Targeted Isolation of ω-3 Polyunsaturated Fatty Acids from the Marine Dinoflagellate Prorocentrum lima Using DeepSAT and LC-MS/MS and Their High Activity in Promoting Microglial Functions

Abstract

In this study, we integrated HSQC-based DeepSAT with UPLC-MS/MS to guide the isolation of omega-3 polyunsaturated fatty acid derivatives (PUFAs) from marine resources. Through this approach, four new (1-4) and nine known (5-13) PUFA analogues were obtained from large-scale cultures of the marine dinoflagellate Prorocentrum lima, with lipidomic profiling identifying FA18:5 (5), FA18:4 (7), FA22:6 (8), and FA22:6 methyl ester (11) as major constituents of the algal oil extract. Structural elucidation was achieved through integrated spectroscopic analyses of IR, 1D and 2D NMR, and HR-ESI-MS data. Given the pivotal role of microglia in Alzheimer's disease (AD) pathogenesis, we further evaluated the neuroprotective potential of these PUFAs by assessing their regulatory effects on critical microglial functions in human microglia clone 3 (HMC3) cells, including chemotactic migration and amyloid-β42 (Aβ42) phagocytic clearance. Pharmacological evaluation demonstrated that FA20:5 butanediol ester (1), FA18:5 (5), FA18:4 (7), FA22:6 (8), and (Z)-10-nonadecenoic acid (13) significantly enhanced HMC3 migration in a wound-healing assay. Notably, FA18:4 (7) also significantly promoted Aβ42 phagocytosis by HMC3 microglia while maintaining cellular viability and avoiding pro-inflammatory activation at 20 μM. Collectively, our study suggests that FA18:4 (7) modulates microglial function in vitro, indicating its potential to exert neuroprotective effects.

Keywords: DeepSAT; Prorocentrum lima; UPLC-MS/MS; amyloid-β42 clearance; microglia migration; omega-3 polyunsaturated fatty acid (ω-3 PUFA); targeted isolation.

Figure 1

DeepSAT-based prioritization of the HSQC spectrum of petroleum ether (PE)-soluble fraction of P. lima and the DeepSAT results (top five structures based on cosine similarity score) for PE fraction of P. lima.

Figure 2

The UPLC-MS/MS results of PE-soluble fraction of P. lima. (A): Extracted ion chromatograms (EICs) in the positive-ion mode. (B): Base peak intensity (BPI) chromatograms in the positive-ion mode. (C): Extracted ion chromatograms (EICs) in the negative-ion mode. (D): Base peak intensity (BPI) chromatograms in the negative-ion mode.

Figure 3

The structures of compounds 1–13 from the cultures of P. lima.

Figure 4

Key correlations of ¹H-¹H COSY and HMBC for compounds 1–4.

Figure 5

Effects of isolated compounds on HMC3 microglial motility and viability. (A): Schematic diagram of scratch wound healing assay: scratch regions in 12-well plates and observation time points. (B): Cell migration microscopy images: yellow dashed lines demarcate scratch boundaries. (C): Scratch healing quantification: percentage of healed area at different time points across treatment groups (n = 5). (D): Cell viability assay: HMC3 cells were treated with DMSO, LPS (1000 ng/mL), or compounds 1–13 (20 μM) for 24 h (n = 5). (E): Data represent the mean values of cell viability (relative to DMSO) and mortality (calculated as 100%—viability) for DMSO (control), LPS (1000 ng/mL), and FA20:5 propanediol ester (2), FA18:4 butanediol ester (3), FA20:5 (6, EPA), FA19:5 (9), and FA18:4 methyl ester (12) (20 μM, 24 h treatment). All significant differences were analyzed versus the DMSO group. Data presented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA, followed by least-significant-difference test).

Figure 6

Aβ42 phagocytic clearance capacity of isolated compounds. (A): FITC-Aβ42 phagocytosis assay workflow: HMC3 cells co-incubated with FITC-Aβ42 for 12 h followed by flow cytometry analysis. (B): Representative flow cytometry plots depicting phagocytosis of FITC-Aβ42 by DAPI-positive cells in LPS (100 ng/mL), FA18:4 (7) (20 μM), FA20:5 (6, EPA, 20 μM), and FA22:6 (8, DHA, 20 μM)-treated groups. The fluorescence signals of cells untreated with FITC-Aβ42 were established as background (labeled as Aβ−) and served as the gating criteria for defining FITC-Aβ42 positive cells (labeled as Aβ+). (C): Phagocytosis rate quantification: flow cytometry detection of Aβ-positive cells (n = 5). (D): Immunofluorescence images of phagocytosis: Iba1 (red), FITC-Aβ42 (green), Hoechst (blue). Scale bar = 20 μm. (E): Inflammatory factor expression: qPCR analysis of Il-6 and Il-1β mRNA levels (DMSO, LPS, FA18:4, and FA22:6 at 20 μM; n = 5). All significant differences were analyzed versus the DMSO group. Data presented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA, followed by least significance difference test).