1. Introduction
Caloric restriction can delay or prevent several age-related disorders,
including neurodegenerative diseases like Alzheimer’s disease (AD) [1, 2, 3, 4, 5]. The
most prominent metabolic alteration due to caloric restriction is the induction
of ketosis (a phenomenon characterized by increased levels of ketone bodies in
blood circulation), decreased oxidative stress, and apoptosis. Ketones, also
called ketone bodies (KBs), are mainly formed by the catabolism of lipids and
comprises -hydroxybutyrate (BHB), acetoacetate, and acetone. The brain
utilizes ketone bodies as an alternate fuel during energy hypometabolism,
reducing its glucose requirement [6]. Moreover, ketone bodies, compared to
glucose, generate more extensive energy due to changes in mitochondrial ATP
production [7].
Given that impaired brain energy metabolism promotes CI/dementia, dietary or
therapeutic intervention to improve energy regulation in the brain would be a
promising approach [8]. As indicated earlier, since energy hypometabolism refers
explicitly to glucose, either a ketogenic diet or administration of
-hydroxybutyrate may overcome the reduced glucose uptake and metabolism,
thereby improve energy deficits in the brain [6, 9]. Additionally, the ketogenic
diet is beneficial in epilepsy, amyotrophic lateral sclerosis, traumatic brain
injury, multiple sclerosis Parkinson’s disease, and other neurological diseases
[10, 11, 12].
Molecular docking by utilizing structure-based drug design is the most widely
used computational method to validate drug action. Moreover, this drug design
technique is suitable to elucidate protein-ligand interactions and predict the
binding poses of hit compounds within the active site of macromolecules such as
enzymes and receptors [13, 14]. Molecular mechanics Generalized Born Surface Area
(MM/GBSA) is another commonly used technique to calculate the free binding energy
and predict binding poses and affinities of ligands [15, 16]. Therefore,
follow-up docking studies and MM/GBSA analysis with the crystal structures of
N-Methyl-D-aspartic acid (NMDA) receptor and acetylcholinesterase (AChE) can
identify the potential critical interactions of -hydroxybutyric acid
within the catalytic binding pocket. Currently, the molecular mechanisms by which
ketogenic diet or ketone body administration improve energy regulation and
cognitive impairment have not been well characterized [17, 18]. Consequently, we
sought to investigate the in vitro neuroprotective effects of
hydroxy-butyric acid on hippocampal neurons (HT-22) and the molecular docking on
the current therapeutic target for cognitive impairment, NMDA receptor AChE.
Furthermore, we also elucidated the effects of hydroxy-butyric acid on
the markers of oxidative stress (prooxidants/antioxidants), mitochondrial
function, and apoptosis because these markers play a vital role in hippocampal
neuronal proliferation or neurodegeneration.
2. Materials and methods
2.1 Chemicals and reagents
Thiazolyl Blue Tetrazolium Bromide (MTT), Dulbecco’s Modification of Eagle’s
Medium (DMEM), Fetal Bovine Serum (FBS), and Penicillin-Streptomycin solution
were purchased from Corning® (Corning, NY). Griess reagent,
Sodium nitrite Phosphate buffer saline (PBS), Dimethyl sulfoxide (DMSO),
-hydroxybutyric acid, Nicotinamide adenine dinucleotide (NADH),
2,7-dichlorofluorescein diacetate (DCFH), Hydrogen Peroxide (H₂O₂),
Phosphoric acid, O-phthalaldehyde (OPT), Glutathione (GSH), Tri-chloroacetic acid
(TCA), Thiobarbituric acid (TBA), and EDTA were purchased from Sigma Aldrich (St.
Louis, MO). Caspase substrates AC-YVAD-AMC and AC-DEVD-AMC were purchased from
Enzo Life Sciences (Farmingdale, NY). Thermo Fisher Scientific Pierce 660 nm
Protein Assay reagent kit was purchased (Pierce, Rock-ford, IL) for protein
quantification.
2.2 HT-22 mouse hippocampal neuronal cell line
HT-22 mouse hippocampal neurons purchased from ATCC (accession number:
CVCL_0321) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 4.5 g/L Glucose, 4 mM L-Glutamine, 10% Fetal Bovine Serum, 100
units/mL Penicillin and 50 g/mL Streptomycin. For the neuronal
proliferation studies, an MTT assay was used. HT-22 neurons were seeded into 96
healthy plates at a density of 1 10 cells/well. Cells were used
within 3–10 passages after they were received.
2.3 Treatment design
To evaluate the effect of -hydroxybutyric acid on the HT-22 cell
proliferation, different concentrations (0 M–10 mM) were incubated
for 24 h. -hydroxybutyrate (250 and 500 M) significantly
increased the HT-22 neuronal proliferation. Hence, to establish the effect on
oxidative stress, mitochondrial function, and apoptosis, the HT-22 neurons were
treated with two different doses (250 and 500 M) of
-hydroxybutyric acid for 12 h. For MTT assay: 24 h cell growth + 24 h
incubation (0 M–10 mM -hydroxybutyric acid). Biochemical
assays: 24 h cell growth + 12 h incubation (250 and 500 M
-hydroxybutyric acid).
2.4 Effect of -hydroxybutyric acid on HT-22 neuronal
proliferation
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) dye-based
cell proliferation assay was performed to study the effect of
-hydroxybutyric acid on the hippocampal neurons compared to the controls
as described by our earlier publications [19]. Additionally, for effect on the
morphological changes, the control and -hydroxybutyric acid-treated
cells were imaged using an Axiovert 25 inverted microscope equipped with a Nikon
Coolpix 4500 camera. As a result, the control and -hydroxybutyric
acid-treated cells were observed for morphological changes.
2.5 Effect of -hydroxybutyric acid on reactive oxygen
species (ROS)
2,7-dichlorofluorescein diacetate (DCF-DA) (DCF, Calbiochem, 287810,
0.5%) dye-based fluorometric assay was performed to study the effect on ROS in
the control and -hydroxybutyric treated HT-22 neurons [20].
2.6 Effect of -hydroxybutyric acid on nitrite content
A colorimetric assay using Griess reagent (Enzo life sciences, ALX-400-004-L050)
was used to quantify the nitrate content in the control and
-hydroxybutyric treated HT-22 neurons [21].
2.7 Effect of -hydroxybutyric acid on glutathione (GSH)
content
Fluorometric-based OPT condensation method was used to quantify the glutathione
content in the control and -hydroxybutyric treated HT-22 neurons [22].
2.8 Effect of -hydroxybutyric acid on catalase activity
A spectrophotometric method using hydrogen peroxide as a substrate was used to
assess the catalase activity in the control and -hydroxybutyric treated
HT-22 neurons [23].
2.9 Effect of -hydroxybutyric acid on Lipid peroxide
content
Colorimetric method using thiobarbituric acid (TBA) (Sigma, T5500, 1%) was used
to measure the thiobarbituric acid reactive substances (TBARS, lipid peroxide)
content in the control and -hydroxybutyric treated HT-22 neurons [24].
2.10 Effect of -hydroxybutyric acid on caspase-1 and
caspase-3 activities
Spectrofluorimetric method using
Ac-Tyr-Val-Ala-Asp-7-amino-4-Trifluoromethlcoumarin (AC-YVAD-AMC,
Enzo-260-024-M005)and N-Acetyl-Asp-Glu-Val-Asp-7-amido-4-Methylcoumarin
(AC-DEVD-AMC) (Enzo-260-031-M005) as substrates were used to assess the caspase-1
and caspase-3 activities in the control and -hydroxybutyric treated
HT-22 neurons [21].
2.11 Effect of -hydroxybutyric acid on Complex-I and
Complex-IV activity
Colorimetric methods using NADH H (VWR, 0384, 1mM) and cytochrome-C (Cytochrome
C oxidase, Sigma C7752) as substrates were used to assess the Complex-I and
Complex-IV activities in the control and -hydroxybutyric treated HT-22
neurons [22, 25].
2.12 Protein quantification
Protein in the control and -hydroxybutyric treated HT-22 neurons were
quantified using Thermo Fisher Scientific Pierce 660 nm Protein Assay reagent
kit.
2.13 Molecular docking
Computational analysis for understanding the pharmacokinetic and pharmacodynamic
properties of -hydroxybutyric acid (Absorption, distribution,
metabolism, elimination (ADME) and Molecular docking profile).
Ligands were sketched in a 2D structure and converted into their 3D structure,
and energy minimization was performed using LigPrep with OPLS3e Force field in
Schrö-dinger software. The NMDA crystal structure (PDB ID 5U8C) and the
acetylcholine esterase (PDB ID 2X8B) were used for docking and molecular
mechanics energies combined with generalized Born and surface area continuum
solvation (MM/GBSA) analysis. Protein Preparation Wizard utility tools
structurally prepare both proteins in the Schrödinger Release 2019-2,
Schrödinger, LLC, New York, NY. All hydrogen atoms were added to the selected
proteins to optimize H-bonding interactions, missing atoms were added, and
missing side chains and loops were filled. N-and C-terminal residues were
specified and charged. Some histidine residues were either flipped or
tautomerized.
In contrast, some residues were only flipped to improve H-bonding and avoid H-H
clashes. Similarly, waters with less than 3 H-bonds to non-waters were removed.
Finally, energy minimization of the protein hydrogens, water, and side chains was
performed by utilizing the OPLS3e Force field. Glide docking (Schrodinger
software) protocols were applied; in this docking program, the flexibility of the
ligands is considered while the protein is considered a rigid structure. The 3D
coordinates of the active site were identified using grid generation. Standard
precision (SP) was selected, and all other parameters were left at the default
settings. The binding free energies (Gbind in kcal/mol) were calculated
for each ligand from the pose view file from glide docking scores using the
MMGBSA (Schrodinger software) and applied in the OPLS3e Force field. The binding
free energy of MMGBSA was predicted for each lig-and-protein complex, as follows:
Gbind = G complex - G-protein - Gligand, where Gbind is the
binding free energy and G complex, G-protein, and Gligand are the free energies
of complex, protein, and ligand, respectively.
2.14 Statistical analysis
All data are expressed as means SEM. Statistical analyses were performed
using Kruskal Wallis non-parametric test followed by an appropriate post-hoc
test, including Dunn’s method (p 0.05 was considered to indicate
statistical significance). All statistical analyses were performed using the
Prism-V software (La Jolla, CA, USA).
3. Results
3.1 Effect of -hydroxybutyric acid on cell viability
The effect of -hydroxybutyric acid on HT-22 cells was determined by MTT
assay and by microscopic images. It was found that -hydroxybutyric acid
at a low dose (10–100 M) had no significant effect on the HT-22
cell viability. Interestingly, -hydroxybutyric acid (250 and 500
M) increased the HT-22 cell viability significantly as compared to
the controls at 24 h (Fig. 1A, p 0.001, n = 12, as seen by the
image, Fig. 1B ). However, -hydroxybutyric acid at a high dose (2.5–10
mM, Fig. 1B) significantly decreased the HT-22 cell viability (Fig. 1A,
*p 0.0001, n = 12). To elucidate the effects of
-hydroxybutyric acid on oxidative stress and mitochondrial functions in
HT-22 cells, we used 250 and 500 M of -hydroxybutyric
acid.
Fig. 1.
Effect of -hydroxybutyric acid on HT-22 cell
viability and morphology. (A) Formazan formed due to the reduction of
tetrazolium dye-MTT was measured colorimetrically at 540 nm. Low dose (10–100
M) -hydroxybutyric acid did not significantly affect the
HT-22 cell viability. Moderate doses (250 and 500 M) of
-hydroxybutyric acid significantly increased the HT-22 cell viability as
compared to the controls at 24 h (p 0.001, n = 12). A high dose
(2.5–10 mM) of -hydroxybutyric acid significantly decreased the HT-22
cell viability as compared to the controls at 24 h (p 0.0001, n =
12). (B) Influence of various concentrations of -hydroxybutyric acid
(0–10 mM) on the morphology of HT-22 cells as observed after 24 hours of
treatment: the control, 10 M, 50 M, 100
M, 250 M and 500 M
-hydroxybutyric acid group displayed spindle or multipolar shaped cells
with transparent cell body with significant growth, whereas from 2.5 mM
-hydroxybutyric acid group showed cell shrinkage and a significant
reduction in neurons.
3.2 Effect of -hydroxybutyric acid on oxidative stress
markers
Lipid peroxidation occurs do the increase in prooxidants and diminished
antioxidants level and/or activity. Hence, we measured different prooxidants (ROS
& nitrite content) and antioxidants markers (glutathione and catalase activity)
to understand their role in altering lipid peroxide formation.
-hydroxybutyric acid decreased the ROS levels at 250 M by
29.6% (statistically nonsignificant). At 500 M by 41.9% (Fig. 2A), non-significantly decreased nitrite content at 250 M by
11.14% and 500 M by 21.56% (Fig. 2B), significantly increased the
glutathione content at 250 M by 267.3% and 500 M by
357.36% (Fig. 2C) and significantly increased catalase activity at 250
M by 42.64% and 500 M by 153.24% (Fig. 2D). Due to
the increased antioxidant effects and decreased prooxidant contents,
-hydroxybutyric acid significantly decreased the formation of lipid
peroxide at 250 M by 38.42% and at 500 M by 43.54%
as seen by the decrease in the formation of TBARS (Fig. 2E).
Fig. 2.
Influence of -hydroxybutyric acid on oxidative stress.
(A) -hydroxybutyric acid significantly reduced the generation of ROS
content: ROS was quantified spectrofluorimetrically using non-fluorescent DCF dye
and measuring the fluorescent DCF at RFU 492/527 nm. Results are expressed as
change in RFU/mg protein, Mean + SEM, **p 0.01, ***p
0.001, n = 5. (B) -hydroxybutyric acid non-significantly decreased the
nitrite con-tent: Nitrite content was quantified colorimetrically using Griess
reagent at 545 nm. Results are expressed as nitrite (M)/mg protein,
Mean + SEM, *p 0.05, n = 5. (C) -hydroxybutyric acid
significantly augmented the formation of GSH content: GSH was measured
spectrofluorimetrically using OPT condensation method at 327/423 nm. Results are
expressed as GSH (M)/mg protein, Mean + SEM, **p 0.01,
***p 0.001, n = 5. (D) -hydroxybutyric acid significantly
increased the catalase activity: Catalase activity was measured
spectrophotometrically using hydrogen peroxide as substrate at 240 nm. Results
are expressed as hydrogen peroxide catalyzed (M)/mg protein, Mean +
SEM, *p 0.05, **p 0.01, n = 5. (E)
-hydroxybutyric acid significantly reduced lipid peroxide content: TBARS
formed was calculated spectrophotometrically using TBA at 532 nm. Results are
expressed as TBARS formed (M)/mg protein, Mean + SEM, *p
0.05, n = 5.
3.3 Effect of -hydroxybutyric acid on apoptotic markers
Concerning the effect of -hydroxybutyric acid on apoptotic action,
caspase-1 and caspase-3 activity was elucidated. -hydroxybutyric acid at
250 M and 500 M significantly decreased caspase-1 by
43.78% and 53.65% (Fig. 3A) and with respect to caspase-3 activity by 44.77%
and 49.4% (Fig. 3B).
Fig. 3.
-hydroxybutyric acid significantly reduced the
caspases’ activity. (A) Caspase-1 activity was measured spectrofluorimetrically
using AC-YVAD-AMC as a substrate at 326/460 nm. Results are expressed as AMC
formed (M)/mg protein, Mean + SEM, **p 0.01, n = 5.
(B) -hydroxybutyric acid significantly reduced the caspase-3 activity:
Caspase-3 activity was measured spectrofluori-metrically using AC-DEVD-AMC as a
substrate at 326/460 nm. Results are expressed as AMC formed (M)/mg
protein, Mean SEM, *p 0.05, n = 5.
3.4 Effect of -hydroxybutyric acid on mitochondrial
functions
To study the effect of -hydroxybutyric acid on mitochondrial functions,
Complex-I and Complex-IV activities were measured. -hydroxybutyric acid
at 250 M non-significantly increased Complex-I activity by 34.4%
and at 500 M significantly by 68.58% (Fig. 4A). Similarly,
concerning Complex-IV, -hydroxybutyric acid at 250 M,
non-significantly increased Complex-IV activity by 46.97% and at 500
M significantly by 81.98% (Fig. 4B).
Fig. 4.
-hydroxybutyric acid significantly increased
mitochondrial function as seen by increased Complex-I and Complex-IV activities.
(A) -hydroxybutyric acid significantly increased the Com-plex-I
activity: Complex-I activity was measured spectrophotometrically using NADH as a
substrate (340 nm). Results are expressed as NADH oxidized (M)/mg
protein, Mean + SEM, *p 0.01, n = 5. (B) -hydroxybutyric
acid significantly increased the Complex-IV activity: Complex-IV activity was
measured spectrophotometrically using cytochrome-C as a substrate (550 nm).
Results are expressed as cytochrome C oxidized (M)/mg protein, Mean
+ SEM, **p 0.01, n = 5.
3.5 Molecular docking
To determine the pharmacokinetic parameters concerning the number of metabolites
formed, CNS activity, and blood-brain barrier permeability, we performed a
computational analysis of -hydroxybutyric acid using a QikProp filter
from Schrö-dinger software. Our in-silico molecular docking results showed a
good absorption, distribution, metabolism, and elimination profile of
-hydroxybutyric acid (Tables 1,2).
Table 1.SASA, FOSA, FISA, PISA #metab, CNS, and QPlog BB values for
-hydroxybutyric acid.
Compound |
SASA(Å) |
FOSA(Å) |
FISA (Å) |
PISA (Å) |
#metab |
CNS |
QPlog BB |
9,12,15-octadecatrienoic_acid |
702.9 |
549.8 |
108.06 |
45.09 |
5 |
−2 |
−1.4 |
9,12-octadecadienoic_acid |
656.08 |
531.3 |
98.1 |
26.6 |
4 |
−2 |
−1.2 |
-Hydroxybutyric acid |
285.7 |
141.6 |
144.1 |
0 |
2 |
−2 |
−0.7 |
Note: The acceptable ranges are as follows: Area is (300–1000), FOSA:
Hydrophobic components of the SASA (0.0–750.0), FISA: Hydrophilic components of
the SASA (7.0–330.0), PISA: (carbon and attached hydrogen) components of
the SASA (0.0–450.0), #metab: Number of likely metabolic reactions (1–8) CNS:
–2 (inactive) to +2 (active), QPlog BB: (–3.0 to –1.2) polar compounds have large
negative values. |
Table 2.Basic pharmacokinetic data of -hydroxybutyric acid.
Compound |
Mol wt. |
Donor HB |
Accept HB |
cLogP |
% Human oral absorption |
Rule of 5 |
9,12,15-octadecatrienoic_acid |
278.43 |
1 |
2 |
5.6 |
89.4 |
1 |
9,12-octadecadienoic acid |
280.45 |
1 |
2 |
5.5 |
90.4 |
1 |
-Hydroxybutyric acid |
104.10 |
1 |
2.7 |
0.8 |
68 |
0 |
Note: The permissible ranges are as follows: Mol wt.: (130–725), Donor HB:
(0.0–6.0), Accept HB: (2.0–20.0), cLogP: (–2.0 to 6.5), % Human oral
absorption: 80% high, 25% low, Rule of five (maximum 4). |
Solvent accessible surface area (SASA) of a molecule is its surface area in
contact with the solvent in the biological system. Lower SASA scores indicate
that more molecules interact with a biomolecule like a protein or a membrane.
Therefore, most of it will likely remain in the unionized form, hence, higher
absorption and bioavailability. On the other hand, higher SASA scores indicate
that more of the molecule interacts with the solvent, such as the aqueous medium
of the stomach (stomach acid), and most of it will likely stay ionized; thus,
lower absorption and bioavailability.
Table 1, -hydroxybutyric acid shows a lower SASA value consistent with
what has been noted elsewhere for -hydroxybutyric acid’s favorable
bioavailability profile. Other parameters like hydro-phobic components of the
SASA (FOSA), hydrophilic components of the SASA (FISA), and (carbon and
attached hydrogen) components of the SASA (PISA) values for
-hydroxybutyric acid are all within the acceptable range. In terms of
metabolic reaction predictions (#metab), -hydroxybutyric acid has a
lower number of metabolic reactions. Lesser negative QPlog BB values indicating
accessibility into the blood-brain barrier and better CNS activity. Other
parameters, including % Human oral ab-sorption and Lipinski’s rule of five, are
within the acceptable range of drug bioavailability (Table 2).
We performed computational molecular docking to determine if
-hydroxybutyric acid has any interactions with receptors involved in
neurotransmission. Docking score and free binding energy values of
-hydroxybutyric acid exhibit a possible interaction with NMDA receptor
and with acetylcholinesterase to exhibit a potent pharmacological effect to
enhance cognitive functions (Table 3 and Table 4). The results are comparable to
some known agonists and antagonists of NMDA receptors and AChE enzyme activators
and inhibitors. However, since this is an in-silico prediction model, further
mechanistic studies need to be performed to validate our preliminary findings.
Table 3.Docking score and free binding energy (MM/GBSA) of
-hydroxybutyric acid, Glutamate, Quinolinic acid, and Riluzole with
NMDA.
|
NMDA |
Compound |
Docking Score |
MM/GBSA Binding energy (kacl/mol) |
-hydroxybutyric acid |
−5.5 |
−26.2 |
Glutamate |
−6.4 |
−30.6 |
Quinolinic acid |
−6.1 |
−26.6 |
Riluzole |
−4.4 |
−19.2 |
, Natural ligand; , Agonist; , Antagonist. |
Table 4.Docking score and free binding energy (MM/GBSA) of
-hydroxybutyric acid, Acetylcholine, Tyrosine, and Rivastigmine with
Acetylcholinesterase (AChE).
|
Acetylcholinesterase (AChE) |
Compound |
Docking Score |
MM/GBSA Binding energy (kacl/mol) |
-hydroxybutyric acid |
−3.3 |
−3.4 |
Acetylcholine |
−4.4 |
−24.3 |
Tyrosine |
−6.9 |
−40.7 |
Rivastigmine |
−4.2 |
−43.8 |
, Natural ligand; , Activator; , Inhibitor. |
4. Discussion
We established the effects of -hydroxybutyric acid, a ketone, on HT-22
hippocampal neurons to mitigate oxidative stress and improve mitochondrial
functions. Accordingly, -hydroxybutyric acid, at a moderate dose of 250
and 500 M, increased the HT-22 cell viability, which could be
attributed to the decreased oxidative stress due to decreased prooxidants and
increased antioxidants content/activity (dose-dependently decreased ROS,
decreased nitrite content, increased glutathione content, and increased catalase
activity) leading to reduced neuronal damage as seen by decreased neuronal lipid
peroxidation [17, 18]. Furthermore, the apoptotic pathway reduced caspase-1 and
caspase-3 activity by -hydroxybutyric acid, resulting in improved
neuronal viability. Additionally, enhanced mitochondrial functions attributed to
Complex-I and Complex-IV activities demonstrated by -hydroxybutyric acid
can cause increased HT-22 cell viability. The valid values such as
FOSA-Hydrophobic components of the SASA, FISA-Hydrophilic components of the SASA,
PISA- carbon and attached hydrogen components of the SASA, number of
metabolites, number of likely metabolic reactions, CNS action, Log P values, and
percentage of human oral absorption favored the bioavailability of
-hydroxybutyric acid (Tables 1,2) for prophylactic and/or therapeutic
use.
Several studies utilizing -hydroxybutyric acid to improve human
cognition have been conducted with varying results [8]. Jensen et al.
[26] investigated the effect of -hydroxybutyric acid on cognition in
Type 2 diabetes mellitus patients. It improved working memory performance without
any change in global cognition. Similarly, Alzheimer’s patients showed
improvements in cognition in response to acute elevations of
-hydroxybutyric acid [27]. The improvement in cognitive functions is
attributed to ketones acting as an alternative fuel for neurons in MCI or AD
patients. In patients with MCI or AD, defects in brain glucose utilization occur,
attributed to amyloid deposition, inflammation, or altered lipid homeostasis
[28].
The neuroprotective effects of -hydroxybutyric acid have drawn
additional interest due to the current hypothesis of energy deficiency in various
neurodegenerative disorders. Most of the neurons with high energy demand do not
efficiently produce high-energy phosphates from fatty acids. Still,
-hydroxybutyric acid can undergo oxidation during deficiency of
glucose/carbohydrates leading to increased energy regulation [8, 29, 30]. Thus,
the neuroprotective effects of -hydroxybutyric acid are considered
notably essential for future prophylactic and therapeutic treatment [31, 32].
Mitochondria are critical for several neuronal functions such as synaptic
plasticity, neurotransmission, and energy regulation of neurons [33]. We found
improvement in mitochondrial complex I and IV with -hydroxybutyric acid
treatment. Similarly, -hydroxybutyrate has been shown to improve
mitochondrial biogenesis and bioenergetics in cultured rat hippocampal neurons
[34]. In addition, previous studies have shown to inhibit mitochondrial ROS
production following glutamate excitotoxicity predominantly with acetoacetate
[35, 36] and alleviate oxidative stress by decreasing ROS and malondialdehyde in
an animal model of epilepsy [37].
Oxidative stress has been predominantly associated with all neurodegenerative
disorders. Oxidative stress is a condition in which there is an increase in the
generation of intracellular ROS, hydroxyl radicals, superoxide anion, and
hydrogen peroxide responsible for damage to lipids, mainly leading to lipid
peroxides associated with neuronal membrane damage [38, 39]. To counteract the
negative impact of oxidative stress, there is a valid molecular defense mechanism
consisting of enzymes, proteins and low molecular weight molecules. These
antioxidants molecular defense mechanisms can catalytically remove the
prooxidants. For instance, superoxide dismutase dismutases superoxide anions into
hydrogen peroxide, which in turn is broken down by catalase. Glutathione
(tripeptide), by scavenging the ROS, neutralizes the neurotoxic effect and
reduces the ROS’s impact to participate in any form of chemical reaction to
complete or partial destruction of DNA, proteins and lipids leading to
neurodegeneration. Interestingly, -hydroxybutyric acid has been shown to
exhibit neuronal solid antioxidant activity [40, 41].
Increased oxidative stress and decreased mitochondrial functions can lead to
heightened expression and/or activity of several pro-apoptotic markers. Caspases
are mainly associated with DNA damage resulting in decreased neuronal
proliferation. Caspase-1 and caspase-3 are cysteine proteases that have been
shown to increase the apoptotic cascade in mostly all neurodegenerative
disorders. We showed a reduction in both caspase-1 and caspase-3 activity with
-hydroxybutyric acid. -Hydroxybutyric acid has been shown to
offer hippocampal neuron proliferative effects through its anti-apoptotic effects
[37]. Caspase inhibitors have been shown to reduce the initiation and/or
progression of neurodegeneration [42].
-hydroxybutyric acid, like the other existing neuroprotectants, also
exhibits increased mitochondrial functions and decreased apoptosis, which might
lead to cognitive enhancement.
Despite the beneficial effects of -hydroxybutyric acid on cognition,
several studies have reported the adverse effects of ketones [43]. For instance,
long-term adverse effects include hepatic steatosis, hypoproteinemia, renal
stones, vitamin and mineral deficiencies [44]. Furthermore, hypoglycemia is a
common manifestation, especially in diabetic patients on treatment. Additionally,
ketones are contraindicated in patients with pancreatitis, liver failure, lipid
and fat metabolism disorders, and porphyria, limiting their utility.
Only limited studies have investigated the dose of -hydroxybutyric acid
required to confer neuroprotection. The uptake of -hydroxybutyric acid
by brain cells occurs either by diffusion or a carrier-mediated process.
Furthermore, there is a high likelihood of a saturable dependent mechanism for
carrier-mediated transport that could alter the amount of
-hydroxybutyric acid reaching the brain. For instance, the affinity of
the carrier transporters is lowest for -hydroxybutyric acid [45], which
indicates that sufficient (50 uM) concentrations are required to attain
sufficient concentrations in the brain [46]. One of the limitations is we
investigated the effects of -hydroxybutyric acid under normal conditions
in vitro cell culture and found a concentration of
250–500 M to offer beneficial effects. However, this concentration
might not be sufficient to reach adequate levels in the brain due to the
saturable dependent mechanism of transporters. Further studies in animal models
to investigate the pharmacokinetics of blood-brain barrier permeability and
concentrations attained in the brain tissue need to be performed.
Although the specific neuron proliferative mechanisms of
-hydroxybutyric acid in the treatment of neurodegenerative diseases are
still uncertain, it is predictable that all neurodegenerative disorders can
affect human health through oxidative damage mitochondrial dysfunction, and
apoptotic pathways. Furthermore, neurodegenerative diseases often involve
multiple mechanisms; hence -hydroxybutyric acid may also exhibit
multipotent neuron proliferative molecular processes. In summary, most of the
previous reports associated protective effects of -hydroxybutyric acid
have been validated. However, our work specifically targeted the hippocampal
neuroprotective effects of -hydroxybutyric acid. Hence, either
-hydroxybutyric acid can play a prophylactic, supportive, or facilitate
the therapeutic effects in various neurodegenerative diseases to improve the
symptoms and quality of life in patients. Thus, -hydroxybutyric acid has
excellent clinical application potential, which further requires immediate
exploration of the neuroprotective effects of invalid cognitive deficient animal
models and humans.
5. Conclusions
Although ketone bodies have been previously studied to evaluate its
neuroprotective effects, our work has validated the role of the
-hydroxybutyric acid at a dose of 250 and 500 M
concentration in neuroprotection. The hippocampal neuron proliferative role can
reduce oxidative stress, maintain energy metabolism, improve mitochondrial
function, and modulate apoptosis. Although the specific mechanisms of
-hydroxybutyric acid in treating neurological diseases are still
uncertain, it is predictable that most neurogenerative diseases could affect
human health through oxidative damage, energy metabolism disorders, mitochondrial
dysfunction, and impaired apoptosis. Therefore, -hydroxybutyric acid can
be used as an adjunct facilitating the disease’s therapy, improving patients’
symptoms and quality of life. Future studies are necessary further to specify the
roles of various components in ketone bodies, identifying therapeutic targets and
related molecular signaling pathways to improve the approach and effectiveness of
ketone bodies.
Author contributions
MM, SR, MA, MG, JS, MAI, FS, TT, MD performed experiments, statistical analysis,
computational docking. In addition, VS, MR, TM, MD were involved in planning,
funding, reviewing, editing, and writing the manuscript.
Ethics approval and consent to participate
Not applicable.
Acknowledgment
We sincerely thank the CNSi, Auburn University for their support and the anonymous reviewers for their excellent criticism and valuable input of the article.
Funding
This research received no external funding.
Conflict of interest
The authors declare no conflict of interest.