Medical/biological study (experimental study)

Neuroprotective effects of lotus seedpod procyanidins on extremely low frequency electromagnetic field-induced neurotoxicity in primary cultured hippocampal neurons med./bio.

By: Yin C, Luo X, Duan Y, Duan W, Zhang H, He Y, Sun G, Sun X

Published in: Biomed Pharmacother 2016; 82: 628-639

Aim of study (acc. to author)

The neurotoxic effects of exposure of primary cultured hippocampal neurons to a 50 Hz magnetic field and the protective effects of lotus seedpod procyanidins should be investigated.

Background/further details

Lotus seedpod procyanidins (LSPCs) possess a broad spectrum of biological, pharmacological and chemoprotective properties against free radicals and oxidative stress. In a previous study by the authors (Duan et al. 2013), it was found that LSPCs had a protective effect against oxidative damage after ELF-EMF exposure in in vivo experiments.
Cells were divided into 5 groups: 1) exposure to the magnetic field, 2) exposure to the magnetic field and treatment with 2.5 mg/ml LSPCs, 3) exposure to the magnetic field and treatment with 5 mg/ml LSPCs, 4) exposure to the magnetic field and treatment with 10 mg/ml LSPCs, 5) control group.

Endpoint

neurotoxic effects in primary cultured hippocampal neurons

Exposure

- 50 Hz
- 50/60 Hz
- magnetic field
- co-exposure

Exposure	Parameters
Exposure 1: 50 Hz Exposure duration: continuous for 90 minutes	magnetic flux density: 8 mT

Exposure 1

Main characteristics

Frequency	50 Hz
Type	magnetic field
Exposure duration	continuous for 90 minutes

Exposure setup

Exposure source	Helmholtz coils
Chamber	cell culture plates
Setup	a pair of Helmholtz coils (1.0 mm wire diameter, 9 cm internal diameter, 13 cm external diameter) in 400 turns, parallel to the ground, generated a homogeneous field

Parameters

Measurand	Value	Type	Method	Mass	Remarks
magnetic flux density	8 mT	-	measured	-	-

Exposed system:

intact cell/cell culture
primary hippocampal neurons from newborn Sprague-Dawley rats

Methods Endpoint/measurement parameters/methodology

genotoxicity/mutation: DNA strand breaks (alkaline comet assay)
molecular biosynthesis: protein expression of Bcl-2 and Bcl-xl (anti-apoptotic markers) and Bax and Bad (pro-apoptotic markers) (Western blot), total protein concentration (BCA assay)
cell function: antioxidant activity (enzyme activity of superoxide dismutase (SOD), spectrophotometry), intracellular calcium concentration (Fluo-3 AM stain, fluorophotometry and fluorescence microscopy), mitochondrial membrane potential (stain with JC-1, fluorescence microscopy)
cell viability/cell division/proliferation: cell viability (MTT assay), cell morphology and apoptosis (Hoechst 33342 stain, fluorescence microscopy), cell cycle analysis (propidium iodide stain, flow cytometry), apoptosis and necrosis (Annexin V-FITC/propidium iodide stain, flow cytometry)
oxidative stress: lipid peroxidation (reaction with thiobarbituric acid, spectrophotometry), content of intracellular reactive oxygen species (dichlorofluorescein stain, fluorophotometry)

Investigated system:

isolated bio./chem. substance
DNA/RNA
intact cell/cell culture
cell lysates

Time of investigation:

after exposure

Main outcome of study (acc. to author)

Cell viability, SOD enzyme activity and mitochondrial membrane potential were significantly decreased in exposed cells (group 1) compared to the control group, while lipid peroxidation, calcium concentration and the content of reactive oxygen species were significantly increased. However, in exposed groups with LSPCs (group 2-4), all these parameters were significantly restored in a LSPC concentration-dependent manner compared with group 1.
In morphological investigations, cells of group 1 showed a rounder shape and fewer neurites as well as condensed and fragmented nuclei compared to the control group. In a combination with 10 mg/ml LSPCs (group 4), morphology was significantly improved.
DNA damage, apoptosis and necrosis were significantly increased in group 1 compared to the control group. In groups 3 (with 5 mg/ml LSPCs) and 4, these parameters were significantly reduced in a LSPC concentration-dependent manner compared to group 1.
Cell cycle analysis revealed a significantly decreased number of cells in G0 phase/G1 phase and a significant S phase arrest in group 1 when compared with the control group. The LSPCs groups 3-4 showed a concentration-dependent restoration of both parameters compared to group 1.
The protein expressions of Bcl-2 and Bcl-xl were significantly decreased and the levels of Bad and Bax significantly increased in group 1 compared with the control group. LSPCs treatment increased the levels of anti-apoptotic proteins Bcl-2 and Bcl-xl and decreased the levels of pro-apoptotic proteins Bad and Bax.
The authors conclude that exposure to a 50 Hz magnetic field might induce neurotoxicity in hippocampal neurons via oxidative stress and activation of mitochondrial apoptotic pathway and that lotus seedpod procyanidins might protect against these effects.

Study character:

medical/biological study
experimental study
full/main study

Study funded by

Jiangsu Key R&D Program, China
Jiangsu Qing Lan Project, China
Jiangsu Six Talent Peaks Project, China
National Natural Science Foundation (NSFC), China
National Special Research Funds, China
Scientific Research Projects of Chinese Medicine Industry, China
The Priority Academic Program Development of Jiangsu Higher Education Institutions, China

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