Azilsartan Attenuates 3-Nitropropinoic Acid-Induced Neurotoxicity in Rats: The Role of IĸB/NF-ĸB and KEAP1/Nrf2 Signaling Pathways

Abstract

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder characterized by motor, psychiatric and cognitive symptoms. Injection of 3-nitropropionic acid (3-NP) is a widely used experimental model for induction of HD. The current study aimed to inspect the potential neuroprotective properties of azilsartan (Azil), an angiotensin II type 1 receptor blocker (ATR1), in 3-NP-induced striatal neurotoxicity in rats. Rats were randomly allocated into five groups and treated for 14 days as follows: group I received normal saline; group II received Azil (10 mg/kg, p.o.); group III received 3-NP (10 mg/kg, i.p); group IV and V received Azil (5 or 10 mg/kg, p.o, respectively) 1 h prior to 3-NP injection. Both doses of Azil markedly attenuated motor and behavioural dysfunction as well as striatal histopathological alterations caused by 3-NP. In addition, Azil balanced striatal neurotransmitters levels as evidenced by the increase of striatal gamma-aminobutyric acid content and the decrease of glutamate content. Azil also amended neuroinflammation and oxidative stress via modulating IĸB/NF-ĸB and KEAP1/Nrf2 downstream signalling pathways, as well as reducing iNOS and COX2 levels. Moreover, Azil demonstrated an anti-apoptotic activity by reducing caspase-3 level and BAX/BCL2 ratio. In conclusion, the present study reveals the neuroprotective potential of Azil in 3-NP-induced behavioural, histopathological and biochemical changes in rats. These findings might be attributed to inhibition of ATR1/NF-κB signalling, modulation of Nrf2/KEAP1 signalling, anti-inflammatory, anti-oxidant and anti-apoptotic properties.

Keywords: 3-nitropropionic acid; Azilsartan; Huntington’s disease; NF-ĸB; Nrf2; Rat.

Fig. 1

Timeline of the experimental design. 3-NP 3-nitropropionic acid, Azil azilsartan

Fig. 2

Effect of Azil (5 or 10 mg/kg) on 3-NP-induced behavioral abnormalities. A Latency time, B Ambulation frequency, C Rearing frequency and D Grip strength. Each bar represents mean ± S.D. (n = 9). Statistical analysis was carried out by one-way ANOVA followed by Tukey’s multiple comparisons test. a: significantly different from the control group at P ≤ 0.05. b: significantly different from 3-NP-treated group at P ≤ 0.05. Azil azilsartan, 3-NP 3-nitropropionic acid

Fig. 3

Effect of Azil (5 or 10 mg/kg) on 3-NP-induced changes in striatal neurotransmitters A glutamate and B GABA. Each bar represents mean ± S.D. (n = 6). Statistical analysis was carried out by one-way ANOVA followed by Tukey’s multiple comparisons test. a: significantly different from the control group at P ≤ 0.05. b: significantly different from 3-NP-treated group at P ≤ 0.05. c: significantly different from Azil (5 mg/kg)-treated group at P ≤ 0.05. Azil Azilsartan, 3-NP 3-nitropropionic acid, GABA γ-amino butyric acid

Fig. 4

Effect of azilsartan (5 or 10 mg/kg) on 3-NP-induced changes in striatal NF-κB signalling pathway parameters. A Densitometric analysis of the Western blots, B ATR1 mRNA expression, C NF-κB p65 expression, D IκB expression, E TNF-α content, F IL-1β content, G COX-2 content, and H iNOS content. Each bar represents mean ± S.D. (n = 6). Statistical analysis was carried by one-way ANOVA followed by Tukey’s multiple comparisons test. a: significantly different from the control group at P ≤ 0.05. b: significantly different from 3-NP-treated group at P ≤ 0.05. c: significantly different from Azil (5 mg/kg)-treated group at P ≤ 0.05. Azil Azilsartan, 3-NP 3-nitropropionic acid, AT1R angiotensin II receptor type 1, IκB inhibitor of kappa-B, NF-κB nuclear factor kappa-b P65, TNF-α tumor necrosis factor-alpha, IL-1β interleukin-1 beta, COX-2 cyclooxygenase-2 and iNOS inducible nitric oxide synthases

Fig. 5

Effect of Azil (5 or 10 mg/kg) on 3-NP-induced changes in striatal Nrf2 signalling pathway parameters. A Densitometric analysis of the Western blots, B MDA content, C SDH activity, D KEAP1 expression, E Nrf2 expression, F NQO-1 content, and G HO-1 content. Each bar represents mean ± S.D. (n = 6). Statistical analysis was carried by one-way ANOVA followed by Tukey’s multiple comparisons test. a: significantly different from the normal control group at P ≤ 0.05. b: significantly different from 3-NP-treated group at P ≤ 0.05. c: significantly different from Azil (5 mg/kg)-treated group at P ≤ 0.05. Azil Azilsartan, 3-NP 3-nitropropionic acid, MDA malondialdehyde, SDH succinate dehydrogenase, KEAP1 Kelch-like ECH-associated protein -1, Nrf2 nuclear factor erythroid 2-related factor 2, NQO-1: NAD(P)H quinone oxidoreductase-1and HO-1: heme oxygenase-1

Fig. 6

Effect of azilsartan (5 or 10 mg/kg) on 3-NP-induced changes in striatal apoptosis. A caspase-3 content, B BAX content, C BCL2 content, and D BAX/BCL2 ratio. Each bar represents mean ± S.D. (n = 6). Statistical analysis was carried out by one-way ANOVA followed by Tukey’s multiple comparisons test. a: significantly different from the normal control group at P ≤ 0.05. b: significantly different from 3-NP-treated group at P ≤ 0.05, c: significantly different from Azil (5 mg/kg)-treated group at P ≤ 0.05. Azil Azilsartan, 3-NP 3-nitropropionic acid, BAX Bcl-2-associated X protein and BCL2 B-cell lymphoma 2

Fig. 7

Effect of Azil (5 or 10 mg/kg) on 3-NP-Induced Histopathological Changes. Photomicrographs of sections A and B show normal histological structure of the striatum of rats receiving saline (control group). Photomicrographs of sections C and D show the normal histological structure of the striatum of rats receiving Azil alone. Photomicrograph of sections E of 3-NP treated rats shows congested blood vessels (arrow) with focal gliosis and F neuronal and perivascular lymphocytic infiltration and edema (arrow). Photomicrograph of sections G and H of Azil 5 mg-treated rat shows few degenerating cells (arrow) and H mild neuronal edema. Photomicrograph of section I and J of Azil 10 mg-treated rat shows apparently normal striatum with no histopathological changes

Fig. 8

Effect of Azil (5 or 10 mg/kg) on 3-NP-induced changes in GFAP expression. Control (A) and Azil (B) groups exhibited mild GFAP expression in the striatum region. In contrast. 3-NP group (C) exhibited significant increase in GFAP expression. Lower expression levels were detected in 3-NP + Azil 5 (D) and 3-NP + Azil 10 (E) groups. F: GFAP immunostaining area %. Each bar represents mean ± S.D. (n = 3). Statistical analysis was carried out by Kruskal–Wallis ANOVA followed by Dunn’s multiple comparison test. a: significantly different from the normal control group at P ≤ 0.05. b: significantly different from 3-NP-treated group at P ≤ 0.05, c: significantly different from Azil (5 mg/kg)-treated group at P ≤ 0.05. Azil Azilsartan, 3-NP 3-nitropropionic acid, GFAP glial fibrillary acidic protein