An in vivo and in silico probing of the protective potential of betaine against sodium fluoride-induced neurotoxicity

Abstract

Excessive fluoride exposure beyond the tolerable limit may adversely impacts brain functionality. Betaine (BET), a trimethyl glycine, possesses antioxidant, anti-inflammatory and anti-apoptotic functions, although the underlying mechanisms of the role of BET on fluoride-induced neurotoxicity remain unelucidated. To assess the mechanism involved in the neuro-restorative role of BET on behavioural, neurochemical, and histological changes, we employed a rat model of sodium fluoride (NaF) exposure. Animals were treated with NaF (9 mg/kg) body weight (bw) only or co-treated with BET (50 and 100 mg/kg bw) orally uninterrupted for 28 days. We obtained behavioural phenotypes in an open field, performed negative geotaxis, and a forelimb grip test, followed by oxido-inflammatory, apoptotic, and histological assessment. Behavioural endpoints indicated lessened locomotive and motor and heightened anxiety-like performance and upregulated oxidative, inflammatory, and apoptotic biomarkers in NaF-exposed rats. Co-treatment with BET significantly enhanced locomotive, motor, and anxiolytic performance, increased the antioxidant signalling mechanisms and demurred oxidative, inflammatory, and apoptotic biomarkers and histoarchitectural damage in the cerebrum and cerebellum cortices mediated by NaF. The in-silico analysis suggests that multiple hydrogen bonds and hydrophobic interactions of BET with critical amino acid residues, including arginine (ARG380 and ARG415) in the Keap1 Kelch domain, which may disrupt Keap1-Nrf2 complex and activate Nrf2. This may account for the observed increased in the Nrf2 levels, elevated antioxidant response and enhanced anti-inflammatory response. The BET-Keap1 complex was also observed to exhibit structural stability and conformational flexibility in solvated biomolecular systems, as indicated by the thermodynamic parameters computed from the trajectories obtained from a 100 ns full atomistic molecular dynamics simulation. Therefore, BET mediates neuroprotection against NaF-induced cerebro-cerebellar damage through rats' antioxidant, anti-inflammatory, and anti-apoptotic activity, which molecular interactions with Keap1-Nrf2 may drive.

Keywords: Betaine; Molecular docking; Molecular dynamics simulation; Neurotoxicity; Oxidative stress; Sodium fluoride.

Fig. 1

Outlines of the experimental timeline to investigate the effect of Betaine (BET) on fluoride rats Albino Wistar strain for 28 consecutive days (A). Created by Arunsi Uche O. using BioRender, https://app.biorender.com/

Fig. 2

a-c The effect of BET on NaF-induced locomotive decline and explorative behaviour (a); representative rack plot and heat map (b); and anxiety-like behaviour and motor dysfunction (c) in male Wistar rats experimentally treated with BET and NaF for 28 consecutive days. All values represent the mean (n = 6; ± SEM). The statistics were analysed using GraphPad Prism V.10.2 for MacOS. The tests used were one-way ANOVA and Tukey Post Hoc. The designated P-values for all tests were set at p < 0.05 (*), < 0.01 (**), < 0.001 (***), and < 0.0001 (****). BET betaine, NaF Sodium fluoride

Fig. 3

The effect of BET in the cerebrum and cerebellum SOD, CAT, GPx and GST activities of rats experimentally challenged with NaF for 28 consecutive days. All value represents the mean (n = 4; ± SEM). The statistics were analysed using GraphPad Prism V.10.2 for MacOS. The tests used were one-way ANOVA and Tukey Post Hoc. The designated P-values for all tests were set at p < 0.05 (*), < 0.01 (**), < 0.001 (***), and < 0.0001 (****). BET betaine, NaF Sodium fluoride; SOD Superoxide dismutase; CAT Catalase; GPx Glutathione peroxidase, and GST Glutathione-S-transferase

Fig. 4

The effect of BET on GSH, TSH (a) and Trx, Trx-R and activity (b) against NaF-induced antioxidant decline in the cerebrum and cerebellum of rats experimentally treated for 28 consecutive days. All value represents the mean (n = 4; ± SEM). The statistics were analysed using GraphPad Prism V.10.2 for MacOS. The tests used were one-way ANOVA and Tukey Post Hoc. The designated P-values for all tests were set at p < 0.05 (*), < 0.01 (**), < 0.001 (***), and < 0.0001 (****). BET betaine, NaF Sodium fluoride; GSH Glutathione; GST Glutathione S-transferase; Trx Thioredoxin, Trx-R Thioredoxin reductase, and TSH Total sulfhydryl group

Fig. 5

The effect of BET on RONS, XO (activities), NO, and LPO levels on NaF-induced oxidative stress in the cerebrum and cerebellum of rats experimentally treated for 28 consecutive days. All value represents the mean (n = 4; ± SEM). The statistics were analysed using GraphPad Prism V.10.2 for MacOS. The tests used were one-way ANOVA and Tukey Post Hoc. The designated P-values for all tests were set at p < 0.05 (*), < 0.01 (**), < 0.001 (***), and < 0.0001 (****). BET betaine, NaF Sodium fluoride; RONS Reactive oxygen and nitrogen species; XO Xanthine oxidase; NO nitric oxide; and LPO Lipid peroxidation

Fig. 6

The effect of BET on NaF-induced elevated proinflammation MPO, IL-10 (a), and signalling biomarkers Nrf-2 and HO-1 (b) in the cerebrum and cerebellum of rats experimentally treated for 28 consecutive days. All value represents the mean (n = 4; ± SEM). The statistics were analysed using GraphPad Prism V.10.2 for MacOS. The tests used were one-way ANOVA and Tukey Post Hoc. The designated P-values for all tests were set at p < 0.05 (*), < 0.01 (**), < 0.001 (***), and < 0.0001 (****). BET betaine, NaF Sodium fluoride; MPO Myeloperoxidase; IL-10 Interleukin-10; Nrf-2 Nuclear erythroid factor-2; and HO-1 Hemeoxygenase-1

Fig. 7

The effect of BET on NaF-induced increases in apoptotic biomarkers (caspases − 3 and − 9) activities in the cerebrum and cerebellum of rats experimentally treated for 28 consecutive days. All value represents the mean (n = 4; ± SEM). The statistics were analysed using GraphPad Prism V.10.2 for MacOS. The tests used were one-way ANOVA and Tukey Post Hoc. The designated P-values for all tests were set at p < 0.05 (*), < 0.01 (**), < 0.001 (***), and < 0.0001 (****). BET betaine, NaF Sodium fluoride; CASP-3 Caspase-3; CASP-9 Caspase-9

Fig. 8

A-B Representative histological changes in the cerebellar cortex and representative histomorphometry of experimental rats treated with NaF and BET (A). The control group showed the typical architecture of the cerebellar layers (Molecular layer (ML), Purkinje cell layer (PCL), and Granule layer (GL) with their cells intact. The NaF-treated group (NaF) showed severe Purkinje cell loss in the PCL and pyknosis of neurons, signifying neuronal death in the PCL (Red arrowhead). Betaine group only (BET) showed typical architecture of the cerebellar cortex. Meanwhile, the NaF + BET1 and NaF + BET2 groups showed some restoration of the damaged Purkinje cells. B: Histological changes in the cerebellum and histomorphometry of experimental rats treated with NaF and BET (B). The control group (CNT) shows the typical architecture of the pyramidal layer of the cerebral cortex, with its cells intact. The treated group (NaF) showed severe loss of the pyramidal neurons and pyknosis of surviving neurons (Red arrowhead). Betaine group only (BET) showed typical architecture of the cerebellar cortex. Meanwhile, the NaF + BET1 and NaF + BET2 groups showed some restoration of the damaged pyramidal cells. All value represents the mean (n = 3; ± SEM). The statistics were analysed using GraphPad Prism V.10.2 for MacOS. The tests used were one-way ANOVA and Tukey Post Hoc. The designated P-values for all tests were set at < 0.05 (*) vs. control. Normal cells (dark arrowhead), pyknotic cell red (arrow) indicating shrunken or degenerated neuronal cells. H and E Stain, x 400 magnification, scale bar: 50 μm

Fig. 9

Molecular interactions of betaine (purple) and IVX (blue) in the inhibitor binding pocket within the Kelch domain of rat Keap-1. a Cartoon and surface views (b) 3D and 2D interactions view of IVX (c) 3D and 2D interactions of BET

Fig. 10

3D and 2D interactions of ligands with Kelch domain of human Keap-1. Keap1-BET complex (a and b). Keap1-Nrf2-BET complex (c and d)

Fig. 11

Molecular interactions of betaine (purple) and MSI (blue) in the active site of Caspase-3. a Cartoon and surface views (b) Caspase-3- MSI (c) Caspase-3-BET complex

Fig. 12

Structural stability and conformational flexibility ligand-protein complexes (a) Backbone root mean square deviation (RMSD) plots. b Total number of hydrogen bonds (c) Root Mean Square Fluctuation (RMSF)

Fig. 13

Unfolding deviation of ligand-protein complexes. a Radius of gyration (b) Surface accessible surface area

Fig. 14

Molecular mechanisms of betaine on Nrf-2-Keap1 pathway and Caspase-3 activation. A: BET can inhibit the interaction between Nrf-2 and Keap1, thus enhancing the stabilisation of Nrf-2, which can translocate into the nucleus and bind to antioxidant-responsive elements (AREs) and other co-activator (SMaf) to induce the transcription of antioxidant and cytoprotective genes. B: Activation of caspase-3 is triggered by the induction of oxidative stress where ROS can impair the integrity of the mitochondrial membrane, releasing cytochrome C, which binds to apaf-1 and pro-caspase-9, releasing active caspase-9. Caspase-9 (active) cleaves inactive caspase-3 and converts it to active caspase-3. Active caspase-3 mediates the execution of programmed cell death. However, BET can form a strong interaction with the amino acid residues in the active site of caspase 3, thus suppressing its apoptotic function. Created by Arunsi Uche O. using BioRender, https://app.biorender.com/

Fig. 15

Proposed mechanistic mode of action of betaine neuroprotective effect against NaF-induced neurotoxicity in male Wistar rats. BET confers protection to the cerebellum and cerebrum rats treated with NaF by enhancing redox balance and resolving inflammation. BET achieves this by suppressing the accumulation of ROS, as evidenced through increased levels of phase-1 antioxidants, including SOD, CAT, and GPx, which can detoxify O2.⁻ and H₂O₂. Furthermore, BET increased the levels of phase-2 antioxidants GST, GSH, and TSH, crucial in detoxifying lipid hydroperoxides and primary oxidative products like MDA. Failure to upregulate these antioxidant molecules may result in MDA accumulation in rats’ cerebrum and cerebellum. Aside from regulating redox balance, BET also resolves inflammation by suppressing the levels of pro-inflammatory mediators while increasing the level of anti-inflammatory markers. Furthermore, BET prevented the manifestation of apoptosis in the cerebellum and cerebrum of rats by inhibiting the activity of caspase 3. Created by Arunsi Uche O. using BioRender, https://app.biorender.com/