Anti-inflammatory effects of trans-cinnamic acid through modulation of endothelial ICAM-1 expression and neutrophil recruitment
Mark de Sousa Pinheiro Fidelix, Samário Lino Santos, Jordana Rodrigues Santana, Alef Batista Bezerra Barros, Erick Gabriel Alves Ferreira, Graziele Regina Souza Silva, Jamylle Nunes de Souza Ferro, Juliane Pereira Silva, Vincent Lagente, Emiliano Barreto
TL;DR
Trans-cinnamic acid reduces inflammation by lowering ICAM-1 expression and neutrophil adhesion in mice and human cells.
Contribution
The study reveals a novel mechanism of trans-cinnamic acid's anti-inflammatory action via ICAM-1 downregulation.
Findings
Trans-cinnamic acid reduced LPS-induced pleurisy by decreasing neutrophil infiltration and cytokine levels.
It suppressed ICAM-1 expression, reducing neutrophil adhesion to endothelial cells.
The compound did not affect neutrophil chemotaxis or CXCL8 secretion.
Abstract
Natural phenolic acid compounds have been extensively studied for their anti-inflammatory properties, particularly in the context of inflammation-associated diseases. In this study, we investigated the anti-inflammatory effects of trans-cinnamic acid on neutrophil accumulation during inflammatory processes using both in vivo and in vitro approaches. For the in vivo experiments, LPS-induced pleurisy was used in mice pretreated with trans-cinnamic acid. Inflammatory parameters, including plasma leakage, leukocyte infiltration, and proinflammatory cytokine levels (IL-6 and TNF-α), were quantified in the pleural exudate. In vitro, the effects of trans-cinnamic acid on neutrophil chemotaxis toward CXCL1 were assessed using the Boyden chamber assay. Additionally, human endothelial EA.hy926 cells were stimulated with TNF-α to evaluate neutrophil adhesion and the expression of the adhesion…
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Figure 6- —Universidade Federal De Alagoas
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Taxonomy
TopicsNatural Antidiabetic Agents Studies · Wound Healing and Treatments · Phytochemicals and Antioxidant Activities
Introduction
Inflammation is the response of vascularised tissue elicited by harmful factors of both endogenous and exogenous origin, with the objective of identifying, containing, and eliminating these factors to promote tissue repair [1]. It is orchestrated by a complex network of cellular and molecular events, including the release of proinflammatory mediators, recruitment of leukocytes, and activation of resident tissue cells [2]. While regulated inflammation supports tissue repair, uncontrolled or persistent inflammatory responses contribute to the onset and progression of several chronic inflammatory diseases [3].
One of the critical events in the initial stage of the inflammatory response involves the release of cytokines such as tumour necrosis factor-α (TNF-α) and interleukin (IL)−6, which activate endothelial cells [4]. When activated, the endothelium expresses additional proinflammatory factors, including cytokines and chemokines (e.g. CXCL8 and CXCL1), and multiple families of adhesion molecules, such as selectins and intercellular adhesion molecule-1 (ICAM-1) [5]. These endothelial cell-associated molecules facilitate the rolling, adhesion, and migration of circulating neutrophils across the endothelial cell barrier to sites of inflammation through a multistep process [5]. Consequently, the maintenance of neutrophils within tissues aggravates and prolongs inflammatory responses [6]. The acute inflammatory response has been reported to be less severe when induced in mice deficient in ICAM-1 compared with their normal counterparts [7]. Thus, although neutrophils are crucial for host defence, excessive neutrophil recruitment and ICAM-1 expression can exacerbate tissue damage [8]. Owing to its importance as a key regulator of essential cellular functions in the inflammatory response, ICAM-1 has been considered a pharmacological target of both clinical and therapeutic interest [6].
Inflammatory diseases are a global health problem that affects millions of people, with substantial negative socioeconomic impacts [9]. Current therapeutic strategies for controlling inflammation include glucocorticoids and nonsteroidal anti-inflammatory drugs (NSAIDs). Despite their efficacy, many patients develop drug resistance or experience severe side effects, highlighting the need for new therapeutic alternatives [10]. Natural products, particularly phenolic acids, have been increasingly investigated as potential modulators of inflammatory processes [11, 12]. Among these, chlorogenic acids, a group of natural polyphenol compounds found in plants, have demonstrated the ability to reduce CXCL8 and ICAM-1 expression, thereby limiting leukocyte recruitment and tissue infiltration [13]. It is also important to mention that ethyl 3‘,4‘,5‘-trimethoxycinnamate, a derivative of cinnamic acid, holds diverse pharmacological properties, among which its inhibitory action on TNF-α-induced ICAM-1 in primary human umbilical vein endothelial cells stands out [14].
Cinnamic acid is an aromatic organic acid found in plants in two isomeric forms: trans-cinnamic acid or cis-cinnamic acid [15]. Trans-cinnamic acid has been shown to be the predominant form in nature (> 99%), as it is much more stable than the cis-isomer [16]. This condition favours the use of the trans isomer in cosmetics, perfumes, and flavourings, and it also serves as a precursor for other compounds, such as the sweetener aspartame [17]. In addition to its use in food, trans-cinnamic acid has been described for its wide range of biological effects, including anti-inflammatory effects. Previous studies demonstrated that trans-cinnamic acid reduced the levels of inflammatory cytokines in the foetal forebrain in a rat model of preeclampsia [18]. In another study, the anti-inflammatory effects of trans-cinnamic acid were evidenced by the reduction of neutrophilic infiltration in the inflamed distal colon of rats subjected to dinitrobenzene sulfonic acid (DNBS)-induced rat colitis [19]. However, the molecular mechanisms underlying its anti-inflammatory activity, particularly regarding neutrophil-endothelium interactions, remain poorly understood. In this context, we aimed to investigate the effects of trans-cinnamic acid on acute inflammation, focusing on neutrophil recruitment in vivo and endothelial cell activation and neutrophil-endothelial adhesion in vitro. By integrating animal models and cellular assays, this study provided novel insights into the potential of trans-cinnamic acid as a modulator of leukocyte recruitment and endothelial cell activation.
Materials and methods
Chemicals and reagents
The following substances were obtained from Sigma Chemical Co. (St. Louis, MO, USA): trans-cinnamic acid (≥ 98% purity, Fig. 1), lipopolysaccharide (LPS, from Escherichia coli O55:B5), trypsin, Dulbecco’s Modified Eagle Medium (DMEM), and phosphate-buffered saline (PBS) tablets. Ethylenediaminetetraacetic acid (EDTA) and dimethyl sulfoxide (DMSO) were purchased from Synth (Diadema, SP, Brazil). May-Grünwald-Giemsa stain, NaCl, and ethanol were purchased from Merck (São Paulo, SP, Brazil). Foetal bovine serum (FBS), penicillin, and streptomycin were obtained from GIBCO/Invitrogen (São Paulo, SP, Brazil). Recombinant human TNF-α and CXCL1 were purchased from R&D Systems (Minneapolis, MN, USA). Commercial enzyme-linked immunosorbent assays (ELISA) kits for IL-6, TNF-α, and CXCL8 were purchased from PeproTech (Rocky Hill, NJ, USA) and used according to the manufacturer’s instructions.Fig. 1. Chemical structure of trans-cinnamic acid.
Trans-cinnamic acid was dissolved in sterile saline (0.9% NaCl) to allow administration in a constant volume (10 µL/g body weight). The control animals received equivalent volumes of vehicle alone. Intrathoracic (i.t.) pre-treatment was performed 60 min before LPS injection.
Animals
Male Swiss mice (25–30 g) were obtained from the breeding colonies of the Federal University of Alagoas (UFAL, Maceió, Brazil). Animals were housed under controlled environmental conditions (22 ± 2 °C, 12 h light/12 h dark cycle) with free access to food and water. The experiments were performed during the light phase. Mice were allowed to acclimatise for at least 2 h before testing, and each animal was used in only one experimental procedure. The study was conducted in accordance with the Basic & Clinical Pharmacology & Toxicology policy for experimental and clinical studies involving natural products and animal experimental procedures [20]. All experiments were performed in accordance with the principles of the Brazilian Society of Laboratory Animal Science (SBCAL) and approved by the Institutional Animal Ethics Committee of UFAL (protocol no. 67/2014).
Lipopolysaccharide-induced pleurisy
Animals were pretreated intraperitoneally (i.p.) with trans-cinnamic acid (3, 10, and 30 mg/kg) 1 h before the induction of pleurisy. Pleurisy was induced by intrapleural (i.t.) injection of LPS (250 ng in sterile saline per cavity). A specially adapted needle was introduced into the right side of the thoracic cavity, and an equal volume of sterile saline was injected into the control animals. Six hours after i.t. injection, the animals were euthanised with an overdose of the anaesthetic sodium pentobarbital (500 mg/kg, i.p.), and the thoracic cavity was washed with 1 mL PBS containing 10 mM EDTA. Pleural washes were collected, and the recovered volume was recorded. The exudate volume was calculated by subtracting the injected volume (1 mL) from the total recovered volume. Total leukocyte counts were performed in Neubauer chambers using pleural washes diluted in Turk’s solution (2% acetic acid). Differential leukocyte counts were obtained from cytospin preparations stained with May–Grünwald–Giemsa and examined under a light microscope using an oil immersion objective (×100). Dexamethasone (Dexa, 1 mg/kg, i.p.) was used as a reference drug. Parallel groups of animals were treated with Dexa 1 h before pleurisy induction, and inflammatory parameters were evaluated at the same time points.
Measurement of TNF-α, IL-6, and CXCL8 by ELISA
Levels of TNF-α and IL-6 in pleural fluid from animals treated with trans-cinnamic acid (3, 10 and 30 mg/kg, i.p.) were quantified through sandwich ELISA, using antibody pairs supplied by PeproTech (Rocky Hill, NJ, USA), according to the manufacturer’s instructions. In a separate set of in vitro experiments, CXCL8 levels were measured in the supernatant of TNF-α-stimulated EA.hy926 cells. Cells were seeded in 24-well plate and, after 24 h, were pretreated for 1 h with trans-cinnamic acid (1, 10, and 30 µM) before co-treatment with TNF-α (50 ng/mL) for 24 h. After treatment, supernatants were collected, and cytokine/chemokine concentrations were determined using ELISA according to the manufacturer’s protocol. Data are expressed as picograms per millilitre (pg/mL).
Isolation of human neutrophils
Human neutrophils were isolated from peripheral venous blood of healthy volunteers through Percoll density gradient centrifugation (Sigma-Aldrich, St. Louis, MO, USA), as previously described [21]. Cells were resuspended in DMEM supplemented with 5% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. Morphological assessment (Giemsa staining) and viability testing (trypan blue dye exclusion) confirmed > 97% purity and > 98% viability. All procedures involving human subjects were approved by the Institutional Ethics Committee, and informed consent was obtained from all donors.
Chemotaxis assay
Neutrophil migration was assessed using a 48-well Boyden chamber (NeuroProbe Inc., Cabin John, MD, USA). The lower wells were filled with 30 µL of chemoattractant (CXCL1, 250 nM) or DMEM (control). The upper wells were filled with neutrophils (1 × 10^5^ cells in 50 µL) preincubated for 1 h with trans-cinnamic acid (1, 10, 30, and 100 µM), Dexa (50 nM), or medium. The chambers were separated by a 5 μm polycarbonate membrane (Sigma-Aldrich). The chamber was incubated at 37 °C in humidified 5% CO_2_ for 60 min. Filters were then fixed and stained with Wright-Giemsa, and the migrated neutrophils were counted under light microscopy (100× magnification) in 15 random fields. The results were expressed as the mean number of migrated neutrophils per field.
Culture of human endothelial cells and adhesion assay
EA.hy926 cells, a human endothelial hybrid cell line derived from human umbilical vein endothelial cells (HUVECs), were obtained from the Rio de Janeiro Cell Bank (Rio de Janeiro, Brazil). Cells were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin, under a humidified 5% CO_2_ atmosphere at 37 °C. Cells were subcultured after trypsinisation (0.5% trypsin solution with 0.2% EDTA) and used between passages 2 and 6.
Neutrophil adhesion to endothelial cells was assessed as previously described [21]. Briefly, EA.hy926 cells (6 × 10^5^ cells/well) were seeded in 24-well plates and incubated until confluence. Monolayers were pretreated with trans-cinnamic acid (1, 10, or 30 µM) for 1 h at 37 °C and subsequently stimulated with TNF-α (50 ng/mL) for 6 h. After washing with PBS, neutrophils (3 × 10^6^ cells) were added and allowed to adhere to the TNF-α-activated endothelial cells for 2 h at 37 °C. Non-adherent neutrophils were removed by gentle washing with PBS. Adherent neutrophils were fixed with ethanol, stained with Giemsa, and visualised under an inverted microscope (100× magnification). Adhesion was quantified by direct counting, and the adhesion index (AI) was calculated as: [(endothelial cells with neutrophils/total endothelial cells) × (neutrophils bound to endothelial cells/total endothelial cells)] × 100.
Cell viability assay
The effect of trans-cinnamic acid on EA.hy926 cells and neutrophil viability was assessed using the MTT assay. Cells (6 × 10^3^/well) were plated in 96-well plates and treated with trans-cinnamic acid (1, 10, or 30 µM) for 24 h. Subsequently, the medium was replaced with fresh DMEM containing MTT (5 mg/mL), and cells were incubated for 2 h at 37 °C under 5% CO_2_. After removing the supernatant, DMSO (150 mL/well) was added to solubilise formazan crystals. Absorbance was measured at 540 nm using a microplate reader. Cell viability (%) was calculated as: (absorbance of treated cells/absorbance of control cells) × 100%.
Flow cytometric analysis of ICAM-1 surface expression
ICAM-1 protein expression on the surface of EA.hy926 cells was assessed through flow cytometry 6 h after pretreatment with trans-cinnamic acid and stimulation with TNF-α (50 ng/mL). Cells were detached with trypsin, washed with PBS containing 1% BSA, and incubated with FITC-conjugated anti-CD54 (ICAM-1) or isotype IgG1 control (eBioscience, San Diego, CA, USA) for 20 min at 4 °C in the dark. After staining, cells were washed, centrifuged, and fixed in 2% formaldehyde (VETEC, Duque de Caxias, RJ, Brazil). Data acquisition was performed on a FACSCanto II flow cytometer (BD Biosciences), and the results were analysed using WinMDI software (v2.8).
ICAM-1 RNA isolation and quantitative Real-Time PCR
To evaluate ICAM-1 mRNA expression, total RNA was isolated from EA.hy926 cells and reverse transcribed into cDNA using the GoScript™ Reverse Transcription Kit (Promega, Madison, WI, USA). RT-qPCR was performed using Fast BRYT Green^®^ Dye (Promega) with gene-specific primers. Each 20 µL reaction contained 2 µL cDNA, and 0.2 µM primers. The amplification protocol was 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 58 °C for 25 s, and 72 °C for 35 s. Fluorescence was detected with a QuantStudio™ 5 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). β-actin was used as housekeeping gene, and relative expression levels were calculated using the 2^−^∆∆Ct method. Data were analysed using the RQ Manager and GraphPad Prism v6.0 (GraphPad Software, La Jolla, CA, USA). The following gene-specific primers were used: ICAM-1 (forward, 5’-TTCCTCACCGTGTACTGGAC-3’; reverse, 5’-GGTAAGGTTCTTGCCCACTG-3’) and β-actin (forward, 5’-AGATGTGGATCAGCAAGCAG-3’; reverse, 5’-GCGCAAGTTAGGTTTTGTCA-3’).
Statistical analysis
Data are presented as mean ± standard deviation (SD). Statistical significance was assessed using one-way ANOVA followed by Tukey’s post hoc test (GraphPad Prism v 5.0; San Diego, CA, USA). Differences were considered statistically significant at P < 0.05.
Results
Effects of trans-cinnamic acid on LPS-induced leukocyte accumulation
Intrathoracic injection of lipopolysaccharide (LPS, 250 ng/cavity) in mice induced a marked increase in plasma protein extravasation, an index of vascular permeability, along with a significant (P < 0.001) increase in total leukocyte counts, characterised by the accumulation of neutrophils and mononuclear cells 6 h after LPS stimulation (Fig. 2A). Pretreatment with trans-cinnamic acid (3, 10, and 30 mg/kg, i.p.), administered 1 h prior to LPS administration, significantly reduced plasma protein extravasation (Fig. 2A). As expected, the reference drug Dexa (1 mg/kg, i.p.) also markedly inhibited protein extravasation compared with the LPS group (Fig. 2A). Under the same experimental conditions, trans-cinnamic acid significantly inhibited total leukocyte infiltration into the pleural cavity at all tested doses (Fig. 2B). This effect was primarily attributed to the reduction in neutrophil recruitment (Fig. 2C), whereas mononuclear cell migration was not significantly affected (Fig. 2D). Dexa, used as a positive control, reduced total leukocyte, neutrophil, and mononuclear cell counts by approximately 62.5%, 55.6, and 38.6%, respectively (Fig. 2B, C and D).Fig. 2. Effect of trans-cinnamic acid on protein extravasation (A), total leukocytes (B), neutrophils (C), and mononuclear cells (D) in the pleural cavity of LPS-stimulated mice. Animals were pretreated intraperitoneally (i.p.) with trans-cinnamic acid (3, 10, and 30 mg/kg), dexamethasone (Dexa, 1 mg/kg, i.p.), or saline (NaCl, 0.9%), 60 min before intrathoracic (i.t.) injection of LPS (250 ng/cavity). Bars represent the mean ± S.D. (n = 5). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. +P < 0.001 vs. saline group, *P < 0.05, **P < 0.01, and ***P < 0.001 vs. untreated LPS group.
Trans-cinnamic acid reduces LPS-induced production of TNF-α and IL-6 in pleural exudates
As shown in Tables 1 and 6 h after LPS injection, there was a significant increase in TNF-α and IL-6 levels in pleural exudates from LPS-injected mice compared with the saline-injected mice. Treatment with trans-cinnamic acid (10 and 30 mg/kg, i.p.) significantly reduced TNF-α concentration relative to the LPS-stimulated group. Similarly, IL-6 levels in pleural exudates were significantly decreased in animals treated with the two higher doses of trans-cinnamic acid (10 and 30 mg/kg, i.p.) (Table 1). As expected, Dexa (1 mg/kg, i.p.) markedly inhibited the LPS-induced production of TNF-α and IL-6 in pleural fluid (Table 1).
Table 1. Effect of trans-cinnamic acid on TNF-α and IL-6 levels in pleural exudates of LPS-challenged miceTreatment(i.p.)LPS(250 ng/cavity)Cytokines (pg/mL)TNF-αIL-6Saline-66.67 ± 14.457.08 ± 0.82Saline+598.56 ± 21.06+107.19 ± 4.79+trans-cinnamic acid(3 mg/kg)+550.71 ± 31.6195.3 ± 2.21trans-cinnamic acid(10 mg/kg)+436.73 ± 13.4976.04 ± 4.22**trans-cinnamic acid(30 mg/kg)+322.65 ± 18.5559.44 ± 1.69Dexamethasone(1 mg/kg)+276.66 ± 12.5435.45 ± 2.11Animals received trans-cinnamic acid (3, 10, and 30 mg/kg, i.p.), dexamethasone (Dexa, 1 mg/kg, i.p.), or saline (NaCl, 0.9%), 60 min before thoracic injection of LPS (250 ng/cavity). Pleural exudates were collected 6 h after LPS challenge for cytokine quantification. Values represent the mean ± S.D. (n = 5). Statistical differences were determined using ANOVA followed by Tukey’s multiple comparisons test. +P < 0.001 vs. saline-treated groupP* < 0.05P < 0.01***P < 0.001 vs. untreated LPS-injected group
Effect of trans-cinnamic acid on neutrophil chemotaxis in vitro
We further investigated whether trans-cinnamic acid directly affects human neutrophil function. To this end, the in vitro chemotaxis response of neutrophils was assessed using the Boyden chamber assay. As shown in Fig. 3, neutrophils exhibited a significant increase in migration toward CXCL1 (250 nM) compared with unstimulated controls. Pretreatment of neutrophils with trans-cinnamic acid (1, 10, 30, and 100 µM) did not alter their migratory response at any of the concentrations tested. In contrast, Dexa, used as a reference drug, significantly inhibited CXCL1-induced neutrophil migration (Fig. 3).Fig. 3. Effect of trans-cinnamic acid on CXCL1-induced neutrophil chemotaxis in vitro. Neutrophils (10^6^ cells) were pretreated with trans-cinnamic acid or dexamethasone (Dexa, 1 µM) for 1 h and then placed in the upper chamber of a Boyden apparatus, while CXCL1 (250 nM) was added to the lower chamber. After 1 h, migrated cells were counted under light microscopy. Each bar represents the mean ± S.D. of three independent experiments performed in triplicate. Statistical differences were determined using ANOVA followed by Tukey’s multiple comparisons test.+++P < 0.001 vs. control group (cells maintained in DMEM); ***P < 0.01 vs. CXCL1-stimulated group.
Trans-cinnamic acid did not affect the production of CXCL8 in TNF-α-stimulated EA.hy926 cells
Next, we evaluated the secretion of the proinflammatory chemokine CXCL8 in the supernatant of TNF-α-stimulated EA.hy926 cells treated with or without trans-cinnamic acid. As expected, TNF-α stimulation markedly increased CXCL8 production after 24 h (Table 2). Treatment of endothelial cells with trans-cinnamic acid (1, 10, or 30 µM) did not significantly alter the amount CXCL8 produced by endothelial cells (Table 2). In contrast, the reference drug Dexa (50 nM) significantly inhibited TNF-α-induced production of CXCL8 by approximately 55% (Table 2).
Table 2. Effect of trans-cinnamic acid on CXCL8 production in TNF-α-stimulated EA.hy926 cellsTreatmentTNF-α (50 ng/mL)CXCL8 (pg/mL)Saline-113.92 ± 34.26Saline+468.17 ± 25.28+trans-cinnamic acid (1 µM)+529.20 ± 150.91trans-cinnamic acid (10 µM)+425.22 ± 27.35trans-cinnamic acid (30 µM)+502.92 ± 87.91Dexamethasone (50 nM)+237.70 ± 30.20*Cells were pretreated with DMEM or trans-cinnamic acid (1, 10, and 30 µM) for 1 h and subsequently stimulated with TNF-α (50 ng/mL) for 24 h. Supernatants were collected, and CXCL8 levels were quantified through ELISA, following the manufacturer’s protocol. Data are expressed as pg/mL and represent the mean ± standard deviation (S.D.) of three independent experiments performed in triplicate. Statistical differences were determined using one-way ANOVA followed by Tukey’s multiple comparisons test. +P < 0.001 vs. unstimulated cells (saline-treated group)
Effect of trans-cinnamic acid on neutrophil-endothelium adhesion
We demonstrated that trans-cinnamic acid reduced in vivo neutrophils recruitment, as well as the production of proinflammatory cytokines and chemokines. However, this compound did not affect neutrophil migratory responses to a chemotactic stimulus in vitro. Since the interaction of neutrophils with endothelial cells represents a critical step in leukocyte recruitment to sites of inflammation, we next evaluated whether trans-cinnamic acid could modulate neutrophil adhesion to TNF-α-primed endothelial cells. As shown in Fig. 4A, neutrophils exhibited significantly higher adhesion to EA.hy926 cells stimulated with TNF-α (50 ng/mL) compared with untreated control endothelial cells (DMEM). Treatment with trans-cinnamic acid significantly reduced neutrophil adhesion, but only at the highest concentration tested (30 µM) (Fig. 4A). Importantly, exposure of neutrophils and epithelial cells to trans-cinnamic acid (1, 10, or 30 µM) for 24 h did not affect cell viability, as assessed through the MTT assay (Fig. 4B). These findings suggested that the inhibitory effect of trans-cinnamic acid on neutrophil adhesion was likely mediated by interference with endothelial cell activation or adhesion molecule expression.Fig. 4. Effect of trans-cinnamic acid on neutrophil-endothelial cell adhesion and cell viability. A EA.hy926 cells were pretreated with DMEM or trans-cinnamic acid (1, 10, and 30 µM) for 1 h and subsequently stimulated with TNF-α (50 ng/mL) for 6 h. Neutrophils were then added and allowed to adhere to EA.hy926 cells for 2 h. B For cell viability assessment, EA.hy926 cells were plated and treated with trans-cinnamic acid (1, 10, and 30 µM) for 24 h, and viability was determined through the MTT assay. Bars represent the mean ± standard deviation (S.D.) of three experiments performed in triplicate. Statistical differences were determined using ANOVA followed by Tukey’s multiple comparisons test. +++P < 0.001 vs. control group (cells maintained in DMEM); **P < 0.05 vs. TNF-α-stimulated group.
Trans-cinnamic acid reduces TNF-α-induced ICAM-1 protein and mRNA expression in EA.hy926 cells
As shown in Fig. 5A, flow cytometry analysis revealed low basal expression of ICAM-1 protein in unstimulated EA.hy926 cells (DMEM-treated). Stimulation with TNF-α (50 ng/mL, 6 h) markedly upregulated ICAM-1 protein expression (Fig. 5A-B). Treatment with trans-cinnamic acid (30 µM) significantly reduced ICAM-1 expression by approximately 39% compared with TNF-α-stimulated cells (Fig. 5A-B). To determine whether this inhibition occurred at the transcriptional level, ICAM-1 mRNA expression was assessed using RT-qPCR. As shown in Fig. 5C, TNF-α stimulation induced a significant increase in ICAM-1 mRNA expression, which was reduced by approximately 50% following treatment with trans-cinnamic acid (30 µM) (Fig. 5C). These data indicated that trans-cinnamic acid suppressed ICAM-1 expression in TNF-α-stimulated endothelial cells at both transcriptional and protein levels.Fig. 5trans-cinnamic acid reduced ICAM-1 protein and mRNA expression in TNF-α-stimulated EA.hy926 cells. A Representative histogram of ICAM-1 protein expression: black curve, DMEM-treated cells; red curve, cells stimulated with TNF-α (50 ng/mL); blue curve, trans-cinnamic acid-treated cells stimulated with TNF-α (50 ng/mL). B Quantification of fluorescence intensity corresponding to ICAM-1 protein expression. C ICAM-1 mRNA levels in EA.hy926 cells treated with DMEM or trans-cinnamic acid (30 µM) for 1 h, followed by stimulation with TNF-α (50 ng/mL) for 6 h. mRNA levels were quantified using RT-qPCR and normalized to β-actin. Bars represent the mean ± standard deviation (S.D.) of three independent experiments performed in triplicate. Statistical differences were determined using ANOVA followed by Tukey’s multiple comparisons test. +++P < 0.001 vs. control group (cells maintained in DMEM); ***P < 0.001 vs. TNF-α-stimulated group.
Discussion
The present study was designed to assess whether trans-cinnamic acid exerts anti-inflammatory effects by reducing vascular permeability and leukocyte accumulation in an LPS-induced pleurisy model. Our results showed that trans-cinnamic acid effectively inhibited LPS-induced pleurisy by attenuating protein extravasation, neutrophil infiltration, and levels of proinflammatory cytokines TNF-α and IL-6. We also evaluated the direct effects of trans-cinnamic acid on neutrophil and endothelial cell function. On neutrophils, we noted that trans-cinnamic acid did not affect the migratory response, as assessed through CXCL1-induced chemotaxis using a Boyden chamber. When evaluating the effects of trans-cinnamic acid on endothelial cells, we observed that it markedly reduced neutrophil adhesion to TNF-α-activated endothelial cells, a phenomenon associated with downregulation of ICAM-1 expression at both the mRNA and protein level. However, the same treatment did not affect the production of the chemokine CXCL8 by TNF-α-stimulated endothelial cells.
LPS-induced pleurisy is a well-characterised experimental model of inflammation that allows the quantification and correlation of both exudate and cellular migration with other inflammatory parameters. The major characteristic of this model in mice is the profile of the acute inflammatory reaction, in which neutrophil migration and exudation are clearly observed in the stimulated tissue 6 h after LPS challenge. Thus, this model constitutes a relevant biological system for evaluating the therapeutic efficacy of various drugs and phytochemical compounds that are useful for treating acute inflammation.
In the present study, LPS-induced pleurisy results revealed that, 6 h after LPS stimulation, the characteristic protein-rich plasma leakage was effectively inhibited by pretreatment with trans-cinnamic acid. This result may reflect the possible role of trans-cinnamic acid in the early vascular events of the inflammatory response involving endothelial cells. In the same LPS-induced pleurisy model, we observed intense neutrophilic exudation into the pleural space, which is another important hallmark of the acute inflammatory response [22]. In this study, we demonstrated that intrapleural injection of LPS in mice provoked an inflammatory reaction characterised by intense migration of neutrophils to the pleural cavity, a phenomenon that was substantially inhibited by previous treatment with trans-cinnamic acid. Likewise, previous studies have also demonstrated that topical treatment with trans-cinnamic acid can attenuate the inflammatory response in both cutaneous damage and rheumatoid arthritis [23, 24]. Moreover, the systemic administration of trans-cinnamic acid in patients with asthma was shown to effectively reduce leukocyte infiltration and suppress proinflammatory cytokine production, thus mitigating pulmonary damage in these patients [24]. This evidence strongly supports our claim that the use of trans-cinnamic acid reduces both plasma leakage and migration of neutrophils into tissues to protect against LPS-induced inflammation.
It is widely accepted that proinflammatory cytokines, such as TNF-α and IL-6, play a critical role in acute inflammation by propagating the inflammatory process through the increase of leukocyte activity [25]. In our study, LPS induced a remarkable increase in TNF-α and IL-6 levels in the pleural exudates compared with the control group. Treatment with trans-cinnamic acid suppressed the levels of these mediators compared with the LPS-stimulated group. This result indicated that the protective effects of trans-cinnamic acid on the inflammatory response may be due to its ability to inhibit the release of these mediators. Regarding this result, it is worth mentioning that both TNF-α and IL-6 are cytokines known to activate endothelial cells at inflammatory sites, in particular by increasing the expression of adhesion molecules, like ICAM-1, which contribute to leukocyte recruitment to sites of inflammation [26]. In this context, it is plausible to suggest that the reduced levels of TNF-α and IL-6 at the inflammatory site may limit ICAM-1 expression in endothelial cells, attenuating neutrophil influx to the tissue.
Since our results showed that trans-cinnamic acid reduced LPS-induced pleural inflammation in mice, we evaluated whether treatment with this compound would also affect the migratory capacity of neutrophils. We used the classical Boyden chamber method for the in vitro measurement of neutrophil migration induced by CXCL1. Our results revealed that the ability of human neutrophils to migrate toward CXCL1 was not affected after trans-cinnamic acid treatment, indicating that neutrophil function remained preserved. Similarly, previous studies have reported that neither oxidative metabolism nor myeloperoxidase (MPO) activity in human neutrophils is affected by exposure to trans-cinnamic acid [27]. In this context, as neutrophils were shown to be refractory to the effects of trans-cinnamic acid and given the importance of endothelial cells for the mobilisation of neutrophils during inflammation, we decided to evaluate whether endothelial cells would be sensitive to treatment with trans-cinnamic acid.
It is widely recognised that activated endothelial cells, in addition to secreting proinflammatory mediators such as chemoattractant molecules, also express adhesion molecules involved in the attachment of leukocytes to their surface [28]. Thus, in a typical inflammatory scenario, endothelial cells contribute to neutrophil mobilisation by producing mediators such as CXCL8, while also favouring neutrophil adhesion through the expression of cell adhesion molecules. ICAM-1 expressed in endothelial cells is key for both adhesion and transendothelial migration of neutrophils [29]. Under physiological conditions, ICAM-1 is expressed at low levels in endothelial cells, and its expression dramatically increases in response to inflammatory cytokines. Experiments using human endothelial cells in vitro have shown that this up-regulation can reach 50- to 100-fold [30]. Indeed, the results obtained in our experiments are in line with this information, as we also observed that neutrophils in contact with endothelial cells treated with TNF-α showed greater adhesion than the control group. We also demonstrated that neutrophil-endothelial cell adhesion was markedly inhibited by trans-cinnamic acid. It is important to highlight that treatment with trans-cinnamic acid did not alter cell viability at the doses tested, nor did it alter CXCL8 secretion by TNF-α-stimulated endothelial cells, showing an absence of cytotoxicity and the selective inhibitory effects of this compound. Based on this result, we verified whether trans-cinnamic acid affected the expression of ICAM-1 by endothelial cells and consequently prevented the adhesion and entry of neutrophils into sites of inflammation. Mechanistically, our results revealed that the elevated mRNA and protein levels of ICAM-1 expressed by TNF-α-activated endothelial cells were markedly reduced by trans-cinnamic acid. Previous studies have shown that trans-cinnamic acid derivatives, such as cinnamaldehydes, are able to suppress expression of ICAM-1 at the transcriptional level in TNFα-stimulated human endothelial cells [31]. Moreover, trans-cinnamic acid downregulated TNFα-induced ICAM-1 mRNA expression in both fibroblast and monocyte cell lines [32]. Furthermore, treatment with trans-cinnamic acid, which reduces ICAM-1 expression in ischaemia–reperfusion injury, has been shown to protect tissues from inflammatory damage [33]. Therefore, it is possible to propose that both the reduction in infiltrated neutrophils in vivo and those adhering to the surface of endothelial cells stimulated in vitro, may have been influenced by the reduction in ICAM-1 expression induced by trans-cinnamic acid.
The present study demonstrated that trans-cinnamic acid exerted protective effects in an acute model of inflammation by reducing vascular leakage, cytokine production, and neutrophil recruitment. These effects were primarily associated with the downregulation of ICAM-1 expression in endothelial cells, leading to impaired neutrophil adhesion and infiltration. Importantly, trans-cinnamic acid did not directly affect neutrophil chemotaxis or endothelial cytokine production, highlighting its selective action on leukocyte–endothelium interactions. Overall, these findings expand current knowledge of the pharmacological properties of phenolic acids and identify trans-cinnamic acid as a promising natural compound with potential therapeutic applications in the control of neutrophil-driven inflammatory diseases. However, further investigation is required to determine the specific mechanism by which trans-cinnamic acid accomplishes its effects.