Abstract
Squalene (SQ), a natural precursor of many steroids, can inhibit tumor progression and decrease serum cholesterol levels. However, it is difficult to discern the effect of highly active molecules in the treatment of diseases because not enough active compounds reach the site of pathology in crowded biosystems. Therefore, it is necessary to design artificial probes that work effectively within crowded systems. In this study, to facilitate cell penetration, the ethylene glycol moiety (used as a probe) was chemically added to SQ to form 2-(2-hydroxyethoxy)-3-hydroxysqualene (HEHSQ). HEHSQ was prepared from 2,3-epoxysqualene and characterized by Rf, FT-IR, 1H NMR, 13C NMR, and high-resolution mass spectrometry. We then evaluated the anti-inflammatory effects of SQ and HEHSQ on lipopolysaccharide- (LPS-) stimulated RAW264.7 murine macrophages. To determine the effect of SQ and HEHSQ on the viability of RAW264.7 cells, an MTT assay was performed. To quantify the anti-inflammatory effect of SQ and HEHSQ, we measured nitric oxide (NO) production, gene expression, and secretion of the proinflammatory cytokine tumor necrosis factor α (TNF-α) and chemokine C-C motif chemokine 2 (CCL2) in LPS-stimulated RAW264.7 cells using an in vitro inflammatory model. 2,3-Epoxysqualene was prepared according to a reported methodology. The reaction of 2,3-epoxysqualene and ethylene glycol in 2-propanol produced 49% HEHSQ. MTT results showed that 10 and 100 µg/mL HEHSQ treatment decreased cell viability, whereas SQ treatment (1–100 µg/mL) did not have any effect on viability. SQ (100 µg/mL) and HEHSQ (1 µg/mL) treatment significantly reduced the production of LPS-stimulated NO and decreased the expression and secretion of proinflammatory TNF-α and CCL2. Therefore, our results suggested that the anti-inflammatory effects of HEHSQ are 100 times higher than that of unmodified SQ. To the best of our knowledge, this study has demonstrated for the first time that HEHSQ can be potentially used as a safe alternative treatment to anti-inflammatory drugs.
Highlights
Inflammation is a protective response against trauma, infection, and tissue injury [1]
A molecular design was created to enhance the pharmacological activity of SQ in a crowded cellular environment. e introduction of the ethylene glycol moiety as a probe at the 2-position of SQ allows it to penetrate biological tissues because HEHSQ is highly soluble in methanol, tetrahydrofuran, and tumor necrosis factor α (TNF-α) level C-C motif chemokine 2 complementary DNA (cDNA) (CCL2) level
Our results showed that LPS treatment significantly increased the expressions of TNF-α, CCL2, and iNOS compared with untreated RAW264.7 cells
Summary
Inflammation is a protective response against trauma, infection, and tissue injury [1]. Chronic inflammation is a long-term phenomenon, causing tissue destruction and organ dysfunction. Macrophages play a critical role during inflammation. Following tissue injury or infection first, the responder macrophages are activated and exhibit an inflammatory phenotype [2]. Activated macrophages produce numerous inflammatory mediators, such as cytokines (tumor necrosis factor-α: TNF-a), chemokines (CCL2), and nitric oxide (NO), which are highly toxic for microorganisms and can be harmful to surrounding healthy tissues and lead to aberrant inflammation [3, 4]. CCL2, which belongs to the C-C class of chemokines, is a critical modulator of inflammation, regulating macrophage recruitment during wound healing, infections, and autoimmune diseases [8]
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