation and Nitrite Levels
The method used by Ohkawa et al., [36] was incorpo-
rated with few modifications to test tissue lipid peroxidation.
Foremost, 10% tissue homogenate was added to 10% SDS
and then mixed with 20% acetic acid. Subsequently, the re-
action mixture was kept in a boiling water bath for 1 h after
adding 0.8% TBA. Later, when the mixture was cooled and
centrifuged to extract out supernatant, the absorbance was
recorded at 532 nm in reference to control. Peroxidation of
lipids was measured in terms of micromoles of malondialde-
hyde (MDA) released per milligram protein. The level of
nitrite synthesis was assessed according to the standard pro-
cedures with slight modifications [37]. The 10% tissue ho-
mogenate was taken and mixed with ammonium chloride
and Griess reagent. The solution was allowed to stand for
half an hour at 37oC, and then the absorbance was taken at
540 nm. The nitrite content was calculated using a standard
curve for sodium nitrite (10-100 mM) in terms of mi-
cromoles per milliliter.
2.5.2. Estimation of Antioxidant Levels
Reduced GSH content in the brain homogenate was de-
termined using the method described by Moron et al., (1979)
and values were expressed as µg/mg protein [38]. The rate of
decomposition of hydrogen peroxide (substrate) was exam-
ined using a spectrophotometer to assess the catalase activity
[35]. To do this, the striatal tissue was mixed with potassium
dichromate and acetic acid (1:3) in a boiling water bath for
10 min, and OD was taken at 570 nm. Measurement of the
CNS & Neurological Disorders - Drug Targets, 2020, Vol. 19, No. 0 3
enzyme activity was done in n moles/min/mg protein. By
using its substrate NADH, the activity of SOD was evaluated
[39]. Ultimately, the absorbance was read at 560 nm against
reagent blank. The difference between reference and experi-
mental OD of the sample gave the inhibition of Nitro Blue
Tetrazolium chloride (NBT) reduction by an enzyme source.
From the enzyme source, the protein was also estimated.
Under the assay conditions, the unit of SOD enzyme activity
was defined as the amount of enzyme required to inhibit the
optical density at 560 nm of NBT reduction by 50% in 1
min. The activity of SOD was expressed as unit per milli-
gram of protein.
2.6. Immunohistochemical Staining
For the histopathological assessment, mice from each
group were anesthetized with diethyl ether, perfused intra-
cardially initially with 0.9% saline (chilled) followed by 4%
paraformaldehyde (chilled) prepared in 0.1 M phosphate-
buffered saline (PBS), pH 7.4, and then decapitated. Brains
were extracted and post-fixed in 10% paraformaldehyde
overnight and later cryoprotected in 30% sucrose solution.
Immunohistochemical staining of TH, α-Synuclein, Akt-1,
ERK1/2, TNF-α and NF-κB was performed in SNpc using
the standard procedure [40]. Coronal sections of 20 µm
thickness passing through SNpc were cut using a cryomicro-
tome (Leica, Wetzlar, Germany). The sections were rinsed
thrice for 10 min each with 0.01 M PBS (pH 7.4), and then
blocked with 10% NGS in PBS 0.3% Triton-X 100 and 1%
BSA in phosphate-buffered saline with Triton X-100
(PBST), as blocking reagent for about 1 h. The sections were
further incubated with primary antibody (polyclonal anti-
mouse antibody against TH in 1:1000 dilution, polyclonal
anti-rabbit α-Synuclein in 1:1500 dilution, monoclonal anti-
mouse TNF-α in 1:700 dilution, monoclonal anti-rabbit
pAkt1 (1:1000), polyclonal anti-rabbit antibody pERK1/2
(1:1000), polyclonal anti rabbit NF-κB p65 antibody in
1:1000 dilution) for 16 h at 4oC. Further, in order to remove
unbound primary antibodies, rinsing in PBS and 1% BSA-
PBS was done twice for each well. It was then incubated with
Cy2-conjugated secondary antibody (for anti-mice primary)
and Cy3-conjugated secondary antibody (for anti-rabbit pri-
mary), prepared in 1% BSA-PBS, for 1 h at room temperature.
Then, sections were rinsed thrice with 1% BSA-PBS at 3-min
interval, and 4’,6-diamidino-2-phenylindole (DAPI) was add-
ed (1 mg/ml). Ultimately, sections were rinsed thrice with
PBS and then mounted on slides using polyvinyl alcohol
mounting medium with DABCO anti-fading (Fluka analyti-
cal). Visualisation was performed under Nikon fluorescent
microscope (Thermo Fisher Scientific). Percentage area val-
ue and cell counting were determined by using Image J soft-
ware 1.52a (NIH, United States).
2.7. Real-Time Polymerase Chain Reaction (PCR) Analy-
sis
Total RNA from the frozen tissues was extracted using
the TRIzol RNA isolation reagent (Invitrogen, Carlsbad, CA,
United States) according to the manufacturer’s instructions.
The RNA yield was quantified on Nanodrop 1000, and the
RNA purity was determined based on the A260/A280 ratio.
For cDNA preparation, reverse transcription was carried out
4 CNS & Neurological Disorders - Drug Targets, 2020, Vol. 19, No. 0 Zahra et al.

using 2 mg total RNA (kept equal for each amplification)
and 20U M-MLV reverse transcriptase (Fermentas, Germa-
ny), 1 X reverse transcriptase (RT) buffer, 20mM dNTPs
(New England Biolabs, United States), 20 U RNasin (Fer-
mentas, Germany), 0.1 M dithiothreitol (DTT) with dieth-
ylpyrocarbonate (DEPC)-treated water, and 100 ng of ran-
dom hexamers (Fermentas, Germany). Gene expression pro-
file analysis was done on the ABI7500 Fast system. Glycer-
aldehyde 3-phosphate dehydrogenase (GAPDH) was taken
as an endogenous control. PCR was operated according to
the previously reported studies, with few modifications. The
reactions were operated by using 10 ml of a real-time mix
containing 5 ml of SYBER green master mix (Applied Bio-
system), 1 ml cDNA, 2 ml nuclease-free water, 0.5 ml each
of forward and reverse primers, and 1 ml RNase inhibitor.
The samples were initially incubated at 50oC for 2 min, fol-
lowed by denaturation at 95oC for 10 min. This was pro-
gressed by 40 cycles at 95oC for 15 s, 60oC for 1 min, and
72oC for 40 s [41]. The abundance or decline of mRNA was
normalized to the geometric average of endogenous control
GAPDH for ΔCt. The fold change was calculated using 2-Δ Δ
Ct
method and reported as fold change unit.
2.8. Western Blot Analysis
Western blot analysis was done according to the method
described by Gupta et al. in 2010 with slight modifications
[42]. Primary antibody dilutions included TH (1:4000), α-
Synuclein (1:1000) and β-actin (1:500). The blots obtained
were developed by using 3,3’-Diaminobenzidine (DAB) and
H2O2 as substrates. The relative band density was calculated
with respect to the expression of β-actin, and the fold change
indicated the resultant expressions of proteins.
2.9. Statistical Analysis
One-way analysis of variance (ANOVA) was used to
analyze the data by applying the Student-Newman-Keuls
test, whereas the fold changes of mRNA were analyzed by
Student’s two-tailed t-test through Graph Pad Prism 5.0
software. The results are expressed as the means ± SEM, and
p-values less than 0.05 (p < 0.05) were considered statistical-
ly significant.
3. RESULTS
3.1. Behavioural Studies
3.1.1. Effect of UA on Motor Coordination
It was seen that, compared to control mice, the duration
of Rotenone-intoxicated mice in the hanging test was signifi-
cantly poorer (p<0.0001; time reduced to 0.58-fold (Rote-
none) as compared to control). On the other hand, the UA
treatment given to the Rotenone group significantly in-
creased the hanging time (p<0.001; time was increased to
0.63 fold (Rotenone+UA) as compared to Rotenone), as
compared to the hanging time Rotenone-intoxicated group
(Fig. 1A). It was observed that the time duration of Rote-
none-intoxicated mice on rotarod significantly reduced,
compared to control (p<0.0001; time reduced to 0.5-fold
(Rotenone) as compared to control). On the other hand, Ro-
tenone-intoxicated mice that were given UA treatment stayed
significantly longer on the rotarod than Rotenone mice
(p<0.001; time was increased to about 0.6 fold (Rote-
none+UA) as compared to Rotenone) (Fig. 1B).
Moreover, in narrow beam walking test, rotenone-
induced mice showed a higher increase in time duration in
comparison with control mice (p<0.0001; time increased to
2.6 folds (Rotenone) as compared to control). However, the
UA-treated rotenone-induced group showed a significant
reduction in the narrow beam walking time, as compared to
Rotenone mice (p<0.001; time was reduced to 0.76 fold (Ro-
tenone+UA) as compared to control) (Fig. 1C).

Fig. (1). Effect of UA on behavioral parameters. (A) A significant improvement in hanging time was observed upon UA administration in
Rotenone intoxicated group. Hanging time in Rotenone-induced Parkinsonian mice was observed to be comparatively reduced as compared
to control. (B) Rotenone-intoxicated mice group has stayed on the rotarod for a lesser time interval as compared to that of the control group.
UA administered-Rotenone group has stayed significantly more on the rotarod as compared to the untreated Rotenone-induced PD mice
group. (C) As compared to control, narrow beam walking time was found to be comparatively increased in Rotenone-intoxicated mice.
While UA treatment has reduced the walking time in the Rotenone-intoxicated group, as shown in comparison with untreated Rotenone-
intoxicated mice. Data are expressed in terms of mean ± SEM (*p < 0.01, **p < 0.001, ***p < 0.0001, n = 8). UA, Ursolic acid; SEM, stand-
ard error of mean. (A higher resolution/colour version of this figure is available in the electronic copy of the article).
Neuroprotection of Rotenone-Induced Parkinsonism by Ursolic Acid CNS & Neurological Disorders - Drug Targets, 2020, Vol. 19, No. 0 5

Fig. (2). Estimation of Catalase, MDA, SOD, GSH and nitrite in SNpc of mice. Rotenone intoxication to the mice has significantly reduced
the antioxidants; Catalase (A), SOD (C) and GSH (D) and increased the level of oxidative stress mediators MDA (B) and Nitrite (E) in com-
parison to control group. Whereas, UA administration to the Rotenone-induced PD mice group has concomitantly increased the level of
Catalase (A), SOD (C) and GSH (D) and decreased the level of MDA (B) and Nitrite (E) as compared to the Rotenone-intoxicated group.
Values are expressed as mean ± SEM (*p < 0.01, **p < 0.001, ***p < 0.0001, n = 4). SOD, Superoxide dismutase; MDA, malondialdehyde;
GSH, glutathione; SEM, standard error of mean. (A higher resolution/colour version of this figure is available in the electronic copy of the
article).

3.2. Biochemical Studies
3.2.1. Effect of UA on Catalase, SOD and Nitrite
The results revealed that in comparison to the control
group, the Rotenone-intoxicated mice showed a significant
reduction in the activities of Catalase (Fig. 2A) and SOD
(Fig. 2C) (p<0.0001), and a significant increase in the nitrite
content (Fig. 2E) (p<0.0001). Although, as compared to the
Rotenone-treated group, UA-treated rotenone-induced mice
showed increased activity of catalase (p<0.01) (Fig. 2A),
SOD (p<0.001) (Fig. 2C), and decreased nitrite level
(p<0.0001) (Fig. 2E).
3.2.2. Effect of UA on MDA and Glutathione (GSH) Con-
tent
Significant elevation (Fig. 2B) in the level of lipid perox-
idation product, MDA, was seen in the Rotenone-intoxicated
group as compared to the control group (p<0.0001). On the
other hand, GSH level showed a significant decline (Fig. 2D)
in the Rotenone-intoxicated group, when compared to the
control group (p<0.0001). However, compared to the Rote-
none group, MDA level (Fig. 2B) was observed to be signif-
icantly reduced (p<0.0001), while GSH levels (Fig. 2D) sig-
nificantly enhanced (p<0.0001), after the administration of
UA to the Rotenone-intoxicated group.
3.3. Effect of UA on the Expression of TH, α-Synuclein
and TNF-α in SNpc
In comparison to the control group mice, in the rotenone-
intoxicated group, the expression of α-Synuclein (p<0.0001;
percentage area increased from about 2% (Control) to 6.5%
(Rotenone)) (Fig. 3A) and TNF-α (p<0.001; percentage area
increased from about 4% (Control) to 7.5% (Rotenone))
(Fig. 3C) was found to be significantly enhanced. Although
on treatment with UA (Rotenone+UA), a fall in the expres-
sion of α-Synuclein (p<0.001; percentage area reduced from
about 6.5% (Rotenone) to 3% (Rotenone+UA)) (Fig. 3A)
and TNF-α (p<0.01; percentage area reduced from about 7.5%
(Rotenone) to 5% (Rotenone+UA)) (Fig. 3C) was noticed in
comparison with Rotenone-intoxicated mice. Furthermore, in
comparison to control group, Rotenone-intoxication reduced
the number TH-positive neurons (p<0.001; number of TH-
positive neurons reduced from about 200 (Control) to 125
(Rotenone)) (Fig. 3B), whereas, in comparison to Rotenone
6 CNS & Neurological Disorders - Drug Targets, 2020, Vol. 19, No. 0 Zahra et al.

Fig. (3). Immunofluorescence expression of α-Synuclein, TH and TNF-α in SNpc of Control, Drug only Control (Control+UA), Rotenone
and Rotenone+UA mice groups by using Image J Software at 20x magnification. Rotenone intoxication has led to a significantly increased
expression of α-Synuclein (A) and TNF-α (C) and reduced immunoreactivity of TH (B) as compared to the control group. While, UA admin-
istration in PD mice has significantly alleviated the expression of α-Synuclein (A) and TNF-α (C) and enhanced the immunoreactivity of TH
(B) as compared to Rotenone-intoxicated mice. Values are expressed as mean ± SEM of percentage area (*p < 0.01, **p < 0.001, ***p <
0.0001, n = 4). TNF-α, tumor necrosis factor alpha; TH, Tyrosine hydroxylase; SNpc, substantia nigra pars compacta; UA, Ursolic acid. The
arrows represent the expression of the respective antibodies in SNpc. Scale bar represents the degree of magnification of the image (For 20X=
100µm). (A higher resolution/colour version of this figure is available in the electronic copy of the article).

group, the UA treatment group showed significantly increased
numbers of TH-positive neurons in SNpc of mice brain
(p<0.001; number of TH-positive neurons increased from
about 125 (Rotenone) to 180 (Rotenone+UA)) (Fig. 3B).
3.4. Effect of UA on the Expression of p-Akt-1 and p-
ERK1/2
The intoxication of Rotenone in mouse has led to the
decreased phosphorylation of Akt (p<0.0001; percentage
area reduced from about 14% (Control) to 6% (Rotenone))
(Fig. 4A) and ERK1/2 (p<0.0001; percentage area reduced
from about 15% (Control) to 7% (Rotenone)) (Fig. 4B) when
compared with the control group, whereas, treatment of UA
to the PD mouse model has shown the elevated level of
phosphorylation for both Akt (p<0.0001; percentage area
increased from about 6% (Rotenone) to 8% (Rotenone+UA))
(Fig. 4A) and ERK1/2 (p<0.0001; percentage area increased
from about 7% (Rotenone) to 9% (Rotenone+UA)) (Fig. 4B)
as compared to the disease group.
3.5. Effect of UA on the Nuclear Translocation of NF-κB
(p65) in SNpc
When compared to the control group (Fig. 5A), it was
observed that the nuclear translocation of NF-κB in Rote-
none-intoxicated mice occurred (p<0.001; Integrated Fluo-
rescence Value (IFV) increased from about 3 to 12) (Fig. 5B)
as a result of Rotenone intoxication. On the other hand, UA
treatment (p<0.01; Integrated Fluorescence Value (IFV) re-
duced from about 12 to 7.5) (Fig. 5C) significantly inhibited
the nuclear translocation of NF-κB in Rotenone-intoxicated
mice.
3.6. Real-Time PCR Analysis: Effect of UA on mRNA
Expression of Proinflammatory Cytokine, IL-1 β;
Anti-inflammatory Cytokine, IL-10; α-Synuclein and
β-Synuclein
When compared to control group, the mRNA expressions
of α-Synuclein (p<0.0001) (Fig. 6A) and the pro-inflammatory
cytokine, IL-1β (p<0.01) (Fig. 6C) were found to be signifi-
cantly elevated, while the mRNA expressions of β-Synuclein
(p<0.01) (Fig. 6B) and IL-10 (p<0.001) (Fig. 6D) were com-
paratively reduced in Rotenone-intoxicated group. UA ad-
ministration to the Rotenone-induced Parkinsonian mice
group, on the other hand, had significantly reduced mRNA
levels of α-Synuclein (p<0.0001) (Fig. 6A) and IL-1β
(p<0.0001) (Fig. 6C) and significantly enhanced mRNA
levels of β-Synuclein (p<0.0001) (Fig. 6B) and IL-10
(p<0.0001) (Fig. 6D).
Neuroprotection of Rotenone-Induced Parkinsonism by Ursolic Acid CNS & Neurological Disorders - Drug Targets, 2020, Vol. 19, No. 0 7

Fig. (4). Immunohistochemical expression of p-Akt-1 and p-ERK1/2 in SNpc of Control, Drug only Control (Control+UA), Rotenone and
Rotenone+UA mice groups by using Image J Software at 20x magnification. Phosphorylation of both Akt-1 (A) and ERK1/2 (B) was de-
creased upon rotenone intoxication in the PD group when compared to the control group. While, UA administration in PD mice has signifi-
cantly elevated the phosphorylation of both the kinases Akt-1 (A) and ERK1/2 (B) as compared to the disease group. Values are expressed as
mean ± SEM of percentage area (*p < 0.01, **p < 0.001, ***p < 0.0001, n = 4). Akt, Serine/Threonine-protein kinase; ERK, Extracellular
signal-related kinase. The arrows represent the expression of the respective antibodies in SNpc. Scale bar represents the degree of magnifica-
tion of the image (For 20X= 100µm). (A higher resolution/colour version of this figure is available in the electronic copy of the article).

Fig. (5). Effect of UA on the nuclear translocation of NF-κB in SNpc with 40x magnifications after immunostaining. Rotenone intoxication
has increased the nuclear translocation of NF-κB (B) in substantia nigra pars compacta (SNpc) in Rotenone as compared to the control group
(A). Although, the nuclear translocation of NF-κB was significantly reduced in Rotenone+UA (C) as compared to the Rotenone-intoxicated
mice group (B). Values are expressed as mean ± SEM of Integrated Fluorescence Value (IFV) (*p < 0.01, **p < 0.001). NF-κB, Nuclear Fac-
tor-κB. The arrows represent the expression of the antibody in SNpc. Scale bar represents the degree of magnification of the image (For 40X=
150µm). (A higher resolution/colour version of this figure is available in the electronic copy of the article).
8 CNS & Neurological Disorders - Drug Targets, 2020, Vol. 19, No. 0 Zahra et al.

Fig. (6). Effect of UA on mRNA expression levels of α-Synuclein, β-Synuclein, IL-1 β and IL-10 in the Rotenone-intoxicated PD mouse
model. The mRNA levels of α-Synuclein (A) and pro-inflammatory cytokine, IL-1 β (C) were significantly elevated and of β-Synuclein (B)
and anti-inflammatory cytokine, IL-10 (D) concomitantly reduced upon Rotenone intoxication as compared to control group. Whereas, UA
treatment after Rotenone-intoxication has significantly attenuated the mRNA levels of α-Synuclein (A) and IL-1 β (C) and increased the
mRNA levels of β-Synuclein (B) and IL-10 (D) as compared to Rotenone-induced PD mouse group. Values in the graph are represented as
mean ± SEM with the level of significance *p < 0.01, **p < 0.001 and ***p < 0.0001. UA, Ursolic acid; IL-1 β, Interleukin-1 β; IL-10, Inter-
lekin-10; SEM, standard error of mean. (A higher resolution/colour version of this figure is available in the electronic copy of the article).

3.7. Western Blot Analysis of TH and α-Synuclein
The expression levels of α-Synuclein (17 KDa) and TH
(62 KDa) (Fig. 7) were also assessed using the Western blot
analysis in tissue lysates isolate of the SNpc region. An en-
hanced α-Synuclein (p<0.0001) (A) and reduced TH
(p<0.0001) (B) expression levels were seen in Rotenone-
intoxicated mice, as compared to the control group. In con-
trast, for treatment with UA, a reduced level of α-Synuclein
(p<0.001) (A) and increased expression of TH level
(p<0.001) (B) were observed when compared to Rotenone-
intoxicated mice.
4. DISCUSSION
The pesticide Rotenone is widely used by gardeners and
is a potent mitochondrial complex I inhibitor that induces the
accumulation of α-Synuclein [15, 16]. Most of the clinical
and pathological hallmarks of PD are successfully recapitu-
lated by Rotenone-intoxication [43, 44]. Substantial evidence
also indicates the reduced phosphorylation of two important
kinases of the PI3K and MAPK pathways, viz. Akt and ERK
by Rotenone exposure [45-48]. Our study also reveals that
Rotenone has induced motor deficits, DA neuronal death,
oxidative stress, neuroinflammation; reduced the phosphory-
lation of Akt and ERK and increased the expression of α-
Synuclein in the mice model of PD.
Previous studies have implicated the anti-cancerous [49],
anti-oxidative [27, 50] and neuroprotective activities of UA
[48-51]. In our previous study, we have demonstrated the
neuroprotective activity of UA in MPTP-induced Parkin-
sonian mice model [27] while our present study further dis-
cusses the neuroprotective potential of UA in Rotenone-
induced PD mouse model by inhibiting the overexpression of
α-Synuclein, oxidative stress and neuroinflammation and
altering the phosphorylation of the cell- survival-related ki-
nases (Akt and ERK).
The symptoms like the slow movement, postural imbal-
ance, and resting tremor are commonly observed in patients
of PD. These symptoms are attributed to the loss of coordi-
nation between the brain and the muscles due to the depletion
of the neurotransmitter, dopamine. Remarkably, the results of
our study have also shown the characteristic behavioural im-
pairment from Rotenone intoxication, similar to that of the
patients of PD [27, 55, 58]. On the other hand, UA administra-
tion to the Parkinsonian mice significantly alleviated these
Neuroprotection of Rotenone-Induced Parkinsonism by Ursolic Acid CNS & Neurological Disorders - Drug Targets, 2020, Vol. 19, No. 0 9

Fig. (7). Western blot analysis to detect the expression level of α-Synuclein and tyrosine hydroxylase (TH) in the SNpc tissue. Values are
expressed as mean ± SEM (**p < 0.001, ***p < 0.0001). The expression of α-Synuclein (17 KDa) (p<0.0001) (A) was increased and TH (62
KDa) (p<0.0001) (B) was decreased in the Rotenone group as compared to the Control group. While, UA treatment to the Rotenone-induced
Parkinsonian mice model has downregulated the expression of α-Synuclein (p<0.001) (A) and upregulated the expression of TH (p<0.001)
(B) as compared to the Rotenone group. (A higher resolution/colour version of this figure is available in the electronic copy of the article).

symptoms, as shown by the assessment of the different be-
havioural parameters in our study.
PD pathogenesis is seen to be triggered by two major
factors, oxidative stress and mitochondrial dysfunction [57].
Stressful environment leads to the production of free radi-
cals, which further react with free oxygen molecules present
on membrane lipids to generate peroxy radicals responsible
for lipid peroxidation. The diagnostic marker for lipid perox-
idation, MDA, is used as a lipid biomarker of PD [58, 59].
As suggested in previous studies, our study shows the ele-
vated levels of MDA in the nigrostriatal region of Rotenone
intoxicated mice. The chemical messenger involved in the
pathogenesis of PD is Nitric Oxide (NO), which - in combi-
nation with superoxide anion (O2-) - forms highly toxic Per-
oxynitrite (ONOO-), liable for the blockage of the respiratory
chain. Besides this, NO is also associated with increased
levels of iron, MDA, and aggravated DNA damage account-
able for neurodegeneration in PD brains [60-62]. Rotenone-
intoxication concomitantly leads to an increased level of NO,
which is also seen in our study. Conversely, UA administra-
tion has attenuated the increased level of NO in the Rote-
none-induced Parkinsonian mice model.
The oxidative damage is prevented by an endogenous
antioxidant defence network, which constitutes enzymes
such as SOD, catalase, and non-enzymatic molecule (such as
GSH, which helps in scavenging the oxygen free-radicals).
Brains of PD patients have been well-documented to show
alteration in redox system [63-65]. Moreover, an imbalance
in redox potential leads to the decline in GSH levels in the
brain tissues of Rotenone-intoxicated mice, as similarly re-
flected by our study. In contrast, GSH level was significantly
restored by the UA treatment in Parkinsonian mice, as shown
by the results of our study. Also, low levels of anti-oxidant
enzymes such as Catalase and SOD make the brain more
prone to oxidative damage. Rotenone exposure leads to the
significant reduction of SOD and Catalase in the striatum of
the basal ganglia, as observed in our study. H2O2 produced
during oxidative stress is responsible for inactivating these
enzymes; thus, their anti-oxidant activity is reduced [66].
However, UA administration to the Rotenone-induced Par-
kinsonian mice model has significantly enhanced the anti-
oxidant activity of SOD and Catalase due to its anti-
oxidative and free radical scavenging properties. Hence, the
UA in our study has shown its potent role by offering protec-
tion against oxidative damage and balancing the redox po-
tential of the cell.
Experimental evidence suggests the alteration in PI3K
and MAPK signaling pathways linked to the pathobiogenesis
of PD [8-10, 67]. These two pathways play an important role
in the proliferation, survival and maintenance of the neurons
[7]. Overexpression of α-Synuclein has been linked with
reduced phosphorylation of both Akt and ERK, which are
10 CNS & Neurological Disorders - Drug Targets, 2020, Vol. 19, No. 0
the key modulators of PI3K and MAPK pathways, respec-
tively [12-14]. Moreover, studies have also suggested the
reduced phosphorylation of Akt and ERK as a result of in-
creased oxidative stress and neuroinflammation [68]. Re-
searchers have demonstrated the protective effect of natural-
ly occurred compounds by regulating the phosphorylation of
these kinases in different neurotoxin-induced model of PD,
including Rotenone [8-10, 69]. Thus, we have tried to study
whether the neuroprotective potential of UA is dependent
upon the activation of these kinases (Akt and ERK) or not.
Our results have suggested reduced phosphorylation of both
these kinases upon Rotenone-intoxication linked to the over-
expression of α-Synuclein; and increased oxidative stress
and neuroinflammation. At the same time, UA treatment
mitigated the effect of Rotenone and offered neuroprotection
by alleviating the symptoms of PD by enhancing the phos-
phorylation of Akt and ERK in accordance with other studies
[8, 10, 45, 47].
Neurotoxicity in PD is also influenced by another major
factor, neuroinflammation [70]. Moreover, numerous studies
implicate the role of NF-κB activation in PD pathogenesis.
NF-κB activation is widely seen in the SN of PD patients, as
well as in animal models [71, 72]. NF-κB, on activation,
translocates into the nucleus and stimulates the transcription
of various pro-inflammatory cytokines, such as IL-1β, and
TNF-α, by the microglia, which further initiates the neuroin-
flammatory cascade [74, 75]. In this study, we estimated the
levels of these inflammatory molecules and tried to explore
the effects of UA on the inflammatory pathways. Rotenone
administration led to the activation and translocation of NF-
κB, which subsequently increased the expression of TNF-α
and IL-1β and decreased level of IL-10 in SNpc, in agree-
ment with previous studies [76–78]. Interestingly, UA sup-
plementation to Rotenone-intoxicated mice has significantly
reduced the activation and translocation of NF-κB, and in-
hibited the expression of TNF-α and IL-1β level. In contrast,
the level of IL-10 is increased on UA administration in Par-
kinsonian mice. IL-10 being an anti-inflammatory cytokine,
is found to inhibit the production of TNF-α and microglial
activation, thereby showing neuroprotective effect by pro-
tecting the DA neurons. This result also suggests that UA
might counteract with the activation of microglia by increas-
ing the level of IL-10, thereby controlling the levels of IL-1β
and TNF-α in accordance with previous studies [79, 80].
The enzyme TH plays a major role in the synthesis of
Dopamine and is the characteristic marker of DA neurons.
Thus, the activity of this enzyme is directly associated with
the functionality of DA neurons in the SN of the brain [15,
79]. The present study also demonstrates the decreased im-
munoreactivity of TH due to Rotenone intoxication, while
upon UA treatment, the reduction of TH neurons was found
to be less in the disease group.
Although there are multiple factors responsible for DA
neuronal loss in the case of PD, the formation of intracyto-
plasmic inclusions, oxidative stress, and neuroinflammation
are major contributors to the disease pathogenesis [44, 81,
82]. Overexpression of α-Synuclein is the pathological hall-
mark of PD and is associated with other factors such as
apoptosis, oxidative stress, and neuroinflammation, and their
interrelation initiates the pathogenic cascade of PD. α-
Zahra et al.
Synuclein, when it gets overexpressed, hinders the function
of complex I of mitochondria and induces the production of
ROS while blocking the respiratory chain. The overexpres-
sion of α-Synuclein in Parkinsonian mice model is similar to
that observed clinically [83]. Besides, the brain is remarka-
bly more susceptible to oxidative stress in comparison to
peripheral organs due to the abundance of polyunsaturated
fats and moderately lowered anti-oxidant activity [84].
Moreover, there is a bi-directional relationship between oxi-
dative stress and overexpression of α-Synuclein and oli-
gomerization, as suggested by different studies [16, 85, 86].
As such, the increased oxidative stress associated with α-
Synuclein accumulation triggers the activation of microglial
cells. Studies have suggested that α-Synuclein aggregates
present in extracellular space, either as a result of neuronal
apoptosis or cellular release, are engulfed by the activated
microglial cells [87-89]. Thus, because the microglial activa-
tion plays an important role in maintaining the brain homeo-
stasis, its activation due to α-Synuclein aggregation and in-
creased production of ROS leads to the activation of the
transcription factor NF-κB and production of proinflammato-
ry molecules like TNF-α and IL-1β. Also, the phosphoryla-
tion of Akt and ERK is reduced by α-Synuclein overexpres-
sion, increased oxidative stress and neuroinflammation,
thereby increasing the neurotoxicity [12-14, 90, 91]. Thus,
this gives us evidence that reducing the expression of α-
Synuclein might slow the progression of the disease. Simi-
larly, in our study, the overexpression of α-Synuclein was
observed in Rotenone-intoxicated mice, while UA treatment
significantly lowered the overexpression of α-Synuclein. β-
Synuclein expression, on the other hand, is associated with
counter-inhibiting the expression and aggregation of α-
Synuclein [92]. Also, β-Synuclein shows neuroprotection by
enhancing the phosphorylation of Akt and reducing the α-
Synuclein overexpression [13]. In this regard, we have also
checked the mRNA level of β-Synuclein in the Rotenone-
intoxicated Parkinsonian mice model. The level of β-
Synuclein has decreased for Rotenone-intoxication, while
UA administration has increased the mRNA level of β-
Synuclein. Thus, our study suggests that the elevated level of
β-Synuclein by UA decreases the overexpression and aggre-
gation of α-Synuclein.
Overall, the study suggests that Rotenone-induced PD
phenotype results from the oxidative stress, neuroinflamma-
tion, degeneration of DA neurons, reduced phosphorylation
of Akt and ERK and α-Synuclein overexpression. However,
UA has alleviated these factors through its anti-inflammatory
and anti-oxidant activities.
CONCLUSION
Hence, through our current study, we have tried to justify
the neuroprotective role of UA in the Rotenone-induced Par-
kinsonian mouse model. Moreover, our study suggests that
there is a relation between oxidative stress, neuroinflamma-
tion, an aberration in the phosphorylation of Akt and ERK,
over expression of α-Synuclein, and DA neuronal loss in
Parkinsonian mice model (which can be simultaneously tar-
geted by UA). Hence, UA appears to be a potential drug
candidate for preventing neurodegeneration in PD. However,
further research is needed to explore the mechanistic target
of UA in PD for its therapeutic intervention.
Neuroprotection of Rotenone-Induced Parkinsonism by Ursolic Acid
AUTHOR CONTRIBUTIONS
WZ: planned and executed the study and wrote the man-
uscript. SNR, SSS, ASR, RS and HD: performed the behav-
ioural tests, biochemical tests, and IHC experiments. HB and
CK: helped in designing the experiments, statistical analysis,
and manuscript writing. RKS: performed the qRT-PCR anal-
ysis. SPS: designed, guided and directed throughout the
study.
ETHICS APPROVAL AND CONSENT TO PARTICI-
PATE
All procedures involving animals were performed in ac-
cordance with the ethical standards approved by the Animal
Ethics Committee of Banaras Hindu University, Varanasi,
India.
HUMAN AND ANIMAL RIGHTS
No humans were used in the study that is the basis of this
research. All experiments on animals were in agreement with
the guidelines for animal experiments established by the Insti-
tutional Animal Ethics Committees (IAECs) of the Laboratory
Animal Research (Reg. No. 1802/GO/Re/S/15/CPCSEA) at
Banaras Hindu University, India.
CONSENT FOR PUBLICATION
Not applicable.
AVAILABILITY OF DATA AND MATERIALS
Not applicable.
FUNDING
None.
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
otherwise.
ACKNOWLEDGEMENTS
The authors would also like to acknowledge Ms. Jenan
Husain, Department of Psychology, California State Univer-
sity, Fullerton, USA for assistance in language correction;
Mr. Chandra Prakash Patel, Interdisciplinary School of Life
Sciences (ISLS), BHU for his assistance in the fluorescence
microscopy and Mr. Ashok Kumar Yadav, Lab attendant, for
his help in the animal care and other support. The authors
also would like to acknowledge UGC Dr. D.S. Kothari Post-
doctoral scheme for awarding the fellowship to Dr. Sach-
chida Nand Rai (Ref. No-F.4-2/2006 (BSR)/BL/19-20/0032).
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