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. 2022 Dec 24;13(1):33.
doi: 10.3390/biom13010033.

First Evidence of the Protective Effects of 2-Pentadecyl-2-Oxazoline (PEA-OXA) in In Vitro Models of Acute Lung Injury

Aniello Schiano Moriello  1   2 , Fiorentina Roviezzo  3 , Fabio Arturo Iannotti  1 , Giuseppina Rea  4 , Marco Allarà  1   2 , Rosa Camerlingo  5 , Roberta Verde  1 , Vincenzo Di Marzo  1   6 , Stefania Petrosino  1   2
Affiliations

First Evidence of the Protective Effects of 2-Pentadecyl-2-Oxazoline (PEA-OXA) in In Vitro Models of Acute Lung Injury

Aniello Schiano Moriello et al. Biomolecules. .

Abstract

Acute respiratory distress syndrome (ARDS) is a serious inflammatory lung disorder and a complication of SARS-CoV-2 infection. In patients with severe SARS-CoV-2 infection, the transition to ARDS is principally due to the occurrence of a cytokine storm and an exacerbated inflammatory response. The effectiveness of ultra-micronized palmitoylethanolamide (PEA-um) during the earliest stage of COVID-19 has already been suggested. In this study, we evaluated its protective effects as well as the effectiveness of its congener, 2-pentadecyl-2-oxazoline (PEA-OXA), using in vitro models of acute lung injury. In detail, human lung epithelial cells (A549) activated by polyinosinic-polycytidylic acid (poly-(I:C)) or Transforming Growth Factor-beta (TGF-β) were treated with PEA-OXA or PEA. The release of IL-6 and the appearance of Epithelial-Mesenchymal Transition (EMT) were measured by ELISA and immunofluorescence assays, respectively. A possible mechanism of action for PEA-OXA and PEA was also investigated. Our results showed that both PEA-OXA and PEA were able to counteract poly-(I:C)-induced IL-6 release, as well as to revert TGF-β-induced EMT. In addition, PEA was able to produce an "entourage" effect on the levels of the two endocannabinoids AEA and 2-AG, while PEA-OXA only increased PEA endogenous levels, in poly-(I:C)-stimulated A549 cells. These results evidence for the first time the superiority of PEA-OXA over PEA in exerting protective effects and point to PEA-OXA as a new promising candidate in the management of acute lung injury.

Keywords: 2-pentadecyl-2-oxazoline; acute respiratory distress syndrome; anti-inflammatory; endocannabinoids; fibrosis; lung epithelial cells; palmitoylethanolamide.

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Conflict of interest statement

A.S.M., M.A. and S.P. are employees of the Epitech Group SpA. S.P. and V.D. are co-inventors on patents on Adelmidrol and/or PEA, respectively, which are unrelated to the present study. The other authors declare no other conflict of interest.

Figures

Figure 1
Figure 1
PEA-OXA and PEA reduce IL-6 release from poly-(I:C)-stimulated A549 cells. (a) IL-6 release was measured after stimulation of A549 cells with poly-(I:C) (100 μg mL−1) in the presence or absence of PEA-OXA (0.1, 1 and 10 μM) for 6 h at 37 °C in a 5% CO2 atmosphere; (b) IL-6 release was measured after stimulation of A549 cells with poly-(I:C) (100 μg mL−1) in the presence or absence of PEA (0.1, 1 and 10 μM) for 6 h at 37 °C in a 5% CO2 atmosphere. Each bar shows the mean ± SEM of independent experiments (n = 4). p-values were determined by ANOVA followed by Tukey’s multiple comparisons test. **** p < 0.0001 and * p < 0.05.
Figure 2
Figure 2
mRNA expression levels of PEA targets (PPARα, TRPV1, CB2) and PEA-catabolizing enzyme (NAAA) in A549 cells. Bar chart with individual points showing the mRNA expression levels of the indicated proteins (PPARα, TRPV1, CB2 and NAAA) measured in A549 cells. Each bar shows the mean ± SEM of 3 independent biological samples. Data are expressed using the 2−Δct formula.
Figure 3
Figure 3
TRPV1 and PPAR-α antagonists do not revert the anti-inflammatory effect of PEA-OXA and PEA in poly-(I:C)-stimulated A549 cells. (a) IL-6 release was measured after that A549 cells were stimulated with poly-(I:C) (100 μg mL−1) and treated with IRTX (0.1 μM) or GW6471 (1 µM) in the presence or absence of PEA-OXA (10 μM), for 6 h at 37 °C in a 5% CO2 atmosphere; (b) IL-6 release was measured after A549 cells were stimulated with poly-(I:C) (100 μg mL−1) and treated with IRTX (0.1 μM) or GW6471 (1 µM) in the presence or absence of PEA (10 μM), for 6 h at 37 °C in a 5% CO2 atmosphere. Each bar shows the mean ± SEM of independent experiments (n = 4). The p-values were determined by ANOVA followed by Tukey’s multiple comparisons test. **** p < 0.0001 and * p < 0.05.
Figure 4
Figure 4
Variation of the levels of PEA, AEA and 2-AG in poly-(I:C)-stimulated A549 cells treated with PEA and PEA-OXA. PEA (a), AEA (b) and 2-AG (c) levels were quantified by LC–MS; after that, A549 cells were stimulated with poly-(I:C) (100 μg mL−1) in the presence or absence of PEA (10 μM) and PEA-OXA (10 μM) for 6 h at 37 °C in a 5% CO2 atmosphere. Each bar shows the mean ± SEM of independent experiments (n = 3). The p-values were determined by ANOVA followed by Tukey’s multiple comparisons test. **** p < 0.0001, *** p < 0.001 and * p < 0.05.
Figure 5
Figure 5
TGF-β1-induced epithelial–mesenchymal transition in A549 cells as evidenced by immunofluorescence approaches is avoided by the treatment with PEA-OXA or PEA. (a) Immunofluorescence staining was examined for the following markers: cytokeratin, an epithelial marker (green fluorescence, panels A–F), and vimentin, a mesenchymal marker (red fluorescence, panels G–L). DAPI staining was included to visualize the cell nucleus (blue fluorescence, panels M–R). In the merged images the co-expression and co-distribution of the markers are visualized (panels S–X). Quantifications of cytokeratin (b) and vimentin (c). The cells were captured with a 40 × microscope objective (Bar = 20 µm). Each bar shows the mean ± SEM of independent experiments (n = 3). The p-values were determined by ANOVA followed by Tukey’s multiple comparisons test. **** p < 0.0001, *** p < 0.001, ** p < 0.01 and * p < 0.05.
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