The interplay between inflammatory and redox processes is a ubiquitous and critical phenomenon in cell biology that involves numerous biological factors. Among them, secretory phospholipases A₂ (sPLA₂) that catalyze the hydrolysis of the sn-2 ester bond of phospholipids are key players. They can interact or be modulated by the presence of truncated oxidized phosphatidylcholines (OxPCs) produced under oxidative stress from phosphatidylcholine (PC) species. The present study examined this important, but rarely considered, sPLA₂ modulation induced by the changes in biophysical properties of PC vesicles comprising various OxPC ratios in mono- or poly-unsaturated PCs. Being the most physiologically active OxPCs, 1-palmitoyl-2-(5′-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) have been selected for our study. Using fluorescence spectroscopy methods, we compared the effect of OxPCs on the lipid order as well as sPLA₂ activity in large unilamellar vesicles (LUVs) made of the heteroacid PC, either monounsaturated [1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)], or polyunsaturated [1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC)] at a physiological temperature. The effect of OxPCs on vesicle size was also assessed in both the mono- and polyunsaturated PC matrices. Results: OxPCs decrease the membrane lipid order of POPC and PDPC mixtures with PGPC inducing a much larger decrease in comparison with POVPC, indicative that the difference takes place at the glycerol level. Compared with POPC, PDPC was able to inhibit sPLA₂ activity showing a protective effect of PDPC against enzyme hydrolysis. Furthermore, sPLA₂ activity on its PC substrates was modulated by the OxPC membrane content. POVPC down-regulated sPLA₂ activity, suggesting anti-inflammatory properties of this truncated oxidized lipid. Interestingly, PGPC had a dual and opposite effect, either inhibitory or enhancing on sPLA₂ activity, depending on the protocol of lipid mixing. This difference may result from the chemical properties of the shortened sn-2-acyl chain residues (aldehyde group for POVPC, and carboxyl for PGPC), being, respectively, zwitterionic or anionic under hydration at physiological conditions.

Oxidative stress induces lipid peroxidation within the membrane bilayer, thereby compromising membrane integrity. Polyphenols (PPs), renowned for their antioxidant properties, have been shown to mitigate oxidative damage. Here, we investigated the structural and antioxidant effects of PPs—specifically flavonoid glycosides (FGs) and phenolic acids (PAs)—extracted from Inula oculus-christi using steady-state fluorescence spectroscopy in both model and cell membranes. Membrane lipid order was evaluated using DPH and Laurdan spectroscopy, while DPH-TEMPO fluorescence quenching was employed to quantify raft-like domain formation in model systems. The antioxidant capacity of the PP extracts was assessed via fluorescence quenching of cis-parinaric acid. Both FGs and PAs conferred approximately 2-fold antioxidant protection, with FGs showing a 1.13-fold greater effect than PAs. In addition, both PP classes promoted lipid raft formation, particularly in cholesterol-rich membranes. PPs increased order in the liquid-disordered (L_d) phase while inducing disorder in the liquid-ordered (L_o) phase, depending on the lipid-to-PP ratio. Notably, FGs enhanced membrane fluidity more strongly in A549 than in MDCKII cells, as reflected by a ~5.7-fold decrease in Laurdan GP in A549 (from 0.04 to −0.17) versus a ~1.4-fold decrease in MDCKII at 200 μg/mL. These findings highlight the dual structural and antioxidative roles of FGs and PAs in preserving membrane integrity under oxidative stress.

The phenyl glycoside myconoside, extracted from Balkan endemic Haberlea rhodopensis, has a positive effect on human health, but the exact molecular mechanism of its action is still unknown. The cell membrane and its associated junctional complex are the first targets of exogenous compound action. We aimed to study the effect of myconoside on membrane organization and cytoskeleton components involved in the maintenance of cell polarity in the MDCKII cell line. By fluorescent spectroscopy and microscopy, we found that at low concentrations, myconoside increases the cell viability by enhancing membrane lipid order and adherent junctions. The opposite effect is observed in high myconoside doses. We hypothesized that the cell morphological and physicochemical changes of the analyzed cell compartments are directly related to cell viability and cell apical-basal polarity. Our finding contributes to a better understanding of the beneficial application of phytochemical myconoside in pharmacology and medicine.

Resveratrol (Resv) is considered to exert a beneficial impact due to its radical scavenger, anti-microbial and anti-inflammatory properties through several mechanisms that could include its interaction with the cell plasma membrane. To address this issue, we investigated the influence of Resv on membrane lipid order and organization in large unilamellar vesicles composed of different lipids and ratios. The studied lipid membrane models were composed of phosphatidylcholine (PC) species (either palmitoyl-docosahexaenoyl phosphatidylcholine (PDPC) or palmitoyl-oleoyl phosphatidylcholine (POPC)), sphingomyelin (SM) and cholesterol (Chol). This study found that the addition of Resv resulted in complex membrane reorganization depending on the degree of fatty acid unsaturation at the sn-2 position, and the Lipid/Resv and SM/Chol ratios. Resv rigidified POPC-containing membranes and increased liquid-ordered (L_o) domain formation in 40/40/20 POPC/SM/Chol mixtures as this increase was lower at a 33/33/34 ratio. In contrast, Resv interacted with PDPC/SM/Chol mixtures in a bimodal manner by fluidizing/rigidifying the membranes in a dose-dependent way. L_o domain formation upon Resv addition occurred via the following bimodal mode of action: L_o domain size increased at low Resv concentrations; then, L_o domain size decreased at higher ones. To account for the variable effect of Resv, we suggest that it may act as a “spacer” at low doses, with a transition to a more “filler” position in the lipid bulk. We hypothesize that one of the roles of Resv is to tune the lipid order and organization of cell plasma membranes, which is closely linked to important cell functions such as membrane sorting and trafficking.

Abnormal accumulation of amyloid-beta (Aβ) peptides in the brain is a hallmark of Alzheimer’s disease (AD). Importantly, the peripheral blood cells are also exposed to the effects of pathological peptides that accumulate in AD. Herein, the interaction of Aβ42 oligomers (Aβ42) with human red blood cells (RBCs) and erythrocyte ghosts as in vitro models for AD is studied combining fluorescence spectroscopy, fluorescence microscopy, and electrokinetics. The binding of Aβ42 to RBCs was evidenced by the use of a fluorescent-labeled peptide. The membrane lipid order increased with the increase in both the Aβ42 concentration and the incubation time, creating a lipid–protein microenvironment characterized by higher molecular order and reduced heterogeneity in RBC membranes compared to control conditions. Notably, the increase in lipid order was less pronounced in erythrocyte ghosts than in intact RBCs. Furthermore, the ζ-potential measurements revealed Aβ42 induced alteration of the surface potential of RBCs in a concentration- and time-dependent manner, with freshly isolated RBCs exhibiting a highly negative potential that became increasingly negative at higher Aβ42 concentrations. These findings suggest that Aβ42 not only impacts neuronal function but also significantly alters the physical properties of RBCs that might compromise their function, potentially contributing to the systemic effects observed in AD.

Oxidative stress generates oxidized phospholipids (OxPLs) that alter membrane structure and inflammatory lipid signaling, yet the underlying biophysical mechanisms remain poorly understood. Here, we examine how two structurally distinct truncated oxidized phosphatidylcholines (OxPCs), 1-palmitoyl-2-(5′-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC), remodel membrane lateral organization and regulate secretory phospholipase A₂ (sPLA₂) activity. Large unilamellar vesicles composed of sphingomyelin, cholesterol, and either monounsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or polyunsaturated 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC) were used to reconstitute the liquid-ordered/liquid-disordered (L_o/L_d) phase coexistence characteristic of eukaryotic plasma membranes. Fluorescence spectroscopy revealed that OxPLs modulate lipid packing and nanodomain organization in a structure- and composition-dependent manner. POVPC promoted pronounced membrane ordering and L_o domain stabilization compared with PGPC, particularly in monounsaturated membranes with low cholesterol content. In contrast, PDPC-containing membranes, especially at elevated cholesterol, exhibited enhanced structural resilience to OxPL-induced perturbations. These biophysical changes were associated with distinct functional outcomes. Notably, the relationship between membrane structural parameters and sPLA₂ activity was not linear, indicating a decoupling between bulk membrane properties and enzymatic response. sPLA₂ activity was linked to membrane lateral organization: the size of L_o domains modulate hydrolysis by influencing the physicochemical properties of L_o/L_d interfaces, which may represent preferential sites for enzyme activation. Consistent with this, POVPC reduced sPLA₂ activity through stabilization of ordered domains at both low and high cholesterol, while PGPC enhanced hydrolysis at high cholesterol. Importantly, PDPC-containing membranes attenuated sPLA₂ activity and exhibited a protective effect against OxPC-induced enzymatic activation. Together, these findings identify membrane lateral organization as a key regulator of sPLA₂ function and provide mechanistic insight into how oxidative stress can differentially modulate inflammatory lipid signaling depending on membrane composition. This work highlights membrane organization as an active determinant of enzyme activity and a potential target in pathologies associated with oxidative stress, including atherosclerosis, neuroinflammation, and metabolic disease.