Antioxidant and anti-inflammatory potential of spirulina and thymoquinone mitigate the methotrexate-induced neurotoxicity

Abstract

The objective of this study was to investigate whether the neurotoxic effects caused by methotrexate (MTX), a frequently used chemotherapy drug, could be improved by administering Spirulina platensis (SP) and/or thymoquinone (TQ). Seven groups of seven rats were assigned randomly for duration of 21 days. The groups consisted of a control group that was given saline only. The second group was given 500 mg/kg of SP orally; the third group was given 10 mg/kg of TQ orally. The fourth group was given a single IP dose of 20 mg/kg of MTX on the 15^th day of the experiment. The fifth group was given both SP and MTX, the sixth group was given both TQ and MTX, and the seventh group was given SP, TQ, and MTX. After MTX exposure, the study found that AChE inhibition, depletion of glutathione, and increased levels of MDA occurred. MTX also decreased the activity of SOD and CAT, as well as the levels of inflammatory mediators such as IL-1, IL-6, and tumor necrosis factor-α. MTX induced apoptosis in brain tissue. However, when MTX was combined with either SP or TQ, the harmful effects on the body were significantly reduced. This combination treatment resulted in a faster return to normal levels of biochemical, oxidative markers, inflammatory responses, and cell death. In conclusion, supplementation with SP or TQ could potentially alleviate MTX-induced neuronal injury, likely due to their antioxidant, anti-inflammatory, and anti-apoptotic effects.

Keywords: Antioxidants; Brain; Inflammation; Methotrexate; Spirulina; Thymoquinone.

Fig. 1

Effect of spirulina (SP) and/or thymoquinone (TQ) on serum AChE activity and IL-1β, IL-6, TNF-α levels against methotrexate (MTX) induced neurotoxicity in rats (n=7). A (AchE); B (IL-1β); C ((IL-6); D (TNF-α). Statistical significance was determined by asterisks, with * indicating p < 0.05, *** indicating p < 0.001, and **** indicating p < 0.0001, compared to the MTX-treated groups

Fig. 2

Effect of spirulina (SP), thymoquinone (TQ) and methotrexate (MTX) on antioxidant parameters in brain tissues in rats (n=7). A (MDA); B (CAT); C (SOD); D (GSH). Statistical significance was determined by asterisks, with * indicating p < 0.05, ** indicating p < 0.01, *** indicating p < 0.001, and **** indicating p < 0.0001, compared to the MTX-treated groups

Fig. 3

Histopathological examination of the cerebrum was conducted on different groups (H&E stain). The analysis revealed the presence of seemingly normal neurons in the control group (A), as well as in the SP-treated (B) and TQ-treated (C) groups. However, significant abnormalities were observed in the MTX-treated group, characterized by neurofibrillary tangles (indicated by a black arrow) (D), neuronal degeneration (indicated by a red arrow) (E), and neuropile vacuolation basophilic necrotic neuron (indicated by a blue arrow) (F). Nevertheless, a small number of shrunken apoptotic neurons (indicated by yellow arrow) were observed in the SP + MTX-treated (G), TQ + MTX-treated (H), and SP + TQ + MTX combination (I) groups

Fig. 4

Histopathological examination of the cerebellum was conducted in various groups (H&E stain). The histological findings revealed nearly normal Purkinje neurons in the control group (A), as well as in the SP-treated (B) and TQ-treated (C) groups, with minimal degeneration observed in a small number of neurons. Molecular (M), Purkinje (P) and granular (G) layers in the control groups. In contrast, the cerebellum of rats treated with MTX exhibited significant aberrations, including Purkinje cell shrinkage (indicated by black arrows) (D), loss of cells (indicated by red arrows) (E), and reduced presence of dendrites (indicated by blue arrows) (F). However, in the SP (G), TQ (H), and combination (I) groups co-treated with MTX, a notable reduction in degenerated/apoptotic Purkinje cells was observed

Fig. 5

Histopathological changes in the hippocampus (H&E). A, B, and C. Normal hippocampal structure observed in the (control, SP, and TQ groups), comprising the molecular layer (M), pyramidal cell layer (P), and polymorphic layer (Po). Additionally, a few dark cells are discernible. D; MTX-treated group exhibiting pyramidal cell layer (black arrows) degeneration and reduced thickness (P), along with the presence of degenerated dark cells. In the SP + MTX-treated (E), TQ + MTX-treated (F), and SP + TQ + MTX combination (G) groups an amelioration of MTX-induced changes were observed

Fig. 6

Photomicrographs of Bax immunohistochemical staining in the Cerebrum after MTX intoxication. A The normal control group shows BAX neurons. B SP group shows BAX negative neurons. C TQ group shows BAX negative neurons. D, E, and F MTX intoxicated group shows BAX-positive neurons. G SP + MTX treatment group, H TQ + MTX treatment group, and I SP + TQ + MTX treatment group. Black arrows show Bax positive neurons. White arrows indicate Bax -negative neurons

Fig. 7

Immunohistochemical staining of Bax in rat cerebellum neurons. A The normal control group shows BAX neurons. B SP group BAX negative neurons. C TQ group shows BAX negative neurons. D, E, and F MTX intoxicated group shows BAX-positive neurons. G SP + MTX treatment group, H TQ + MTX treatment group, and I SP + TQ + MTX treatment group. Black arrows show Bax positive neurons. White arrows indicate Bax -negative neurons

Fig. 8

Immunohistochemistry staining of Bax in rat hippocampal region. A The normal control group shows BAX-negative neurons. B SP group BAX negative neurons. C TQ group BAX negative neurons. D, E, F The expression of Bax in the hippocampal region after MTX intoxication is shown by immunohistochemistry staining. G SP + MTX treatment group, H TQ + MTX treatment group, and I SP + TQ + MTX treatment group. Black arrows show Bax positive neurons. White arrows indicate Bax -negative neurons

Fig. 9

Scoring of Bax immunostaining intensity in the cerebrum, cerebellum, and hippocampal regions for comparative analysis among all experimental groups. A (cerebrum Bax level score); B (Cerebellum Bax level score); C (Hippocampus Bax level score). Statistical significance was determined by asterisks, with * indicating p < 0.05, ** indicating p < 0.01, *** indicating p < 0.001, **** indicating p < 0.0001, and ns; non-significant compared to the MTX-treated groups