Neuroprotective Effects of n-3 Polyunsaturated Fatty Acid-Enriched Phosphatidylserine Against Oxidative Damage in PC12 Cells

Abstract

Neurodegenerative diseases are defined by progressive loss of specific neuronal cell populations and are associated with protein aggregates. Oxidative stress has been implicated in their pathological processes. Previous studies revealed that docosahexaenoic acid (DHA) is beneficial in neurodegenerative diseases. Phospholipids (PLs) derived from marine products are rich in DHA and eicosapentaenoic acid (EPA). In the present study, we investigated the neuroprotective effects of DHA-enriched and unenriched phosphatidylcholine (PC) and phosphatidylserine (PS) on oxidative stress induced by hydrogen peroxide (H₂O₂) and tert-butylhydroperoxide in PC12 cells. Cell viability and leakage of lactate dehydrogenase results showed that the neuroprotective effect of PS was superior to that of PC. DHA- and EPA-enriched PC and PS were superior to that without DHA or EPA; in addition, the improvement with n-3 polyunsaturated fatty acid-enriched PS (n-3 PS) was dose dependent. Acridine orange/ethidium bromide staining showed that DHA- and EPA-enriched PS (DHA/EPA-PS) could significantly inhibit apoptosis. Mechanistic studies revealed that EPA-PS and DHA-PS were effective to increase superoxide dismutase (SOD) levels by 48.4 and 58.2 % and total antioxidant capacity (T-AOC) level by 51 and 94 %, respectively, in the H₂O₂ model. Similar results for SOD and T-AOC levels were shown in the t-BHP model. EPA/DHA-PS could downregulate the messenger RNA level of Caspase-3, Caspase-9, and Bax, upregulate Bcl-2, inhibit Bax, and increase Bcl-2 at protein level. In conclusion, EPA/DHA-PS could protect PC12 cells from oxidative stress and prevent mitochondrial-mediated apoptosis. Our findings indicate that the neuroprotective effects of DHA/EPA-PLs depend on the molecular form. Further studies are necessary to reveal detailed mechanisms and structure-effect relationships.

Keywords: Antioxidant; Mitochondrial-mediated apoptosis; Oxidative stress; n-3 polyunsaturated fatty acid-enriched phosphatidylserine.

Fig. 1

Structures of phosphatidylcholine and phosphatidylserine. a Phosphatidylcholine (PC) contains a choline head-group; sn-2 can be replaced by EPA and DHA to form EPA-PC and DHA-PC, respectively. b Phosphatidylserine (PS) contains a serine head-group; sn-2 can be replaced by EPA and DHA to form EPA-PS and DHA-PS, respectively

Fig. 2

Effect of phospholipids from different sources on viability of PC12 cells. Cell viability was measured by MTT assay after pretreatment with PC (a) and PS (b) from different sources for 24 h. Results are mean ± SEM of three independent experiments

Fig. 3

Dose-dependent toxicity of H₂O₂ and t-BHP in PC12 cells. PC12 cells were treated with various doses of a H₂O₂ (100, 200, 250, 300, 400, and 500 μmol/L) or b t-BHP (50, 100, 150, 200, 300, and 400 μmol/L) for 4 h, and cell viability was measured by MTT assay. Concentrations of 200 μmol/L H₂O₂ and 150 μmol/L t-BHP were chosen for subsequent experiments. Results are mean ± SEM of three independent experiments

Fig. 4

Effect of different phospholipids on cell survival when treated with H₂O₂. PC12 cells were pretreated with different phospholipids (40 μg/mL) and serine (Ser, 40 μg/mL) for 24 h, then exposed to 200 μmol/L H₂O₂ for 4 h. Cell viability was measured by MTT assay (a), and LDH leakage into medium was determined by assay kit (b). Results are mean ± SEM of three independent experiments. ^## P < 0.01, versus normal group, *P < 0.05, **P < 0.01, versus H₂O₂ group. ^a–f Means with different superscripts among these groups are significantly different (P < 0.05, one-way ANOVA)

Fig. 5

Effect of n-3 PS on survival of PC12 cells pretreated with different concentrations (10 and 40 μg/mL) of EPA-PS and DHA-PS for 24 h then exposed to 200 μmol/L H₂O₂ or 150 μmol/L t-BHP for 4 h. Cell viability was measured by MTT assay (a, c), and leakage of LDH to medium was determined by assay kit (b, d). Results are mean ± SEM of three independent experiments. ^## P < 0.01, versus normal group, *P < 0.05, **P < 0.01, versus model group

Fig. 6

Effect of n-3 PS on cellular morphology of PC12 cells treated with EPA-PS or DHA-PS (10, 40 μg/mL) for 24 h then exposed to 200 μmol/L H₂O₂ for 4 h. Cellular morphology was observed by inverted microscope

Fig. 7

Effects of n-3 PS on apoptosis determined morphologically after staining cells with AO/EB followed by fluorescence microscopy inspection. PC12 cells were pretreated with phospholipids prior to incubation with H₂O₂. At the end of incubation, the cells were harvested, washed with PBS, and adjusted to density of 10⁶ cells/mL of PBS. AO/EB solution was added to the cell suspension in final concentration of 100 μg/mL

Fig. 8

Effect of n-3 PS on SOD activity and T-AOC in PC12 cells pretreated with EPA-PS or DHA-PS (40 μg/mL) for 24 h then exposed to 200 μmol/L H₂O₂ or 150 μmol/L t-BHP for 4 h. SOD activity (a, b) and T-AOC (c, d) were measured by assay kit. Results are mean ± SEM of three independent experiments. ^## P < 0.01, versus normal group. ^a–cMeans with different superscripts among these groups are significantly different (P < 0.05, one-way ANOVA)

Fig. 9

Effect of n-3 PS on Bcl-2, Caspase-9, Caspase-3, Akt-2, and GSK-3β mRNA level in PC12 cells pretreated with EPA-PS or DHA-PS (40 μg/mL) for 24 h then exposed to 150 μmol/L t-BHP for 4 h. The mRNA levels of Caspase-3 (a), Caspase-9 (b), Bcl-2 (c), Akt-2 (d), and GSK-3β (e) were determined by quantitative RT-PCR and normalized against β-actin. Results are mean ± SEM of three independent experiments. ^# P < 0.05, ^## P < 0.01, versus normal group. ^ab Means with different superscripts among these groups are significantly different (P < 0.05, one-way ANOVA)

Fig. 10

Effect of n-3 PS on Bcl-2 and Bax protein level in PC12 cells pretreated with EPA-PS or DHA-PS (40 μg/mL) for 24 h then exposed to 150 μmol/L t-BHP for 4 h. Expression of Bcl-2 and Bax level were detected by Western blot analysis and normalized against β-actin. Results are mean ± SEM of three independent experiments. ^## P < 0.01, versus normal group. ^a–cMeans with different superscripts among these groups are significantly different (P < 0.05, one-way ANOVA)