Ameliorative effects and possible molecular mechanisms of action of fibrauretine from Fibraurea recisa Pierre on d-galactose/AlCl3-mediated Alzheimer's disease

Abstract

Fibrauretine is one of the main active ingredients from the rattan stems of Fibraurea recisa Pierre It exhibits a series of significant pharmacological effects. The present study aimed to evaluate the potential anti Alzheimer's disease (AD) effects of fibrauretine on a d-galactose/AlCl₃-induced mouse model, and the underlying mechanisms of action were further investigated for the first time. Our results showed that pretreatment with fibrauretine significantly improved the ability of spatial short-term working memory in the model mice during the Y-maze test, as well as the abilities of spatial learning and memory during the Morris water maze. The levels of brain tissue amyloid (Aβ), P-Tau, Tau and acetylcholinesterase (AchE) were evidently increased in d-galactose/AlCl₃-intoxicated mice, and these effects were reversed by fibrauretine. In contrast, a significant increase in the levels of the neurotransmitter acetylcholine (Ach) and choline acetyl transferase (ChAT) was observed in the fibrauretine-treated groups compared with the model group. Neuronal oxidative stress, evidenced by increased malondialdehyde (MDA) and nitric oxide (NO) levels and a decline in glutathione (GSH), catalase (CAT) and superoxide dismutase (SOD) activity, was significantly alleviated by fibrauretine pretreatment. The suppression of the neuroinflammatory response by fibrauretine was realized not only by the decrease in the levels of tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the brain tissues and by the enzyme-linked immunosorbent assay (ELISA) but also by the protein expression levels of nuclear factor-κB (NF-κB), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS), which were measured by immunohistochemistry and western blotting. In addition, the protein expression levels of inflammatory factors interleukin-33 (IL-33) and ST2 in the brain tissues were detected by immunohistochemistry. Furthermore, the effects of western blotting demonstrated that the administration of fibrauretine significantly suppressed the protein expression levels of caspase-3, cleaved caspase-3, and Bax and increased the protein expression levels of Bcl-2, and the results of the H&E and TUNEL assay all suggested the inhibition of apoptosis in the neurons. The results clearly suggest that the underlying molecular mechanisms of action of the fibrauretine-mediated alleviation of d-galactose/AlCl₃-induced Alzheimer's disease may involve antioxidant, anti-inflammatory, and anti-apoptotic effects.

Fig. 1. Effect of fibrauretine and donepezil on total arm entries (A); spontaneous alternation (B); escape latency (C); crossing times (D); time in target quadrant (E) from d-galactose/AlCl₃-induced AD mice. All data were expressed as the mean ± SD, n = 10. *p < 0.05, **p < 0.01 vs. control group; ^#p < 0.05, ^##p < 0.01 vs. model group.

Fig. 2. Effects of fibrauretine and donepezil on the level of brain Aβ (A); the level of brain Ach (B); the level of brain AchE (C); the level of brain Tau (D); the level of brain P-Tau (E); and the level of brain ChAT (F) in d-galactose/AlCl₃-induced AD. All data are expressed as the mean ± SD, n = 10. *p < 0.05, **p < 0.01 vs. control group; ^#p < 0.05, ^##p < 0.01 vs. model group.

Fig. 3. Effects of fibrauretine and donepezil on the levels of malondialdehyde (MDA) (A); peroxide dismutase (SOD) (B); catalase (CAT) (C); nitric oxide (NO) (D); and glutathione (GSH) (E) in d-galactose/AlCl₃ induced AD. All data are expressed as the mean ± SD, n = 10. *p < 0.05, **p < 0.01 vs. control group; ^#p < 0.05, ^##p < 0.01 vs. model group.

Fig. 4. Morphological changes in d-galactose/AlCl₃ induced AD mice. Brain tissue sections stained by H&E (×400) (A); brain tissue sections stained by TUNEL (×400) (B); the presence of TUNEL positive cells measured by the image analyser (C); all data are expressed as the mean ± SD, n = 10. *p < 0.05, **p < 0.01 vs. control group; ^#p < 0.05, ^##p < 0.01 vs. model group.

Fig. 5. Effects of fibrauretine and donepezil on the levels of TNF-α (A); IL-1β (B). Effects of fibrauretine and donepezil on the expression of: IL-33 (C); ST2 (D); NF-κB (E); COX-2 (F); iNOS (G); Bax (H) and Bcl-2 (I). The column chart shows the antibodies stained area of: IL-33 (J); ST2 (K); NF-κB (L); COX-2 (M); iNOS (N); Bax (O) and Bcl-2 (P). The protein expression was determined by immunohistochemistry in brain tissues from all groups. *p < 0.05, **p < 0.01 vs. control group; ^#p < 0.05, ^##p < 0.01 vs. model group.

Fig. 6. Effects of fibrauretine and donepezil on the protein expression of COX-2, iNOS (A); Bax, Bcl-2 (B); caspase-3, cleaved caspase-3 (G); and NF-κB (H). The column chart shows antibody relative intensity of COX-2 (C); iNOS (D); Bax (E); Bcl-2(F); cleaved caspase-3 (I); NF-κB (J). The protein expression was examined by the western blotting analysis in brain tissues from all experimental groups. *p < 0.05, **p < 0.01 vs. control group; ^#p < 0.05, ^##p < 0.01 vs. model group.