Characterization of lactic acid bacteria derived exopolysaccharides for use as a defined neuroprotective agent against amyloid beta1-42-induced apoptosis in SH-SY5Y cells

Abstract

Alzheimer's disease (AD) is a disease characterized by cerebral neuronal degeneration and loss in a progressive manner. Amyloid beta (Aβ) in the brain is toxic to neurons, being a main risk factor for initiation and continuation of cognitive deterioration in AD. Neurotoxicity of Aβ origin is also linked to oxidative stress characterized by excessive lipid peroxidation, protein oxidation, changes in antioxidant systems, and cerebral DNA damage in AD. Furthermore, Aβ can induce oxidative neuronal cell death by a mitochondrial dysfunction. Cellular injury caused by oxidative stress can be possibly prevented by boosting or promoting bodily oxidative defense system by supplying antioxidants in diet or as medications. However, most synthetic antioxidants are found to have cytotoxicity, which prevents their safe use, and limits their administration. For this reason, more attention has been paid to the natural non-toxic antioxidants. One of the most promising groups of non-toxic antioxidative compounds is thought to be polysaccharides. This study investigated the characterization and protective action exerted by exopolysaccharides (EPSs) originated from Lactobacillus delbrueckii ssp. bulgaricus B3 and Lactobacillus plantarum GD2 to protect from apoptotic activity exerted by Aβ_1-42 among SH-SY5Y cells. We characterized EPSs by elemental analysis, FTIR, AFM, SEM, and XRD. The antioxidant effects of EPSs were determined by the DPPH free radical scavenging activity, hydroxyl radical scavenging activity, metal ion chelating activity, lipid peroxidation inhibitory activity, and superoxide anion scavenging activity method. The protective effects of EPSs were determined by flow cytometry and RT-PCR. Mannose ratio, molecular weight, functional groups, surface morphology, and amorphous character structure of EPSs are thought to play a role in the protective effect of EPSs. EPSs reduced apoptotic activity of Aβ_1-42 in addition to their depolarizing effect on mitochondrial membrane potential in concentration-dependent manner. These observations contribute the inclusion of EPSs among the therapeutic options used to manage various neurological disorders in the traditional medicine in a scientific manner, indicating that EPSs may be promising natural chemical constituents that need advanced research and development for pharmacological therapy of AD.

Figure 1

FTIR spectrum of l-EPS_S [l-EPS_B3 (A) and l-EPS_GD2 (B)].

Figure 2

AFM images of l-EPSs [l-EPS_B3 (A,C) and l-EPS_GD2 (B,D); three (A,B) and two dimensional views (C,D)].

Figure 3

SEM images of l-EPS_B3. Fig (A–G) showing SEM images of l-EPS_B3 at 100×, 1000×, 2000×, 5000×, 10.000×, 20.000×, respectively.

Figure 4

SEM images of l-EPS_GD2. Fig (A–G) showing SEM images of l-EPS_GD2 at 100×, 1000×, 2000×, 5000×, 10.000×, 20.000×, respectively.

Figure 5

XRD pattern of l-EPSs [l-EPS_B3 (A) and l-EPS_GD2 (B)].

Figure 6

Dose-dependent inhibition of Aβ_1-42 aggregation by l-EPSs (A), time course of inhibition of Aβ_1-42 aggregation by l-EPSs (B) using the ThT assay. Values are expressed as mean ± SD. Pearson’s correlation test, if p < 0.05. *significant difference from the Aβ_1-42 (n:3 for each bar).

Figure 7

AFM images of Aβ_1-42 fibrils formed when Aβ_1-42 was incubated alone (A) or in the presence of l-EPS_B3 (B) and l-EPS_GD2 (C) at 37 °C for 24 h. The figure size was 10 µm × 10 µm.

Figure 8

Effect of l-EPSs on Aβ_1-42-induced cell apoptosis (A) and the loss of Δψm and the release of mitochondrial cytochrome c into the cytosol (B) in SH-SY5Y cells using flow cytometry. Values are expressed as mean ± SD. Pearson’s correlation test, if p < 0.05. #significant difference from the control (n:3 for each bar). *significant difference from the Aβ_1-42 (n:3 for each bar).

Figure 9

The mRNA levels of Bax (A), Bcl-2 (B), caspase-3 (C), caspase-7 (D), caspase-8 (E), caspase-9 (F), and cyctochrome c (G) in SH-SY5Y cells. Values are expressed as mean ± SD. Pearson’s correlation test, if p < 0.05. #significant difference from the control (n:3 for each bar). *significant difference from the Aβ_1-42 (n:3 for each bar).