Protective effects of blueberry anthocyanin extracts on hippocampal neuron damage induced by extremely low-frequency electromagnetic field

Xiyun Sun,Zihn Xu,Yuehu Wng,Ning Liu

a College of Food,Shenyang Agricultural University,120 Dongling Rd,Shenyang City 110866,China

b College of Land and Environment,Shenyang Agricultural University,China

ABSTRACT The protective effects of blueberry anthocyanin extracts against damage induced by extremely lowfrequency electromagnetic field(ELF-EMF)were investigated in a rat model.Wistar rats were exposed to ELF-EMF with or without the administration of blueberry anthocyanin extracts(50,100,and 200 mg/kg per day intragastrically once a day)for 30 days.Blueberry anthocyanin extracts supplementation inhibited the decrease in Nissl substance levels, cell membrane integrity, and mitochondrial membrane potential induced by ELF-EMF; prevented the increase in nitric oxide, malondialdehyde, and Ca2+ concentrations; suppressed superoxide dismutase and glutathione depletion; and enhanced the cognitive ability of the rats exposed to ELF-EMF.The protective effects of blueberry anthocyanin extracts against hippocampal neuron injury caused by ELF-EMF were dose-dependent.These results demonstrated that blueberry anthocyanin extracts suppress hippocampal neuron injury caused by ELF-EMF by inhibiting cell membrane damage and oxidative stress pathways,and suggested that blueberry anthocyanin treatment potentially prevents hippocampal neuron injury.

Keywords:Blueberry anthocyanin extracts Protective effects Extremely low-frequency electromagnetic field Hippocampal neuron

1.Introduction

Extremely low-frequency electromagnetic fields (ELF-EMFs),with the frequency of 0-100 kHz, are ubiquitous in everyday life.Previous studies have suggested the involvement of ELF-EMF in diseases, including immune disorders and cancer [1].ELF-EMF exposure could prolong the lifetime or increase concentration of free radicals in living cells[2-4].Reactive oxygen species(ROS)are generated in free-radical chain reactions,causing oxidative stress in tissues accompanied by the increase in nitric oxide(NO)and malondialdehyde(MDA)content[5,6].In addition,Martínezsámano and Jesús et al.[7]indicated that chronic exposure to ELF-EMF is similar to physiological stress,and induce changes on brain lipid profile.A 7-day exposure to ELF-EMF might have anxiogenic effects on rats[8].Exposure to ELF-EMF causes working memory(WM)task performance decline, and this effect is associated with hippocampal function[9].

In recent years, some natural plant extracts have shown beneficial effects in preventing chronic diseases, such as cancer and cardiovascular diseases.Ilhan et al.[10]indicated that Ginkgo biloba extract prevented ELF-EMF-induced oxidative stress in the rat brain by regulating the activity of antioxidant enzymes.Additionally,zinc and selenium supplementation modulated the increased activities of antioxidant enzymes caused by ELF-EMF [11,12].Despite these efforts, high efficiency and low side effects agents counteracting ELF-EMF-caused damage remains controversial.Therefore,development of treatment options for preventing or alleviating ELF-EMF-induced damage is required.

Blueberry(Vaccinium corymbosum)is recognized worldwide as a valuable fruit because of multiple biological activities including anti-aging and antioxidant activity [13].Anthocyanins are abundant in blueberry and are the main bioactive compounds of the fruit.Blueberry was shown to protect hippocampal cells against damage induced by dopamine (DA), amyloid beta peptide, or lipopolysaccharide[14].

However, to the best of our knowledge, the effects of blueberry anthocyanins on the ELF-EMF-induced damage have not been assessed.In this study, a rat model of damage to cognitive functions and hippocampal neurons was established,in order to test the protective mechanisms of blueberry anthocyanin extracts against ELF-EMF-induced hippocampal damage, which provides insight into the functional effects of blueberry anthocyanins on hippocampal cells.

2.Materials and methods

2.1.Extraction of blueberry anthocyanins

Frozen blueberries were thawed for 1 h and homogenized.The homogenate(60 g)was extracted with 900 mL of acidized methanol(0.1%HCl)for 1 h at 40°C.The material was subjected to vacuum filtration and the residue was re-extracted by repeating the extraction procedure until the solvent remained clear.The methanol extracts were combined for rotary evaporation at 40°C for 1 h.The collected residue was dried under vacuum at 50°C.The dry extract(10 g)was dissolved with 95%ethanol to a final volume of 1000 mL,for column chromatography.The column (?6 cm×110 cm) was loaded with XAD macroporous resin and regarded as 1 bed volume (BV).The mobile phase flow rate was 1 BV/h.The elution gradient scheme used was as follows: water 1 BV, 30% ethanol 3 BV, 60% ethanol 3-4 BV, 85%-90% ethanol 3 BV.The 60% ethanol elution fraction was collected and lyophilized.

2.2.Identification of anthocyanins

Anthocyanins were identified using HPLC-ESI-MS2systems(Agilent 1100, Palo Alto, CA, USA) at 520 nm.The detailed conditions were as reported in our previous study [15].Individual anthocyanins were quantified based on the calibration curves of structurally related external standards(cyanidin-3-glucoside).

2.3.Animal treatment

Wistar rats (50 animals, 25 male, 25 female) were randomly divided into 5 groups.Group 1 rats received a standard diet.Rats in groups 2,3,4,and 5 were exposed to ELF-EMF(repetition frequency 50 Hz, average field strength 0.3 mT) for 30 days (8 h daily) and were fed standard diet, 50, 100, and 200 mg/kg per day blueberry anthocyanin extracts, respectively, for the next 20 days.All animals received humane care,and the study protocols were approved by the Committee for Care of Laboratory Animals of the Shenyang Agricultural University.

After anesthesia, the rats were sacrificed and bilateral hippocampus was extracted.After washing with PBS buffer, the hippocampal tissue was cut into 1 mm×1 mm×1 mm slices and digested with 0.25% trypsin for 30 min.The digested tissues were filtered to a single cell suspension using a 300 mesh and diluted to 105cells/mL for determination of Ca2+, mitochondrial membrane potential, and cell membrane integrity.Tissue homogenate(10%)was prepared for NO,superoxide dismutase(SOD),MDA,and glutathione(GSH)content analysis.

2.4.Analysis of memory function

The effects of blueberry anthocyanin extracts on memory function of rats were examined using the Morris water maze test, as described by Duda et al.[16], with slight modifications.Morris water maze system automatically recorded time with the accuracy of 0.1 s.The differences in cognitive and memory abilities of rats were determined through statistical analysis of escape latency.Cognitive and memory abilities of rats were determined 4 times at 1-day intervals.Before the Morris water maze experiment, the rats were allowed to swim freely for 2 min.Afterwards each rat was trained 4 times for 60 s.An entry point was randomly selected from four quadrants,and the rats were placed in the water facing the wall.Escape latency time,defined as the time the rats required to find and climb up the hidden platform,was recorded.

3.Analysis of hippocampal neuron damage

3.1.Analysis of nissl substance content

Nissl body is a representative structure of brain neurons and plays an important role in the synthesis of protein.Nissl staining can specifically reflect the size,number,morphology,location and survival of neurons [17].After washing with distilled water,paraffin sections of hippocampal tissue (5 μm) were stained for 10 min with 0.1%toluidine blue.The excess dye was removed with water.The samples were dehydrated with ethanol and fixed with glycerin-gelatin.The number of neurons and neuronal optical density were determined using NIS-Elements BR 2.30 image analysis software.

3.2.Analysis of cell membrane integrity

Cell membrane integrity was determined using the method of Hiraoka and Kimbara[18].Briefly,following the addition of 10 μL of propidium iodide (1 g/L), the cells were incubated for 10 min and observed under a microscope.Three horizons were selected at random to determine the average number of damaged cells.

3.3.Histological analysis

Hippocampal tissues were fixed in 4%paraformaldehyde for 2 h at room temperature and sliced.Next,paraffin sections were prepared, stained with haematoxylin and eosin (H&E), and observed under a light microscope.

3.4.Determination of mitochondrial membrane potential

Suspension of hippocampus cells was prepared using 0.125%trypsin digestion.Cell density was adjusted to 5×105/mL with PBS.Fluo3/AM was added to final concentration of 10 μmol/L,and the cells were incubated for 1 h in the dark.Afterward, the cells were centrifuged and the supernatant was removed.The cells were washed 3 times with PBS and resuspended in 0.5 mL.The average value of fluorescence intensity was determined on 104neurons using flow cytometry with the excitation and emission wavelengths of 488 and 530 nm,respectively.

3.5.Changes in intracellular Ca2+ concentration

Hippocampus cells suspension was prepared using 0.125%trypsin digestion.The cells were resuspended in culture medium and cell density was adjusted to 5×106/mL.Rhodamine 123 was added to a final concentration of 10 μg/mL,and the cells were incubated for 30 min in a carbon dioxide incubator at 37°C and 5%CO2.The average value of fluorescence intensity was determined on 104neurons using flow cytometry with the excitation and emission wavelengths of 506 and 526 nm,respectively.

3.6.Determination of NO,MDA,SOD,and glutathione levels

NO, MDA, SOD and GSH levels, which are important indicators of response to oxidative stress, were determined using thecorresponding commercial kits(Nanjing Jiancheng Bioengineering Institute,Nanjing,China)according to the manufacturer’s instructions.

Table 1 The identification of major anthocyanins in blueberry extracts.

3.7.Statistical analysis

Data were expressed as mean±SD.Statistical analyses were performed using the SPSS Statistics v.16.0 software (SPSS Inc.,Chicago, IL, USA) with t-test P ＜0.05 was considered statistically significant.

4.Results and discussion

4.1.Anthocyanins composition

The individual compositions and contents of anthocyanins detected in blueberry anthocyanins extracts are shown in Table 1.A chromatogram and mass spectrum are presented in Fig.1.A total of 13 anthocyanins were identified in blueberry anthocyanin extracts.Among these individual monomeric anthocyanins, delphinidin 3-galactoside, delphinidin 3-arabinoside, cyanidin 3-galactoside,petunidin 3-galactoside, petunidin 3-arabinoside, malvidin 3-galactoside, malvidin 3-glucoside and malvidin 3-arabinoside appeared to be the main monomer anthocyanins in blueberry anthocyanin extracts, accounting for 25.26%, 22.66%, 13.11%,10.08%, 9.09%, 5.14%, 6.95% and 50.13%, respectively.The contents of cyanidin 3-acetylhexoside, petunidin 3-acetylglucoside,delphinidin 3-glucoside, and cyanidin 3-glucoside were low,accounting for 0.38%,0.16%,0.02%and 0.03%,respectively.Cyanidin 3-arabinoside can only be traced by MS.

4.2.Effects of blueberry anthocyanin extracts on memory function of rats

Morris water maze test is a classic method to evaluate the memory function of rats.As shown in Table 2, in the present study, compared to the control group, ELF-EMF exposure significantly reversed the memory function of rats at different stages,as indicated by a longer escape latency.Blueberry anthocyanin treatment prevented ELF-EMF-induced memory damage.On day 20 after ELF-EMF exposure for 30 days, escape latency of rats supplemented with blueberry anthocyanin extracts (200 mg/kg per day) was significantly shorter than that of the untreated rats.Our results suggest that blueberry anthocyanin extracts significantly restore the memory function of rats exposed to ELF-EMF, in agreement with previous work [19].Similar reports have previously shown that Curcuma comosa hexane extract improved the performance of rats in the Morris water maze, and that D-camphor-crataegus berry extract combination enhanced the cognitive ability of human participants aged 50-80 years[20].

Fig.1.Identification of major anthocyanins.A, Delphinidin 3-galactoside; B, Delphinidin 3-arabinoside; C, Cyanidin 3-galactoside; D, Petunidin 3-galactoside; E,Cyanidin 3- arabinoside; F, Petunidin 3-arabinoside; G, Malvidin 3-galactoside; H,Malvidin 3-glucoside; I, Malvidin 3-arabinoside; J, Cyanidin 3-acetylhexoside; K,Petunidin 3-acetylglucoside;L,Delphinidin 3-glucoside;M,Cyanidin 3-glucoside.

4.3.Effects of blueberry anthocyanin extracts on hippocampal neuron damage

After dispersion of Nissl substance or contained intracytoplasmic vacuoles,neurons appear hyperchromatic or pale and shrunken[21].In order to evaluate the protective effects of blueberry anthocyanin extracts on ELF-EMF-induced hippocampal neuron damage,Nissl substance content and cell membrane integrity were determined.As shown in Fig.2,Nissl substance content in rats exposed toELF-EMF without blueberry anthocyanin extracts supplementation was significantly lower compared to control.However, blueberry anthocyanin treatment inhibited the decrease in Nissl substance content,particularly at doses of 100 and 200 mg/kg per day.No significant difference in Nissl substance content was found between the control group and the high dose group (200 mg/kg per day),indicating that blueberry anthocyanin extracts suppressed the decline of Nissl substance content caused by ELF-EMF exposure in a dose-dependent manner.

Table 2 Escape latency of rats at different stages.

Fig.2.The effects of blueberry anthocyanins on nissl substance and cell membrane integrity.Results are presented as mean±SD(n=10).aP ＜0.05,compared with control group; 1P ＜0.05,compared with model group.

Cell membrane integrity is a key indicator for evaluating cell damage[22].The results of cell membrane integrity are presented in Fig.2.Cell membrane integrity of hippocampal neurons of rats exposed to ELF-EMF was 91.6%, significantly lower than control.Treatment with blueberry anthocyanin extracts suppressed the decrease.Cell membrane integrity of hippocampal neurons of rats treated with blueberry anthocyanin extracts at 200 mg/kg per day was significantly higher than that of rats in the ELF-EMF model group.These results suggest that blueberry anthocyanin extracts suppress ELF-EMF-induced hippocampal neuron damage by maintaining cell membrane integrity,potentially by inhibiting oxidative stress[23,24].

Histological analysis of the control group revealed normal tissue structure: clear organization layers, neat arrangement, and complete shape(Fig.3).Tissues exposed to ELF-EMF without blueberry anthocyanin extracts post-treatment showed a significant reduction in observable tissue layers.Interestingly, the 200 mg/kg per day blueberry anthocyanin extracts post-treatment significantly reduced ELF-EMF-induced damage.

In the central nervous system, the hippocampus is a key brain region for learning and memory.By analyzing the Morris water maze test and the analysis of nissl substance, the results showed that ELF-EMF exposure could cause hippocampal neuron damage and affect the learning and memory ability of rats.The damage of hippocampal neurons is mainly reflected in the reduction of nissl substance content,the decrease of cell membrane integrity and the reduction of tissue structure.Blueberry anthocyanin extracts have a certain protective effect on this damage mechanism,and have a recovery effect on memory damage in rats.

Fig.3.Histological changes of hippocampal neurons.Magnification factor:400×.

4.4.Effects of blueberry anthocyanin extracts on mitochondrial membrane potential

Mitochondrial membrane potential is important in maintaining mitochondrial integrity [25].As shown in Fig.4, ELF-EMF exposure significantly decreased mitochondrial membrane potential.The decrease was inhibited by blueberry anthocyanin extracts,particularly at a dose of 200 mg/kg per day.Moreover, mitochondrial membrane potential of hippocampal neurons of rats treated with 200 mg/kg per day blueberry anthocyanin extracts was significantly different compared to untreated rats exposed to ELF-EMF.These results demonstrated that blueberry anthocyanin extracts protect hippocampal neurons from ELF-EMF damage by preventing the decrease of mitochondrial membrane potential.Our results are consistent with a previous report by Li et al.[26],who suggested that tea polyphenols ameliorated the changes in mitochondrial membrane potential.

4.5.Effects of blueberry anthocyanin extracts on intracellular Ca2+ concentration

The imbalance of intracellular Ca2+contributes to ROS generation and is associated with cellular responses including growth,death,and gene expression[27-29].The changes in Ca2+concentration determined in this study are presented in Table 3.The highest Ca2+concentration was found in untreated rats exposed to ELF-EMF(26.81 μmol/L), and was 5.23 μmol/L higher than that of control rats.Blueberry anthocyanin extracts application inhibited the ELFEMF-induced increase in Ca2+concentration(Fig.4).In a previous report, Yin et al.[30] suggested that lotus seedpod procyanidins inhibited the elevation of intracellular Ca2+and prevented the disruption of mitochondrial membrane potential caused by ELF-EMF exposure.

Fig.4.The effects of blueberry anthocyanins on mitochondria membrane potential and Ca2+levels.Results are presented as mean±SD (n=10).aP ＜0.05, compared with control group; 1P ＜0.05,compared with model group.

Table 3 The effects of blueberry anthocyanins on mitochondria membrane potential and Ca2+ levels.

ELF-EMF exposure can lead to damage of hippocampal neurons,damage of cell structures and functions, decrease of mitochondrial membrane potential and hindrance of neuronal energy metabolism.Mitochondria are the main part of cell energy metabolism.Due to the increase of intracellular Ca2+contributes,the protective uptake of Ca2+by mitochondria leads to calcium overload, which further leads to the decrease of mitochondrial membrane potential and changes in the morphology and function of mitochondria.This exacerbates nerve cell damage, which triggers apoptosis.The above results further confirmed that the preventive effect of blueberry anthocyanin extracts on hippocampal rat neurons from ELF-EMF-caused damage was related to the improvement of mitochondrial function.

4.6.Effects of blueberry anthocyanin extracts on oxidative stress

NO, MDA, SOD, and GSH are important indicators of oxidative stress.The effects of blueberry anthocyanin extracts on ELF-EMFcaused oxidative stress were evaluated by determining NO, MDA,SOD, and GSH levels.Generation of NO, a highly reactive oxidant,is involved in formation of the lipid peroxidation product MDA[31].In order to examine the mechanisms involved in protective effects of blueberry anthocyanin extracts on hippocampal neurons,the levels of NO and MDA were determined (Fig.5).NO levels in rats exposed to ELF-EMF were significantly increased compared to control; an effect suppressed by blueberry anthocyanin extracts.Similarly, blueberry anthocyanin extracts treatment attenuated the ELF-EMF-induced increase in MDA content.Moreover, MDA content of ELF-EMF exposed rats treated with 200 mg/kg per day blueberry anthocyanin extracts was significantly decreased compared to untreated rats exposed to ELF-EMF.These results suggest that blueberry anthocyanin extracts treatment inhibits ELF-EMFcaused oxidative stress in hippocampal neurons via attenuation of NO and MDA generation,and are in agreement with the results of Hassan, Serage, and Gad [32], who reported that blackberry juice decreased aluminum-and fluoride-induced NO and MDA increase.

SOD and GSH prevent lipid peroxidation and are important physiological antioxidants[33,34].As shown in Fig.5,SOD and GSH activities were significantly decreased by ELF-EMF exposure.The effect was attenuated by blueberry anthocyanin extracts supplements,with greatest results obtained at 200 mg/kg per day.These results demonstrate that blueberry anthocyanin extracts suppress the decrease in SOD and GSH activities caused by ELF-EMF exposure, consistent with a previous report by Freitas et al.[35], who suggested that agmatine maintained hippocampal antioxidant balance in mice by increasing the activities of SOD and GSH.

Fig.5.The effects of blueberry anthocyanins on NO, MDA, SOD and GSH levels.Results are presented as mean±SD (n=10).aP ＜0.05, compared with control group; 1P ＜0.05,compared with model group.

Under normal circumstances, the production of ROS and the scavenging of ROS in the body are in a state of dynamic equilibrium.The results showed that ELF-EMF exposure could upset this equilibrium and lead to oxidative stress.Blueberry anthocyanin extracts treatment can inhibit oxidative stress and protect hippocampal neurons.

5.Conclusions

The results of this study suggest for the first time that blueberry anthocyanin extracts protect hippocampal rat neurons from ELFEMF-caused damage.This finding is corroborated by higher levels of Nissl substance,improved cell membrane integrity,and increased mitochondrial membrane potential,compared to the model group.Possible mechanism involved is suppression of oxidative stress,indicated by reduced NO,MDA,and Ca2+levels and increased SOD and GSH activities.In addition, Morris water maze test revealed that blueberry anthocyanin extracts enhanced the cognitive ability of rats exposed to ELF-EMF.These findings provide a basis for the use of blueberry anthocyanins in suppressing hippocampal damage induced by ELF-EMF exposure.

Declaration of Competing Interest

The authors declare that they have no conflict of interest.

Acknowledgments

This work was supported by the Natural Science Foundation Key Program of Liaoning Province(20170540803),the Liaoning Provincial Department of Education Project(LSNJC201911).