Drug-induced anti-inflammatory response in A549 cells, as detected by Raman spectroscopy: a comparative analysis of the actions of dexamethasone and p-coumaric acid.

Corticosteroids are widely used as effective treatments for the control of chronic inflammatory diseases. However, because their long-term administration carries serious consequences, there is a need to investigate alternative therapies to reduce or even replace their use. In this regard, phenolic compounds have been presented as an alternative for the treatment of inflammatory diseases. p-Coumaric acid, a natural phenolic compound found throughout nature, exhibits antioxidative and anti-inflammatory properties. Herein, using a combination of Raman spectroscopy with principal component analysis and hierarchical cluster analysis, the inflammatory process induced by cigarette smoke extract (CSE) in epithelial cells treated with either a corticosteroid or p-coumaric acid was monitored in vitro. Our findings showed that p-coumaric acid had a significant anti-inflammatory effect in CSE-activated epithelial cells, and thus may be a useful alternative to corticosteroids for the treatment of airway inflammation in chronic obstructive pulmonary disease. In addition, multivariate analysis of the cell spectral data indicated that the mechanisms of action of the two drugs occur through different routes.

Application of Raman spectroscopy for characterization of the functional polarization of macrophages into M1 and M2 cells.

Macrophages are key cells in the immune inflammatory response that can be differentiated into M1 and M2 phenotypes. Polarization has a critical therapeutic value, especially in diseases in which an M1/M2 imbalance plays a pathophysiological role. Raman spectroscopy has proven to be a promising bioanalytical technique for discriminating different cell types. However, to our knowledge, its application to identify the functional polarization of macrophages into M1 or M2 cells is yet to be investigated. In this work, Raman spectroscopy was applied to the analysis of macrophage polarization, and the spectral datasets were analyzed using principal component analysis (PCA). In vitro, resting J774.1 macrophages were treated with LPS/IFN-γ to induce the M1 phenotype or with IL-4 to induce the M2 phenotype. The resulting Raman spectra showed sufficient biochemical information to distinguish between M1 and M2 phenotypes when analyzed by PCA, reflecting the changes in cell markers caused by differentiation. The Raman spectra collected from LPS-stimulated M1 and M2 macrophages were more intense. The functional phenotype of M1 macrophages was confirmed by IL-6 secretion and TNF-α mRNA expression, while M2 macrophages produced IL-10 and Arg-1 mRNA, as well as by the morphological changes observed by scanning electron microscopy. Taken together, the results indicate that Raman spectroscopy combined with PCA analysis is a useful tool to identify the functional phenotypes of macrophages, providing an alternative way to distinguish between cells in distinct differentiation stages.

Exosome-Enriched Plasma Analysis as a Tool for the Early Detection of Hypertensive Gestations.

Hypertensive disorders of pregnancy are closely associated with prematurity, stillbirth, and maternal morbidity and mortality. The onset of hypertensive disorders of pregnancy (HDP) is generally noticed after the 20th week of gestation, limiting earlier intervention. The placenta is directly responsible for modulating local and systemic physiology by communicating using mechanisms such as the release of extracellular vesicles, especially exosomes. In this study, we postulated that an analysis of exosome-enriched maternal plasma could provide a more focused and applicable approach for diagnosing HDP earlier in pregnancy. Therefore, the peripheral blood plasma of 24 pregnant women (11 controls, 13 HDP) was collected between 20th and 24th gestational weeks and centrifuged for exosome enrichment. Exosome-enriched plasma samples were analyzed by Raman spectroscopy and by proton nuclear magnetic resonance metabolomics (1H NMR). Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to analyze the Raman data, from the spectral region of 600-1,800 cm-1, to determine its potential to discriminate between groups. Using principal component analysis, we were able to differentiate the two groups, with 89% of all variances found in the first three principal components. In patients with HDP, most significant differences in Raman bands intensity were found for sphingomyelin, acetyl CoA, methionine, DNA, RNA, phenylalanine, tryptophan, carotenoids, tyrosine, arginine, leucine, amide I and III, and phospholipids. The 1H NMR analysis showed reduced levels of D-glucose, L-proline, L-tyrosine, glycine, and anserine in HDP, while levels of 2-hydroxyvalerate, polyunsaturated fatty acids, and very-low-density lipoprotein (VLDL) were increased. 1H NMR results were able to assign an unknown sample to either the control or HDP groups at a precision of 88.3% using orthogonal partial least squares discriminant analysis and 87% using logistic regression analysis. Our results suggested that an analysis of exosome-enriched plasma could provide an initial assessment of placental function at the maternal-fetal interface and aid HDP diagnosis, prognosis, and treatment, as well as to detect novel, early biomarkers for HDP.

Uvaol Prevents Group B Streptococcus-Induced Trophoblast Cells Inflammation and Possible Endothelial Dysfunction.

Group B Streptococcus (GBS) infection during pregnancy is involved in maternal sepsis, chorioamnionitis, prematurity, fetal infection, neonatal sepsis, and neurodevelopmental alterations. The GBS-induced chorioamnionitis leads to a plethora of immune and trophoblast cells alterations that could influence endothelial cells to respond differently to angiogenic mediators and alter placental vascular structure and function in pregnant women. In this context, preventive measures are needed to reduce such dysfunctions. As such, we evaluated the effects of a non-lethal exposure to inactivated GBS on trophoblast cells and chorionic villi explants, and if the treatment with uvaol would mitigate these effects. The concentration of 106 CFU of GBS was chosen since it was unable to reduce the HTR-8/SVneo cell line nor term chorionic villi explant viability. Raman spectroscopy of trophoblast cells showed significant alterations in their biochemical signature, mostly reverted by uvaol. GBS exposure increased HTR-8/SVneo cells IL-1ß and IFN-γ production, phagocytosis, oxidative stress, and decreased trophoblast cell migration. The Ea.hy926 endothelial cell line produced angiopoietin-2, CXCL-8, EGF, FGF-b, IL-6, PlGF, sPECAM-1, and VEGF in culture. When co-cultured in invasion assay with HTR-8/SVneo trophoblast cells, the co-culture had increased production of angiopoietin-2, CXCL-8, FGF-b, and VEGF, while reduced sPECAM-1 and IL-6. GBS exposure led to increased CXCL-8 and IL-6 production, both prevented by uvaol. Chorionic villi explants followed the same patterns of production when exposed to GBS and response to uvaol treatment as well. These findings demonstrate that, even a non-lethal concentration of GBS causes placental inflammation and oxidative stress, reduces trophoblast invasion of endothelial cells, and increases CXCL-8 and IL-6, key factors that participate in vascular dysregulation observed in several diseases. Furthermore, uvaol treatment prevented most of the GBS-provoked changes. Hence, uvaol could prevent the harmful effects of GBS infection for both the mother and the fetus.