Molecular Docking, Bioinformatic Analysis, and Experimental Verification for the Effect of Naringin on ADHD: Possible Inhibition of GSK-3β and HSP90

Abstract

Background/Objectives: One of the most abundant and growing neurodevelopmental disorders in recent decades is attention deficit hyperactivity disorder (ADHD). Many trials have been performed on using drugs for the improvement of ADHD signs. This study aimed to detect the possible interaction of naringin with Wnt/β-catenin signaling and its putative anti-inflammatory and protective effects in the mouse ADHD model based on bioinformatic, behavioral, and molecular investigations. Furthermore, molecular docking was applied to investigate possible interactions with the GSK-3β and HSP90 proteins. Methods: Male Swiss albino mice were divided into four groups, a normal control group, monosodium glutamate (SGL) control, SGL + naringin 50 mg/kg, and SGL + naringin 100 mg/kg. The psychomotor activity of the mice was assessed using the self-grooming test, rope crawling test, and attentional set-shifting task (ASST). In addition, biochemical analyses were performed using brain samples. Results: The results of the SGL group showed prolonged grooming time (2.47-folds), a lower percentage of mice with successful crawling on the rope (only 16.6%), and a higher number of trials for compound discrimination testing in the ASST (12.83 ± 2.04 trials versus 5.5 ± 1.88 trials in the normal group). Treatment with naringin (50 or 100 mg per kg) produced significant shortening in the grooming time (31% and 27% reductions), as well as a higher percentage of mice succeeding in crawling with the rope (50% and 83%, respectively). Moreover, the ELISA assays indicated decreased dopamine levels (0.36-fold) and increased TNF-α (2.85-fold) in the SGL control group compared to the normal mice, but an improvement in dopamine level was observed in the naringin (50 or 100 mg per kg)-treated groups (1.58-fold and 1.97-fold). Similarly, the PCR test showed significant declines in the expression of the Wnt (0.36), and β-catenin (0.33) genes, but increased caspase-3 (3.54-fold) and BAX (5.36-fold) genes in the SGL group; all these parameters were improved in the naringin 50 or 100 mg/kg groups. Furthermore, molecular docking indicated possible inhibition for HSP90 and GSK-3β. Conclusions: Overall, we can conclude that naringin is a promising agent for alleviating ADHD symptoms, and further investigations are required to elucidate its mechanism of action.

Keywords: ADHD; Wnt/β-catenin signaling; molecular docking; monosodium glutamate; mouse; naringin.

Figure 1

Proposed binding mode of naringin in the ATP-binding site of GSK-3β (A) and HSP90 (B). Ligand is displayed as cyan sticks and the important protein residues are displayed as gray sticks with a cartoon backbone. Polar contacts are shown as red dashed lines.

Figure 2

(A) Two-dimensional representation of the binding mode of naringin in the ATP-binding site of GSK-3β. The interaction stability over the MD simulation is displayed as a percentage beside each interaction. (B) RMSD of the backbone and the ligand during the MD simulation. (C) Interaction fraction diagram during the 100 ns simulation.

Figure 3

(A) Two-dimensional representation of the binding mode of naringin in the ATP-binding site of HSP90. The interaction stability over the MD simulation is displayed as a percentage beside each interaction. (B) RMSD of the backbone and the ligand during the MD simulation. (C) Interaction fraction diagram during the 100 ns simulation.

Figure 4

Wnt signaling pathway [04310]. The pathway was obtained from the KEGG database and shows that Wnt/β-catenin is involved in the nuclear translocation of β-catenin and the activation of target genes via TCF/LEF transcription factors, making this pathway crucial for the self-renewal of cells.

Figure 5

Role of Wnt//β-catenin signaling. (A) Wnt/β-catenin interactions with many proteins, such as GSK3B, HSP90, Casp3, BAX, and Bcl2. (B) Co-expression analysis of interacting proteins. (C) The colored lines indicate different evidence types. For example, red is fusion evidence, light blue is a database, green is neighborhood, blue is co-occurrence, purple is experimental evidence, black is co-expression, and yellow is text mining evidence. (D) The intensity of the color indicates the level of confidence that two proteins are functionally associated. (E) Gene ontology shows the molecular processes most associated with Wnt/β-catenin signaling. Wnt1: proto-oncogen Wnt-1, CASP3: caspase-3 subunit p-12, BAX: apoptosis regulator BAX, BCL2: BCL2-like protein 2, Hsp90ab1: heat shock protein HSP 90-beta1, Ctnnb1: catenin beta-1, Gsk3b: glycogen synthase kinase-3 beta.

Figure 6

(A) A ShinyGo 0.8 dot plot representing the top 15 diseases related to naringin target genes. (B) A Venn diagram showing the target genes of naringin (yellow circle) and ADHD (violet circle) and the common pathways between them (brown part). The diagram was created using FunRich. (C) A heatmap demonstrating the model of gene expression of naringin target genes. The FunRich 3.1.3 bioinformatic tool was used. The color code of the heatmap ranges from 3 to −3, with 3 (most red) being the most expressed and −3 (most green) being the least expressed.

Figure 7

The behavior of mice in the self-grooming test and rope crawling test, and the number of trials in ASTT. (A) The time taken for self-grooming. (B) The percentage of mice (out of six) crawling on the rope. (C) The SD phase, (D) the CD phase, (E) the RV1 phase, and (F) the IDS phase. Data are means ± SD except for Panel (B), which is the % of animals. At p < 0.05, * vs. normal and # vs. SGL control.

Figure 7

The behavior of mice in the self-grooming test and rope crawling test, and the number of trials in ASTT. (A) The time taken for self-grooming. (B) The percentage of mice (out of six) crawling on the rope. (C) The SD phase, (D) the CD phase, (E) the RV1 phase, and (F) the IDS phase. Data are means ± SD except for Panel (B), which is the % of animals. At p < 0.05, * vs. normal and # vs. SGL control.

Figure 8

Naringin modulates the brain levels of glutamate, dopamine, and inflammation markers in mice fed with SGL. (A) Dopamine, (B) glutamate, (C) TNF-α, and (D) NFκB. Data are mean ± SD. At p < 0.05, * vs. normal, # vs. SGL control, and $ vs. SGL + naringin-50.

Figure 9

RT-PCR analysis of the expression of the target genes. (A) Wnt, (B) β-catenin, (C) caspase-3, (D) Bcl2, and (E) BAX. Data are means ± SD, * vs. normal control and # vs. SGL control, ^$ vs. the SGL/naringin-50 group at p < 0.05.

Figure 10

H&E-stained sections of the mice groups. The normal group shows neuron cell bodies arranged in a compacted form and regular nuclei, indicated by a black arrow, with intact fibrillary cytoplasmic processes (red arrow). The cortex shows normal neurons (black arrow) and astrocytic cells (red arrow). The SGL control CA2 region shows smudged nuclei of neurons with pericellular vacuolation and a mildly disrupted arrangement (black arrow) with decreased cellularity, and there are moderately disturbed fibrillary processes in multiple areas (red arrows). The cortex displays degenerate neurons (black arrow) and increased vacuolation of astrocytic cells (red arrow). The SGL + naringin 50 CA2 region shows focal pericellular vacuolation (black arrow) and scattered fibrillary process degeneration (red arrow). The cortex shows mild degenerative changes to neurons (black arrow) and mild vacuolation of astrocytic cells (red arrow). The SGL + naringin 100 CA2 region shows a regular arrangement of neurons with cell bodies showing normal chromatin patterns and nuclei with minimal vacuolation (black arrow), and intact fibrillary processes (red arrow). The cortex shows regular neurons (black arrow) with astrocytic cells showing minimal vacuolation (red arrow).

Figure 11

Immunohistochemical staining for Bcl2 in the hippocampi of the experimental groups. The normal group showed organized neurons with moderate-to-strong nuclear staining in most of the cells (CA2 and CA3 regions). The SGL control group showed less organized neurons with weak-to-absent staining for Bcl2 in most of the cells (CA2 and CA3 regions). The SGL + naringin-50 group showed mild-to-moderate staining in a few cells (CA2 and CA3 regions). The SGL + naringin-100 group showed improvements in the neuronal structures and moderate-to-strong staining for Bcl2 in most of the cells (CA2 and CA3 regions). Bcl2 immunostaining at ×400 magnification for both images in each group.

Figure 12

A diagram of the attention set-shifting task (ASST) for mice.