Porcine liver decomposition product-derived lysophospholipids promote microglial activation in vitro

Abstract

Cognitive impairments such as dementia are common in later life, and have been suggested to occur via a range of mechanisms, including oxidative stress, age-related changes to cellular metabolism, and a loss of phospholipids (PLs) from neuronal membranes. PLs are a class of amphipathic lipids that form plasma membrane lipid bilayers, and that occur at high concentrations in neuronal membranes. Our previous study suggested that a porcine liver decomposition product (PLDP) produced via protease treatment may improve cognitive function at older ages, by acting as a rich source of PLs and lysophospholipids (LPLs); however, its specific composition remains unclear. Thus, the present study used a novel liquid chromatography electrospray ionization tandem mass spectrometric (LC-MS/MS) protocol to identify the major PLs and LPLs in PLDP. Furthermore, it assessed the effect of identified LPLs on microglial activation in vitro, including cell shape, proliferation, and cell morphology. The results of the conducted analyses showed that PLDP and PLDP-derived LPLs concentration-dependently modulate microglial activation in vitro. In particular, lysophosphatidylcholine (LPC) concentration-dependently promotes cell morphology, likely via effects mediated by the enzyme autotaxin (ATX), since inhibiting ATX also promoted cell morphology, while conversely, increasing ATX production (via treatment with high levels of LPC) abolished this effect. These findings suggest that LPC is likely neuroprotective, and thus, support the importance of further research to assess its use as a therapeutic target to treat age-related cognitive impairments, including dementia.

Figure 1

Modulation of neuronal microglial cell size by porcine liver decomposition product (PLDP) treatment. (A) BV-2 cells (≥3 fields/sample) were analyzed and counted as described. (B) Quantification of cell surface after treatment with the indicated concentrations of porcine liver decomposition product (PLDP). Cell surface area was quantified by ImageJ software. Data are expressed as mean ± S.E. n = 3, **P < 0.01. Bars, 100 μm.

Figure 2

Porcine liver decomposition product (PLDP) induced morphological change (microglial processes) in microglia. (A) Representative images of BV-2 microglial cells treated with 1% PLDP for 12 h. Bright field (upper figures) and representative drawings of the morphological change (lower figures) are shown. Bars, 100 μm (B) The number of cells exhibiting morphological change was measured after 1% PLDP for 12 h, before morphological change (in ≥ 3 fields/sample) was analyzed as described.

Figure 3

Liquid chromatography electrospray ionization tandem mass spectrometric (LC-MS/MS) analysis of porcine liver decomposition product (PLDP) phospholipid (PL) composition. PL species in PLDP were identified (via selected reaction monitoring), including (A) phosphatidylcholine (PC), (B) phosphatidylethanolamine, (C) phosphatidylinositol (PI), (D) phosphatidylserine (PS), and (E) phosphatidic acid (PA).

Figure 4

Liquid chromatography electrospray ionization tandem mass spectrometric (LC-MS/MS) analysis of porcine liver decomposition product (PLDP) lyso-phosholipid (LPL) composition. LPL species in PLDP were identified (via selected reaction monitoring), including (A) lysophosphatidylcholine (LPC), (B) lysophosphatidylethanolamine (LPE), (C) lysophosphatidylinositol (LPI), (D) lyophosphatidylserine (LPS), and (E) lysophosphatidic acid (LPA).

Figure 5

Effect of porcine liver decomposition product (PLDP)-derived lyso-phosholipid (LPLs) on SIM-A9 and BV-2 cell proliferation. Microglial cells were assess using an MTT colorimetric assay after treatment with (A,G) PLDP, (B,H) lysophosphatidylcholine (LPC)18:1, (C,I) lysophosphatidylethanolamine (LPE)18:0, (D,J) lysophosphatidylinositol (LPI)18:0, (E,K) lyophosphatidylserine (LPS)18:0, or (F,L) lysophosphatidic acid (LPA)18:1 for 24 h. Data are expressed as the mean ± S.E. n = 3, **P < 0.01 or *P < 0.05.

Figure 6

Induction of morphological change by lysophosphatidylcholine (LPC) or lysophosphatidylethanolamine (LPE) in SIM-A9 cells. (A) Microglial cells were exposed to serum-free medium containing vehicle, PLDP (1%), charcoal-dextran-treated 1% PLDP (CD-PLDP) or 10 μM each LPLs (LPA, LPE, LPS, LPI, and LPC for 12 h. The number of cells exhibiting morphological change was measured. Representative data from three independent experiments are shown. Magnification, ×200. (B) The number of cells exhibiting morphological change was measured after vehicle or 30 µM LPC or LPE for 12 h. Cells were exposed to serum-free medium containing vehicle or the indicated concentration of LPC or LPE for 12 h. Representative data from three independent experiments are shown. Magnification, ×200. Data are expressed as the mean ± S.E. *P < 0.01).

Figure 7

The autotaxin (ATX) inhibitor BI-2545 promotes lysophosphatidylcholine (LPC) -mediated cell morphology. (A) Enzymatic pathways controlling lysophosphatidic acid (LPA) and LPC levels. ATX cleaves LPC to generate LPA, and then PLA₁ catalyzes the hydrolysis of PC to LPC. (B) LPC (18:1) inhibited ATX activity. Both LPC and LPA (each 10 μM) inhibition of ATX were significantly decreased. Data are expressed as the mean ± S.E. *P < 0.01) (C) ATX levels in a concentrated (∼30-fold) microglial-conditioned culture medium (upper panel) and cell lysate (lower panel) were analyzed via western blotting using a rabbit anti-ATX polyclonal antibody. (D) BV-2 and SIM-A9 both cell line was treated with or without LPC (30 μM) for 12 h. LPC-mediated ATX expression was analyzed via western blotting using a rabbit anti-ATX polyclonal antibody. (E) Thin-layer chromatography (TLC) analysis of NBD-LPA levels after treatment with concentrated conditioned medium, with or without BI-2545 (10 nM) for 12 h. Samples aliquots (10 μl) were spotted onto the TLC plate. NBD-LPC 18:1 or NBD-LPA 18:1 were detected using blue LED irradiation. The microglial-conditioned medium generated NBD-LPA18:1 at expected levels, but this effect was completely abolished by the addition of BI-2545. (F) BI-2545 treatment promoted LPC-mediated (10 μM) cell morphology. Neurite lengths were measured for 100 cells, and the total neurite length/cell was calculated. Data are presented as the mean ± S.E. *P < 0.01.

Figure 8

Pro- and anti-inflammatory cytokine mRNA expression by SIM-A9 cells after exposure to lipopolysaccharide (LPS), with or without lysophosphatidylcholine (LPC). (A) SIM-A9 cells were treated with PLDP-derived PLs (0.3 μg/ml) for 24 h, before their relative mRNA expression of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-4, IL-6, IL-10, IL-12, and transforming growth factor (TGF)-β was assessed. Data are expressed as the mean ± S.E. n = 3, **P < 0.01. (B) SIM-A9 cells were treated with LPS (10 ng/ml) with or without PLDP-derived PLs (0.3 μg/ml) for 24 h, before their relative mRNA expression of IL-6 was assessed. Data are expressed as the mean ± S.E. n = 3, **P < 0.01. (C) SIM-A9 cells were treated with LPS (10 ng/ml) with or without 0, 1, 3, 10, or 30 μM LPC for 24 h, before their relative mRNA expression of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-4, IL-6, IL-10, IL-12, and transforming growth factor (TGF)-β was assessed. Data are expressed as the mean ± S.E. n = 3, **P < 0.01. (D) SIM-A9 cells were treated with LPS (10 ng/ml) with or without 0, 3, 10, or 30 μM LPC for 24 h. LPS-mediated IL-6 expression was analyzed via western blotting using a rabbit anti-IL-6 polyclonal antibody.