Effects of the probiotic formulation SLAB51 in in vitro and in vivo Parkinson's disease models

Abstract

Parkinson is a common neurodegenerative disorder, characterized by motor and non-motor symptoms, including abnormalities in the gut function, which may appear before the motor sign. To date, there are treatments that can help relieve Parkinson' disease (PD)-associated symptoms, but there is no cure to control the onset and progression of this disorder. Altered components of the gut could represent a key role in gut-brain axis, which is a bidirectional system between the central nervous system and the enteric nervous system. Diet can alter the microbiota composition, affecting gut-brain axis function. Gut microbiome restoration through selected probiotics' administration has been reported. In this study, we investigated the effects of the novel formulation SLAB51 in PD. Our findings indicate that this probiotic formulation can counteract the detrimental effect of 6-OHDA in vitro and in vivo models of PD. The results suggest that SLAB51 can be a promising candidate for the prevention or as coadjuvant treatment of PD.

Keywords: 6-OHDA; BDNF; Parkinson's disease; probiotics; tyrosine hydroxylase.

Figure 1

Dopaminergic phenotype of SH-SY5Y neuroblastoma cells. Contrast phase microscopy of differentiated with ATRA/TPA and not differentiated SH-SY5Y cells and histogram showing dopamine production. Western blotting for TH. Immunofluorescence of β-tubulin III and TH. Western blotting and immunofluorescence for GAP43. Results are mean ± SE of 3 different experiments (n=3). *p< 0,05, **p< 0,005 vs. ATRA/TPA.

Figure 2

MTS assay of cells treated with different concentration of SLAB51 (left). MTS assay of cells treated with 35 μM 6-OHDA and 35 μM 6-OHDA and SLAB51 0.1mg/ml (right). Data are mean ± SE of three different experiments run in quadruplicate (n=3). *** p< 0.0005 vs Ctr; +++ p< 0.0005 vs 6-OHDA.

Figure 3

WB and relative densitometric analysis for Ctr, 6-OHDA and 6-OHDA+SLAB51 for mBDNF, p-TrkB, p-ERK5, p-CREB. Results are mean ± SE of 3 different experiments (n=3). *p< 0.05; ** p< 0.005 vs Ctr; ++ p< 0.005, +++ p< 0.0005 vs 6-OHDA. Representative WB figures are shown.

Figure 4

WB and relative densitometric analysis for Ctr, 6-OHDA and 6-OHDA+SLAB51 for PI3K, p-Akt, and PSD95. Results are mean ± SE of 3 different experiments (n=3). ** p< 0.005, ***p< 0.0005 vs Ctr; ++ p< 0.005, +++ p< 0.0005 vs 6-OHDA. Representative WB figures are shown.

Figure 5

WB and relative densitometric analysis for Ctr, 6-OHDA and 6-OHDA+SLAB51 for pro-BDNF, p-JNK, p-ERK1,2 and P75. Results are mean ± SE of 3 different experiments (n=3). ** p< 0.005 vs Ctr; ++ p< 0.005, +++ p< 0.0005 vs 6-OHDA. Representative WB figures are shown.

Figure 6

WB and relative densitometric analysis for Ctr, 6-OHDA and 6-OHDA+SLAB51 for PPARγ and 4-HNE proteins adducts. Results are mean ± SE of 3 different experiments (n=3). ** p< 0.005, ***p< 0.0005 vs Ctr; ++ p< 0.005, +++ p< 0.0005, vs 6-OHDA. Representative WB figures are shown.

Figure 7

(A) Procedural timeline with specific timepoints. (B) Body weight and behavioral tests in SHAM, SLAB51, 6-OHDA and 6-OHDA+SLAB51 animals. ** p< 0.005, *** p< 0.0005 vs Ctr; ++ p< 0.005, +++ p< 0.0005 vs 6-OHDA.

Figure 8

Immunostaining of TH in dopaminergic neurons. Transverse section taken through the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA), immunostained for TH to evaluate the dopaminergic-induced injury by stereotaxic injection of 6-OHDA in the right side. Histograms shows the percentage of TH+ fibers loss in striatum (CPu) and TH+ cell bodies in substantia nigra (SN) (expressed in percentage of unlesioned side). * p< 0.05, *** p< 0.0005 vs Ctr; + p< 0.05, ++ p< 0.005 vs 6-OHDA.

Figure 9

Immunofluorescence for DAT in mice Substantia nigra. Images were taken at confocal microscope at 20x magnification.

Figure 10

Triple immunostaining at 20x magnification for Iba1, TH and GFAP, nuclei were counterstained with DAPI. On the left it is possible to appreciate mosaic figures, while on the right inset at higher magnification for TH, Iba1 and GFAP staining and merge figures were reported. Histograms for Iba1 show the number of Iba1 + cells, while for GFAP the fluorescence intensity, as % of controls, is plotted. ** p< 0.005 vs Ctr; + p< 0.05, ++ p< 0.005 vs 6-OHDA.

Figure 11

Immunofluorescence analysis for PPARγ in substantia nigra. On the left, the mosaic images obtained using confocal microscopy at 20x magnification were shown. In the center, double immunostaining at 40x magnification with TH and PPARγ as well as the merge figures were reported. On the right it is possible to observe the inset of the indicated boxes.

Figure 12

Western blotting and relative densitometric analysis for mBDNF, p-TrkB and PPARγ in substantia nigra (SN) and striatum (CPu). Results are mean ± SE of 3 experiments (n=3). * p< 0.005, ** p< 0.005, *** p< 0.0005 vs Ctr; + p< 0.005, +++ p< 0.0005 vs 6-OHDA. Representative WB images are shown.

Figure 13

Western blotting and relative densitometric analysis for p-Nfr2, HO-1 and NF-KB in SN and CPu. Results are mean ± SE of 3 experiments (n=3). ** p< 0.005, *** p< 0.0005 vs Ctr; ++ p< 0.0005, +++ p< 0.0005 vs 6-OHDA. Representative WB images are shown.

Figure 14

TUNEL assays in mice substantia nigra. Figures were taken at confocal microscope at 40x magnification. The graph shows apoptotic index obtained by counting positive nuclei. * p< 0.005, ** p< 0.005 vs ctr; ++< 0.005 vs 6-OHDA. Representative figures are reported.