A Comparative Study About the Neuroprotective Effects of EPA-Enriched Phosphoethanolamine Plasmalogen and Phosphatidylethanolamine Against Oxidative Damage in Primary Hippocampal Neurons

ZHU Yunfang, ZHANG Tiantian, DING Lin, SHI Haohao, XUE Changhu, 3),XIE Wancui, CHE Hongxia, , , and WANG Yuming, 3),

A Comparative Study About the Neuroprotective Effects of EPA-Enriched Phosphoethanolamine Plasmalogen and Phosphatidylethanolamine Against Oxidative Damage in Primary Hippocampal Neurons

ZHU Yunfang1), ZHANG Tiantian1), DING Lin1), SHI Haohao1), XUE Changhu1), 3),XIE Wancui2), CHE Hongxia1), 2),*, and WANG Yuming1), 3),*

1),,266003,2),,,266042,3),,266237,

s Oxidative stress is involved in the progression of neurodegenerative diseases. Previous evidences showed that plasma- logens could improve neurodegenerative diseases. In this study, we investigated the function of phosphoethanolamine plasmalogens enriched with EPA (EPA-pPE) and phosphatidylethanolamine enriched with EPA (EPA-PE) on oxidative damage prevention after hy- drogen peroxide (H2O2) and tert-butylhydroperoxide (t-BHP) challenge in primary hippocampal neurons.Results showed that neu- rons pretreated with EPA-pPE and EPA-PE demonstrated the ability to alleviate oxidative damage, which was proved by the in- creased cell viability. Moreover, the shape and number of neurons were more similar to those of the control group. Antioxidant acti- vity, apoptosis, as well as TrkB/ERK/CREB signaling pathway were investigated to explore the mechanisms. The results suggested that EPA-PE was superior to EPA-pPE in regulating mitochondrial apoptosis. EPA-pPE was more prominent than EPA-PE in upre- gulating TrkB/ERK/CREB signaling pathway. Phospholipids with EPA exerted neuroprotective effectsinhibiting oxidative stress, suppressing apoptosis, and regulating TrkB/ERK/CREB signaling pathway. Therefore, the results provide a scientific basis for utili- zation of phospholipids enriched with EPA on the treatment of neurodegenerative disease.

oxidative stress; EPA-enriched phosphoethanolamine plasmalogens; EPA-enriched phosphatidylethanolamine; primary hippocampal neurons

1 Introduction

Neuronal damage is one of the characteristics of Al- zheimer’s disease (AD). Oxidative stress is supposed to play an important role in this process (Singh., 2019). Excessive free radicals can cause cell dysfunction, and even lead to cell deathattacking proteins, DNA, lipids and other biological macromolecules (Tonnies and Trushina,2017). Therefore, improving the oxidative stress is beli- eved to be an effective therapeutic strategy to prevent and treat neurodegenerative diseases.

Mounting evidences have verified that fish oil can main- tain neuronal function, improve cognitive deficit, and alle- viate neurodegenerative disease (Shahidi and Ambigai- palan, 2018). Various investigations have shown that DHA/EPA enriched glycerophospholipids (DHA/EPA-PL) exhi- bit better bioactivity than triglycerides enriched with DHA/ EPA(DHA/EPA-TG) and DHA/EPA-enriched ethyl esters (DHA/EPA-EE) (Burri and Johnsen, 2015; Wen., 2019).Previous studies have reported that the protective effects of DHA/EPA-PL were more significant than other forms of DHA in relieving neuronal apoptosis induced by MPTP (Wang., 2018). Though the neuroprotective effects of DHA have been proven well, few studieshave highlight- ed the protective effects of EPA. Previous studies reported that EPA-phosphatidylcholine, rather than EPA-EE, was equivalent to DHA in improving cognitive deficit (Wen., 2016). Wu. (2014) showed that phospholipids enriched with EPA protected PC12 cells from oxidative damage.Our studies also showed that phosphatidylserine enriched with DHA/EPA protected PC12 cells against oxi- dative damage (Che., 2018).

Phosphoethanolamine plasmalogens are special glycer- ophospholipids, which are featured by vinyl ether bond at the sn-1 position (Paul., 2019). Previously, we found that phosphoethanolamine plasmalogens enriched with EPA (EPA-pPE) were more effective than phosphatidylethano- lamine enriched with EPA (EPA-PE) in decreasing Aβcon- centration (Che., 2018).As endogenous antioxidant,plasmalogens can effectively scavenge free radicals due tothe vinyl ether linkage. This supports the presumption thatplasmalogens can exert beneficial effects in the preven- tion and treatment of neurodegenerative diseases.

Our previous studies were entirely conducted in PC12 cells, which are ‘neuron-like’ cells and are not considered to be equivalent to neurons. The hippocampus, mainly re- sponsible for learning and memory, is one of the most vul- nerable parts of the brain. Moreover, the dysfunction of hippocampal is involved in numerous pathological condi- tions. In this study, we evaluated the neuroprotective func- tion of glycerophospholipids enriched with EPA (EPA-pPE and EPA-PE) against H2O2/t-BHP-induced oxidative da- mage and the possible mechanism in primary hippocam- pal neurons. The results suggest that EPA-PL can relieveneuronal cell damage induced by H2O2/t-BHP, as indicated by the increased cell viability. We further explored the pro- tective effects of EPA-pPE and EPA-PE on antioxidation, apoptosis, and TrkB/ERK/CREB pathway to explain the underlying mechanisms. All data might provide interest- ing insights into the utilization of EPA-enriched phospho- lipids on the prevention and treatment of neurodegenera- tive disease.

2 Materials and Methods

2.1 Materials

was obtained from Nanshan aqua- tic market of Qingdao (Qingdao, China). Superoxide dis- mutase (SOD) and total antioxidant capacity (T-AOC) as- say kits were purchased from Nanjing Jiancheng Bioen- gineering Institute (Nanjing, China). Antibodies of Bax,iNOS, Caspase 3, Cyt-c, phosphorylated TrkB (Y515), phos- phorylated ERK and phosphorylated CREB, synaptophy- sin (SYN), PSD-95 were obtained from Cell Signaling Tech- nology (Boston, USA).

2.2 Extraction and Purification of EPA-PC and EPA-pPE

was used for the extraction of to- tal lipids. After removing neutral lipids and glycolipids, EPA-PLs were obtained by silica gel column chromato- graphy (Wu., 2014). EPA-pPE and EPA-PC were se- parated and extracted from EPA-PL (Ding., 2020). EPA-PE and Egg-PE were enzymatic synthesized accord- ing to our previous study(Che., 2018). EPA-PE and Egg-PE were separated and extracted from the above re- action system by silica-gel column chromatography. Thepurity of EPA-pPE (93.4%), EPA-PE (92.6%) and Egg-PE (96.8%) were detected according to HPLC-ELSD. EPAin EPA-pPE, EPA-PE and Egg-PE account for 45.6%,49.3% and 0.82%, respectively. There was no obvious dif- ference of the fatty acids composition between these two EPA groups as shown in our previous research (Che., 2017). To prevent the peroxidation, EPA-pPE, EPA-PE and Egg-PE were stored in ?80℃ before further analyses.

2.3 Isolation of Primary Hippocampal Neurons

P0 pups of Sprague-Dawley (SD)rats were provided by Qingdao Lukang Pharmaceutical Experimental Animal Center. Hippocampal neurons were isolated following the methods in previous study (Calvo-Rodriguez., 2017).Briefly, the hippocampi of the P0 pups was separated quick-ly. Then the tissues were put into trypsin solution (0.125%) after they were cut into pieces on ice box. The hippocam- pi pieces in trypsin were placed in CO2incubator (37℃) with gentle shaking every 5min. After 20min, the hippo- campi pieceswere centrifuged to discard the trypsin. Sub- sequently, medium with Fetal Bovine Serum (9:1) was add- ed to stop enzymatic hydrolysis. After 10min the cell pel- lets were collected after centrifugation, and suspended in neurobasal medium and placed for 5min, hippocampal neu- rons suspension was obtained. After the cells adhered to the dish wall, the medium was discarded, and the cells were washed twice with PBS. The cells were cultured in neurobasal medium with 2% B-27, 100UmL?1penicillin, and 100μgmL?1streptomycin. The medium was replaced every other day. When primary hippocampal neurons form- ed a denser neural fiber network, they were used for ex- periments. The purity of neurons (>95%) was detected by immuno-fluorescence staining (Che., 2020).

2.4 Measurement of Cell Viability

EPA-pPE, EPA-PE and Egg-PE liposome were freshly prepared and diluted to the appropriate concentration by medium for following experimentsMTT assay was adopt- ed to detect the viability of hippocampal neurons. Hippo- campal neurons suspension (200μL)was seeded on 96- well (3.5×105mL?1) plate. Once the neural network was formed, hippocampal neurons were treated with different concentrations of H2O2(0, 200, 400, 600μmL?1) for 24h, or with t-BHP (0, 50, 100, 150, 200μmL?1) for 2h and 4h to determine the optimal concentrations.

The neurons were pre-treated with 10μgmL?1EPA-pPE, EPA-PE and Egg-PE for 24h to explore the neuroprotec- tive function against the oxidative damage induced by H2O2and t-BHP. The MTT detection method was the same as our previous experiment(Che., 2018).

Inverted microscope was used to observe the morpho- logy of hippocampal neurons to determine whether EPA- PLs could protect hippocampal neurons from t-BHP in- duced damage.

2.5 Determination of SOD and T-AOC Activity

Primary hippocampal neurons (2mL, 1×106cellsmL?1) were seeded in a six-well plate. Following 4h t-BHP in- cubation, the medium was removed. After washed with PBS, the neurons were then scraped from the six-well plate, and placed into 200μL cold PBS. Cell homogenate was obtained using an ultrasonic cell disruptor. The su- pernatant was collected after centrifugation (4000, 20min) at 4℃. The SOD and T-AOC activities were quanti- fied by corresponding assay kits, respectively.

2.6 Western Blotting Analysis

DNA-RNA-Protein Kit (Omega Bio-Tek, USA) was used to extract protein. Subsequently, BCA protein assay kit (Be- yotime, China) was adopted to determine the total protein concentration. The western blotting experiments were con- ducted according to our previous study(Che., 2020). The protein expression of iNOS, Bax, Cyt-c, Caspase 3, phosphorylated TrkB, phosphorylated ERK, PSD95, syn- aptophysin, phosphorylated CREB and β-actin were de- tected using western blotting.

2.7 Statistical Analysis

Data were presented as mean±SEM. Variance of all the groups was analyzed using SPSS software (version 18.0). Statistical significance between the model group and con- trol group was tested by t-test. Duncan’s multiple-range test was used to compare the difference between EPA-PLs groups and model group.When<0.05,it is considered statistically significant.

3 Results

3.1 Effects of H2O2 and t-BHP on Cell Viability of Primary Hippocampal Neurons

The survival of hippocampal neurons under oxidative stress was quantified using MTT method (Fig.1). The 50% inhibitory concentration of H2O2was approximately 400μmolL?1when primary hippocampal neurons were treat- ed for 24h. The 50% inhibitory concentration of t-BHP was about 100μmolL?1when neurons were incubated for 4h. Therefore, 400μmolL?1H2O2and 100μmolL?1t- BHP were used to induce injury of primary hippocampal neurons for the subsequent experiments in the present study.

Fig.1 Effects of H2O2 and t-BHP on viability of hippocampal neurons. Primary hippocampal neurons were treated with various doses of (A) H2O2 for 24h or (B) t-BHP for 2h and 4h. Results are expressed as mean±SEM of three indepen- dent experiments.

3.2 EPA-PLs Improved Neuronal Survival

EPA-pPE, EPA-PE and Egg-PE (5, 20, 60μgmL?1) were added to the primary cultured hippocampal neurons. No to-xic effects of all phospholipids (less than 10μgmL?1) wereobserved on the hippocampal neurons within 24h (data not shown). The concentration of 10μgmL?1was selected to in-vestigate the protective functions of EPA-pPE, EPA-PE and Egg-PE (Fig.2). From the results, EPA-pPE, EPA-PE and Egg-PE could remarkably decrease the cell cytotoxicity in-duced by H2O2or t-BHP,and the protective function of pho- spholipids enriched with EPA were superior to Egg-PE.

Fig.2 Effects of different phospholipids on cell survival when treated with H2O2 (A) and t-BHP (B). Results are expressed as mean±SEM of three independent experiments. *P<0.01, versus control group. Different superscripts among these groups are significantly different (P<0.05, one-way ANOVA).

3.3 Phospholipids Enriched with EPA Improved Neuronal Morphology

The protective function of phospholipids enriched with EPA on t-BHP induced oxidative damage of hippocampal neurons were investigated. Inverted microscope was used to observe the cellular morphology of neurons. The neu- rons in model group was heterogeneous and round, and the dendrite length was obviously decreased when treated with t-BHP. Interestingly, EPA-pPE and EPA-PE pretreatments could significantly alleviate oxidative damage as proved by the re-formation of neural network structure (Fig.3).

Fig.3 Effects of EPA-enriched phospholipids on cellular morphology of primary hippocampal neurons treated with EPA- pPE or EPA-PE for 24h then exposed to 100μmolL?1 t-BHP for 4h. Cellular morphology was observed by inverted micro- scope.

3.4 EPA-pPE and EPA-PE Increased SOD and T-AOC Activities

SOD and T-AOC activities as well as the expression of iNOS were measured to explore whether EPA-pPE and EPA-PE could improve oxidative damage by increasing anti-oxidant activity and decreasing oxidant activity. As shown in Fig.4, when exposed to t-BHP, SOD and T-AOC activitiesin neurons significantly decreased to 62.25 and 0.91Umg?1protein, respectively. Interestingly, EPA-pPE and EPA-PE pretreatment could markedly relieve the re- duction of SOD and T-AOC activities, and no significant difference was observed between these two groups. Com- pared with model group, the SOD activities in EPA-pPE and EPA-PE group were increased by 41.37% and 28.51%,respectively. The T-AOC activitieswere increased by 31.87%and 57.14%, respectively.

Fig.4 Effects of EPA-pPE and EPA-PE on SOD activity (A), T-AOC activity (B) and iNOS protein level (C). Primary hip- pocampal neurons were pretreated with EPA-pPE and EPA-PE for 24h and then exposed to 100μmolL?1 t-BHP for 4h. Results are expressed as mean±SEM of three independent experiments. *P<0.01, versusmodel group. Different super- scripts among these groups were significantly different (P<0.05).

As shown in Fig.4C, t-BHP exposure obviously increasedthe relative expression of iNOS (<0.05). Accordingly, both EPA-pPE and EPA-PE could evidently suppress the protein level of iNOS. No obvious difference was found between these two groups.

3.5 EPA-pPE and EPA-PE Downregulated Bax, Cyt-c and Caspase 3 Expressions

Oxidative stress can induce apoptosis.Thus we detect- ed the expression of apoptosis-related proteins by western blotting. As shown in Fig.5, t-BHP treatment obviously ele- vated the protein expression of Bax, Cyt-c and Caspase 3 in model group (t-BHP). Interestingly, the pretreatment with EPA-pPE and EPA-PE markedly reduced their expression levels. Importantly, EPA-PE showed a more prominent im- provement effect than EPA-pPE in decreasing the pro-apo- ptotic proteins’ expressions (Bax and Cyt-c). While no sta-tistical difference was found between these two EPA groups in downregulating Caspase 3 expression.

Fig.5 Effects of EPA-pPE and EPA-PE on Bax, Cyt-c and Caspase 3 protein levels in t-BHP treated neurons. The expres- sion levels of Bax, Cyt-c and Caspase 3 were detected by Western blotting analysis and normalized with β-actin. Results were expressed as mean±SEM of three independent experiments. *P<0.01, versusmodel group. Different superscripts among these groups are significantly different (P<0.05).

3.6 Different Phospholipids Treatment Promoted p-TrkB/p-ERK/p-CREB

Western blotting was conducted to further explore the expressions of p-TrkB (Thr515), p-ERK and p-CREB pro- teins, which were related to synaptic plasticity. An obvi- ous decreasing trend was observed after H2O2treatment. Interestingly, the expressions of p-TrkB and p-ERK were significantly upregulated in these two EPA groups. More- over, the protective function of EPA-pPE was superior to EPA-PE. Notably, pretreatment with EPA-pPE and EPA-PEcould evidently upregulate the expression of p-CREB, and no significant difference was observed between these two EPA groups (Fig.6).

3.7 Phospholipids Enriched with EPA IncreasedExpressions ofSynaptophysin and Postsynaptic Density Protein 95

Synaptic vesicle-related proteins synaptophysin (SYN) and postsynaptic density protein 95 (PSD95) play impor- tant roles in regulating neuronal function. Thus we detect- ed the expression of SYN and PSD95 proteins via west- ern blotting (Fig.7). As for the expression of PSD95 pro- tein, no statistical difference was observed among these four groups. Pretreatment of EPA-pPE and EPA-PE could visibly alleviate the reduction of SYN induced by H2O2, and no significant difference was found between these two EPA groups.

4 Discussion

Increasing studies have verified that phospholipids can exert beneficial effects on age-related cognitive decline, such as Alzheimer’s disease. Marine-derived phospholi- pids, sufficient in DHA/EPA, have aroused much atten- tion of scholars due to their biological activities. In ma- rine foods, DHA/EPA-phospholipids mainly exist in four forms: phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol (Zhang., 2019).

Fig.6 Effects of EPA-pPE and EPA-PE on TrkB/CREB signaling in H2O2-treated neurons. The expressions of phospho- rylated TrkB (Y515), phosphorylated ERK and phosphorylated CREB were detected by Western blotting analysis and normalized with β-actin. Results are expressed as mean±SEM of three independent experiments. * P<0.05, versus model group. Different superscripts among these groups are significantly different (P<0.05).

Fig.7 Effects of EPA-pPE and EPA-PE on the expressions of PSD95 and SYN in H2O2-treated neurons. (A),western blot- ting analysis; (B),relative expression of SYN; (C),relative expression of PSD95. Data are presented as mean±SEM. Different letters indicate significant differences among the groups.

The difference between polar group and fatty acid com- position lead to different health efficacy of phospholipids. Marine foods, such as sea cucumbers, ascidians and mus- sels are rich in phosphoethanolamine plasmalogens. Ester bond and ether bond at the sn-1 position was the main dif- ference between phosphoethanolamine plasmalogens and phosphatidylethanolamine. Dietary supplementation of etha- nolamine glycerophospholipids can increase the concen- trations of phosphoethanolamine plasmalogens in the plas- ma and liver in AD rats(Yamashita., 2017). The re- sults above indicated that plasmalogens might improve cog- nitive deficit by regulating brain lipid composition.

In this study, we used primary hippocampal neurons to study the effects of EPA-pPE and EPA-PE on oxidative stress. The results indicated that pretreatment with EPA- pPE and EPA-PE provided protection to hippocampal neu-rons against H2O2/t-BHP-induced damage. Consistent with our previous study, H2O2and t-BHP are cytotoxic to neu- ronal cell in a dose-dependent pattern. EPA-pPE and EPA- PEpretreatments could obviously improve the cell viabi- lity of hippocampal neurons. In addition, EPA-pPE and EPA-PE can improve the abnormal cellular morphology of neurons and promote the re-formation of neural net- work structure. which further suggests that EPA-pPE and EPA-PE have a protective effect on H2O2/t-BHP-induced damage.

It has been confirmed that oxidative stress participatesin the occurrence and development of Alzheimer’s dis- ease(Singh., 2019). Mounting researches revealed that oxidative stress is implicated in neuronal cell death(Uddin., 2020). H2O2and t-BHP are widely used to induce oxidative damage in the study of various neuro- degenerative diseases(Wu., 2014; Che., 2018). SOD plays an important role in maintaining oxida- tive stress balance (Chiang., 2018). T-AOC repre-sents the total antioxidant capacity. The reduced T-AOC and SOD activities can reflect the degree of cell damage. Both EPA-pPE and EPA-PE can remarkably relieve thedecreased T-AOC and SOD activities induced by t-BHPin hippocampal neurons, suggesting that the protective function of EPA-pPE and EPA-PE might partly depend on its anti-oxidative function.

Neuronal death caused by mitochondria-dependent apop- tosis contributes to the development of neurodegenerative (Uddin., 2020). Caspases are key regulatory factors in most death signals, which can be activated by cyto-chrome c (Bortolotti., 2018). In the present study, H2O2and t-BHP significantly enhanced the expressions of Bax, Cyt-c and Caspase 3. Treatments with EPA-PE andEPA-pPE markedly inhibited oxidative stress-mediated mi-tochondrial dysfunction. These data indicated that EPA- PE and EPA-pPE exerted their neuroprotective properties by inhibiting the neuronal apoptosis.

It has been demonstrated that cyclic AMP-dependent re-sponse element-binding protein (CREB) plays pivotal role in neuronal plasticity, which is regulated by neurotrophins, especially the levels of BDNF(Duan., 2014). The binding of BDNF to tropomyosin receptor kinase B (TrkB) leads to TrkB autophosphorylation and activation of its downstream enzymes, including extracellular signal-regu- lated kinases (ERK) and phosphatidylinositol 3 kinase (PI3K)/Akt pathways(Ibrahim., 2020). Our results showed that H2O2obviously downregulated the phospho- rylation of TrkB, ERK and CREB, which indicated that oxidative stress could result in the dysfunction of the TrkB/ ERK/CREB signaling. The present results support the hy- pothesis that both EPA-pPE and EPA-PEcan exert protec- tive function by inducing TrkB autophosphorylation, acti- vating ERK and enhancing CREB phosphorylation in H2O2- treated neurons. Moreover, EPA-pPE was superior to EPA- PE in regulating the TrkB/ERK/CREB signaling. The re- sults demonstrate that phosphoethanolamine plasmalogens might have the capacity affecting neuroplasticity by regu- lating TrkB/ERK/CREB signaling pathway. The present study only primarily compared the effects of EPA-PE and EPA-pPE on oxidative stress with a single dose. Future stu- dies are necessary to clarify the dose-response and struc- ture-activity relationships, as well as the exact mechanism involved in the oxidative stress.

5 Conclusions

In summary, phospholipids enriched with EPA have out-standing effects in regulating oxidative stress-induced neu- ronal damage, as proved by the increased cell viability. Moreover, cells pretreated with EPA-pPE and EPA-PE de-monstrated ability to prevent oxidative damage, as the shape and number of neurons are more similar to those of the control group. Underlying mechanisms including antioxi- dant activity, mitochondrial apoptosis, as well as TrkB/ ERK/CREB signaling pathway were investigated. The re- sults suggest that EPA-PE exerted better effects than EPA- pPE in regulating the mitochondrial apoptosis pathway. EPA-pPE was superior to EPA-PE in upregulating TrkB/ ERK/CREB signaling pathway (Fig.8).

Fig.8 Primary cultured hippocampal neurons are used to compare the effects of EPA-pPE and EPA-PE on TrkB/ ERK/CREB signaling pathway and neuronal apoptosis in oxidative damage model. Our results indicate EPA-PE ex- erted better effects than EPA-pPE in regulating the mitochondrial apoptosis pathway. EPA-pPE was superior to EPA-PE in upregulating TrkB/ERK/CREB signaling path-way. EPA-pPE and EPA-PE exerted neuroprotective ef- fects of primary hippocampal neurons via suppressing apop- tosis and regulating TrkB/ERK/CREB signaling pathway.

Acknowledgement

This work was supported by the National Natural Sci- ence Foundation of China (No. 31901688).

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. E-mail: chechehongxia@163.com E-mail: wangyuming@ouc.edu.cn

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