Transcript of κB Inhibitors Attenuate MCAO Induced Neurodegeneration and ...
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Inhibitors Attenuate MCAO Induced Neurodegeneration NF- B
Inhibitors Attenuate MCAO Induced Neurodegeneration
and Oxidative Stress—A Reprofiling Approach and Oxidative Stress—A
Reprofiling Approach
Awais Ali Riphah International University
Fawad Ali Shah Riphah International University
Alam Zeb Riphah International University
Imran Malik Riphah International University
Arooj Mohsin Alvi Riphah International University
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Recommended Citation Recommended Citation Ali, Awais; Shah, Fawad
Ali; Zeb, Alam; Malik, Imran; Alvi, Arooj Mohsin; Alkury, Lina
Tariq; Rashid, Sajid; Hussain, Ishtiaq; Ullah, Najeeb; Khan, Arif
Ullah; Koh, Phil Ok; and Li, Shupeng, "NF-κB Inhibitors Attenuate
MCAO Induced Neurodegeneration and Oxidative Stress—A Reprofiling
Approach" (2020). All Works. 2507.
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doi: 10.3389/fnmol.2020.00033
Edited by:
Technology, Pakistan
ERA’s Lucknow Medical College, India
Weirong Fang, China Pharmaceutical University,
China
‡Present address: Fawad Ali Shah
Robarts Research Institute, Schulich School of Medicine &
Dentistry,
Western University, London, ON, Canada
Received: 29 August 2019 Accepted: 12 February 2020 Published: 24
March 2020
Citation: Ali A, Shah FA, Zeb A, Malik I, Alvi
AM, Alkury LT, Rashid S, Hussain I, Ullah N, Ullah Khan A, Koh PO
and
Li S (2020) NF-κB Inhibitors Attenuate MCAO Induced
Neurodegeneration and Oxidative Stress—A Reprofiling
Approach.
Front. Mol. Neurosci. 13:33. doi: 10.3389/fnmol.2020.00033
NF-κB Inhibitors Attenuate MCAO Induced Neurodegeneration and
Oxidative Stress—A Reprofiling Approach Awais Ali1†, Fawad Ali
Shah1* ‡, Alam Zeb1, Imran Malik1†, Arooj Mohsin Alvi1†, Lina Tariq
Alkury2, Sajid Rashid3, Ishtiaq Hussain 4, Najeeb Ullah5,7, Arif
Ullah Khan1, Phil Ok Koh6 and Shupeng Li7,8*
1Riphah Institute of Pharmaceutical Sciences, Riphah International
University, Islamabad, Pakistan, 2College of Natural and Health
Sciences, Zayed University, Abu Dhabi, United Arab Emirates,
3National Center for Bioinformatics, Quaid-i-Azam University,
Islamabad, Pakistan, 4Department of Pharmacy, Abbottabad University
of Science and Technology, Khyber Pakhtunkhwa, Pakistan, 5Institute
of Basic Medical Sciences, Khyber Medical University, Peshawar,
Pakistan, 6Department of Anatomy, College of Veterinary Medicine,
Research Institute of Life Science, Gyeongsang National University,
Jinju, South Korea, 7State Key Laboratory of Oncogenomics, School
of Chemical Biology and Biotechnology, Peking University Shenzhen,
China, 8Centre for Addiction and Mental Health, Campbell Research
Institute, Toronto, ON, Canada
Stroke is the leading cause of morbidity and mortality worldwide.
About 87% of stroke cases are ischemic, which disrupt the
physiological activity of the brain, thus leading to a series of
complex pathophysiological events. Despite decades of research on
neuroprotectants to probe for suitable therapies against ischemic
stroke, no successful results have been obtained, and new
alternative approaches are urgently required in order to combat
this pathological torment. To address these problems, drug
repositioning/reprofiling is explored extensively. Drug repurposing
aims to identify new uses for already established drugs, and this
makes it an attractive commercial strategy. Nuclear factor-kappa
beta (NF-κB) is reported to be involved in many physiological and
pathological conditions, such as neurodegeneration,
neuroinflammation, and ischemia/reperfusion (I/R) injury. In this
study, we examined the neuroprotective effects of atorvastatin,
cephalexin, and mycophenolate against the NF-κB in ischemic stroke,
as compared to the standard NF-κB inhibitor caeffic acid phenethyl
ester (CAPE). An in-silico docking analysis was performed and their
potential neuroprotective activities in the in vivo transient
middle cerebral artery occlusion (t-MCAO) rat model was examined.
The percent (%) infarct area and 28-point composite neuro score
were examined, and an immunohistochemical analysis (IHC) and
enzyme-linked immunosorbent assay (ELISA) were further performed to
validate the neuroprotective role of these compounds in stroke as
well as their potential as antioxidants. Our results demonstrated
that these novels NF-κB inhibitors could attenuate ischemic
stroke-induced neuronal toxicity by targeting NF-κB, a potential
therapeutic approach in ischemic stroke.
Keywords: reprofiling, MCAO stroke, p-NF-κB, atorvastatin,
cephalexin, mycophenolate, caeffic acid phenethyl ester
Frontiers in Molecular Neuroscience | www.frontiersin.org 1 March
2020 | Volume 13 | Article 33
INTRODUCTION
Ischemic stroke is characterized by a decrease in cerebral blood
flow (CBF) and deprivation of both glucose and oxygen, which are
required to maintain the metabolic demands of the brain
(Siniscalchi et al., 2014). Reliable for infarct production, focal
cerebral ischemia models, such as transient or permanent middle
cerebral artery occlusion (MCAO), closely resemble human ischemic
stroke (Fluri et al., 2015), and these are majorly probed for
ischemic pathophysiological processes or related neuroprotective
strategies (Shah et al., 2019c). Ischemia-induced brain detriment
may be partially recovered if appropriate treatment is given within
a certain time frame, i.e., approximately 3–4.5 h. Thrombolytics,
such as tissue-type plasminogen activator (t-PA), and embolectomy,
such as craniectomy, are the approved treatments for ischemic
stroke (Drieu et al., 2018). However, t-PA application is limited
by its side effects and short therapeutic window (Khan et al.,
2007). According to the stroke treatment academy industry
roundtable (STAIR) guidelines, stroke treatment should be initiated
within 3–4.5 h after symptom onset. Moreover, the effectiveness of
t-PA reduces and the danger of complications increases even within
this time frame (Yperzeele et al., 2014). The most notable
complication with t-PA is hemorrhagic stroke-like intracranial
hemorrhage, which further compromises the clinical scope of t-PA in
ischemic stroke (Liu et al., 2004). Furthermore, t-PA
administration after this time window may cause neurovascular
damage, such as neuroinflammation and oxidative stress, which
facilitate leukocyte infiltration, increase reactive oxygen species
(ROS), and cause an intracellular calcium surge (Zhang et al.,
2005).
Successful management of stroke remains a major challenge in the
clinical setup. Pre-clinical studies from the last decade have
identified hundreds of neuroprotective agents, but not a single
moiety successfully passed the clinical trials. A long list of
perplex factors, such as complex brain structure, longer time
window, diverse or different outcome strategies, no precise target,
heterogeneity of stroke, and a lack of optimal duration of drug
administration, are among the major variants of drug failure in
clinical trials (Cheng et al., 2004). As a result, drug development
is severely hampered due to high expenditure, which rose from US$ 4
billion in 1975 to US$ 40 billion in 2009 (Schuhmacher et al.,
2016). To address these problems, drug repositioning, also known as
reprofiling, is increasingly explored (Padhy and Gupta, 2011) as an
alternative attractive strategy.
Nuclear factor-kappa beta (NF-κB) is ubiquitously expressed in the
cytoplasm and executes various functions, ranging from cell
proliferation and cell differentiation to cell death (Manavalan et
al., 2010). Previous studies demonstrated the involvement of NF-κB
in Alzheimer’s disease (AD), Parkinson’s disease (PD), and ischemic
stroke (Sivandzade et al., 2019), while NF-κB inhibition played a
key role in reversing these disease pathologies. NF-κB is composed
of several subunits, such as p65 (RelA), RelB, c-Rel, p105/p50
(NF-κB1), and p100/p52 (NF- κB2), which, upon activation, assemble
to form homo- or hetero- dimerized transcription factor complexes
(Chen et al., 2007). It is present in the cytoplasm as an inactive
dimer with the
inhibitory subunit of κB (IκB), a pro-inflammatory mediator.
Oxidative stress, cytokines, carcinogens, viruses, and bacterial
toxins induce the transition of NF-κB from the inactive to the
active state. This is due to breakage of the NF-κB/IκB complex and
further proteasomal degradation of cytosolic IκB set free NF- κB
for nuclear translocation. In the nucleus, NF-κB regulates
transcription of different inflammatory and apoptotic genes
(Stephenson et al., 2000). Residues of NF-κB-p50 involved in
interactions with DNA are: tyrosine-36, arginine-54, arginine-56,
tyrosine-57, cysteine-59, glutamic acid-60, histidine-64, lysine-
144, lysine-145, lysine-272, glutamine-274, and arginine-305 (Chen
et al., 1998; Berkowitz et al., 2002).
Caffeic acid phenethyl ester (CAPE) is a potential NF-κB inhibitor,
as demonstrated by several studies (Russo et al., 2002; Li et al.,
2017). CAPE is reported to have anti-inflammatory, antioxidant,
immunomodulatory, and anticancer properties (Murtaza et al., 2014).
Furthermore, CAPE exhibited protective effects in a rat MCAO model,
and it also attenuated infarction and neuroinflammation by the
NF-κB-dependent pathway (Khan et al., 2007). In the present study,
we docked CAPE with NF-κB, and the subsequent interactions were
taken as a reference for FDA drug-NF-κB complex. Atorvastatin,
mycophenolate, and cephalexin were selected from about 580 drugs
compare to the referenced CAPE-NF-κB complex. Moreover, in silico
molecular docking results were further validated by in vivo
experiments. Taken together all these, our results suggested that
drug repurposing could be a safe and time and cost-effective option
for drug development in ischemic stroke.
EXPERIMENTAL PROCEDURES
Bioinformatics Resources Ligand Preparation About 580 FDA-approved
drugs were selected from https://www.drugbank.ca/. The mol and SDF
files were saved from online databases such as Chemspider1 and
PubChem2. The files were then changed to PDB format with the help
of the Biovia Discovery Studio software (DSV). We used the
Auto-Dock program to investigate ligand–protein affinity. Affinity
was determined by the ligand-receptor complex’s E-value or the
binding power value (Kcal/mol) of the best pose. Ligands were
docked against the single target NF- κB. The 3D-structure of target
protein was obtained from http:/www.rcsb.org/pdb/home/home.do in
PDB format with PDB ID: ILE5/ILE9, which was then purified using
Discovery Studio Visualizer. Discovery Studio Visualizer was also
utilized for post-docking analysis and a creating a schematic
representation of hydrogen bonds, hydrophobic interactions, and
amino acid residues involved in ligand–protein complexes (Shah et
al., 2018; Al Kury et al., 2019).
Active Site Identification of NF-κB The crystal structure of NF-κB
(PDB ID: ILE9/ILE5) was retrieved online from
(http://www.rcsb.org). The NF-κB active
1http://www.chemspider.com/
2https://pubchem.ncbi.nlm.nih.gov/
Frontiers in Molecular Neuroscience | www.frontiersin.org 2 March
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site (tyrosine 36, arginine-54, arginine-56, tyrosine-57, cysteine-
59, glutamic acid-60, histidine-64, lysine-144, lysine-145, lysine-
272, glutamine-274, and arginine-305) was determined through a
literature survey (Chen et al., 1998; Berkowitz et al., 2002). The
structure of NF-κB was then purified using DSV.
Docking Studies In the present study, about 580 approved drugs were
docked for their potential role in stroke using NF-κB as a target
by the in-silico approach. Each drug was virtually docked against
NF-κB through an AutoDock Vina 4.2 (Trott and Olson, 2010) suit of
PyRx to achieve an optimal complementarity of steric and
physicochemical properties. The number of runs for each docking was
set to 100. It has a grid map to aid the actual docking process
with exhaustiveness of eight. To attain the best compounds using
the comparative docking approach, these drugs were also used for
docking through PatchDock. Afterward, hydrophobic and electrostatic
interactions were mapped using Discovery Studio Visualizer (Meng et
al., 2006). Three drugs—atorvastatin, cephalexin, and
mycophenolate—were selected for in vivo analysis and biochemical
studies.
Chemicals and Reagents PBS tablets and proteinase K were obtained
from (MP Bio USA). Formaldehyde, hydrogen peroxide (H2O2), reduced
glutathione (GSH), trichloroacetic acid (TCA),
1-chlor-2,4-dinitrobenzene (CDNP), N-(1-Naphthyl) ethylenediamine
dihydrochloride, 5,5′-dithio-bis-(2- nitro benzoic acid; DTNB), and
caffeic acid phenethyl ester (CAPE) were purchased from (Sigma-
Aldrich, USA). Atorvastatin, cephalexin, and mycophenolate were
obtained from the local pharmaceutical industry with the highest
analytical grade (99% HPLC grade). Mouse monoclonal anti-TNF-α
(SC-52B83), mouse monoclonal anti- p-NF-κB (SC-271908), mouse
monoclonal anti-COX-2 (SC- 514489), mouse monoclonal anti-GFAP
(SC-33673), ABC Elite kit (SC-2018), and 3,3′-diaminobenzidine
peroxidase (DAB; SC- 216567) were purchased from (Santa Cruz
Biotechnology, USA). The horseradish peroxidase-conjugated
secondary antibody (ab- 6789) and mounting media (ab-10431) were
obtained from Abcam UK. The p-NF-κB Elisa kit (Cat # SU-B28069) and
p- JNK (Cat # SU-B30586) Elisa kit were purchased from (Shanghai
Yuchun Biotechnology, China).
Animals and Drug Treatment Sprague Dawley (SD) male rats (220–300
g) were kept under circadian lighting conditions, and food and
water were allowed ad libitum. During this period, the bodyweight
of each animal was measured on alternative days. All the study
protocols were approved by the research and ethical committee
(REC/RIPS/2018/09) of Riphah Institute of Pharmaceutical Sciences,
Riphah International University, Islamabad. The rats were
arbitrarily divided into six experimental groups (n = 10):
Sham-treated group; diseased group/MCAO group: atorvastatin (20
mg/kg) treated group/Atorvastatin + MCAO; cephalexin (15
mg/kg)-treated group/Cephalexin + MCAO; mycophenolate (200
mg/kg)-treated group/Mycophenolate + MCAO; and the CAPE (10
mg/kg)-treated group/CAPE + MCAO. These drugs
were administered intraperitoneally 30 min after occlusion and
before reperfusion, and then daily for 3 days. Rats were sacrificed
6 h after the last dose, and brain tissue was extracted and stored
at −80C. Twenty rats died during surgical procedures, including
five from the MCAO group, five from Atorvastatin + MCAO, four from
Cephalexin + MCAO, three from Mycophenolate + MCAO, and three from
CAPE + MCAO, which we further adjusted by adding more rats (Figure
1).
Middle Cerebral Artery Occlusion (MCAO) Surgery MCAO was performed
as previously described (Shah et al., 2014, 2016, 2018, 2019a).
Briefly, rats were anesthetized with ketamine and xylazine (3.2:1,
I/P) mixture. The right common carotid artery (CCA) and external
carotid artery (ECA) were dissected through a midline cervical
incision without damaging the vagus nerve, and a temporary suture
as well as a permanent suture were made around CCA and ECA,
respectively, using (6/0) silk. A microvessel clip was placed
around the internal carotid artery (ICA) and the pterygopalatine
artery (PPA). A small incision was made in the ECA, and a nylon
filament (3/0) about 30 mm with a blunted rounded tip was inserted
(Figure 1). The filament was guided from the ECA into the ICA and
progressed more into the origin of the middle cerebral artery until
the resistance was felt. The filament was remained in place for 90
min and gently pulled back after due time to recanalize the vessel,
and the suture was made around the ECA open end. The incision was
closed by continuous surgical suture. The sham group was exposed to
the same experimental procedure except for filament insertion.
After 72 h of reperfusion, the animals were decapitated, and brain
samples were collected.
Neurobehavioral Testing To evaluate the sensorimotor functions of
the rats undergoing MCAO, a 28-point composite neuro score was
used. This neuro score consisted of 11 tests with a cumulative
maximum score of 28 (Shah et al., 2019b). These tests included: (1)
circling (maximum four points); (2) motility (maximum three
points); (3) general condition (maximum three points); (4) righting
reflex (maximum one point); (5) paw placement (maximum four
points); (6) horizontal bar (maximum three points); (7) inclined
platform (maximum three points); (8) grip strength (maximum two
points); (9) contralateral reflex (maximum one point); (10)
contralateral rotation when held by the base of tail (maximum two
points); and (11) visual forepaw reaching (maximum two points). A
0-score indicated severe neurological impairment, while a
cumulative score of 28 indicated healthy functioning.
TTC (2,3,4-triphenyltretrazolium Chloride) Staining At the end of
the 72-h period, rats were executed under anesthesia. The brain
tissues were carefully removed and washed with phosphate buffer
solution (PBS). The brain tissues were stored at −80C for few
minutes. Thick coronary slices (2 mm) were made using the sharp
blade from the frontal lobe and then incubated in 2%
2,3,4-triphenyl tetrazolium chloride (TTC) solution for 20–30 min
until a full demarcation was observed for MCAO-operated rats, while
deep red staining
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FIGURE 1 | Schematic presentation of experiment: rats
acclimatization, middle cerebral artery occlusion (MCAO) surgery,
drug administration, neurobehavioral studies, euthanization of rats
for tissue collection to carry out biochemical analysis, i.e.,
glutathione-S-transferase (GST), glutathione (GSH), catalase and
lipid peroxidation (LPO), morphological analysis (2,3,4-triphenyl
tetrazolium chloride staining, hematoxylin and eosin staining, and
immunohistochemistry), and biochemical assay (enzyme-linked
immunosorbent assay ELISA). The red arrow indicates the blue nylon
silk (3/0) approximately 3 cm in length with a bulging tip.
was used for sham-operated rats (Park et al., 2018). Later, a 4%
paraformaldehyde solution was used to fix these coronal slices, and
they were kept at 4C until utilized. The slices were then
photographed for % infarct area using ImageJ software. Moreover,
the corrected brain infarction was measured as follow:
Corrected infarct area = left hemisphere area − (right hemisphere
area− infarct area)/total area.
These coronal slices were then embedded in paraffin wax, and thin 4
µm coronal sections were made by rotary microtome for further
morphological analysis.
Hematoxylin Eosin (H&E) Staining First, slides were
de-paraffinized with absolute xylene (100%), rehydrated with
absolute ethanol in gradient concentrations starting from 100% to
70%, and lastly washed with distilled water. Then, slides were
dipped in hematoxylin for 10 min, before they were retained in a
glass jar under running water for 5 min. These slides were dipped
in ammonia water (1%) and HCl (1%) for a short interval and
immediately rinsed with water (Khan et al., 2019). Slides were then
immersed into eosin solution for 5–10min, rinsed with water, and
air-dried. Graded ethanol (70%, 95%, and 100%) was used to
dehydrate the dried slides and then mounted with coverslips. An
Olympus light microscope (Olympus, Japan) was used for capturing
pictures and ImageJ was used for the analysis of pictures. A total
of five images were captured and analyzed per group while focusing
specifically on neuronal cells morphological integrity, such as
size, shape, infiltrated cells, and vacuolation (Ouh et al.,
2014).
Immunohistochemical Staining and Analysis Immunohistochemical
staining and analysis were done as described previously with little
modifications (Sung et al., 2012; Ali et al., 2018). Slides were
first de-paraffinized with absolute xylene (100%) followed by
rehydration with absolute ethanol in gradient series starting from
100% to 70%. The slides were then treated with proteinase K, an
enzymatic process of antigen retrieval, and then washed with 0.1 M
PBS. To block the peroxidase activity, 3% hydrogen peroxide (H2O2)
diluted in methanol was used. Later on, slides were washed with PBS
and then incubated with normal goat serum (3%–5%) that contained
0.1% Triton X-100. Overnight incubation was performed with primary
antibodies, such as TNF-α, p-NF-κB, COX-2, and GFAP (Dilution 1:
100, Santa Cruz Biotechnology, USA). The following day, 0.1 M PBS
was used twice for washing the slides and incubated for 90 min with
secondary antibody [biotinylated secondary antibody (dilution
factor 1:50)] and then with ABC Elite kit (Santa Cruz
Biotechnology, USA) in a humidified chamber for 1 h. Then slides
were stained in DAB solution after rinsing with 0.1M PBS, and
dehydration was done with graded ethanol series. The slides were
covered with mounting media after fixing with xylene. A light
microscope (Olympus, Japan) was used for capturing the
immunohistochemical TIFF images. p-NF-κB, TNF-α, COX-2, and GFAP
were quantitatively analyzed by ImageJ software. The intensity is
expressed as the relative integrated density to the sham.
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Enzyme-Linked Immunosorbent Assay (ELISA) p-NF-κB and p-JNK
expression were measured using rat p-NF- κB and rat p-JNK ELISA
kits according to the manufacturer’s instructions (Shanghai Yuchun
Biotechnology, China). The supernatants of brain tissue homogenates
were treated with designated antibodies in 96-well plates to
quantify p-JNK and p-NF-κB. The concentration of p-JNK and p-NF-κB
was determined by using ELISA microplate reader (BioTekELx808- USA)
after the reaction between enzyme and substrate. Values were
expressed as pictograms of cytokines per milliliter (pg/ml). All
the procedures were repeated at least three times.
Determination of the Lipid Peroxidation (LPO) in Tissue Lipid
peroxidation (LPO) in the rat brain was determined as demonstrated
previously (Callaway et al., 1998). Homogenization of Rat forebrain
was done in 20 mM Tris- HCl, pH 7.4 (10 ml) at 4C using a polytron
homogenizer. The supernatant was collected after centrifugation of
homogenate at 1,000 g for 10 min at 4C. Freshly prepared ferric or
ferrous ammonium sulfate was added to brain homogenate (40 µl) to
perform lipid peroxidation and incubated for 30 min at 37C.
Afterward, 75 µl of 2-thiobarbituric acid (TBA; 0.8%) was added
which was prepared by dissolving TBA (400 mg) in water (50 ml). The
absorbance was measured at 532 nm using a plate reader.
GST, GSH and Catalase Activity The levels of reduced glutathione
(GSH) were measured by Moron et al. (1979). The activity of
catalase was analyzed by the procedure described by Sinha (1972).
The activity of glutathione- S-transferase (GST) was described by
the method of Surapaneni and Jainu (2014).
Statistical Analysis ImageJ software (Image J 1.301) was used to
analyze the data morphologically. Means ± SEM was used to represent
the data. One-way ANOVA was used to analyze the TTC, neurobehavior
results, and oxidative stress, and this was followed by post hoc
Bonferroni Multiple Comparison tests using graph pad prism-5
software. For all other morphological analysis and protein
quantification by Elisa, a two-way analysis of variance (ANOVA) was
used. Significant difference values p< 0.05 were represented by
symbol * or # while **or ## represented p < 0.01 and ∗∗∗ or ###
represented p < 0.001. A significant difference relative to
shamwas represented by the symbol * while significant difference
relative to MCAO was represented by symbol #.
RESULTS
Docking Analysis About 580 FDA drugs were screened by in silico
approach for possible NF-κB inhibition relative to the referenced
CAPE, used as a standard inhibitor. Each compound was virtually
docked against NF-κB through AutoDock Vina 4.2 suit of PyRx to
achieve an optimal complementarity of steric and physicochemical
properties. Based on the more precise docking parameters, about 360
drugs were screened out from a total of 580 drugs that were showing
polar and non-polar
interactions with the crucial residues of NF-κB (Supplementary
Figure). Twenty drugs were then scrutinized, namely, methyldopa,
metyrapone, aminocaproic acid, aminolevulinic acid, atorvastatin,
butalbital, caprylic acid, carbamoylcholine, cephalexin,
cytarabine, diazolidinyl urea, dolasetron, famciclovir, fenoprofen,
flucytosine, hydroxychloroquine, isradipine, lenvatinib,
L-carnitine, and mycophenolate, as shown in Table 1, and the list
was further reduced to atorvastatin, cephalexin, and mycophenolate
(Figure 2A, Table 2). The structures of atorvastatin, cephalexin,
mycophenolate, and CAPE were
TABLE 1 | E-value (Kcal/mol) and post-docking analysis
(interactions) of methyldopa, metyrapone, aminocaproic acid,
aminolevulinic acid, atorvastatin, butalbital, caprylic acid,
carbamoylcholine, cephalexin, cytarabine, diazolidinyl urea,
dolasetron, famciclovir, fenoprofen, flucytosine,
hydroxychloroquine, isradipine, lenvatinib, L-carnitine, and
mycophenolate with nuclear factor kappa beta (NF-κB).
FDA approved drugs Hydrogen bonding
Van Dar Vaal forces
Abbreviations: TYR, tyrosine; GLU, glutamic acid; HIS, histidine;
ARG, arginine; CYS, cysteine.
Frontiers in Molecular Neuroscience | www.frontiersin.org 5 March
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FIGURE 2 | 3D structures of atorvastatin, cephalexin,
mycophenolate, caeffic acid phenethyl ester (CAPE), and the nuclear
factor kappa beta (NF-κB). (A) Atorvastatin, cephalexin,
mycophenolate, and (CAPE) were retrieved from
https://www.drugbank.ca/ and converted to PDB format. (B) The
crystal structure of NF-κB (PDB ID: ILE9/ILE5) was retrieved from
RCSB. NF-κB active pocket composed of (tyrosine 36, arginine-54,
arginine-56, tyrosine-57, cysteine-59, glutamic acid-60,
histidine-64, lysine-144, lysine-145, lysine-272, glutamine-274,
and arginine-305) is shown by a red square.
TABLE 2 | E-value (Kcal/mol) and post-docking analysis of best pose
of atorvastatin, cephalexin, mycophenolate, and CAPE with
NF-κB.
FDA drugs E-Value Kcal/mol
Cephalexin −7.1 6 GLU-60 GLY-61 PRO-62 HIS-64 GLY-65
ASN-136
SER-110
SER-110
Atorvastatin and mycophenolate respectively form four and three
hydrogen bonds with NF-κB. Abbreviations: TYR, tyrosine; GLU,
glutamic acid; PRO, proline; HIS, histidine; SER, serine; GLY,
glycine; ASN, asparagine. Cephalexin forms six hydrogen bonds with
a minimum E value (-7.1).
retrieved from the drug bank database3, converted into PDB format
using DSV as shown in Figure 2A. The structure of p-NF-κB (PDB ID:
ILE5/ILE9) was downloaded from the RCSB website4 and shown in
Figure 2B, and the active pocket of p-NF-κB (shown by a red square)
was docked with these ligands. Table 2 showed the binding energies
and binding
3https://www.drugbank.ca/ 4http://www.rcsb.org/
residues of mycophenolate, cephalexin, atorvastatin, and CAPE after
docking with NF-κB, while the best-docked poses of mycophenolate,
cephalexin, atorvastatin, and CAPE were shown in Figure 3. A
docking analysis showed that atorvastatin fitted to NF-κB with a
bond distance of 1.93–3.09 Å, and indicating high polar contacts
(Figures 3A,B). Furthermore, four hydrogen bonds were formed
between atorvastatin and NF-κB residues (TYR-57, GLU-60, PRO-62,
and HIS-64). In addition, Van der Waal and electrostatic
interactions were also observed which further provided stability to
the atorvastatin/NF-κB binding complex. The docking results of
mycophenolate and NF-κB are shown in Figures 3D,E. Here, three
hydrogen bonds were observed with NF-κB residues (TYR-57, HIS-64,
and SER-110). Post-docking interactions for cephalexin are shown in
Figures 3G,H. Cephalexin fitted to NF-κB with a bond distance of
2.09–3.24 Åand the corresponding polar and other electrostatic
interactions are shown. Also, the docking results of CAPE are shown
in Figures 3J,K.
Effect of Test Drugs on Brain Infarction and Neurodegeneration
Sensorimotor functions before and after MCAO (24 h, 48 h, and 72 h)
surgery were assessed with a 28-point neuro score method as
described previously (Lenzlinger et al., 2004; Table 3, Figure 4A).
Severe neurological defect was observed in the MCAO group relative
to sham control (p < 0.001), as shown by a lesser composite
score (Figure 4A). This neuro score was significantly improved by
treatment with atorvastatin (p< 0.01), cephalexin (p < 0.01),
and mycophenolate (p < 0.01) compared to the MCAO group.
Interestingly, a significant increase in the neurological
cumulative score was found overtime on
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Ali et al. Repurposing of FDA Drugs Against MCAO
FIGURE 3 | Docking results and best pose of atorvastatin,
mycophenolate, cephalexin, and CAPE. The post-docking analysis was
visualized by discovery studio software (DSV) in both 2D and 3D
poses. Interaction between atorvastatin and NF-κB is shown by the
panels (A,B), mycophenolate and NF-κB by the panels (D,E),
cephalexin and NF-κB by the panels (G,H), and CAPE and NF-κB by the
panels (J,K). 3D poses are shown by panels (A,D,G,J) and 2D by
panels (B,E,H,K). Panels (C,F,I,L) represent the best pose of
atorvastatin, mycophenolate, cephalexin, and CAPE fitted to the
active pocket of NF-κB. Abbreviations: TYR, tyrosine; GLU, glutamic
acid; PRO, proline; HIS, histidine; SER, serine; GLY, glycine; ASN,
asparagine.
TABLE 3 | Twenty-eight-point composite neuro score.
Test Test name Score
1 Circling 4 2 Motility 3 3 General condition 3 4 Righting reflex 1
5 Paw placement 4 6 Horizontal bar 3 7 Inclined platform 3 8 Grip
strength 2 9 Contralateral reflex 1 10 Contralateral rotation 2 11
Visual forepaw reaching 2
Accumulative score 28
days 2 and 3 after MCAO in rats treated with atorvastatin,
cephalexin, and mycophenolate, indicating that a spontaneous
sensorimotor functional recovery occurred after MCAO (data not
shown).
TTC staining was performed to examine the neuroprotective effects
of atorvastatin, cephalexin, and mycophenolate. Considerable
infarct changes were observed in MCAO group (39.1 ± 4) as compared
to sham-operated group
(p < 0.001), with the corrected infarcted area being reduced by
20.2± 3.76% (p< 0.05), 27.33± 3.8% (p< 0.05), 16.29± 6.54% (p
< 0.01), and 14.40 ± 5.6% (p < 0.01) for mycophenolate,
atorvastatin, cephalexin, and CAPE respectively. Cephalexin and
mycophenolate treated group showed a significant reduction in
corrected infarct area when compared with atorvastatin as shown in
Figure 4B.
H and E staining was used to determine the extent of neuronal
injury in cortex and striatum. We found aberrant morphological
features, variations in color staining, such as cytoplasmic
pyknosis and nuclear basophilia, distorted neuronal size and shape,
such as swelling and scalloped angular neuronal cells, as well as
inflammatory infiltrated cells in the ipsilateral cortex and
striatum (Figure 4C). Treatment with atorvastatin, cephalexin, and
mycophenolate significantly attenuated this damage both in the
cortex and striatal tissue (p < 0.01), similarly to the
CAPE-administered group (p< 0.01; Figure 4C).
Effect of Test Drugs on Oxidative Stress Malondialdehyde (MDA) is a
reactive aldehyde produced by lipid peroxidation, which has been
demonstrated to be an
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Ali et al. Repurposing of FDA Drugs Against MCAO
FIGURE 4 | Effect of test drugs on brain infarction and
neurodegeneration. (A) 28 points composite scoring. Data are
presented as Mean ± SEM, analyzed by one-way ANOVA (n = 10/group).
***Indicates p < 0.001, and ## represent p < 0.01. Symbols
*shows significant difference relative to sham control while #
shows significant difference relative to the MCAO group. (B) Brain
coronal sections were stained with TTC which distinguishes between
ischemic and non-ischemic areas (n = 5/group). ***Indicates p <
0.001, while symbol ## represent p < 0.01 significant difference
and # indicates p < 0.05. The symbol *shows significant
difference relative to sham control while # shows significant
difference relative to MCAO. The parietal cortex, striatum,
hippocampus and thalamus region are respectively shown by green,
blue, black, and yellow arrows. (C) Representative photomicrographs
of hematoxylin and eosin staining, showing the extent of neuronal
injury. Data are presented as Mean ± SEM, analyzed by two-way
analysis of variance (ANOVA). scale bar 50 µm, magnification 40X.
Slides were made from TTC sections, fixed
(Continued)
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FIGURE 4 | Continued initially in 4% paraformaldehyde. From these
sections, paraffin blocks were made and later 4 µm thin coronal
sections were prepared by a rotary microtome (n = 5/group). The
neuropil of the ipsilateral cortex is characterized by intensive
vacuolation, swelling and scalloped neurons with intense
cytoplasmic eosinophilia and nuclear basophilia along with pyknotic
nuclei. Treatment with test drug substances attenuated the extent
of neuronal death and more surviving neurons could be seen in the
ipsilateral cortex and striatum. ***Indicates p < 0.001, while
## represent p < 0.01 significant difference; # indicates p <
0.05. The symbol * shows significant difference relative to sham
control while # shows significant difference relative to
MCAO.
important indicator of oxidative stress and can be measured by
thiobarbituric acid reactive substances (TBARS). A TBARS test was
performed, and peroxides showed a drastic increase in the MCAO
group, an effect that could be rescued by atorvastatin, cephalexin,
and mycophenolate treatment. The LPO content in the cortex of the
MCAO group was (45.9 ± 1.79) as compared to the sham group (25.9 ±
0.28; p < 0.001, Table 4). Atorvastatin, cephalexin, and
mycophenolate attenuated this increased contents of TBRAS with 20.1
± 1.28 (p < 0.01), 19.4 ± 1.49 (p < 0.01), and 18.85 ± 1.26
(p < 0.01), and the effects that could match to that of CAPE
group 18.85 ± 1.26 (p< 0.01) in the cortex. Similarly, peroxide
levels were decreased in the striatum of rats treated with
atorvastatin (18.8 ± 1, p < 0.01), cephalexin (17.15 ± 1, p <
0.01), and mycophenolate (16.45 ± 1.04, p < 0.01) compared to
the MCAO group (35.53± 1.71) as shown in Table 5. Furthermore, the
antioxidant effect of atorvastatin, cephalexin, and mycophenolate
were examined using GST, GSH, and catalase levels both in cortex
and striatum (Tables 4, 5). Non-enzymatic antioxidant GSH and
enzymatic antioxidant catalase and GST were significantly
increased in atorvastatin-, cephalexin-, and mycophenolate- treated
groups compared to that of MCAO. Atorvastatin + MCAO group showed a
marked increase in the levels of GSH (60.35 ± 1.86 and 32.8 ±
1.65), GST (54.4 ± 1.59 and 25.315 ± 2.56), and catalase (23.15 ±
1.43 and 17.57 ± 1.53) in the cortex and striatum, respectively
(Tables 4 and 5).
Effect of Test Drugs on the Expression of p-NF-κB To determine the
possible effects of atorvastatin, cephalexin, and mycophenolate on
p-NF-κB levels, an ELISA was performed in the cortex and striatum
according to the manufacturer’s guidelines (Shanghai Yuchun
Biotechnology, China). As shown in Figure 5A, all three drugs
showed statistically significant decreased when compared with the
MCAO group in both the cortex and striatum (p < 0.05
atorvastatin, p < 0.05 cephalexin, and p < 0.05 mycophenolate
in the cortex and in the striatum. CAPE (10 mg/kg) treatment also
attenuated p- NF-κB expression in the cortex (p < 0.01) and
striatum (p < 0.05), as shown in Figure 5A. These results were
further validated by immunohistochemical analysis (Figure 5B).
Together, these results suggested that atorvastatin, cephalexin,
and mycophenolate could modulate NF-κB phosphorylation levels, and
this accounts for its neuroprotective effects.
Effect of Test Drugs on the Inflammatory Cascade NF-κB is comprised
of p65 (RelA), RelB, c-Rel, p105/p50 (NF-κB1), and p100/p52
(NF-κB2; Oeckinghaus and Ghosh, 2009). The inactive complex of
NF-κB is bound by an inhibitory subunit of κB inhibitors (IκB),
which is present in
TABLE 4 | Effect of atorvastatin, mycophenolate, cephalexin, and
CAPE on oxidative enzymes in the cortex.
Groups GST (µM CDNB conjugate/ min/mg of protein)
GSH (µM/mg of protein) Catalase (µM H2O2/ min/mg of protein)
L-PO nmol/MDA/ mg of protein
Sham 62.7 ± 1.98 65.55 ± 1.59 38.25 ± 1.76 25.9 ± 0.28 MCAO 25 ±
1.72*** 20.65 ± 1.58*** 18.65 ± 2.31*** 45.9 ± 1.79*** Atorvastatin
+ MCAO 54.4 ± 1.59## 60.35 ± 1.86## 23.15 ± 1.43# 20.1 ±
1.28##
Cephalexin + MCAO 49.4 ± 1.57# 52.25 ± 1.39# 19.1 ± 1.56# 19.4 ±
1.49##
Mycophenolate + MCAO 44.65 ± 0.44# 48.1 ± 1.41# 18.05 ± 1.62# 18.85
± 1.26##
CAPE + MCAO 60.25 ± 1.02## 63.2 ± 1.34## 34.25 ± 1.30## 18.85 ±
1.26##
Data were presented as Mean ± SEM, n = 5/group. Data were analyzed
by one way ANOVA followed by post-hoc Bonferroni Multiple
Comparison test using graph-pad prism-5 software. ***p < 0.001
shows a significant difference compared to sham, while ## or #show
respectively p < 0.01 or p < 0.05 relative to MCAO. After
euthanasia, brain tissue samples were collected and preserved at
-80C. Abbreviations: GST, glutathione S-transferase; GSH,
glutathione; LPO, lipid peroxidase.
TABLE 5 | Effect of atorvastatin, mycophenolate, cephalexin, and
CAPE on oxidative enzymes in the striatum.
Groups GST (µM CDNB conjugate/ min/mg of protein)
GSH (µM/mg of protein) Catalase (µM H2O2/ min/mg of protein)
L-PO nmol/MDA/ mg of protein
Sham 41.95 ± 0.77 63.9 ± 1.77 21.6 ± 1.37 12.15 ± 1.61 MCAO 8.2 ±
2.12*** 12.85 ± 1.64*** 7.11 ± 0.56*** 35.53 ± 1.71*** Atorvastatin
+ MCAO 25.315 ± 2.56# 32.8 ± 1.65# 17.57 ± 1.53## 18.8 ±
1.0##
Cephalexin + MCAO 22.95 ± 1.34# 14.16 ± 1.28 16.47 ± 1.79## 17.15 ±
1.07##
Mycophenolate + MCAO 15.835 ± 1.74 11.55 ± 1.76 12.35 ± 1.72# 16.45
± 1.04##
CAPE + MCAO 28.28 ± 1.81## 42.25 ± 1.40## 19.92 ± 1.90## 20.85 ±
1.09##
Data were presented as means ± standard error of the mean (SEM).
Data were analyzed by one way ANOVA followed by posthoc Bonferroni
Multiple Comparison test using graph-pad prism-5 software. ***p
< 0.001 shows a significant difference compared to sham, while
## or # show respectively p < 0.01 or p < 0.05 relative to
MCAO. After euthanasia, brain tissue samples were collected and
preserved at -80C. Abbreviations: GST, glutathione S-transferase;
GSH, glutathione; LPO, lipid peroxidase.
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Ali et al. Repurposing of FDA Drugs Against MCAO
FIGURE 5 | Effect of test drugs on p-NF-κB. Panel (A) illustrates
the effect of atorvastatin, cephalexin, mycophenolate, and CAPE on
p-NF-κB expression in cortex and striatum by Elisa. Two-way
analysis of variance (ANOVA) was used to analyze the results. The
expression level of p-NF-κB was significantly decreased in the
treatment vs. MCAO group. ** indicates p < 0.01, while ##
represent p < 0.01 significant difference, and # indicates p
< 0.05. Symbols * shows significant difference relative to sham
control while # shows significant difference relative to MCAO. (B)
Effect of atorvastatin, cephalexin, mycophenolate, and CAPE on
immunohistochemistry of p-NF-κB in cortex and striatum. Two-way
ANOVA was used to analyze the data, scale bar 20 µm, magnification
40X and (n = 6/group). p-NF-κB exhibits nuclear localization, ***
indicates p < 0.001, while symbol ** represent p < 0.01, and
# indicates p < 0.05 significant difference. The symbol * shows
significant difference relative to sham control while # shows
significant difference relative to MCAO.
the cytoplasm. Pro-inflammatory cytokines, including tumor necrosis
factor-alpha (TNF-α) and interleukin-1 beta (IL-1β), bind to their
corresponding receptors that further link to downstream molecules,
thus involving the sequential activation of ASK1, SEK1, and p-JNK.
These lead to IκB phosphorylation and proteasomal degradation,
result in NF-κB release and nuclear translocation, and subsequently
induce the transcription of inflammatory proteins such as inducible
nitric oxide (i-NOS) and cyclo-oxygenase (COX-2; Yao et al.,
2013).
Moreover, this inflammatory pathway was further examined with an
ELISA assay for p-JNK (Figure 6A), which showed higher expression
in the MCAO group, whereas the level of p- JNK was significantly
ameliorated in the drug treatment groups (p < 0.05 cephalexin, p
< 0.05 mycophenolate in the cortex as well as p < 0.05
mycophenolate, p < 0.001 atorvastatin, and p < 0.01
cephalexin in the striatum). Immunohistochemistry was performed to
further validate the ELISA results, which showed elevated
expression of GFAP, COX-2, TNF-α in cortex and striatum after
ischemic injury (Figures 6B–D). Atorvastatin, cephalexin, and
mycophenolate significantly decreased the expression of COX-2,
TNF-α, and GFAP.
DISCUSSION
Ischemic brain injury involves the disturbance of stable networks
involving multiple signaling pathways and a variety of genes (Sung
et al., 2016). Among them, NF-κB has been considered
as a prominent controller of a variety of pathological or cell
death cascades (Russo et al., 2002; Li et al., 2017). In numerous
animal models of acute ischemic injury, NF-κB inhibition has been
proved to be protective (Manna et al., 2000; Wang et al., 2010). In
this regard, we performed a comparative analysis of three
FDA-approved drugs—atorvastatin, cephalexin, and mycophenolate—in
ischemic brain injury, to evaluate their possible neuroprotective
role based on drug repurposing. The bioinformatics analysis was
further supported with experiments in MCAO models, where all three
drugs showed anti-inflammatory and neuroprotective
properties.
In silico studies are a fast and cost-effective technique for new
drug development (Prakhov et al., 2010). Auto-Dock vina, patch
dock, gromacs, and gold suite are widely used for ligand docking,
while lower binding values (kcal/mol) reveal reduced energy of
desolvation, which depicts the stability of ligand–protein
complexes (Pecsi et al., 2010). It is necessary to mention that
binding energy is a cumulative result of hydrogen bonds, covalent
bonds, hydrophobic interactions, and weak interactions, like van
der Waals forces, between a ligand and receptor. Steric bumps in a
ligand–receptor complex are responsible for instability, while
hydrogen bonds play a significant role in the stability of a
complex and strong affinity (Zhao and Huang, 2011). A comparative
study was conducted to compare the results of the test ligands to
that of standard drug, which was obtained from RCSB and PubChem. In
our study, test compounds atorvastatin,
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Ali et al. Repurposing of FDA Drugs Against MCAO
FIGURE 6 | Effect of test drugs on the inflammatory cascade. Effect
of atorvastatin, cephalexin, mycophenolate, and CAPE on
inflammatory markers. (A) Elisa analysis of p-JNK. The data are
expressed as the Mean ± SEM. ***p < 0.001 relative to sham while
## or # show respectively p < 0.01 or p < 0.05 relative to
MCAO, with n = 5/group, After euthanasia, brain tissues were
collected and preserved at -80C and later processed for protein
analysis. The p-JNK was significantly expressed in the MCAO group
relative to sham, while treatment with test drugs attenuated the
MCAO induced hyperexpression of JNK in cortex and striatum. (B)
Immunohistochemistry results for astrocytes (GFAP) in the cortex
and striatum tissues of the brain (C) Immunohistochemistry of COX2
in the cortex and striatum tissues of the brain and (D)
Immunohistochemistry of TNF-α in the cortex and striatum tissues of
the brain. Immunoslides were processed from stained TTC coronal
sections. From the thick coronal TTC sections, paraffin blocks were
made and later 4 µm thin coronal sections were prepared by a rotary
microtome.
(Continued)
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Ali et al. Repurposing of FDA Drugs Against MCAO
FIGURE 6 | Continued Scale Bar 50 µm, magnification 40X, (n =
5/group). GFAP exhibits a characteristic morphological shape, while
COX2 and TNF-α exhibited cytoplasmic localization. ***p < 0.001
shows a significant difference compared to sham, while ## or # show
respectively p < 0.01 or p < 0.05 relative to MCAO. Data are
presented as Mean ± SEM and analyzed by GraphPad Prism 8
software.
cephalexin, and mycophenolate showed a high affinity to the NF-κB
receptor as compared to a standard drug, i.e., CAPE. Our study
found cephalexin and mycophenolate manifested the best binding
score with the lowest E-value against NF-κB. Based on the E-value,
the order of ligand affinity was found as cephalexin >
mycophenolate > CAPE > atorvastatin. Previous studies
demonstrated that atorvastatin markedly reduced brain edema and
infarct size at 24 h of ischemic injury and significantly
suppressed the upregulation of NF-κB (Cheng et al., 2018).
Atorvastatin may also prevent an NF-κB shift from the cytoplasm to
the nucleus, which could be attributed to the downregulation of
HMGB1 (Wang et al., 2010). Also, previous studies with statin
showed a decrease in infarct volume by both pretreatment and
posttreatment (Baryan et al., 2012). In another study, simvastatin
significantly reduced the infarct volume size (38.18%), as compared
to atorvastatin (23.71%) and rosuvastatin (13.88%; Garcia-Bonilla
et al., 2014). Similarly, previous studies showed that post MCAO
treatment with mycophenolate (200 mg/kg) for three successive days
resulted in a significant reduction in infarct volume size. In
agreement with these results, our study demonstrated that
atorvastatin reduced the infarct volume, decreased p-NF-κB
expression in the cortex and striatum.The role of oxidative stress
in I/R injury
is well established (Behn et al., 2007). Therefore, a therapeutic
strategy having an antioxidant effect may be considered an
effective approach. Treatment with mycophenolate at 200 mg/kg dose
reduced the oxidative stress, and this was similar to previous
findings. Likely, we observed that different oxidative damage
parameters are the critical determinants in I/R brain injury, which
causes a significant increase in LPO and protein oxidation
accompanied by significant depletion in brain GSH as well as a
decrease in GST and catalase. Therefore, scavenging free radicals
and preventing lipid and protein peroxidation by CAPE and tested
drugs can suppress oxidative damage and inflammatory responses.
GSH, GST, and catalase levels were reduced in theMCAOgroup,
whichmay render the susceptibility of plasma membranes towards
peroxide and other free radicals’ attacks. Accordingly, we observed
an elevated level of TBARS, accompanied by depleted GSH, GST, and
catalase level inMCAO where post-treatment with CAPE and test drugs
reversed these effects, is concomitant with the previous
observations where antioxidants were used as a remedy in
experimental stroke models (Meng et al., 2006; Khan et al., 2009;
Wang et al., 2010). The observed neuroprotective effect of
mycophenolate could be partially attributed to its anti-oxidant
activity, as mycophenolate treatment at doses of 100 and 200 mg/kg
ameliorated the raised NO activity after 24 h of ischemia (Bilginer
et al., 2009).
Our results demonstrated that cephalexin, mycophenolate, and
atorvastatin reduced the expression of proinflammatory cytokines
and oxidative stress-related factors like TNF-α, GFAP, and COX-2.
Moreover, our results also indicated that cephalexin can be a
strong scavenger of ROS and a potent inhibitor of NF-κB derived
inflammation, which may deserve further exploration as of its
future application. Our results provided the first targeting
FIGURE 7 | Schematic diagram depicting pathological events
following MCAO that mediate post-ischemic inflammation. NF-κB
activation regulates the inflammatory cytokines and adhesion
molecules, and this leads to peripheral inflammatory cells
migrating to the ischemic area and enhance ischemic brain
injury.
Frontiers in Molecular Neuroscience | www.frontiersin.org 12 March
2020 | Volume 13 | Article 33
In conclusion, we demonstrated that cephalexin, mycophenolate, and
atorvastatin were identified as NF-κB inhibitors as referred by
CAPE with molecular docking. Further experiments showed that
treatment with cephalexin, mycophenolate, atorvastatin, and CAPE
reduced infarct area and improved motor deficits. Furthermore, CAPE
inhibited NF-κB activation and thus preventing the expression of
NF-κB dependent genes including cytokines i.e., p-JNK, TNF-α and
COX2 as depicted by Figure 7.
DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the
article/Supplementary Material.
ETHICS STATEMENT
All the study protocols and guidelines were approved by the
research and ethical committee (REC/RIPS/2018/09) of the Riphah
Institute of Pharmaceutical Sciences, Riphah International
University, Islamabad.
AUTHOR CONTRIBUTIONS
AA: conceptualization. FS: methodology. AA, AMA, SR and IM:
validation. FS, LA, PK, NU and IH: investigation. AZ and AU:
resources. FS and LA: writing. AA, FS and SL: review and editing.
FS and SL: supervision. SL: funding acquisition.
FUNDING
ACKNOWLEDGMENTS
We are thankful to the Higher Education Commission (HEC) Pakistan
for the Startup grant support (1615/SRGP/HEC/2017) and
JCYJ20170810163329510 by Science and Technology Innovation
Committee of Shenzhen; CAS Key Laboratory of Receptor Research, for
providing financial support to execute this study in the Riphah
Institute of Pharmaceutical Sciences, Riphah International
University, Islamabad, Pakistan.
SUPPLEMENTARY MATERIAL
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Conflict of Interest: The authors declare that the research was
conducted in the absence of any commercial or financial
relationships that could be construed as a potential conflict of
interest.
Copyright © 2020 Ali, Shah, Zeb, Malik, Alvi, Alkury, Rashid,
Hussain, Ullah, Ullah Khan, Koh and Li. This is an open-access
article distributed under the terms of the Creative Commons
Attribution License (CC BY). The use, distribution or reproduction
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Frontiers in Molecular Neuroscience | www.frontiersin.org 14 March
2020 | Volume 13 | Article 33
Recommended Citation
NF-B Inhibitors Attenuate MCAO Induced Neurodegeneration and
Oxidative Stress—A Reprofiling Approach
INTRODUCTION
Docking Studies
Neurobehavioral Testing
RESULTS
Effect of Test Drugs on Oxidative Stress
Effect of Test Drugs on the Expression of p-NF-B
Effect of Test Drugs on the Inflammatory Cascade
DISCUSSION