Neuroprotective effects of punicalagin and/or micronized zeolite clinoptilolite on manganese-induced Parkinson's disease in a rat model: Involvement of multiple pathways

Abstract

Background: Manganism, a central nervous system dysfunction correlated with neurological deficits such as Parkinsonism, is caused by the substantial collection of manganese chloride (MnCl₂) in the brain.

Objectives: To explore the neuroprotective effects of natural compounds, namely, micronized zeolite clinoptilolite (ZC) and punicalagin (PUN), either individually or in combination, against MnCl₂-induced Parkinson's disease (PD).

Methods: Fifty male albino rats were divided into 5 groups (Gps). Gp I was used as the control group, and the remaining animals received MnCl₂ (Gp II-Gp V). Rats in Gps III and IV were treated with ZC and PUN, respectively. Gp V received both ZC and PUN as previously reported for the solo-treated plants.

Results: ZC and/or PUN reversed the depletion of monoamines in the brain and decreased acetyl choline esterase activity, which primarily adjusted the animals' behavior and motor coordination. ZC and PUN restored the balance between glutamate/γ-amino butyric acid content and markedly improved the brain levels of brain-derived neurotrophic factor and nuclear factor erythroid 2-related factor 2/heme oxygenase-1 and decreased glycogen synthase kinase-3 beta activity. ZC and PUN also inhibited inflammatory and oxidative markers, including nuclear factor kappa-light-chain-enhancer of activated B cells, Toll-like receptor 4, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 and caspase-1. Bcl-2-associated X-protein and B-cell leukemia/lymphoma 2 protein (Bcl-2) can significantly modify caspase-3 expression. ZC and/or PUN ameliorated PD in rats by decreasing the levels of endoplasmic reticulum (ER) stress markers (p-protein kinase-like ER kinase (PERK), glucose-regulated protein 78, and C/EBP homologous protein (CHOP)) and enhancing the levels of an autophagy marker (Beclin-1).

Discussion and conclusion: ZC and/or PUN mitigated the progression of PD through their potential neurotrophic, neurogenic, anti-inflammatory, antioxidant, and anti-apoptotic activities and by controlling ER stress through modulation of the PERK/CHOP/Bcl-2 pathway.

Keywords: Parkinson's disease; antioxidant; anti‐inflammatory; autophagy; endoplasmic reticulum stress; micronized zeolite clinoptilolite; oxidative stress; punicalagin.

FIGURE 1

Effects of micronized zeolite clinoptilolite and punicalagin, either alone or in combination, on brain neurotransmitter levels (A) DA, (B) NE, (C) 5‐HT, (D) ACHE activity, (E) glutamate, and (F) GABA in MnCl₂‐induced PD. NC, Normal control rats fed a rodent chow diet; Mn: MnCl₂, control group, Rats were treated daily with MnCl₂ for 5 weeks to develop PD; MnCl₂ + ZC, Rats received MnCl₂ + zeolite clinoptilolite nanoparticles; Mn + PUN, Rats received MnCl₂ + punicalagin; MnCl₂ + COMB, Rats received a combination of MnCl₂+ drugs (ZC and PUN); PD, Parkinson's disease; ZC, zeolite clinoptilolite; PUN, Punicalagin; COMB, Combination of ZC and MnCl₂; 5‐HT, 5‐hydroxytryptamine or serotonin; DA, Dopamine; NE, Norepinephrine; ACHE, Acetyl choline esterase; GABA, Gamma‐aminobutyric acid. The data are presented as the means ± SEMs, n = 6/group; ^asignificant difference from NC; ^bsignificant difference from Mn; ^csignificant difference from Mn + ZC; ^dsignificant difference from Mn + PUN. For parametric data, one‐way ANOVA was performed followed by Tukey's multiple comparisons test to assess group variation, with a significance level set at p < 0.05, while the Kruskal–Wallis test was used for nonparametric data, followed by Dunn's post hoc multiple comparison tests.

FIGURE 2

Effect of micronized zeolite clinoptilolite and punicalagin, either alone or in combination, on brain lipid peroxidation, (A) MDA levels and the levels of the oxidative stress markers (B) SOD, (C) TAC, (D) iNOS, (E) Nrf2, and (F) HO‐1 in MnCl₂‐induced PD. NC: Normal control rats, Mn, MnCl₂, control group, Rats treated daily with MnCl₂ for 5 weeks to develop PD, Mn + ZC, Rats that received MnCl₂ + Zeolite clinoptilolite nanoparticles, Mn + PUN: Rats that received MnCl₂ + punicalagin, Mn + COMB, Rats that received MnCl2 + drug combinations (ZC and PUN), PD, Parkinson's disease, ZC, Zeolite clinoptilolite, PUN, Punicalagin, COMB, Combination of ZC and MnCl₂, Nrf2, Nuclear factor erythroid 2‐related factor, TAC, Total antioxidant capacity, MDA, Malondialdehyde, SOD, Superoxide dismutase, iNOS, Inducible nitric oxide synthase, and HO‐1, Heme‐oxygenase‐1. The data are presented as the means ± SEMs, n = 6/group; ^asignificant difference from NC; ^bsignificant difference from Mn. For parametric data, one‐way ANOVA was performed followed by Tukey's multiple comparisons test to assess group variation, with a significance level set at p < 0.05, while the Kruskal–Wallis test was used for nonparametric data, followed by Dunn's post hoc multiple comparison tests.

FIGURE 3

Effect of micronized zeolite clinoptilolite and punicalagin either alone or in combination on brain proinflammatory, inflammatory, and inflammasome activation biomarkers in MnCl₂‐induced PD. NC, Normal control rats fed a rodent chow diet; Mn, MnCl₂, control group, Rats were treated daily with MnCl₂ for 5 weeks to induce PD; Mn + ZC, Rats received MnCl₂ + micronized zeolite clinoptilolite; Mn + PUN, Rats received MnCl₂ + punicalagin; Mn + COMB, Rats received MnCl₂+ drug combinations (ZC and PUN); PD, Parkinson's disease; ZC: Zeolite clinoptilolite; COMB, Combination of ZC and MnCl₂; PUN, Punicalagin; TLR‐4, Toll‐like receptor 4; GSK‐3β, Brain‐derived neurotrophic factor; IL‐Iβ, Interleukin‐1β; TNF‐α, Tumor necrosis factor alpha; COX‐2, Cyclo‐oxygenase 2; NLRP3, NLR family pyrin domain containing 3; NF‐kB, Nuclear factor kappa kβ; PGE‐2, Prostaglandin E2; Caspase‐1, Cysteine‐aspartic protease‐1. The data are presented as the means ± SEMs, n = 6/group; ^asignificant difference from NC; ^bsignificant difference from Mn. For parametric data, one‐way ANOVA was performed followed by Tukey's multiple comparisons test to assess group variation, with a significance level set at p < 0.05, while the Kruskal–Wallis test was used for nonparametric data, followed by Dunn's post hoc multiple comparison tests.

FIGURE 4

Effect of micronized zeolite clinoptilolite and punicalagin either alone or in combination on ER stress, autophagy, apoptosis, and neurodegenerative biomarkers in MnCl₂‐induced PD. NC, Normal control rats fed a rodent chow diet; Mn, MnCl₂, control group, Rats were injected daily with MnCl₂ for 5 weeks to progress PD; Mn + ZC, Rats received MnCl₂ + zeolite‐micronized clinoptilolite; Mn + PUN, Rats received MnCl₂ + punicalagin; Mn + COMB, Rats received MnCl₂ + drug combinations (ZC and PUN); PD, Parkinson's disease; ZC, zeolite clinoptilolite; PUN, Punicalagin; COMB, Combination of ZC and MnCl₂; GRP78, Glucose Regulated Protein 78; p‐PERK, Phospho‐protein kinase RNA‐like ER kinase; Beclin‐1, Bcl‐2‐homology (BH)‐3 domain; Bcl‐2, B‐cell lymphoma‐2; Bax, Bcl‐2‐associated X apoptosis regulator C/EBP‐homologous protein; BDNF, Brain‐derived neurotrophic factor; CHOP, C/EBP homologous protein. Data are represented as the mean ± SEM, n = 6/group; ^asignificant difference from NC, ^bsignificant difference from Mn. For parametric data, one‐way ANOVA was performed followed by Tukey's multiple comparisons test to assess group variation, with a significance level set at p < 0.05, while the Kruskal–Wallis test was used for nonparametric data, followed by Dunn's post hoc multiple comparison tests.

FIGURE 5

Photomicrographs of brain sections (cerebral cortex, subiculum, and fascia dentata in the hippocampus, striatum, and substantia nigra) (magnification 40×). Where (a1, a2, a3, a4, a5) are the normal control group (NC); (b1, b2, b3, b4, b5) are the manganese (Mn) group; (c1, c2, c3, c4, c5) are the zeolite clinoptilolite (ZC) treatment group (Mn + ZC); (d1, d2, d3, d4, d5) are the punicalagin (PUN) treatment group (Mn + PUN); and (e1, e2, e3 e4, e5) are the combination group (Mn + combination of ZC and PUN). In brain sections, the orange arrow indicates nuclear pyknosis and degeneration.