Melatonin Protects against the Side-Effects of 5-Fluorouracil on Hippocampal Neurogenesis and Ameliorates Antioxidant Activity in an Adult Rat Hippocampus and Prefrontal Cortex

Abstract

Melatonin is an endogenous hormone that exhibits antioxidant functions and neuroprotective effects. The hippocampus and the prefrontal cortex (PFC) play an important role linked to working memory. 5-fluorouracil (5-FU) can induce oxidative stress and reduce neurogenesis in the subgranular zone (SGZ) of the dentate gyrus in a rat hippocampus and these alterations are related to working memory deficits. This study aimed to determine the effect of melatonin on 5-FU-induced oxidative stress that interferes with the antioxidant enzymes and protein expression levels in a rat hippocampus and PFC. A total of 68 male Sprague Dawley rats were divided into four groups: vehicle, 5-FU, melatonin and melatonin+5-FU groups. Rats were administered 5-FU (25 mg/kg, i.v.) on days 9, 12, 15, 18 and 21 and received melatonin (8 mg/kg, i.p.) at 19:00 from day 1 to day 21 of the experiment. Lipid peroxidation was assessed by measuring malondialdehyde (MDA) levels. Antioxidant enzyme levels including glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD) were determined. p21 immunofluorescence staining and Western blotting were used to detect the cell cycle arrest and protein expression of the nuclear factor erythroid 2-related factor 2 (Nrf2), doublecortin (DCX) and brain derived neurotrophic factor (BDNF), respectively. The results showed that melatonin reduced the number of p21-positive cells in the SGZ of the dentate gyrus and increased Nrf2, DCX and BDNF protein expression in rats treated with 5-FU. Moreover, melatonin restored antioxidant enzyme levels and reduced oxidative stress in the hippocampus and PFC caused by 5-FU. These findings reveal a mechanism of the neuroprotective properties of melatonin against 5-FU in a rat hippocampus and PFC.

Keywords: 5-fluorouracil; antioxidant enzymes; hippocampus; melatonin; neurogenesis; oxidative stress; prefrontal cortex; rat.

Figure 1

Timeline of drug administration. Red arrows represent each single intravenous (i.v.) injection of a 5-FU/Saline solution. The blue bracket represents the period of time for intraperitoneal (i.p.) injections of a melatonin/ethanol solution. Animals were killed and the brains were removed on day 25 as shown by the black arrow.

Figure 2

Immunofluorescence staining of a cell cycle arrest. Green p21-positive cells are indicated with arrowheads in the SGZ of the dentate gyrus (A–D). Sections were counterstained with red nuclear dye, propidium iodide (PI). Inserted images show p21 immunofluorescence staining under high magnification (Bar scales: 50 μm, 40×). The number of p21-positive cells in the 5-FU group was significantly higher than those in the vehicle group (E). ## p < 0.01 compared with the vehicle group. * p < 0.05, *** p < 0.001 compared with the 5-FU group. Subgranular zone: SGZ; granule cell layer: GCL; molecular layer: ML.

Figure 3

MDA (A), CAT (B), GPX (C) and SOD (D) levels in the hippocampus. ### p < 0.001 compared with the vehicle group. ** p < 0.01 and *** p < 0.001 compared with the 5-FU group. Malondialdehyde: MDA; catalase: CAT; glutathione peroxidase: GPX; superoxide dismutase: SOD.

Figure 4

MDA (A), CAT (B), GPX (C) and SOD (D) levels in the PFC. # p < 0.05 and ### p < 0.001 compared with the vehicle group. * p < 0.05, ** p < 0.01 and *** p <0.001 compared with the 5-FU group. Malondialdehyde: MDA; catalase: CAT; glutathione peroxidase: GPX; superoxide dismutase: SOD.

Figure 5

DCX (A), Nrf2 (B) and BDNF (C) protein expression in the hippocampus. ## p < 0.01 and ### p < 0.001 compared with the vehicle group. * p < 0.05 and *** p < 0.001 compared with the 5-FU group. Doublecortin: DCX; nuclear factor erythroid 2-related factor 2: Nrf2; brain derived neurotrophic factor: BDNF.

Figure 6

Nrf2 (A) and BDNF (B) protein expression in the PFC. # p < 0.05 and ### p < 0.001 compared with the vehicle group. * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with the 5-FU group. Doublecortin: DCX; nuclear factor erythroid 2-related factor 2: Nrf2; brain derived neurotrophic factor: BDNF.