Neuroprotective role of morin hydrate on 3-nitropropionic acid-elicited huntington's disease: in vivo investigation of RIPK1/RIPK3/MLKL necroptosis signaling pathway

Abstract

Background: Huntington's disease (HD) is a rare dominantly inheritable autosomal neurodegenerative disease with unclear pathophysiological pathways. In neurodegenerative disorders, including HD, necroptosis plays a significant role in neuronal death. Morin hydrate (MH), a natural bioactive flavonoid, has various pharmacological properties via orchestrating neuroinflammation, apoptosis, and necroptosis. Up to now, there is no extant data on the impact of MH on the necroptotic pathway in HD.

Aim: This research aimed to scrutinize the effect of MH on neurodegeneration initiated by 3-nitropropionic acid (3-NP) administration in rats via modulating necroptosis and apoptosis signaling pathways and compare it with necrosulfonamide (NSA) as a necroptosis inhibitor.

Methods: HD was triggered in male wistar rats by intraperitoneal injection of 3-NP (10 mg/kg/day) for 14 days. Intraperitoneal injection of MH (20 mg/kg/day, i.p.) or NSA (1.65 mg/kg/day, i.p.) an hour prior to 3-NP administration for 14 days. At the end of study, rats were weighed, and their locomotor activity was assessed via grip strength and open field tests. Striata of rats were investigated histologically and immunohistochemically by evaluation the expression levels of glial fibrillary acidic protein (GFAP). Striatal tumor necrosis factor-alpha (TNF-α), caspase 3, and 8 levels were quantified through the ELISA technique, while striatal expression of necroptosis-associated proteins; phosphorylated form of receptor interacting protein kinase 1/3(p-RIPK1, p-RIPK3) and phosphorylated form of mixed lineage kinase domain-like protein (p-MLKL) were assessed by the Western blot technique. Striatal succinate dehydrogenase (SDH) activity was assayed colorimetrically. Finally, gene enrichment analysis using ShinyGO was employed.

Results: MH and NSA significantly mitigated body weight loss and ameliorated locomotor deterioration, besides reversing histological abnormalities in the striatum of rats. Intriguingly, MH exerted similar effects on specific biomarkers and molecular signals as NSA. MH and NSA inhibited neuroinflammation, apoptosis, and necroptosis by significantly decreasing the striatal (TNF-α), caspase 3, and necroptosis-associated proteins (P-RIPK1, P-RIPK3, and P-MLKL) levels. Besides, MH and NSA also decreased striatal GFAP and increased SDH activity. Gene enrichment analysis revealed a significant interaction between genes. Together, MH exerts a neuroprotective action on 3-NP-elicited HD rats via reducing neuroinflammation, apoptosis, and necroptosis. This study highlights MH as a potential protection against HD, calling for further research to confirm its neuroprotective effects.

Keywords: 3-nitropropionic acid; Huntington’s disease; Morin hydrate; Necroptosis; Necrosulfonamide.

Fig. 1

Schematic diagram of the study design. ELISA: Enzyme-linked immunosorbent assay, GFAP: Glial fibrillary acidic protein, MH: morin hydrate, MLKL: mixed lineage kinase domain-like protein, 3-NP: 3-nitropropionic acid, NSA: necrosulfonamide, RIPK1/3: receptor interacting protein kinase 1/3, SDH: succinate dehydrogenase, TNF-α: tumor necrosis factor-alpha. Created with https://www.BioRender.com

Fig. 2

Effect of NSA or MH on 3-NP- elicited body weight reduction in rats. The results were analyzed by one-way ANOVA, then Tukey’s multiple comparisons test, and revealed as the mean ± SD (n = 10). Significant levels are denoted through the following: ****p < 0.0001, ns: no significance. MH, morin hydrate; 3-NP, 3-nitropropionic acid; NSA, necrosulfonamide; SD, standard deviation

Fig. 3

Effect of NSA or MH on 3-NP- elicited motor aberrations in the grip strength test (A) and behavioral aberrations in open field test (B-G) in rats. (A) grip strength, (B) total distance travelled, (C) mean speed, (D) time immobile, (E) immobile episodes, (F) immobile latency, (G) a representative track plot. The results were analyzed by one-way ANOVA, then Tukey’s multiple comparisons test, and revealed as the mean ± SD (n = 10). Significant levels are denoted through the following order: **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: no significance. gf, gram force; MH, morin hydrate; 3-NP, 3-nitropropionic acid; NSA, necrosulfonamide; SD, standard deviation

Fig. 4

Effect of NSA or MH on histopathological alterations in the striatum region in 3-NP-treated rats. Representative photomicrographs demonstrating H&E staining of the striatum from [A-B] control group; normal histological appearance, [C-D] 3-NP group; congested blood vessel (yellow arrow) with perivascular lymphocytic cuffing (green arrow) and neuronal degeneration (black arrow)[E-F] NSA group; apparently normal striatum, [G-H] MH group; apparently normal striatum [I] blood vessel congestion score, [J] neuronal degeneration score, [K] perivascular lymphocytic score. The results were analyzed by one-way ANOVA, Kruskal–Wallis followed by Dunn’s multiple comparison tests, and revealed as the mean ± SD (n = 3). Significant levels are denoted through the following order: *p < 0.05, **p < 0.01, ns: no significance. H&E, hematoxylin and eosin; MH, morin hydrate; 3-NP, 3-nitropropionic acid; NSA, necrosulfonamide

Fig. 5

Effect of NSA or MH on striatal GFAP immunoreactivity changes in 3-NP-treated rats. Immunostaining results for GFAP in the striatum region. Photomicrographs showing weak immunohistochemical staining of GFAP in the striatum from [A-B] control group; weak GFAP expression, [C-D] 3-NP group; intense GFAP expression, [E-F] NSA group; moderate GFAP expression, [G-H] MH group; moderate GFAP expression, [I] Chart illustrating the quantification of GFAP area %. The results were analyzed by one-way ANOVA, then Tukey’s multiple comparisons test, and revealed as the mean ± SD (n = 3). Significant levels are denoted through the following order: **p < 0.01, ****p < 0.0001, ns: no significance. GFAP, glial fibrillary acidic protein; MH, morin hydrate; 3-NP, 3-nitropropionic acid; NSA, necrosulfonamide; SD, standard deviation.

Fig. 6

Effect of NSA or MH on 3-NP- elicited abnormalities in striatal [A] TNF-α levels, [B] caspase 3 and [C] caspase 8 activities. The results were analyzed by one-way ANOVA, then Tukey’s multiple comparisons test, and revealed as the mean ± SD (n = 6). Significant levels are denoted through the following order: **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: no significance.MH, morin hydrate; 3-NP, 3-nitropropionic acid; NSA, necrosulfonamide; SD, standard deviation; TNF-α, tumor necrosis factor-alpha.

Fig. 7

Effect of NSA or MH on 3-NP- elicited abnormalities in striatal necroptotic markers expression in rats. Expression of striatal proteins, including phosphorylated RIPK1 (Tyr284) [A], phosphorylated RIPK3 (Ser227) [B], and phosphorylated MLKL (Ser125) [C] levels. Western blot images [D]. The results were analyzed by one-way ANOVA, then Kruskal–Wallis followed by Dunn’s multiple comparison tests, and revealed as the mean ± SD (n = 3). Significant levels are denoted through the following order: *p < 0.05, ****p < 0.0001, ns: no significance. MH, morin hydrate; 3-NP, 3-nitropropionic acid; NSA, necrosulfonamide; p-RIP1/3, phosphorylated form of receptor interacting protein 1/3; p-MLKL, phosphorylated form of mixed lineage kinase domain-like protein; SD, standard deviation.

Fig. 8

Effect of NSA or MH on 3-NP- elicited aberrations in striatal SDH activity in rats. The results were analyzed by one-way ANOVA, then Tukey’s multiple comparisons test, and revealed as the mean ± SD (n = 6). Significant levels are denoted through the following order: ***p < 0.001, ****p < 0.0001, ns: no significance. MH, morin hydrate; 3-NP, 3-nitropropionic acid; NSA, necrosulfonamide; SD, standard deviation; SDH, succinate dehydrogenase.

Fig. 9

(A-C) STRING-enriched sets of the top pathways involving our studied genes; (A) biological processes domain, (B) cellular components, and (C) molecular functions. (D) A diagram illustrating the protein-protein interaction network of differentially expressed genes, generated using the STRING database. The circles denote proteins, while the straight lines denote the interactions between different proteins. Abbreviation: MLKL, mixed lineage kinase domain-like protein; CASP3, caspase 3; CASP8, caspase 8; GFAP, glial fibrillary acidic protein; and RIP1/3, receptor-interacting protein 1/3

Fig. 10

Diagrammatic illustration summarizing the neuroprotective effects of MH and NSA in 3NP-elicited HD in rat striata. MH; morin hydrate, MLKL; mixed lineage kinase domain-like protein, 3-NP; 3-nitropropionic acid, NSA; necrosulfonamide, RIPK1/3; receptor interacting protein kinase 1/3, SDH; succinate dehydrogenase, TNF-α; tumor necrosis factor-alpha, TNFR1; tumor necrosis factor receptor-1. Created with https://www.BioRender.com