Mitigating effect of ferulic acid on di-(2-ethylhexyl) phthalate-induced neurocognitive dysfunction in male rats with a comprehensive in silico survey

Abstract

Di-(2-ethylhexyl) phthalate (DEHP) is the most abundant phthalate threatening public health-induced neurotoxicity. This neurotoxicity is associated with behavioral and biochemical deficits in male rats. Our study investigated the neuroprotective effect of ferulic acid (FA) on male rats exposed to DEHP. Thirty-two male Wistar rats were assigned to four groups. Group I control rats received corn oil, group II intoxicated rats received 300 mg/kg of DEHP, group III received 300 mg/kg of DEHP + 50 mg/kg of FA, and group IV received 50 mg/kg of FA, all agents administrated daily per os for 30 days. Anxiety-like behavior, spatial working memory, and recognition memory were assessed. Also, brain oxidative stress biomarkers, including brain malondialdehyde (MDA), reduced glutathione (GSH), nitric oxide (NO), superoxide dismutase (SOD), brain-derived neurotrophic factor (BDNF) as well as heme oxygenase-1 (HO-1) were measured. Moreover, brain histopathology examinations associated with immunohistochemistry determination of brain caspase-3 were also evaluated. Furthermore, docking simulation was adapted to understand the inhibitory role of FA on caspase-3 and NO synthase. Compared to DEHP-intoxicated rats, FA-treated rats displayed improved cognitive memory associated with a reduced anxious state. Also, the redox state was maintained with increased BNDF levels. These changes were confirmed by restoring the normal architecture of brain tissue and a decrement in the immunohistochemistry caspase-3. In conclusion, FA has potent antioxidant and antiapoptotic properties that confirm the neuroprotective activity of FA, with a possible prospect for its therapeutic capabilities and nutritional supplement value.

Keywords: Cognition; DEHP; Docking; Ferulic acid; Neurotoxicity; Oxidative stress.

Fig. 1

Schematic diagram for the experimental design. This study was conducted for 30 days and divided into four groups, including group 1: control rats received corn oil, group 2: DEHP (300 mg/kg), group 3: DEHP (300 mg/kg) + FA (50 mg/kg), and group 4: (50 mg/kg). All agents were given per os, with FA administered 1 h before DEHP. All groups were subjected to behavioral assessments 24 h after the last dose from the 31st to the 35th. Anxiety-like behavior, spatial working memory, and long-term memory were measured using elevated plus maze, Y-maze, and novel object recognition, respectively. On the 36^th, brain sampling was collected for biochemical analysis, histopathological examination, and FTIR spectroscopy. Furthermore, in silico survey was conducted, such as a molecular docking study and ADMET analysis

Fig. 2

Effect of phthalates (DEHP) and ferulic acid (FA) administration on the anxiety-like behavior of male rats. a Elevated plus maze: number of open-arm entry. b Elevated plus maze: duration in open arm. c Elevated plus maze: number of closed-arm entry. d Elevated plus maze: duration in the closed arm. Data are expressed as mean ± standard error (SE) (one-way analysis of variance (one-way ANOVA) followed by Tuckey post hoc test for eight rats in each group. *Significantly different from the control group, P < 0.05. # Significantly different from the DEHP group, P < 0.05

Fig. 3

Effect of DEHP and FA administration on the spatial working memory and recognition memory of male rats. a Y-maze test: number of arm entries. b Y-maze test: spontaneous alternation percentage (SAP). c Novel object recognition test: preference for the novel object. d Novel object recognition test: discrimination ratio. Data are expressed as mean ± standard error (SE) using one-way analysis of variance (one-way ANOVA) followed by Tuckey post hoc test for eight rats in each group. *Significantly different from the control group, P < 0.05. #Significantly different from the DEHP group, P < 0.05

Fig. 4

Effect of DEHP and FA administration on oxidative stress marker level in the male rat’s brain. a Brain-derived neurotrophic factor (BDNF). b Superoxide dismutase (SOD). c Reduced glutathione (GSH). d Nitric oxide (NO). e Malondialdehyde (MDA). f Heme oxygenase-1 (HO-1). Data are expressed as mean ± standard error (SE) using one-way analysis of variance (one-way ANOVA) followed by Tuckey post hoc test for eight rats in each group. *Significantly different from the control group, P < 0.05. #Significantly different from the DEHP group, P < 0.05. &Significantly different from the DEHP + FA group, P < 0.05

Fig. 5

Brain sections of male albino rats. H&E × 400. a Cerebral cortex section of control rats showed normal structure and distribution of neurons and neuroglia on neuropil. b Cerebral cortex sections of DEHP-intoxicated rats revealed shrunken pyknotic pyramidal neurons (black arrow) and neuroglia (yellow arrow) with pericellular spaces. c Cerebral cortex of DEHP + FA rats revealed marked maintenance of normally shaped pyramidal neurons that had vesicular central nuclei (yellow arrows) and few neurons still pyknotic (black arrow). d FA-treated rats had cerebral cortex with normal structure and distribution of neurons and neuroglia on neuropil. e Hippocampus sections of control rats showed the normal structure of molecular layer (M) that consisted of neurons and neuroglia (yellow arrowhead), pyramidal cell layer (P) that formed of linear arranged triangular-shaped neurons with vesicular central nuclei (black arrow), and polymorphic layer (PL) formed of neurons and neuroglia (yellow arrowhead). f Hippocampus of DEHP-intoxicated rats revealed neuropil vacuolation (black arrowhead), degenerated and pyknotic pyramidal neurons (black arrow) with perineural spaces (green star), and disarrangement of triangular-shaped neurons (arched black arrow). g Hippocampus of DEHP + FA showed obvious recovery in the form of diminished neuropil vacuolation (yellow arrowhead), and many pyramidal neurons appeared nearly normal (yellow arrows) except for a few neurons that appeared pyknotic with pericellular space (black arrow). h FA-treated rats had normal hippocampus layers, molecular (M), pyramidal (P) with triangular-shaped neurons (black arrow), and polymorphic (PL) cell layers, respectively. i Cerebellar cortex of control rats consisted of molecular (M) that had stellate neurons (yellow arrowhead), Purkinje (P) that had pear-shaped neurons with large central vesicular nuclei (black arrows), and granular (G) cell layers, respectively. j Cerebellar cortex of DEHP-intoxicated rats revealed pyknotic and degenerated Purkinje cells (black arrow) with perineuronal spaces (black arrowhead) and pericellular spaces (red arrowhead) in the molecular layer. k Cerebellar cortex of DEHP + FA showed marked maintenance of normal pear-shaped Purkinje cells (black arrows); most of the stellate cells appeared nearly normal (red arrowhead), but few Purkinje cells (red arrows) and stellate cells (black arrowhead) still pyknotic with perineural space. l FA-treated rats had normal cerebellar cortex layers, respectively, molecular (M), Purkinje (P), and granular (G) cell layers

Fig. 6

Immunohistochemical caspase-3-stained brain sections × 400. a, e, and i Control rats’ cerebral cortex, hippocampus, and cerebellar cortex had negative caspase-3 immunoexpression. b, f, and j DEHP-intoxicated rats’ cerebral cortex, hippocampus, and cerebellar cortex revealed strong positive caspase-3 immunoexpression (black arrow). c, g, and k DEHP + FA-treated rats’ cerebral cortex, hippocampus, and cerebellar cortex showed mild caspase-3 immunoreaction (black arrow). d, h, and l FA-treated rat’s cerebral cortex, hippocampus, and cerebellar cortex showed negative caspase-3 immunoexpression

Fig. 7

Effect of DEHP and FA administration on the percent area covered by caspase-3-positive immunoreactive cells within the brain of rats. The results were expressed as mean ± SE. *Significantly different from the control group. ^#Significantly different from the DEHP-intoxicated group. P value ≤ 0.05

Fig. 8

Effect of FA on wave number alterations in brain tissue of DEHP-intoxicated rats. Representative infrared spectra of lyophilized brain samples from different experimental groups

Fig. 9

Two-dimensional interaction of FA with the active site of a caspase-3 and b NO synthase. c color codes for interactions