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. 2024 Sep 26;30(1):158.
doi: 10.1186/s10020-024-00917-5.

Parthenolide ameliorates 3-nitropropionic acid-induced Huntington's disease-like aberrations via modulating NLRP3 inflammasome, reducing microglial activation and inducing astrocyte shifting

Mona E Noureldeen  1 , Nancy N Shahin  1 , Hebat Allah A Amin  2 , Maha M El-Sawalhi  1 , Heba R Ghaiad  3
Affiliations

Parthenolide ameliorates 3-nitropropionic acid-induced Huntington's disease-like aberrations via modulating NLRP3 inflammasome, reducing microglial activation and inducing astrocyte shifting

Mona E Noureldeen et al. Mol Med. .

Abstract

Background: Huntington's disease (HD) is a progressive neurodegenerative disease that causes motor, cognitive, and psychiatric abnormalities, with no satisfying disease-modifying therapy so far. 3-nitropropionic acid (3NP) induces behavioural deficits, together with biochemical and histological alterations in animals' striata that mimic HD. The role of nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome in HD pathogenesis remains largely uncharacterized. Parthenolide (PTL), a naturally occurring nuclear factor kappa B (NF-κB) inhibitor, is also known to inhibit NLRP3 inflammasome. Whether PTL is beneficial in HD has not been established yet.

Aim: This study evaluated the possible neuroprotective effects of PTL against 3NP-induced behavioural abnormalities, striatal biochemical derangements, and histological aberrations.

Methods: Male Wistar rats received PTL (0.5 mg/kg/day, i.p) for 3 weeks and 3NP (10 mg/kg/day, i.p) was administered alongside for the latter 2 weeks to induce HD. Finally, animals were subjected to open-field, Morris water maze and rotarod tests. Rat striata were examined histologically, striatal protein expression levels of glial fibrillary acidic protein (GFAP), cluster of differentiation 45 (CD45) and neuron-specific enolase (NSE) were evaluated immunohistochemically, while those of interleukin (IL)-1β, IL-18, ionized calcium-binding adapter molecule-1 (Iba1) and glutamate were determined by ELISA. Striatal nuclear factor erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein (Keap1), NF-κB, NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, S100 calcium-binding protein A10 (S100A10) and complement-3 (C3) were assessed by gene expression analysis.

Results: PTL improved motor, locomotor, cognitive and anxiety-like behaviours, restored neuronal integrity, upregulated Nrf2, and inhibited NLRP3 inflammasome, NF-κB and microglial activation. Additionally, PTL induced astrocyte shifting towards the neuroprotective A2 phenotype.

Conclusion: PTL exhibits neuroprotection against 3NP-induced HD, that might be ascribed, at least in part, to its modulatory effects on Keap1/Nrf2 and NF-κB/NLRP3 inflammasome signaling.

Keywords: 3-Nitropropionic acid; Huntington’s disease; NF-κB; NLRP3; Neuroinflammation; Parthenolide.

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Conflict of interest statement

There are no conflicts of interest to declare.

Figures

Fig. 1
Fig. 1
Timeline schedule for the experimental study. 3NP: 3-nitropropionic acid; PTL: parthenolide; i.p: intraperitoneal
Fig. 2
Fig. 2
Effect of PTL on 3NP-induced neurobehavioural changes in the open-field task. (A – D) show the effect of PTL on motor activity per 3 min in the open-field, where (A) Mean speed, (B) Distance travelled, (C) Number of crossed lines and (D) Rearing frequency. (E – H) show the effect of PTL on anxious behaviour, where (E) Immobility time (F) Thigmotaxis time, (G) Centre time and (H) Distance in centre. (I) Track plots of rats during the open-field test. Differences among groups were analysed by one-way ANOVA followed by Tukey’s post-hoc test in (C), whereas statistical differences among groups were analysed by Kruskal Wallis followed by Dunn’s post-hoc test in (A), (B), (D), (E), (G), and (H). Differences among groups were analysed by Mann Whitney test in (F), while statistical differences between 3NP and PTL + 3NP groups were analysed by Mann Whitney in (G). Each column represents the mean of 10–12 rats ± SEM. a = significant difference from the control group at P < 0.05 and b = significant difference from the 3NP-intoxicated group at P < 0.05. 3NP: 3-nitropropionic acid, PTL: parthenolide, m: meter and sec: second
Fig. 3
Fig. 3
Effect of PTL on 3NP-induced neurobehavioural changes in Morris water maze and the rotarod tests. (A) shows the effect of PTL on motor activity in terms of distance travelled. (B – D) show the effect of PTL on spatial learning and memory where (B) Latency to first platform zone entry, (C) Time spent in target quadrant, (D) Platform zone time. (E) Track plots of rats during Morris water maze test. (F) Fall off time on rotarod apparatus. Differences among groups were analysed by one-way ANOVA followed by post-hoc test in (A) and (B), whereas statistical differences between groups were analysed by Kruskal Wallis followed by Dunn’s post-hoc test in (C), (D) and (F). Each column represents the mean of 10–12 rats ± SEM. a = significant difference from the control group at P < 0.05 and b = significant difference from the 3NP-intoxicated group at P < 0.05. 3NP: 3-nitropropionic acid, PTL: parthenolide, m: meter and sec: second
Fig. 4
Fig. 4
Effect of PTL on 3NP-induced neuronal injury. (A) Representative photomicrographs of the immunohistochemical evaluation of NSE protein expression (magnification x400). Neurons are represented by long arrows, glia by short arrows, and microglia by dashed arrows. (B) Area percentage of immunohistochemical expression of NSE in striatum, and (C) Striatal glutamate levels. Differences among groups were analysed by Kruskal Wallis followed by Dunn’s post-hoc test, while differences between 3NP-intoxicated group and PTL + 3NP group were analysed by Mann Whitney. Each column represents the mean of 6–8 rats ± SEM. a = significant difference from the control group at P < 0.05 and b = significant difference from the 3NP-intoxicated group at P < 0.05. For quantification of NSE immunostaining in the striatum, values are the mean ± SEM of the area percentage of NSE-immune staining to the total area of the microscopic field across five non-overlapping fields/section. NSE: Neuron-specific enolase. 3NP: 3-nitropropionic acid and PTL: parthenolide
Fig. 5
Fig. 5
Effect of PTL on 3NP-induced changes in oxidative stress markers. Gene expression analysis of (A) NRF2 and (B) KEAP 1 in striatum. Differences among groups were analysed by Kruskal Wallis followed by Dunn’s post-hoc test, while statistical differences in NRF2 expression between control group and 3NP group were analysed by Mann Whitney. Each column represents the mean of 6–8 rats ± SEM. a = significant difference from the control group at P < 0.05 and b = significant difference from the 3NP-intoxicated group at P < 0.05. NRF2: Nuclear factor erythroid 2-related factor 2, KEAP 1: Kelch-like ECH-associated protein 1. 3NP: 3-nitropropionic acid, PTL: parthenolide
Fig. 6
Fig. 6
Effect of PTL on 3NP-induced changes in inflammasome activation markers. (A – D) striatal gene expression of (A) NF-κB p65, (B) NLRP3, (C) ASC and (D) Caspase-1. (E & F) show striatal levels of (E) IL-1β and (F) IL-18. Differences among groups were analysed using Kruskal Wallis followed by Dunn’s post-hoc test, Statistical differences between 3NP-intoxicated group and PTL + 3NP group were analysed by Mann Whitney in (E) and (F). Each column represents the mean of 6–8 rats ± SEM. a = significant difference from the control group at P < 0.05 and b = significant difference from the 3NP-intoxicated group at P < 0.05. NF-κB p65: Nuclear factor kappa B, NLRP3: nucleotide-binding domain leucine-rich repeat (NLR) and pyrin domain containing receptor 3, ASC: apoptosis-associated speck-like protein containing a caspase recruitment domain, IL-1β: interleukin 1 beta, IL-18: interleukin 18. 3NP: 3-nitropropionic acid and PTL: parthenolide
Fig. 7
Fig. 7
Effect of PTL on 3NP-induced microglial activation and astrocyte phenotype shifting. (A) Representative photomicrographs of the immunohistochemical evaluation of GFAP protein expression (magnification x400) and (B) Area percentage of immunohistochemical expression of GFAP in striatum (C) Representative photomicrographs of the immunohistochemical evaluation of CD45 protein expression (magnification x400) and (D) Area percentage of immunohistochemical expression of CD45 in striatum. (E) Striatal Iba-1 concentrations. (F & G) Gene expression analysis of striatal S100A10 (F) and complement component-3 (G). Differences among groups were analysed by one-way ANOVA followed by Tukey’s post-hoc test in (B), (D) and (E), whereas statistical differences among groups were analysed using Kruskal Wallis test followed by Dunn’s post-hoc test in (F) and (G). Each column represents the mean of 6–8 rats ± SEM. a = significant difference from the control group at P < 0.05 and b = significant difference from the 3NP-intoxicated group at P < 0.05. For quantification of GFAP and CD45 immunostaining in the striatum, values are the mean ± SEM of the area percentage of GFAP-immune staining and CD45-immune staining, each separately, to the total area of the microscopic field across five non-overlapping fields/section. GFAP: glial fibrillary acidic protein, CD45: cluster of differentiation 45, Iba-1: ionized calcium-binding adapter molecule 1, S100A10: S100 calcium-binding protein A10. 3NP: 3-nitropropionic acid and PTL: parthenolide
Fig. 8
Fig. 8
H&E-stained striatal sections showing effect of PTL on 3NP-induced histopathological alterations. (A) Representative photomicrographs of histological examination (magnification x100) showing control group with preserved neuronal structures (long arrows) and normal glia (short arrows), whereas in 3NP group neuronal damage (long arrows) and proliferating astroglia (short arrows) was evident. PTL control group showed preserved neuronal structures (long arrows) and unremarkable glia (short arrows) where PTL treatment group presented with fairly preserved neuronal structures (long arrows) and mild astroglial proliferation (short arrows). (B) Representative photomicrographs of histological examination (magnification x400), showing control group with preserved neuronal structures (long arrows) and normal glia (short arrows), while 3NP group showed neuronal damage with shrunken neurons (long arrows), and proliferating astroglia (short arrows). Finally, PTL control group showed preserved neuronal structures (long arrows) and normal glia (short arrows) whereas the PTL treatment group presented with fairly preserved neuronal structures (long arrows) and mild astroglial proliferation (short arrows). (C) Neuronal count per high power field of H&E-stained striatal brain sections. Differences among groups were analysed by one-way ANOVA followed by Tukey’s post-hoc test. Each column represents the mean of five non-overlapping fields per slide counted in high power field of H&E-stained striatal brain sections of 6 rats per group. a = significant difference from the control group at P < 0.05 and b = significant difference from the 3NP-intoxicated group at P < 0.05. H&E: haematoxylin and eosin, HPF: high power field, 3NP: 3-nitropropionic acid and PTL: parthenolide
Fig. 9
Fig. 9
Heatmaps summarizing the correlations of astrocyte and microglial markers with the studied parameters. (A) Heatmap for the correlation between astrocyte markers and the studied parameters. (B) Heatmap for the correlation between microglial markers and the studied parameters. Correlations were analyzed by Spearman correlation and Spearman correlation coefficients (r) between the markers were plotted in the heatmap. The dark-blue colour corresponds to a correlation coefficient close to -1 and the light-yellow colour corresponds to values close to 1. NLRP3; nucleotide-binding domain leucine-rich repeat (NLR) and pyrin domain containing receptor, ASC; apoptosis-associated speck-like protein containing a caspase recruitment domain, Caspase-1, NF-κB; Nuclear factor kappa B, Nrf2; Nuclear factor erythroid 2-related factor 2, Keap1; Kelch-like ECH-associated protein 1, IL-1β; interleukin 1 beta, IL-18; interleukin 18, GFAP; glial fibrillary acidic protein, C3; complement component 3, S100A10; S100 calcium-binding protein A10, CD45; cluster of differentiation 45, Iba1; ionized calcium-binding adapter molecule 1
Fig. 10
Fig. 10
Schematic representation summarizing the neuroprotective effects of PTL in 3NP-induced HD-like perturbations in rats. 3NP: 3-nitropropionic acid, PTL: parthenolide, IP: intraperitoneal, DMSO: dimethyl sulfoxide, SDH: succinate dehydrogenase, ROS: reactive oxygen species, Nrf2: nuclear factor erythroid 2-related factor 2, Keap1: Kelch-like ECH-associated protein, NF-κB: nuclear factor kappa B, IκB: inhibitory-κB, IL-1β: interleukin-1β, IL-18: interleukin-18, NSE: neuron-specific enolase, GFAP: glial fibrillary acidic protein, C3: complement component 3, S100A10: S100 calcium-binding protein A10, CD45: cluster of differentiation 45, Iba1: ionized calcium binding adapter molecule-1
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