Modulation of microbiota, inflammation, iron status and gene expression of affected receptors in Parkinson's disease rat model by synbiotic and dark chocolate

Abstract

Parkinson's disease is among the neurodegenerative diseases that have no promising remedies. The present research is dealing with searching the associations between molecular, biochemical and microbiota changes in Parkinson's disease rat model with and without intervention with dark chocolate as prebiotic or a mixture of probiotics (Lactobacillus plantarum EMCC 1039, Lactobacillus acidophilus and Bifidobacterium lactis BB12) during feeding a diet containing oat as prebiotic collectively designated as synbiotic (Sb). Four groups of rats were assigned; a normal control (C), a group given rotenone to induce Parkinson's disease (P), and two test groups treated with rotenone; one received synbiotic (PSb) and the other treated by dark chocolate (PCh). Results showed plasma soluble transferrin receptor /log ferritin ratios that elevated in the P group denoting anemia was reduced in the test groups; with superiority to Sb. The increased brain malondialdehyde (MDA) together with the decreased glutathione (GSH) indicating high oxidative stress in the P group were improved in the test groups. Immune system that was affected in the P group by reduction of plasma CD4 which is the cluster of differentiation 4 and elevation of brain tumor necrosis factor-α (TNF-α) and plasma interferon γ (IFNγ) was improved in the test groups and completely amended concerning IFNγ; Sb showed more promising effect than chocolate concerning TNF-α and CD4. Up-regulation of the brain divalent metal transporter 1 gene (DMT1) in the P group was down-regulated significantly in the test groups till matching that of the C group. Down-regulation of brain dopamine receptor D1 (DRD1) gene expression was significantly up-regulated in the test groups with superiority of Sb. Brain histopathological changes in the P group were improved on treatment with either chocolate or Sb with more promising effect by Sb. The cecum content of Firmicutes (F) showed no difference among the different groups while Bacteroidetes (B) was significantly reduced on treatment with chocolate compared to all groups with significant increase of F/B. Cecum Lactobacilli showed significant increase in the PSb group compared to all other groups. It could be concluded that treatment with dark chocolate and Sb improved experimental PD with variable degrees.

Keywords: Cecum microbiota; DMT1; DRD1; Dark chocolate; Inflammation; Iron status; Oxidative stress; Parkinson’s disease; Rats; Synbiotic.

Fig. 1

Nutritional parameters of different experimental groups. For each parameter, bars with different letters are significantly different at p ≤ 0.05. C: Normal control, P: Parkinson’s control, PCh: Parkinson’s rats with administration of chocolate, PSb: Parkinson’s rats with administration of synbiotic. SE is present above each bar (І).

Fig. 2

Iron status of different experimental groups. (A) Plasma ferritin; (B) Plasma sTfR and sTfR/log ferritin. For each parameter, bars with different letters are significantly different at p ≤ 0.05. C: Normal control, P: Parkinson’s control, PCh: Parkinson’s rats with administration of chocolate, PSb: Parkinson’s rats with administration of synbiotic. SE is present above each bar (І).

Fig. 3

Brain malondialdehyde, reduced glutathione and TNF-α of different experimental groups. For each parameter, bars with different letters are significantly different at p ≤ 0.05. C: Normal control, P: Parkinson’s control, PCh: Parkinson’s rats with administration of chocolate, PSb: Parkinson’s rats with administration of synbiotic. SE is present above each bar (І).

Fig. 4

Plasma INFγ and CD4 of different experimental groups. For each parameter, bars with different letters are significantly different at p ≤ 0.05. C: Normal control, P: Parkinson’s control, PCh: Parkinson’s rats with administration of chocolate, PSb: Parkinson’s rats with administration of synbiotic. SE is present above each bar (І).

Fig. 5

The relative expression of DMT1 and DRD1 genes in brain tissue of different experimental groups. For each parameter, bars with different letters are significantly different at p ≤ 0.05. C: Normal control, P: Parkinson’s control, PCh: Parkinson’s rats with administration of chocolate, PSb: Parkinson’s rats with administration of synbiotic. SE is present above each bar (І).

Fig. 6

Spearman’s correlation coefficient (rho) Heatmap showing correlations between different parameters: Colors range from dark blue (strong positive correlation, i.e. r = 1.0) to dark red (strong negative correlation, i.e. r = − 1.0). The darker the colors the higher is the significance, *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Fig. 7

Brain histopathology of the different experimental groups. (a1) and (a2) Show normal appearance of the brain of the normal control group (X400) with noticeable intact neurons and cortex and normal nuclei (n), meninges (M), perikaryon (p) and myelination (star); (b1–b4) The brain sections of the P group (X400) showed severe changes, (b1) demonstrated neurons atrophy, degeneration and shrunken and bounded by microglial cells (arrow) with vacuolated neutrophil (asterisks) and accompanied by neurofibrillary tangles simulating lewy bodies, (b2) Neuroglial cells (red arrow) were severely accumulated with demyelination, (b3) The hippocampal region (H) displayed hemorrhage with blood vessel congestion in the striatum and midbrain, (b4) Cerebral malacia (Ma) is observed with tissue loss and gliosis accompanied with neuronal apoptosis; (c1–c3) Brain of rats received dark chocolate, (c1) (X400): The cerebral cortex showed decline in karyopyknotic neuronal cells, (c2) (X100), (c3) (X400): Displaying mild vacuolated neurtrophil and congestion (Co). (d1–d3) Rats treated by synbiotic, (d1) (X400): showing modest neuronal necrosis and degeneration with reduction of lesions meanwhile the cerebral cortex pyramidal cells appear normal. (d2) (X100), (d3) (X400) displaying reduced vacuolar spaces around perikaryon (P), and demyelination appears to be minimized.