Memantine/Rosuvastatin Therapy Abrogates Cognitive and Hippocampal Injury in an Experimental Model of Alzheimer's Disease in Rats: Role of TGF-β1/Smad Signaling Pathway and Amyloid-β Clearance

Abstract

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder of complex pathogenesis and multiple interacting signaling pathways where amyloidal-β protein (Aβ) clearance plays a crucial role in cognitive decline. Herein, the current study investigated the possible modulatory effects of memantine/ rosuvastatin therapy on TGF-β1/p-Smad/p21 signaling pathway and their correlation to the blood brain barrier transporters involved in Aβ-clearance and microRNAs as a novel molecular mechanism in AD treatment. AD was induced by a single intracerebroventricular streptozotocin injection (ICV-STZ, 3 mg/kg) in rats and drug therapy was continued for 28 days after AD induction. Efficacy was monitored by applying a battery of behavioral assessments, as well as biochemical, histopathological, molecular and gene expression techniques. The upregulated TGF-β1-signaling in the untreated rats was found to be highly correlated to transporters and microRNAs governing Aβ-efflux; ABCA1/miRNA-26 and LRP1/miRNA-205 expressions, rather than RAGE/miRNA-185 controlling Aβ-influx; an effect that was opposed by the tested drugs and was found to be correlated with the abolished TGF-β1-signaling as well. Combined memantine/rosuvastatin therapy ameliorated the STZ evoked decreases in escape latency and number of crossovers in the Morris water maze test, % spontaneous alternation in the Y-maze test, and discrimination and recognition indices in the object recognition test. The evoked behavioral responses were directly related to the β-amyloid accumulation and the alteration in its clearance. Additionally, drug treatment increased brain glutathione and decreased malondialdehyde levels. These findings were histopathologically confirmed by a marked reduction of gliosis and restoration of neuronal integrity in the CA1 region of the hippocampus of the AD rats. These findings implicated that the memantine/rosuvastatin combination could offer a new therapeutic potential for AD management by abrogating the TGF-β1/p-Smad2/p21 pathway and regulating Aβ-clearance.

Keywords: Alzheimer; Blood-brain barrier transporters; MicroRNA; TGF-β; β-amyloid.

Fig. 1

Schematic diagram for the study timeline. (ICV-STZ: Intracerebroventricularly injection of freshly prepared streptozotocin, MWM: Morris water maze, ORT: The object recognition test)

Fig. 2

Effect of ICV-STZ (3 mg/kg) administration on the mean escape latency (sec) in the MWM test in male rats. Data are presented as mean ± S.E.M (n = 8 control, n = 26 ICV-STZ) and were analyzed statistically using two-way repeated measures ANOVA followed by Bonferroni’s post hoc test (P < 0.05) as compared with the corresponding value of control (*), 1^st day within the same group (a), 2^nd day within the same group (b). Values in parentheses indicate the correlation coefficient using linear regression analysis for control and ICV-STZ-treated rats

Fig. 3

Effect of memantine (30 mg/kg/day), rosuvastatin (20 mg/kg/day), and their combination on the escape latency during acquisition in the ICV-STZ treated rats. Memantine (30 mg/kg/day), rosuvastatin (20 mg/kg/day) and their combination were gavaged orally for 28 days after AD model establishment. Data are presented as mean ± S.E.M (n = 6–8) and were analyzed statistically using two-way repeated measures followed by Bonferroni’s post hoc test (P < 0.05). As compared with (*) control, (#) ICV-STZ, ( $ ) ICV-STZ-memantine, (&) ICV-STZ-rosuvastatin-treated groups; (Ψ and Φ) as compared to day 1 and day 2, respectively, (δ) indicate potentiating interaction using Factorial Design Drug interaction test

Fig. 4

Effect of memantine (30 mg/kg/day), rosuvastatin (20 mg/kg/day), and their combination on amyloid β, TGF-β1/Smad2/p21 in the hippocampus of ICV-STZ rats. (A) Aβ 1−42 , (B) TGF-β1, (C) p-Smad2, and (D) p21 gene. Results were normalized to β-actin as a reference gene. Data are presented as means of 6–8 rats ± S.E.M and were analyzed statistically using one-way ANOVA followed by Tukey’s post hoc test (P < 0.05). As compared with (*) control, (#) ICV-STZ, ( $ ) ICV-STZ-memantine, (&) ICV-STZ-rosuvastatin-treated groups. Memantine (30 mg/kg/day), rosuvastatin (20 mg/kg/day) and their combination were gavaged orally for 28 days after model establishment. (Aβ 1−42 : amyloid β 1−42 ; TGF-β1: transforming growth factor β1; p-Smad2: phospho-Smad2)

Fig. 5

Effect of memantine (30 mg/kg/day), rosuvastatin (20 mg/kg/day), and their combination on gene expression of BBB transporters in rats with ICV-STZ-induced AD. (A-C): Comparison between different groups according to mRNA expression of (A) ABCA1, (B) LRP1 and (C) RAGE. (D): Comparison between the effects of each drug on different BBB transporters. Results were normalized to β-actin as a reference gene. Data are presented as mean ± S.E.M (n = 6–8) and were analyzed statistically using one-way ANOVA followed by Tukey’s post hoc test (P < 0.05). As compared with (*) control, (#) ICV-STZ, ($) ICV-STZ-memantine, (&) ICV-STZ-rosuvastatin-treated groups; a: Statistically significant compared to ABCA1 expression, b: Statistically significant compared to LRP1 expression. λ: indicates additive interaction using Factorial Design Drug interaction test. Memantine (30 mg/kg/day), rosuvastatin (20 mg/kg/day) and their combination were gavaged orally for 28 days after model establishment. (ABCA1: ATP-binding cassette transporter A1, LRP1: LDL receptor–related protein 1, RAGE: receptor for advanced glycation end products)

Fig. 6

Effect of memantine (30 mg/kg/day), rosuvastatin (20 mg/kg/day), and their combination on the expression of miRNAs regulating BBB transporters in rats with ICV-STZ-induced AD. A-C: Comparison between different groups according to the expression of (A) miRNA-26, (B) miRNA-205 and (C) miRNA-185. (D): Comparison between the effects of each drug on different miRNAs. Results were normalized to U6 as a reference gene. Data are presented as means of 6–8 rats ± S.E.M and were analyzed statistically using one-way ANOVA followed by Tukey’s post hoc test (P < 0.05). As compared with (*) control, (#) ICV-STZ, ($) ICV-STZ-memantine, (&) ICV-STZ-rosuvastatin-treated groups; a: Statistically significant compared to 26 expression, b: Statistically significant compared to 205 expression. Memantine (30 mg/kg/day), rosuvastatin (20 mg/kg/day) and their combination were gavaged orally for 28 days after model establishment. (ABCA1: ATP-binding cassette transporter A1, LRP1: LDL receptor–related protein 1, RAGE: receptor for advanced glycation end products)

Fig. 7

Effect of memantine (30 mg/kg/day), rosuvastatin (20 mg/kg/day), and their combination on oxidative stress in rats with ICV-STZ-induced AD. (A) GSH and (B) MDA. Data are presented as means of 6–8 rats ± S.E.M and were analyzed statistically using one-way ANOVA followed by Tukey’s post hoc test (P < 0.05). As compared with (*) control, (#) ICV-STZ, ($) ICV-STZ-memantine, (&) ICV-STZ-rosuvastatin-treated groups. Memantine (30 mg/kg/day), rosuvastatin (20 mg/kg/day) and their combination were gavaged orally for 28 days after model establishment. (GSH: reduced glutathione; MDA: malondialdehyde)

Fig. 8

Photomicrographs and morphometric analysis of hippocampus CA1 region stained with hematoxylin and eosin H&E (X400) showing the effect of different treatments on ICV-STZ rats. Panel I : sections from control rats (A), ICV-STZ rats (B), ICV-STZ-memantine-treated rats (30 mg/kg/day) (C), ICV-STZ-rosuvastatin treated rats (20 mg/kg/day) (D) and ICV-STZ-combination-treated rats (E). Black arrows: Normal nerve cells, yellow arrows: pericellular space, green arrows: degenerated, pyknotic neurons, white arrows: necrotic neurons associated with satellitosis, and red arrows: necrotic neurons associated with neuronophagia. Panel II shows the hippocampal differential count of normal neurons (F), degenerated neurons (G), and glial cells (H) in the ICV-STZ rats. Data are presented as means of 6–8 measures ± S.E.M and were analyzed statistically using one-way ANOVA followed by Tukey’s post hoc test (P < 0.05). As compared with (*) control, (#) ICV-STZ, ($) ICV-STZ-memantine, (&) ICV0-STZ-rosuvastatin-treated groups. Memantine (30 mg/kg/day), rosuvastatin (20 mg/kg/day) and their combination were gavaged orally for 28 days after model establishment