Isoimperatorin therapeutic effect against aluminum induced neurotoxicity in albino mice

Abstract

Background: Although aluminum (Al) is not biologically crucial to the human body, classical studies have demonstrated that excessive human exposure to Al can induce oxidative damage, neuroinflammatory conditions and neurotoxic manifestations implicated in Alzheimer's disease (AD). Exposure to Al was reported to be associated with oxidative damage, neuroinflammation, and to enhance progressive multiregional neurodegeneration in animal models. Several plant-derived natural biomolecules have been recently used to reduce the toxic effects of Al through decreasing the oxidative stress and the associated diseases. A good candidate still to be tested is an active natural furanocoumarin, the isoimperatorin (IMP) that can be extracted from Lemon and lime oils and other plants. Here, we examined the neuroprotective effects of IMP on aluminum chloride (AlCl3)-induced neurotoxicity in albino mice. Methods: Twenty-four male albino mice were used in this study. Mice were randomly devided into 5 groups. The first group was given distilled water as a control, the second group was given AlCl3 orally (10 mg/wt/day) starting from the 2nd week to the end of the 6th week, the third group received AlCl3 orally and IMP interperitoneally, i. p. (30 mg/wt/day) starting from week 2 till week 6 where IMP was supplement 1st and then 4 h later AlCl3 was given to mice. The fourth group received the control (IMP 30 mg/wt, i. p.) from the 2nd week till the end of the experiment. Rodent models of central nervous system (CNS) disorders were assessed using object location memory and Y-maze tests in 6th week began. Essential anti-inflammatory and oxidative stress indicators were evaluated, including interleukin-1 β (IL-1β), tumor necrosis factor α (TNF-α), malondialdehyde (MDA), total antioxidant capacity (TAC), and catalase activity (CAT). In addition, serum levels of brain neurotransmitters such as corticosterone, acetylcholine (ACh), dopamine and serotonin in brain homogenates were measured calorimetrically. Results: The study results revealed that the daily treatment of AlCl3 upregulated the TNF-α and IL-1β levels, increased MDA accumulation, and decreased TAC and CAT activity. In addition, aluminum induced a reduction in concentrations of ACh, serotonin and dopamine in the brain. However, IMP significantly ameliorates the effect of AlCl3 through modulating the antioxidant and regulating the inflammatory response through targeting Nrf2 (NF-E2-related factor 2) and mitogen-activated protein kinase (MAPK). Conclusion: Thus, IMP might be a promising treatment option for neurotoxicity and neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, which are associated with neuro-inflammation and oxidative stress.

Keywords: ALCL; inflammation; isoimperatorin; neurotoxicity; oxidative stress.

FIGURE 1

Effect of IMP on body and brain weight of experimental animals. (A) Body weight of animal starting date (g). (B) Body weight of the animal at the end of experimental date (g). The difference between the treatment and control groups was statistically significant; *p < 0.05, #p < 0.05 was considered statistically significant. Values are expressed as mean ± SD, n = 3. *p < 0.05 indicates significant differences between the AlCl3 alone group and the control group. #p < 0.05 shows significant differences between AlCl3 alone and IMP with AlCl3.

FIGURE 2

Spatial memory learning of mice. (A) Object place recognition test. (B) Y-maze test. The difference between the treatment and control groups was statistically significant; *p < 0.05, #p < 0.05 was considered statistically significant. Values are expressed as mean ± SD, n = 3. *p < 0.05 indicates that there are significant differences between the AlCl3 alone group and the control group. #p < 0.05 shows significant differences between AlCl3 alone and IMP with AlCl3.

FIGURE 3

Effect of IMP on the level of Ach, Serotonin and Dopamine. (A) Level of acetylcholine concentration (nmol/min/mg protein). (B) Level of dopamine (pg/mg protein). (C) Level of serotonin (pg/mg protein). The difference between the treatment and control groups was statistically significant; *p < 0.05, #p < 0.05 was considered statistically significant. Values are expressed as mean ± SD, n = 3. *p < 0.05 indicates that there are significant differences between the AlCl3 alone group and the control group. #p < 0.05 shows significant differences between AlCl3 alone and IMP with AlCl3.

FIGURE 4

Level of pro-inflammatory cytokines in brain homogenates. (A) Level of TNF-α (pg/mg protein). (B) Level of IL-1β (pg/mg protein). (C) Western blot analysis of NF-кB and the pro-inflammatory cytokine (TNF-α and IL-1β) expression in brain homogenates. The difference between the treated groups and the control group was calculated; *p < 0.05, #p < 0.05 was considered statistically significant. Values are expressed as mean ± SD, n = 3. *p < 0.05 indicates significant differences between AlCl3 treated group and the control group. #p < 0.05 shows significant differences between AlCl3 alone and IMP with AlCl3.

FIGURE 5

Activity of antioxidant enzymes. (A) Formation of MDA in brain homogenates (nmol/g tissue). (B) The activity of catalase (µM of H₂O₂ decomposed/min/mg tissue). (C) Total antioxidant capacity (µmol/g tissues). The difference between the treatment and control groups was statistically significant; *p < 0.05, #p < 0.05 was considered statistically significant. Values are expressed as mean ± SD, n = 3. *p < 0.05 indicates that there are significant differences between the AlCl3 alone group and the control group. #p < 0.05 shows significant differences between AlCl3 alone and IMP with AlCl3.

FIGURE 6

Expression of MAPK kinase in brain homogenates. Western blot analysis of brain homogenates determines the expression of pJNK and pP38—total JNK and total p38 as a loading control. The difference between the treatment and control groups was statistically significant; *p < 0.05, #p < 0.05 was considered statistically significant. Values are expressed as mean ± SD, n = 3. *p < 0.05 means that there are significant differences between the AlCl3-alone group and the control group. #p < 0.05 shows significant differences between AlCl3 alone and IMP with AlCl3.

FIGURE 7

Expression of Nrf2 in brain homogenates. Western blot analysis of Nrf2, HO-1, and NQO-1 expression in control and experimental animals. The difference between the treatment and control groups was statistically significant; *p < 0.05, #p < 0.05 was considered statistically significant. Values are expressed as mean ± SD, n = 3. *p < 0.05 indicates significant differences between the AlCl3-alone group and the control group. #p < 0.05 shows significant differences between AlCl3 alone and IMP with AlCl3.

FIGURE 8

Histopathological analysis of brain tissue. The difference between the treatment and control groups was statistically significant; *p < 0.05, #p < 0.05 was considered statistically significant. Values are expressed as mean ± SD, n = 3. *p < 0.05 indicates significant differences between the AlCl3-alone group and the control group. #p < 0.05 shows significant differences between AlCl3 alone and IMP with AlCl3.

FIGURE 9

Aproposed scheme to summarizing the obtained results. Mechanistic interaction between signaling molecules is shown. In the present study when mice are treated with AlCl3, inflammation occurs, which increases expression of inflammatory markers such as TNF-α, IL-1β, and NF-kB. TNF-1β, NF-kB, and MAPK activation are attenuated by supplementation with IMP. IMP supplementation attenuates Nrf2 and antioxidant enzymes activation during AlCl3 treatment.