Saudi Pharmaceutical Journal 30 (2022) 863–873

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Saudi Pharmaceutical Journal

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Original article

Empagliflozin attenuates neurodegeneration through antioxidant,

anti-inflammatory, and modulation of a-synuclein and Parkin levels
in rotenone-induced Parkinson’s disease in rats
Sanaa Ahmed a, Mahmoud M. El-Sayed b, Mohamed A. Kandeil c, Marwa M. Khalaf b,⇑
a
Pharmacology Department, Faculty of Medicine, Sohag University, Sohag 82524, Egypt
b
Pharmacology & Toxicology Department, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt
c
Biochemistry Department, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 62514, Egypt

a r t i c l e i n f o a b s t r a c t

Article history: Sodium-glucose co-transporter 2 (SGLT 2) inhibitors are a relatively new antidiabetic drug with antiox-
Received 23 November 2021 idant and anti-inflammatory properties. Therefore, this study aimed to investigate whether SGLT 2 inhi-
Accepted 10 March 2022 bitors have a neuroprotective effect in PD. Twenty-four Wistar rats were randomized into four groups.
Available online 16 March 2022
The first one (control group) received dimethyl sulfoxide (DMSO) as a vehicle (0.2 mL/48 hr, S.C). The sec-
ond group (positive control) received rotenone (ROT) (2.5 mg/kg/48 hr, S.C) for 20 successive days,
Keywords: whereas the third and fourth groups received empagliflozin (EMP) (1 and 2 mg/kg/day, orally), respec-
Parkinsonism
tively. The two groups received rotenone (2.5 mg/kg/48 hr S.C) concomitantly with EMP for another
Neuroprotection
SGLT-2 inhibitors
20 days on the fifth day. By the end of the experimental period, behavioral examinations were done.
Neurodegeneration Subsequently, rats were sacrificed, blood samples and brain tissues were collected for analysis. ROT sig-
Rotenone nificantly elevated oxidative stress and proinflammatory markers as well as a-synuclein. However, dopa-
a-synuclein mine (DP), antioxidants, tyrosine hydroxylase (TH), and Parkin were significantly decreased. Groups of
(EMP + ROT) significantly maintained oxidative stress and inflammatory markers elevation, maintained
a-synuclein and Parkin levels, and elevated TH activity and dopamine level. In both low and high doses,
EMP produced a neuroprotective effect against the PD rat model, with the high dose inducing a more sig-
nificant effect.
Ó 2022 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Parkinson’s disease (PD) is the second most public neurodegen-

erative disease affecting central and peripheral nervous systems.
The neurodegeneration process appears as a consequence of
Abbreviations: CP, Ceruloplasmin; DP, Dopamine; DMSO, Dimethyl sulphoxide; dopaminergic neurons damage in substantia nigra, inducing a
DOPAC, Dihydrophenyl acetic acid; DOPAL, Dihydroxyphenylacetaldehyde; EMP,
marked decline in the striatum levels of dopamine (DP) (Acar
Empagliflozin; GABA, c-Aminobutyric acid; GSH, Reduced glutathione; IL-1b,
Interlukine 1b; MAO, Monoamine oxidase; MDA, Malondialdehyde; NO, Nitric et al., 2019). Although most research has focused on the basal gan-
oxide; PD, Parkinson’s disease; ROS, Reactive oxygen species; ROT, Rotenone; SGLT glia in Parkinson’s disease pathology, some evidence suggests that
2, Sodium glucose co-transporter 2; SOD, Superoxide dismutase; TNF-a, Tumor the cerebellum also contributes to PD related to motor and cogni-
necrosis factor–a; TH, Tyrosine hydroxylase; a–syn, Alpha-synuclien.
⇑ Corresponding author.
tive decline through the loss of Purkinje cells neurons, which were
present in the cerebellum and were responsible for regulating and
E-mail addresses: [email protected] (S. Ahmed), marwa.khalaf@-
pharm.bsu.edu.eg (M.M. Khalaf). coordinating movement via releasing of GABA neurotransmitter
Peer review under responsibility of King Saud University. (Hasan et al., 2020a; Piras et al., 2021). Globally, PD affects 1% of
people over the age of 60 and 5% of people over the age of 85
(Balestrassi and Silva, 2021). In Egypt, approximately 2500–2750
per lakh of the population
50 years suffer from PD attributed
Production and hosting by Elsevier

https://doi.org/10.1016/j.jsps.2022.03.005
1319-0164/Ó 2022 The Author(s). Published by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
S. Ahmed, M.M. El-Sayed, M.A. Kandeil et al. Saudi Pharmaceutical Journal 30 (2022) 863–873

to the exposure of a high percentage of the workforce to pesticides chew and had free access to tap water ad libitum. This experimental
in agriculture (Rösler et al., 2018). protocol was approved (Approval No. 021-160) according to the
The main feature of Parkinson’s disease (PD) is the accumula- Medical Research Ethics Committee guidelines, Faculty of Phar-
tion of misfolded alpha -synuclein (a-syn) protein aggregates, macy, Beni-Suef University, Egypt, under the recommendations of
which results in the formation of Lewy’s bodies (Recasens et al., the EU strategies 2010/63/EU for experimental animals.
2014). In addition, mutations in Parkin and Dj-1 genes may be
involved in the onset of PD. Parkin mutation causes mitochondrial 2.2. Drugs and chemicals
dysfunction, which is considered a central contributor in PD patho-
genesis, while Dj-1 mutation produces a defect in the protective ROT and interleukin-1B (IL-1B) ELISA kits were purchased from
role of antioxidants (Dodson and Guo, 2007). In PD, oxidative stress Sigma Aldrich (St. Louis, MO, USA), while EMP (Jardiance) was
is thought to be the definite fundamental pathway that initiates obtained from Boehringer Ingelheim (Germany). DMSO and sun-
the dysfunction of neurons cells (Hwang, 2013). The substantia flower oil were purchased from El-Nasr chemical company (Cairo,
nigra of patients with PD demonstrated an elevation in the oxida- Egypt). Glutathione (GSH), superoxide dismutase (SOD), nitric
tion rate of proteins, lipids, and DNA (Nakabeppu et al., 2007). oxide (NO), and blood glucose kits were acquired from the Biodiag-
When there is an imbalance between the generation of reactive nostic Company (Giza, Egypt). Malondialdehyde (MDA) kit was
oxygen species (ROS) and the cellular defense antioxidant system, obtained from Eagle Biosciences, Inc. (Boston, USA). Ceruloplasmin
oxidative stress occurs. Dopaminergic neurons become exclusively (CP) and DP ELISA kits were purchased from Indomedix Egypt
more susceptible to oxidative stress due to the presence of some (Cairo, Egypt). Tumor necrosis factor-alpha (TNF-a), dihydrox-
ROS-generating enzymes such as monoamine oxidase (MAO) and yphenylacetic acid (DOPAC), and MAO-B ELISA kits were gained
tyrosine hydroxylase (TH) (Hwang, 2013; Wei et al., 2018). Inflam- from RayBiotec, (GA, USA), SunLong Biotech Company (China),
mation significantly contributes to the initiation and progression of and MyBioSource, Inc. (San Diego, USA), respectively. Fast Cast
neurodegeneration. It is primarily produced due to microglial acti- acrylamide kit (SDS-PAGE) and ReadyPrep TM protein abstraction
vation, where it secretes many proinflammatory cytokines and kit (total protein) were supplied by Bio-Rad Laboratories Inc. Brad-
neurotoxic substances (Goldstein, 2020). Moreover, insulin resis- ford protein analysis kit (SK3041) for protein investigation quanti-
tance may be associated with PD contributing to the disease pro- tatively was provided by Bio basic Inc (Markham Ontario L3R 8T4
gression and acceleration (Kalampokini et al., 2019). Canada). Rabbit Anti-TH antibody Neuronal Marker (ab112) and
One of the newest hypotheses for PD is the accumulation of immunohistochemical detection system were obtained from Lab
toxic intermediate product 3,4-dihydroxyphenyl acetaldehyde vision Neomarker (USA).
(DOPAL) produced from MAO-catalyzed dopamine oxidation.
DOPAL can interact with a-syn and induce its oligomerization pro- 2.3. Study design
cess forming the major component of Lewy’s bodies (Goldstein,
2020). Rats were randomized into four groups (six rats each). The first
Rotenone (ROT) is a natural pesticide extracted from Legumi- cohort (normal control) received only vehicle (1 mL DMSO diluted
nosae plants. Administration of ROT results in a selective and in sunflower oil to 50 mL) given in a dose of (0.2 mL/48 hr S.C). The
potent complex-I inhibition which is a feature of idiopathic PD. second group (positive control) received ROT (2.5 mL/kg/48 hr S.C)
In addition, ROT administration leads to selective dopaminergic for 20 days (Hassanzadeh et al., 2014). ROT (2.5 mg/mL) was pre-
neurodegeneration, oxidative stress, a-syn accumulation, and pared by dissolving 12.5 mg ROT in a 5 mL vehicle (DMSO + sun-
microglial activation (Anusha et al., 2017). flower oil). The third and fourth groups received EMP 1 and
Like metformin and liraglutide, many hypoglycemic agents 2 mg/kg/day orally, respectively (Han et al., 2017). On the fifth
exhibit antioxidant and anti-inflammatory potential at the central day of EMP treatment, animals received ROT (2.5 mg/kg/48 hr S.
level (Oliveira et al., 2016; Fanget al., 2012). Sodium-glucose co- C) concomitant with EMP for additional 20 days. After finishing
transporter 2 (SGLT 2) is responsible for the reabsorption of more the experiment, the behavioral tests were performed, and then rats
than 90% of the total renal-filtered glucose expressed in proximal were sacrificed, blood samples were collected, and brains were
tubules. Empagliflozin (EMP) is an SGLT 2 inhibitor recently removed for assay.
approved as an antidiabetic drug due to its additional antioxidant
and anti-inflammatory benefits (Li et al., 2019; Lai et al., 2020). 2.4. Induction of PD
It is well known that all medications used in the treatment of
PD are symptomatic, and their efficacy diminishes with time For PD induction, rats were injected with ROT (2.5 mg/kg/48 hr
(Mao et al., 2020). Therefore, one of the best strategies in treating S.C) for 20 days (Hassanzadeh et al., 2014). The rotarod test was
PD is to search for new effective drugs to prevent neuronal death performed every 48 hr to ensure that induction was done.
and slow down the PD disease progression. Therefore, the current
study attemoted to investigate the potential protective effect of 2.5. Behavioral investigations
EMP as a new SGLT 2 inhibitor against rotenone-induced PD in rats.
2.5.1. Open field test
In order to assess locomotor performance, open-field assess-
2. Material and methods ment was applied. The open field gadget is a square field manufac-
tured of wood (100  100  40 cm) with a ground divided into 25
2.1. Animals identical-size squares with webs. After being placed in the center
of the apparatus, a video camera was fixed on the top of the box
Twenty-four male albino Wistar rats 180–220 gm in weight to record animal ambulation (number of quadrangles overpassed
were used in this investigation. Animals were obtained from the by the animals with its all paws in one minute) (Neto et al., 2013).
animal house, Faculty of Medicine, Sohag University, Egypt. Then,
they were kept under suitable environmental conditions (temper- 2.5.2. Rotarod test
ature around 25 ± 2 °C and 12 h light/dark). Rats were left one Rotarod test is a behavioral test used to assess motor coordina-
week before starting the experiment for accommodation. Through- tion in the PD rat model. Before starting the test, animals were
out the experiment, animals were nourished with standard pellet allowed to train for three successive days on apparatus until reach-
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ing stable performance. Animals were placed on a circling rod with procedure of the manufacturer was followed. The method is based
15 rpm speed. Each rat could stopover was listed as the latency on the response of a chromogenic reagent, N-methyl-2- phenylin-
period to drop. The test was repeated three times for each rat, with dole, to malondialdehyde (MDA) at 45 °C. One particle of MDA
a 10-minute rest interval in between to avoid lethargy and tension, reacts with 2 particles of chromogenic reagent to produce a con-
and the mean was calculated for each animal (Voss et al., 2003). stant chromophore with maximal absorbance at 586 nm
(Uchiyama and Mihara, 1978).
2.6. Sample preparation NO was evaluated using a kit: NO 25 33 from Biodiagnostic Co.,
Giza, Egypt according to the procedure provided by the manufac-
2.6.1. Blood collection and serum separation turer. It can be measured as the final product of NO in vivo which
After the end of the experiment, blood samples were collected is nitrite (NO2). Colorimetric nitrite determination depends on
from all animals under anesthesia from the retro-orbital venous addition of Griess reagent which convert nitrite into deep purple
plexus in sterile tubes. Samples were then centrifuged at 2000 r. azo compound. The photoabsorbance of this compound accurately
p.m. for 10 min for separation of clear serum to be kept at determines nitrite (Montgomery and Dymock, 1961).
20 °C until the analysis time. The serum was used for the estima-
tion of blood glucose levels. 2.9. Determination of IL-1b, TNF-a, DP, and DOPAC contents as well as
MAO-B activity in brain tissue
2.6.2. Preparation of brain tissue homogenate
The brain tissue containing striatum, substantia nigra, and cere- The brain content of IL-1b, TNF-a, DP, and DOPAC, and the brain
bellum were quickly removed and cleaned using normal cold sal- activity of MAO-B were measured by using the monoclonal double-
ine and weighed. One portion of brain tissue was homogenized antibody sandwich ELISA method as described by the manufactur-
in 10 mL cold phosphate buffer (100 mM potassium phosphate, ers’ prescriptions. Concentrations were measured using 2600
PH 7.0, containing 2 mM EDTA) per gram brain tissue then cen- microplate State Fax reader (Awareness Technologies, Palm City,
trifuged at 4000 r.p.m. for 15 min at 4 °C. Finally, the clear super- USA). This assay employs antibodies specific for Rat IL-1b, TNF-a,
natant was separated for the biochemical assessment of the brain DP, DOPAC or MAO-B, respectively coated on a specific provided
content of GSH, SOD, MDA, NO, IL-1b, TNF-a, MAO-B, CP, DP, and well plate. Standards and samples are pipetted into the wells and
its metabolites. The second part was lysed in RIPA buffer for west- IL-1b, TNF-a, DP, DOPAC or MAO-B present in the sample is
ern blotting estimation of a-syn and Parkin protein expression. The bounded to the wells by the immobilized antibody. The wells were
third part of the tissue containing substantia nigra was immersed washed and biotinylated antiRat IL-1b, TNF-a, DP, DOPAC or MAO-
in liquid nitrogen and preserved at –80 °C for immunohistochem- B antibody is added. After washing of any unbound biotinylated
ical investigation of TH enzyme activity. The remaining part, which antibody, HRP conjugated streptavidin is pipetted to the wells.
contains the cerebellum, was incubated in formalin until the The wells are again washed and tetramethylebenzidine (TMB) sub-
histopathological analysis. strate solution is added to the wells and color develops in propor-
tion to the amounts of IL-1b, TNF-a, DP, DOPAC or NAO-B bound.
2.7. Determination of blood glucose level The Stop Solution changes the color from blue to yellow, and the
intensity of the color is measured at 450 nm (Finch et al., 1995;
Blood glucose level was measured colorimetrically using NO GL Laczko et al., 2020; Chakraborty and Diwan, 2019).
13 20 kit acquired from Biodiagnostic Co, Giza, Egypt. The hydro-
gen peroxide produced from glucose by glucose oxidase action at 2.10. Determination of ceruloplasmin (CP) content in brain tissue
pH 7 is determined by the peroxidase oxidative coupling of N, N-
diethylaniline with 4-amino-antipyrine. A 20-ml sample of blood According to the producer’s directions, CP was estimated using
serum reacted with a colorimetric kit containing (phosphate buf- the competitive ELISA technique (Saraf et al., 2016). Samples were
fer, phenol, 4-amino-antipyrin and glucose oxidase) for 10 min at added to Horseradish Peroxidase (HRP) conjugated antibody
room temperature and pH 7. A red violet color was formed and preparation specific for CP. The absorbance was measured using
was measured at 553 nm (Kabasakalian et al., 1974). a 2600 microplate State Fax reader (Awareness Technologies, Palm
City, USA).
2.8. Determination of antioxidant and oxidative stress biomarkers in
brain tissue 2.11. Western blotting analysis of a-syn and Parkin proteins

GSH concentration in brain tissue homogenate was measured The brain tissue was homogenized in RIPA lysis buffer and cen-
colorimetrically using NO GR 25 11 kit obtained from Biodiagnostic trifuged at 4 °C for 20 min at 12,000 rpm, and then the supernatant
Co., Giza, Egypt. The procedure was performed according to the was taken. The total concentration of protein was assessed by the
manufacturers’ instructions provided. GSH estimation depends Bradford protein assay kit (SK3041) (Bio basic Inc Canada) (Gilda
on the reduction of Ellman’s reagent [5,50 -dithiobis (2- and Gomes, 2013). 20 ug/mL protein concentration of each sample
nitrobenzoic acid)] by glutathione to yield a yellow-colored pro- was overloaded with an equivalent volume of Laemmli buffer (4%
duct (Ellman, 1959). The absorbance of this reduced chromogen SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol
was measured at 405 nm and it was directly related to GSH blue, and 0.125 M Tris HCL. These mixtures were incubated at
concentration. 95 °C for 5 min to confirm protein denaturation. Samples with an
SOD activity in brain tissue homogenate was also determined equal amount of proteins were parted with 10% SDS-PAGE. Poly-
colorimetrically, using NO SD 25 21 kit purchased from Biodiag- acrylamide gels were done using TGX Stain-free. The SDS-PAGE
nostic Co., Giza, Egypt. The procedure was performed according TGX Stain-Free Fast Cast was prepared following the producer’s
to the manufacturers’ instructions provided. Determination of instructions. The gel was collected in a transfer PVDF film blocked
SOD is based on the ability of this enzyme to inhibit phenazin in tris-buffered saline (3% bovine serum albumin and Tween 20)
methosulphate-mediated reduction of nitroblue tetrazolium dye for one hour at temperature. Afterward, the blot was run for
(Marklund and Marklund, 1974). 7 min at 25 V to allow protein bands to transfer from the gel to film
Lipid peroxides were evaluated using a kit purchased from using BioRad Trans-Blot Turbo. The film was incubated with an
Eagle bioscince Co. with Catalog Number: LIP39-K01. The provided anti-a-syn and anti-Parkin antibody. The objective proteins were
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identified by ChemidocTM XRS + system with Image Lab software

(Bio-Rad, USA), and band intensity was quantified by the Image Lab
program (Peferoen et al., 1982).

2.12. Immunohistochemical study of TH enzyme

After deparaffinization, tissue slices were gained by xylene

overnight then hydrated in alleviated concentrations of alcohol
100, 90, 70, and 50% for 5 min each. Antigen retrieval was carried
out by warming slides in citrate buffer (pH 6) in the microwave for
5 min and washed twice in PBS for 20 min. 3% hydrogen peroxide
solution was used for 10 min for blocking the activity of endoge-
nous peroxide, then washed twice in PBS (pH 7.2) for 5 min each.
The sections were treated for one hour. in a moisture compartment Fig. 1. Effect of EMP on blood glucose level in ROT-induced PD in male Wistar
with rabbit polyclonal antibody against TH (Lab vision Neomarker rats. Data were presented as mean ± SEM n = 6. group I = control group. group
USA) branded as a dark brown reaction inside the cytoplasm. The II = ROT group. group III = EMP1mg + ROT group. group IV = EMP2mg + ROT group.
slides were washed in PBS, and biotinylated secondary antibody
(Universal Multilink) was applied for 10 min and washed twice.
the dose of 2 mg/kg EMP showed a significant improvement in
The bound antibodies were established using streptavidin peroxi-
motor coordination than 1 mg/kg dose, while no significant differ-
dase 2 drops on the sections for 30 min and washed for 5 min, then
ence between the two doses was observed regarding the locomotor
exposed for 10 min to freshly prepared diaminobenzidine (DAB)
activity (Fig. 2 A and B).
and chromogen. After the appearance of the brown color, the slides
were instantly washed in tap water. The sections were stained
with hematoxylin for the detection of tissue architecture. The 3.3. Effect of EMP on the brain content of antioxidant and oxidative
slides were dehydrated, and the cover slipped using Flotex perma- stress biomarkers
nent mountain media (Renshaw, 2006).
As revealed in Table 1, ROT administration showed a significant
2.13. Histopathological examination of cerebellum reduction in the brain GSH content and SOD enzyme activity with a
significant promotion in the brain content of MDA and NO com-
Following fixation in neutral buffered formalin and dehydrated
in ascending grades of alcohol, tissues were fixed in Paraffin; slices
of 5 lm thickness were established using Micro-Teck microtome
(Germany). Subsequently, slides were stained in Erlich’s hema-
toxylin for 5 min and blued in tap water for 3 min after they were
stained in 1% aqueous eosin solution for 1 min then washed in the
water finally dehydrated in alcohol and cleared in xylol then stud-
ied by an Olympus (model CX21) Japan, light microscope (Suvarna
et al., 2018).

2.14. Statistical analysis

Data were presented as mean ± SEM, and quantitative variables

were compared using a one-way ANOVA using (IBM-SPSS version
22.0) software package, followed by Tukey Kramer’s post hoc test
for multiple comparisons. The level of significance was determined
at a P-value  0.05.

3. Results

3.1. Effect of EMP on blood glucose level

Regarding blood glucose level, no significant difference was

observed between the different groups at p < 0.05 as shown in
Fig. 1.

3.2. Behavioral study

The results of the open field and rotarod tests revealed that ROT
had a significant reduction in both locomotor activity and motor
coordination compared to the control group. The reduction in these Fig. 2. Effect of EMP on (A) locomotor activity determined by open field test and
activities confirmed PD induction in the ROT group. However, (B) motor coordination determined by rotarod test in ROT-induced PD in male
when EMP was administered concurrently with ROT in both low Wistar rats. Data were presented as mean ± SEM n = 6. d Significantly different
when compared to the control group at p < 0.05. # Significantly different when
and high doses, it significantly improved locomotor activity and compared to ROT group at p < 0.05. * Significantly different when compared to EMP
motor coordination compared to the ROT group. When the results (low dose) group at p < 0.05. group I = control group. group II = ROT group. group
of 1 and 2 mg/kg EMP doses were compared, it was discovered that III = EMP1mg + ROT group. group IV = EMP2mg + ROT group.

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Table 1
Effect of EMP on the brain content of antioxidant and oxidative stress biomarkers in ROT-induced PD in male Wistar rats.

Parameters Control ROT EMP(1 mg/kg) + ROT EMP(2 mg/kg) + ROT

#
GSH)mg/g tissue) 564 ± 42.42 144 ± 12.92 d
426 ± 23.42 568 ± 18.93#*
SOD (U/g tissue) 750 ± 43.67 266.5 ± 18.20d 605 ± 24.42# 748 ± 20.73#*
MDA (nmol/g tissue) 0.3113 ± 0.02 3.17 ± 0.02d 0.76 ± 0.03# 0.367 ± 0.01#*
NO mmol/g tissue)) 2.48 ± 0.16 9.22 ± 0.74d 4.39 ± 0.07# 3.56 ± 0.09#*

Data were presented as mean ± SEM n = 6.

d Significantly different when compared to the control group at p < 0.05.
# Significantly different when compared to ROT group at p < 0.05.
* Significantly different when compared to EMP (low dose) group at p < 0.05.
EMP = empagliflozin ROT = rotenone GSH = glutathione.
SOD = superoxide dismutase MDA = malonaldehyde NO = nitric oxide.

pared to the control group. In contrast, EMP administration either

in 1 mg or 2 mg concurrent with ROT led to a significant upturn in
GSH content and SOD activity with a significant decline in MDA
and NO’s brain content compared to the ROT group. Besides,
administration of EMP in high doses induced significant improve-
ment in the previously measured parameters compared to low
doses.

3.4. Effect of EMP on the brain content of proinflammatory cytokines

(IL-1B and TNF-a)

Administration of ROT caused a significant upsurge in the brain

content of IL-1B and TNF-a versus the control group, while admin-
istration of EMP either in a dose of 1 or 2 mg/kg concurrent with
ROT led to a significant fixing in these parameters versus the
ROT group. In comparison between the two EMP doses, there
was more significant improvement with the high dose than the
low dose, as depicted in (Table 2).

3.5. Effect of EMP on the brain content of DP and DOPAC

Administration of ROT led to a significant reduction in DP and

DOPAC content in the brain tissue homogenate compared to the
control group. On the contrary, administration of EMP either in a
dose of 1 or 2 mg/kg concurrent with ROT resulted in a significant
saving in DP and DOPAC contents compared to the ROT group.
Administration of the high dose of EMP produced a significant
decrease in these parameters compared to the low dose, as demon-
strated in (Fig. 3 A and B). Fig. 3. Effect of EMP on the brain content of (A) dopamine and (B) DOPAC in
ROT-induced PD in male Wistar rats. Data were presented as mean ± SEM n = 6. d
Significantly different when compared to the control group at p < 0.05. #
3.6. Effect of EMP on the brain monoamine oxidase-B (MAO-B) activity Significantly different when compared to ROT group at p < 0.05. * Significantly
and ceruloplasmin (CP) content in brain tissue different when compared to EMP (low dose) group at p < 0.05. group I = control
group. group II = ROT group. group III = EMP1mg + ROT group. group
IV = EMP2mg + ROT group. DOPAC = dihydrophenyl acetic acid.
ROT administration resulted in a significant increase in MAO-B
activity and a significant decrease in CP content compared to the
control group, as displayed in (Table 3). However, administering in CP content compared to the ROT group. When comparing the
EMP at a dose of 1 or 2 mg/kg concurrently with ROT resulted in two EMP doses, the high dose resulted in more significant
a significant reduction in MAO-B activity and a significant increase improvement than the low dose.

Table 2
Effect of EMP on the brain content of proinflammatory cytokines in ROT-induced PD in male Wistar rats.

Parameters Control ROT EMP(1 mg/kg) + ROT EMP(2 mg/kg) + ROT

IL-1B (ng/g tissue) 491.8 ± 4.74 8352 ± 462.90d 2066 ± 176.06# 579.50 ± 15.45#*
TNF-a (ng/g tissue) 321.2 ± 5.93 4755 ± 298.30d 2152 ± 139.00# 578 ± 22.65#*

Data were presented as mean ± SEM n = 6.

d Significantly different when compared to the control group at p < 0.05.
# Significantly different when compared to ROT group at p < 0.05.
* Significantly different when compared to EMP (low dose) group at p < 0.05.
EMP = empagliflozin ROT = rotenone.
IL-1b = interlukin- 1b TNF-a = tumor necrosis factor –a.

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Table 3
Effect of EMP on the brain MAO-B activity and CP content in ROT-induced PD in male Wistar rats.

Parameters Control ROT EMP(1 mg/kg) + ROT EMP(2 mg/kg) + ROT

#
MAO-B U/mg tissue)) 31.32 ± 1.73 156.5 ± 5.20 d
66.48 ± 4.88 42.23 ± 3.21#*
CP ng/g tissue)) 320.8 ± 18.35 74.33 ± 3.24d 221.33 ± 18.23# 307.33 ± 22.04#*

Data were presented as mean ± SEM n = 6.

d Significantly different when compared to the control group at p < 0.05.
# Significantly different when compared to ROT group at p < 0.05.
* Significantly different when compared to EMP (low dose) group at p < 0.05.
EMP = empagliflozin ROT = rotenone.
MAO-B = monoamineoxidase-B CP = ceruloplasmin.

3.7. Effect of EMP on a-syn and Parkin protein expression Pioglitazone, which can assist in reducing inflammation after a
traumatic brain injury (Deng et al., 2020) or in a Parkinson’s dis-
ROT administration resulted in a significant increase in total - ease model caused by 6-hydroxydopamine (Machado et al.,
syn, but a significant decrease in Parkin protein expression com- 2019). Furthermore, exendin-4, liraglutide, and lixisenatide are
pared to the control group. However, when compared to the ROT GLP-1RAs that raise striatal DP and TH protein levels in the 1-me
group, groups that received EMP at a dose of 1 or 2 mg/kg concur- thyl-4-phenyl-1,2,3,6-tetrahydropyridine animal model of PD (Liu
rently with ROT showed a significant restriction in a-syn and a sig- et al., 2015).
nificant saving in Parkin protein expression. As depicted in Fig. 4 The current study revealed that EMP did not produce hypo-
high dose EMP caused a significant change in a-syn and Parkin pro- glycemia, as evidenced by the blood glucose level estimation. This
tein expression than low dose, as demonstrated in Fig. (4 A, B and, result is consistent with a previous study which stated that EMP
C). does not produce severe hypoglycemia either alone or in combina-
tion with other antidiabetic drugs (metformin, sulphonylurea, or
3.8. Effect of EMP administration on tyrosine hydroxylase (TH) Pioglitazone) except with insulin (Ndefo et al., 2015).
immunohistochemistry Open field test is a behavioral test used to assess locomotion
activity in PD models. Changes in locomotion are used as an indi-
The immunohistochemical analysis of substantia nigra in the cator for alterations in the neurological state (Kraeuter et al.,
control group showed positive staining of TH as a brown cytoplas- 2019). In addition, the rotarod test is a popular method used to
mic reaction (Fig. 5 A). (ROT group): revealed a marked decrease in investigate motor coordination and equilibrium in PD models
TH immunoreactivity compared to the control group (Fig. 5 B). Pro- (Hung and Hsueh, 2021). Our study revealed that ROT administra-
nounced preservation was observed in TH immunoreactivity in the tion to experimental animals produced deterioration of behavioral
EMP + ROT treated groups and there is a dose-dependent improve- function (locomotor activity by 62% and motor coordination by
ment compared to the ROT group. (Fig. 5 C and D). These photomi- 54.42%), which is in harmony with previous research (Zaitone
crographs correlate with the values of mean A% displayed in et al., 2019). However, rats that received EMP concurrent with
(Fig. 6). ROT showed improvement in these functions compared with the
ROT group (locomotor activity by 91.5%, 112% and motor coordina-
3.9. Effect of EMP on histopathological examination for the cerebellum tion by 66.22%, 100% in low and high doses, respectively). This
finding is in line with previously published studies that proved that
As demonstrated in Fig. 7, histological examination of the cere- SGLT 2 inhibitor, EMP, enhances open field performance in
bellar tissue of the control group revealed normal histological ischemic stroke (Amin et al., 2020) and dapagliflozin which
structure. With regard to the ROT group, it demonstrated a improves these functions in ROT induced PD (Arab et al., 2021).
decreased number of Purkinje cells with shrunken darkly stained Oxidative stress is an imbalance between ROS generation and
nuclei and dilated perineural space. Co-administration of antioxidant defense mechanisms. It is believed to be the definite
EMP1 + ROT; cerebellar tissue showed many nerve fibers and den- underlying cause that leads to cellular dysfunction (Hwang,
drites with few numbers of Purkinje cells with dilated perineural 2013; Wei et al., 2018). To assess oxidative state in this study,
space. Co-administration EMP2 + ROT, cerebellar cortex showed we measured GSH, MDA, NO content, and SOD activity in brain
restoration of the normal histological structure. homogenate. Our study revealed that ROT produced significant
deterioration in the levels of these parameters (lowering in GSH
content and SOD activity by 74% and 64%, respectively, while
4. Discussion MDA and NO content showed an elevation by 918% and 272%,
respectively) where these results are consistent with results of
This study was directed to evaluate the potential neuroprotec- ‘‘Kaur et al. (2011) and Alabi et al. (2019)”. The finding can explain
tive effect of EMP on rats model of ROT-induced PD. EMP is a selec- these effects that ROT is a high-affinity inhibitor of complex-I of
tive inhibitor of SGLT2 that reduces hyperglycemia in type 2 mitochondrial electron transport chain leading to increased pro-
diabetic patients by inhibiting glucose renal reabsorption, leading duction of free radicals resulting in oxidative damage ‘‘Sherer
to increased glucose renal excretion (Zinman et al., 2015). Glucose et al. (2003) and Chen et al. (2008)”. On the other hand, our study
level regulation in the brain occurred by SGLTs may contribute to revealed that EMP intake produced significant improvement in
the expectation of EMP central neuroprotective effect (Yu et al., these parameters (an increase in GSH by 196%, 294% and SOD by
2010). 127%, 180%, while a decrease in MDA by 76%, 88%, and NO by
Metformin is a common glucose-lowering drug that has been 52%, 61% was observed in low and high doses respectively) where
found to protect neurons in animal and cellular models by decreas- our findings align with another study (Shin et al., 2016). The
ing a-syn aggregation, preventing mitochondrial dysfunction, and antioxidant effect of EMP may be due to stimulation of the nuclear
giving antioxidant effects (Lin et al., 2021). Other antidiabetic med- factor erythroid 2 (Nrf2)- related factor 2 pathway (Hasan et al.,
ications with neuroprotective effects include a peroxisome 2020b). This pathway has antioxidant function due to its vital role
proliferator-activated receptor-gamma (PPAR) agonists such as
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group group group group (A)

parkin

-actin

-syn

(B)

(C)

Fig. 4. Effect of EMP on a-syn and parkin protein expression in ROT-induced PD in male Wistar rats. Western blot bands (A), Densitometric quantitation of a-syn (B)
and parkin (C) protein expressions. Data were presented as mean ± SEM n = 6. d Significantly different when compared to the control group at p < 0.05. # Significantly
different when compared to ROT group at p < 0.05. * Significantly different when compared to EMP (low dose) group at p < 0.05. group I = control group. group II = ROT group.
group III = EMP1mg + ROT group. group IV = EMP2mg + ROT group. EMP = empagliflozin ROT = rotenone a-syn = a-synuclien.

in the expression of many genes for antioxidant enzymes such as (Abdelsalam and Safar, 2015). IL-1b and TNF-a are neuroinflamma-
SOD, glutathione-s-transferase (GST), NADPH quinone tory markers produced due to ROT-activated microglia and/or as a
dehydrogenase-1 (NQO1). In addition, induction of this pathway response to a-syn aggregation (Hoffmann et al., 2016). These
was found to protect against cellular apoptosis (Ahmed and cytokines can trigger and extend neuroinflammation, resulting in
Mohammed, 2021). blocking the induction of brain-derived neurotrophic factor
ROT was previously reported to exert severe inflammatory (BDNF), an essential protein for dopaminergic neurons develop-
potential that can be explained by its activation of NF-KB, which ment and survival (Zhou et al., 2019). In our findings, ROT admin-
results in inflammatory cytokines elevation in microglia istration resulted in an increase in proinflammatory biomarkers IL-

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to the diminished activity of NF-KB, which suppresses proinflam-

matory mediators TNF-a and IL-1b expression. In addition, this
anti-inflammatory potential can be related to its antioxidant effect
(Abdelhamid et al.,2020).
DP is metabolized enzymatically by MAO-B in the brain, giving
rise to DOPAC, which is further reduced to homovanillic acid
(Jenner and Olanow, 1996). It is well known that MAO-B has a
pathological role in PD via some toxin activation and free radicals
formation (Thakur and Nehru, 2013). Our results demonstrated
that ROT significantly increased MAO-B activity by 400%, reducing
DP and DOPAC concentrations by 77% and 90%, respectively. These
results are the same as the previously mentioned findings (Jenner
and Olanow, 1996). This finding can be explained by the ROT-
induced C/EBPb pathway, which upregulates both MAO-B and a-
syn transcription. In addition, dopamine metabolism by MAO-B
strongly activates the d-secretase enzyme leading to a positive
feedback circuit in dopaminergic neurodegeneration (Wu et al.,
2021). On the other hand, the administration of EMP + ROT
resulted in the significant preservation of these parameters.
CP, a ferroxidase enzyme, plays a vital role in iron metabolism.
It converts ferrous (Fe2+) to less-toxic ferric iron (Fe3+) (Liu
et al.,2019). Our study showed a significant decrease in the brain
CP content in the ROT-treated group by 77%. This result agrees
with (Hineno et al.,2011) who owed this to CP consumption in pre-
venting the oxidation and subsequent neurodegeneration caused
by ROT. However, EMP administration revealed significant
enhancement in CP by 198% and 314% for low and high EMP doses,
respectively, which can be attributed to its antioxidant effect
(Sugizaki et al.,2017) and its ability to regulate the copper home-
ostasis, which acts as a cofactor for the antioxidant Cu/Zn-SOD
enzymes activity (Montes et al., 2014).
Our result showed that ROT led to a significantly upregulated a-
syn level by 239% compared to the control group, which agrees
with (Li et al., 2020) and significantly down-regulated parkin pro-
Fig. 5. (A) Immunohistochemical staining of striatal TH expression of the control tein by 67%, which is compatible with (Rahimmi et al.,2015), while
group (400 X) showing the positive immune reaction of TH. (B): the ROT group EMP + ROT resulted in significant preservation in these protein
revealed almost negligible immune reaction compared to the control group. (C and
D): EMP treated groups showed restoration of staining of TH compared to the ROT
levels. a-syn is a fundamental contributor in PD, which can initiate
group with a dose-dependent effect. and potentiates the neurodegeneration process by many path-
ways; inhibition of autophagy, induction of mitochondrial dys-
function, and increased oxidative stress in addition to microglial
activation causing neuroinflammation and an increase in proin-
flammatory cytokines (Segura-Aguilar, 2017).
However, the lack of Parkin protein promotes dopaminergic
neurodegeneration by affecting the mitochondrial morphology
and function with potential membrane reduction (Pinto et al.,
2018). Moreover, Parkin overexpression is shown to reduce MAO
expression leading to further reduction in ROS produced through
MAO catalyzed dopamine oxidation (Jiang et al., 2006).
All previous results are supported by the immunohistochemical
TH estimation in substantia nigra, which referred to its decline
with ROT administration. This result is in harmony with the result
of (Pan et al., 2020). Moreover, a significant enhancement after
Fig. 6. Quantitative image analysis for TH immunohistochemical staining EMP administration was observed in TH immunoreactivity. This
presented as mean area% (A%). Data were presented as mean ± SEM n = 6. d result is in agreement with a study on dapagliflozin (Matthews
Significantly different when compared to the control group at p < 0.05. # et al., 2017). TH catalyzes the rate-limiting step in dopamine syn-
Significantly different when compared to ROT group at p < 0.05. * Significantly thesis, and its decline in PD is thought to be a secondary effect to
different when compared to EMP (low dose) group at p < 0.05. group I = control
group. group II = ROT group. group III = EMP1mg + ROT group. group
dopaminergic neurons degeneration. TH may not directly function
IV = EMP2mg + ROT group. in the degeneration process due to its dopamine feedback regula-
tion (Nagatsu et al., 2019).
Our biochemical results were supported by histopathological
1B and TNF-a by more than ten times for each, which is consistent examination of the cerebellum, which showed a decreased number
with (Mansour et al., 2018), while EMP + ROT administration of Purkinje cells with shrunken darkly stained nuclei with dilated
resulted in significant restriction in IL-1b by 75%, 93% and 55%, perineural space, which aligns with (Khadrawy et al., 2016). Purk-
88% for TNF-a after low and high doses administration, respec- inje cells contribute to PD pathology via loss of GABA due to necro-
tively, compared to ROT group which is consistent with (Jojima tic changes in Purkinje cells (Khadrawy et al., 2016). These changes
et al., 2016). The anti-inflammatory effect of EMP can be attributed were prevented in EMP + ROT groups where Purkinje cells resem-
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S. Ahmed, M.M. El-Sayed, M.A. Kandeil et al. Saudi Pharmaceutical Journal 30 (2022) 863–873

Fig. 7. A photomicrograph of a section in the cerebellar cortex of a rat (A): (control group) showing; molecular layer (M) with few scattered cells (black arrow), Purkinje cell
layer (P), Purkinje cells appear as large pyriform cells arranged in a single row (red arrows) and granular cell layer (G) appears as tightly packed small cells with deeply stained
nuclei (white arrow). (B): ROT group showing; molecular layer (M) few scattered cells (black arrow), Purkinje cell layer (P) with deeply stained Purkinje cells with pyknotic
nuclei surrounded with vacuolated neuropil (red arrows), and granular layer (G) having cells with dense nuclei white arrow. (C): EMP1mg + ROT group showing; molecular
layer (M) few scattered cells (black arrow), Purkinje cell layer (P) few numbers of Purkinje cells with dilated perineural space (red arrows), and granular layer (G) having cells
with dense nuclei white arrow. (D): EMP2mg + ROT group showing nearly normal molecular layer (M) with few scattered cells (black arrow), Purkinje cell layer (P), Purkinje
cells appear as large pyriform cells arranged in a single row (red arrows) and granular cell layer (G) appears as tightly packed small cells with deeply stained nuclei (white
arrow). ROT = Rotenone. EMP1 = Empagliflozin 1 mg. EMP2 = Empagliflozin 2 mg. TH = tyrosine hydroxylase.

bled those in the control group. Elfeber et al., 2004 explained this Declaration of Competing Interest
effect by the expression of SGLT mRNA in the Purkinje cells and
blood vessels of brains of humans and rodents, which play a crucial The authors declare that they have no known competing finan-
role as an energy supply source for neurons on increased glucose cial interests or personal relationships that could have appeared
demand, such as in hypoxemia and hypoglycemia (Sano et al., to influence the work reported in this paper.
2020).
Acknowledgment

5. Conclusion
The authors would like to thank Dr. Sahar Gebril, Histology
Department, Faculty of Medicine, Sohag University, for assistance
Results of the current study revealed that, ROT administration
in fulfilling this research.
resulted in oxidation of dopamine generating of hazardous
metabolites. This is mediated by induction of oxidative stress and
inflammation besides elevating MAO-B enzyme activity. Dopamin-
ergic neuron degeneration was accelerated by overexpression of a- References
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