The Beneficial Effect of Carvacrol in HL-1 Cardiomyocytes Treated with LPS-G: Anti-Inflammatory Pathway Investigations

Carvacrol (CAR), a natural phenolic monoterpene, possesses different biological activities, such as anti-inflammatory and antioxidant activities. The current study aimed to evaluate the response of HL-1 cardiomyocytes to an inflammatory stimulus triggered by lipopolysaccharide from Porphyromonas gingivalis (LPS-G), alone or in co-treatment with CAR, to investigate the potential protective role of CAR in the inflammatory process through modulation of the TLR4/NFκB/NALP3/IL-1β pathway and ROS production. In an in vitro experiment, HL-1 cardiomyocytes were exposed to LPS-G and incubated with CAR. We evaluated the anti-inflammatory effect of CAR by the reduction in TLR4, NFκB, NALP3, and IL-1β expression using immunofluorescence staining. Western blot analysis also validated the modulation of the TLR4/NFκB/NALP3/IL-1β pathway. ROS analyses confirmed the protective effects of CAR. Our results suggest that CAR could provide a significant protection role against inflammatory stimulus generated by LPS-G, involving the suppression of the TLR4/NFκB/NALP3/IL-1β signaling pathway.


Introduction
Carvacrol (CAR) is a natural compound isolated from essential oils, and several studies have reported its antibacterial, antioxidant, anti-inflammatory, fungicidal, insecticidal, and chemopreventive activities [1,2].
For a long period, CAR has largely been utilized as a food or food additive in the food industry. CAR and thymol, the main constituents of oregano extract essential oil, are responsible for its antioxidant and antibacterial activities [8]. Natural products, such as essential oils and their constituents, have also been shown to be potential and promising drug molecules for the development of therapeutic compounds against cardiovascular diseases [9]. These outcomes have revealed novel and unidentified cardiovascular properties of these natural molecules, opening a new frontier with respect to their common utilization [10]. The cardioprotective role of CAR has already been recognized, with previous papers reporting a decrease in infarction in Wistar rats, and a decrease in cardiac injury markers, such as creatine kinase, creatine kinase-MB, cardiac troponin T, and lactate dehydrogenase, in animals treated with CAR [11,12].
Several studies have already demonstrated that CAR possesses anti-inflammatory properties. In a previous paper, CAR was shown to have the capability to reduce the levels of inflammatory cytokines and the expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 in ischemic cortical tissues [13]. In a paper published by Canbek et al., the role of CAR in preserving the liver against ischemia/reperfusion damage in rats was shown [14]. These findings suggest that CAR could potentially have a cardioprotective effect against myocardial ischemia/reperfusion injury [15]. It is well known that NALP3 inflammasome plays a central role in the development and progression of cardiovascular diseases [16].
Based on the literature, heart failure is one of the complications that may be linked with periodontal disease [17]. P. gingivalis is one of the bacteria correlated with periodontal illness and its invasion into the blood results in sepsis and systematic inflammation [18]. Moreover, after P. gingivalis invasion, circulating endotoxin is largely linked with the development of heart damage. Lipopolysaccharide from P. gingivalis (LPS-G) represents an endotoxin and virulence factor.
Based on this knowledge, the purpose of the current work was to analyze the response of the HL-1 cell line to LPS-G, alone or in co-treatment with CAR, to evaluate the potential protective role of CAR in the inflammatory process through modulation of the TLR4/NFκB/NALP3/IL-1β pathway.
For a long period, CAR has largely been utilized as a food or food additive in the food industry. CAR and thymol, the main constituents of oregano extract essential oil, are responsible for its antioxidant and antibacterial activities [8]. Natural products, such as essential oils and their constituents, have also been shown to be potential and promising drug molecules for the development of therapeutic compounds against cardiovascular diseases [9]. These outcomes have revealed novel and unidentified cardiovascular properties of these natural molecules, opening a new frontier with respect to their common utilization [10]. The cardioprotective role of CAR has already been recognized, with previous papers reporting a decrease in infarction in Wistar rats, and a decrease in cardiac injury markers, such as creatine kinase, creatine kinase-MB, cardiac troponin T, and lactate dehydrogenase, in animals treated with CAR [11,12].
Several studies have already demonstrated that CAR possesses anti-inflammatory properties. In a previous paper, CAR was shown to have the capability to reduce the levels of inflammatory cytokines and the expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 in ischemic cortical tissues [13]. In a paper published by Canbek et al., the role of CAR in preserving the liver against ischemia/reperfusion damage in rats was shown [14]. These findings suggest that CAR could potentially have a cardioprotective effect against myocardial ischemia/reperfusion injury [15]. It is well known that NALP3 inflammasome plays a central role in the development and progression of cardiovascular diseases [16].
Based on the literature, heart failure is one of the complications that may be linked with periodontal disease [17]. P. gingivalis is one of the bacteria correlated with periodontal illness and its invasion into the blood results in sepsis and systematic inflammation [18]. Moreover, after P. gingivalis invasion, circulating endotoxin is largely linked with the development of heart damage. Lipopolysaccharide from P. gingivalis (LPS-G) represents an endotoxin and virulence factor.
Based on this knowledge, the purpose of the current work was to analyze the response of the HL-1 cell line to LPS-G, alone or in co-treatment with CAR, to evaluate the potential protective role of CAR in the inflammatory process through modulation of the TLR4/NFκB/NALP3/IL-1β pathway.

Experimental Study Design
All experiments were performed in triplicate with HL-1. The study design is reported as follows:

Cell Viability Assay
The HL-1 cardiomyocyte cells were seeded at a cell density of 6000/well into a 96-well tissue culture plate. The cell metabolic activity of HL-1 cell lines was evaluated after 24, 48, and 72 h of treatment with CAR at 6.25, 12.5, 25, and 50 µM and at 6.25 and 50 µM alone or in co-treatment with LPS (5 µg/mL) on a 96-well polystyrene plate using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2Htetrazolium (MTS) assay (CellTiter 96 ® Aqueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA). At the established time points, 20 µL per well of MTS dye reagent were placed in the culture medium, and HL-1 cells were placed in an incubator for 3 h at 37 • C [19]. The amount of formazan product was measured at the 490 nM wavelength with a Synergy™ HT Multi-detection microplate reader (Biotech, Winooski, VT, USA), which measured the number of live cells [20]. Data are representative of three independent experiments performed in triplicate.

Reactive Oxygen Species (ROS) Evaluation
HL-1 cells were seeded at 85000/well in a 35 mm imaging dish (µ-Dish, ibidi GmbH, Gräfelfing, Germany). Then, HL-1 cells were placed in culture medium containing 6.25 and 50 µM CAR alone or in co-treatment with LPS (5 µg/mL) for 24 h. After 24 h, the medium was replaced with Normal External Solution (NES) containing (in mM) 125 NaCl, 5 KCl, 1 MgSO 4 , 1 KH 2 PO 4 , 5.5 glucose, 1 CaCl 2 , and 20 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES), pH 7.4 and then incubated with 10 µM of 2 ,7dichlorodihydrofluorescein diacetate (H2DCFDA, Thermo Fisher, Waltham, MA, USA) in a humidified incubator for 30 min at 37 • C, keeping the appropriate culture medium administration for the entire processes. After the incubation with the dye, HL-1 cells were rinsed with NES and detected in NES alone (HL-1) or maintained in NES plus LPS-G, LPS-G, and CAR at 6.25 or 50 µM or CAR alone. Using a confocal microscope, images were casually taken for each experimental condition through a motorized table SMC 2009 and the multiple single position acquisition function (Tiles-Advanced setup, carrier 35 mm petri dish) of Zen Blue software (Zen 3.0 SR, Carl Zeiss) utilizing a Zeiss LSM800 microscope (Carl Zeiss) furnished with an inverted microscope Axio-obserber D1 (Carl Zeiss) and an objective W-Plan-Apo 40 X/1.3 DIC (Carl Zeiss). At 488 nm, the excitation and emission were detected with the filter set over 505-530 nm. During the acquisition of the sample images, the acquisition settings were unchanged. The analyses of the offline pictures were executed utilizing Fiji distribution of ImageJ (version 1.53c, National Institutes of Health, Bethesda, MD, USA) quantifying the mean of fluorescence intensity (arbitrary units, F) and the area of the HL-1 measured (µm 2 ) for each obtained cell [24]. Quantitative data of the amount of ROS are expressed as ratio F/µm 2 .

Statistical Analysis
Statistical significance was established with GraphPad 5 (GraphPad, San Diego, CA, USA) software utilizing the t-test and ordinary one-way ANOVA and then by post hoc Bonferroni's multiple comparisons analysis. Values of p < 0.05 were considered as statistically significant.

Effects of CAR Alone or in Co-Treatment with LPS-G on HL-1 Cells' Metabolic Activity
The effects of CAR alone or in combination with LPS-G on cardiomyocytes of the HL-1 cell line were evaluated using the MTS assay. Initially, CAR was tested individually at concentrations ranging from 6.25 to 50 µM to evaluate the effect on cell viability. CAR appears to be well tolerated in HL-1 cell lines considering that the percentage of viable cells was not affected by CAR treatment at 6.25, 12.5, 25, and 50 µM after 24, 48, and 72 h ( Figure 2). Instead, cell metabolic activity was reduced when treated with LPS-G at 5 µg/mL compared to the untreated cells after 72 h of treatment with CAR. Then, CAR was tested at 6.25 and 50 µM alone or in combination with LPS-G (5 µg/mL). After 72 h of treatment, the co-treatment of CAR/LPS-G showed a higher percentage of viable cells compared to the cells treated with LPS-G (5 µg/mL) (Figure 3). appears to be well tolerated in HL-1 cell lines considering that the percentage of viable cells was not affected by CAR treatment at 6.25, 12.5, 25, and 50 μM after 24, 48, and 72 h ( Figure 2). Instead, cell metabolic activity was reduced when treated with LPS-G at 5 μg/mL compared to the untreated cells after 72 h of treatment with CAR. Then, CAR was tested at 6.25 and 50 μM alone or in combination with LPS-G (5 μg/mL). After 72 h of treatment, the co-treatment of CAR/LPS-G showed a higher percentage of viable cells compared to the cells treated with LPS-G (5 μg/mL) (Figure 3).

Expression Levels of TLR4/NFκB/NALP3/IL-1β in CAR, LPS-G, and in Combination CAR/LPS-G-Treated Cells
The immunofluorescence results showed that the TLR4/NFκB/NALP3/IL-1β pathway was expressed significantly in HL-1 treated with LPS-G for 24 h compared to the untreated cells. Cells treated with CAR alone at 6.25 and 50 μM or in co-treatment with CAR and LPS-G reported a severe decrease in proteins involved in the inflammation process (Figures 4 and 5). These results were confirmed by Western blot analysis, where the HL-1 cell line treated with LPS-G showed higher expression of TLR4/NFκB/NALP3/IL-1β compared to the CTRL sample. On the contrary, CAR administration alone did not influence the protein levels similar to the untreated HL-1 cells (Figure 6). cells was not affected by CAR treatment at 6.25, 12.5, 25, and 50 μM after 24, 48, and 72 h ( Figure 2). Instead, cell metabolic activity was reduced when treated with LPS-G at 5 μg/mL compared to the untreated cells after 72 h of treatment with CAR. Then, CAR was tested at 6.25 and 50 μM alone or in combination with LPS-G (5 μg/mL). After 72 h of treatment, the co-treatment of CAR/LPS-G showed a higher percentage of viable cells compared to the cells treated with LPS-G (5 μg/mL) (Figure 3).

Expression Levels of TLR4/NFκB/NALP3/IL-1β in CAR, LPS-G, and in Combination CAR/LPS-G-Treated Cells
The immunofluorescence results showed that the TLR4/NFκB/NALP3/IL-1β pathway was expressed significantly in HL-1 treated with LPS-G for 24 h compared to the untreated cells. Cells treated with CAR alone at 6.25 and 50 μM or in co-treatment with CAR and LPS-G reported a severe decrease in proteins involved in the inflammation process (Figures 4 and 5). These results were confirmed by Western blot analysis, where the HL-1 cell line treated with LPS-G showed higher expression of TLR4/NFκB/NALP3/IL-1β compared to the CTRL sample. On the contrary, CAR administration alone did not influence the protein levels similar to the untreated HL-1 cells ( Figure 6).

Expression Levels of TLR4/NFκB/NALP3/IL-1β in CAR, LPS-G, and in Combination CAR/LPS-G-Treated Cells
The immunofluorescence results showed that the TLR4/NFκB/NALP3/IL-1β pathway was expressed significantly in HL-1 treated with LPS-G for 24 h compared to the untreated cells. Cells treated with CAR alone at 6.25 and 50 µM or in co-treatment with CAR and LPS-G reported a severe decrease in proteins involved in the inflammation process (Figures 4 and 5). These results were confirmed by Western blot analysis, where the HL-1 cell line treated with LPS-G showed higher expression of TLR4/NFκB/NALP3/IL-1β compared to the CTRL sample. On the contrary, CAR administration alone did not influence the protein levels similar to the untreated HL-1 cells ( Figure 6).

CAR Reduces Reactive Oxygen Species Production in LPS-G-Treated HL-1 Cells
The amount of ROS generation promoted by the treatment with LPS-G was assessed in the HL-1 cell line loaded with cell-permeable H2DCFDA. This fluorescent probe, once activated by intracellular esterases, became active to react with oxidative species. Here, the H2DCFDA is transformed to the extremely fluorescent 2 ,7 -dichlorofluorescein (DCF) by ROS and the increase in fluorescence signals. Pictures were taken of live cells using confocal microscopy as evidenced in Figure 7A and the fluorescence signal was analyzed offline. The quantitative results ( Figure 7B) show a notable increase in the ROS amount in HL-1 cells treated with 5 µg mL −1 LPS-G compared to the control condition. Interestingly, the co-incubation of LPS-G together with CAR at 6.25 and 50 µM counteracted the LPS-G increase in ROS production while CAR alone did not affect the production of ROS.

CAR Reduces Reactive Oxygen Species Production in LPS-G-Treated HL-1 Cells
The amount of ROS generation promoted by the treatment with LPS-G was assessed in the HL-1 cell line loaded with cell-permeable H2DCFDA. This fluorescent probe, once activated by intracellular esterases, became active to react with oxidative species. Here, the H2DCFDA is transformed to the extremely fluorescent 2′,7′-dichlorofluorescein (DCF) by ROS and the increase in fluorescence signals. Pictures were taken of live cells using confocal microscopy as evidenced in Figure 7A and the fluorescence signal was analyzed offline. The quantitative results ( Figure 7B) show a notable increase in the ROS amount in HL-1 cells treated with 5 μg mL −1 LPS-G compared to the control condition. Interestingly, the co-incubation of LPS-G together with CAR at 6.25 and 50 μM counteracted the LPS-G increase in ROS production while CAR alone did not affect the production of ROS.

Discussion
It is well known that cardiovascular disease might be induced by long-term inflammation, with the latter being controlled by different molecules that promote inflammation, such as cytokines and angiotensin receptors.
Research on essential oils has attracted significant interest from the scientific com-

Discussion
It is well known that cardiovascular disease might be induced by long-term inflammation, with the latter being controlled by different molecules that promote inflammation, such as cytokines and angiotensin receptors.
Research on essential oils has attracted significant interest from the scientific community due to its potential regarding the identification of novel active molecules for the treatment of cardiovascular illnesses, such as myocardial infarction, arterial hypertension, angina pectoris, and heart breakdown. Different mechanisms have been identified regarding the role of essential oils and their main active components in supporting the health of the cardiovascular system [25].
In a paper published by Lee et al. (2006), it was evidenced that Porphyromonas gingivalis impairs cardiomyoblasts H9c2 through the promotion of hypertrophy factors and stimulation of the MMP9 fibrosis molecule. A previous work demonstrated that cardiomyopathy stimulated by Porphyromonas gingivalis in part includes p38 MAPK induction [26].
Porphyromonas gingivalis pathogenesis widely depends on its different virulence factors, comprising its specific structure, such as lipopolysaccharide, heat shock, fimbriae, and proteins, and secretory factors, such as gingipains and outer membrane vesicles [27]. The inflammatory response induced by LPS-G is a widespread in vitro model.
Numerous signaling pathways are implicated in different cellular processes that can eventually promote the release of inflammatory molecules and could instigate tissue damage, which can be stimulated by outer membrane endotoxins.
In our work, LPS-G stimulation was responsible for promoting an inflammatory response in an in vitro model of the HL-1 cell line, which activated the inflammatory TLR4/NALP3/NFκB/IL-1β pathway and ROS production. Based on the literature, LPS-G plays a key role in inducing inflammation and stimulating cells to secrete proinflammatory cytokines, such as IL-1β, TNF-α, and IL-6. LPSs are known to instigate proinflammatory cytokine formation, mainly through TLR4 and NFkB activation [28].
NALP3 is a member of NOD-like receptors (NLRs; set of intracellular PRRs), which, upon activation, assemble into a high-molecular-weight multi-protein caspase-1-activating platform known as the NALP3 inflammasome that orchestrates maturation of highly potent proinflammatory cytokines, such as IL-1β and IL-18 [29]. The NALP3 inflammasome is controlled by the existence of molecular patterns that are correlated with damage and starts or intensifies the inflammatory reaction throughout IL-1β and/or IL-18 formation.
Furthermore, NALP3, being the most fully characterized inflammasome, is implicated in intracellular inflammation; it can be stimulated by several bacterial ligands, including LPS-G [30]. Hence, unraveling the cellular and molecular aspects of NALP3 inflammasome activation and inhibition are crucial to the discovery and development of therapeutic candidates targeting NALP3 in different inflammatory disease conditions. The inflammatory reactions can be instigated by a sensor factor belonging to the NALP3 inflammasome. In tissue damage, the NALP3 inflammasome and TLR4 signaling have a crucial role [31]. TLR4 acts as a vital factor that stimulates the inflammatory reaction in the body. The suppression or downregulation of the NALP3/TLR4 signaling pathway represents a possible target in different long-term illnesses [32]. TLR4 stimulation instigates the myeloid differentiation factor 88 (MyD88) signaling pathway, which promotes rapid activation of NFκB, which consequently leads to an increase in IL-18, IL-6, IL-1β, tumor necrosis factor-α (TNF-α), and monocyte chemotactic protein-1 (MCP-1) [33]. ROS were proposed as the common signal for NALP3 inflammasome activation since most NALP3 stimuli can instigate ROS in treated cells, and lysosomal NADPH oxidase was primarily assumed to be the cause of ROS generation.
Our in vitro data suggest that HL-1 cells treated with LPS-G alone show increased ROS production and enhanced expression of TLR4/NALP3/NFκB/IL-1β. Instead, when the HL-1 cells were treated with CAR or in co-treatment with CAR/LPS-G, CAR being a natural antioxidant, the total ROS production in LPS-G-treated HL-1 cells was reduced and a reduction in the release of inflammatory mediators, such as TLR4/NALP3/NFκB/IL-1β, Antioxidants 2022, 11, 386 9 of 11 was shown as reported by confocal microscopy and Western blot analysis. These results obtained may suggest the possible pharmacological benefit of maintaining mitochondrial function with CAR, a natural antioxidant that attenuates NALP3 activation.
The present results suggest that CAR instigates anti-inflammatory outcomes in HL-1 cells through the generation of inflammatory factors that modulate the TLR4/NALP3/NFκB/ IL-1β pathway and by decreasing ROS production. The current work aimed to investigate the possible therapeutic benefit of CAR in a cardiomyocyte cellular model, modulating the inflammatory effects induced by LPS-G through TLR4/NALP3/NFκB/IL-1β and ROS production. This biological outcome was, for the first time, investigated in HL-1 cells, whereas data for other terpenoids are not available yet. Conversely, CAR and some other structurally similar phenolic components of plant essential oils, such as thymol, eugenol, vanillin, and guaiacol, were tested on an L-type Ca 2+ current in isolated ventricular cardiomyocytes in the concentration range of 10-1000 µM. Among these natural compounds, CAR was the most effective in the concentration-dependent and reversible suppression of cardiac Ca 2+ currents, thus suggesting that this effect on cardiomyocytes is strongly related to the chemical functionalization of the core nucleus of terpenoids (CAR > thymol > eugenol) [34] (thymol (a regioisomer of CAR) also displayed concentration-dependent inhibitory effects on K + currents in ventricular cardiomyocytes) [35]. Lastly, thymol and CAR were shown to act as inhibitors of protein and lipid oxidation induced by hydroxyl radicals to myofibrillar proteins; the mechanism was related to the reduction of lipid-derived volatile carbonyls and the formation of protein crosslinking [36].

Conclusions
CAR displayed a beneficial effect in an in vitro model on HL-1 cardiomyocytes treated with LPS-G. The protective role revealed by CAR may be attributed to its antioxidant and anti-inflammatory properties. Our work suggests that CAR could provide a significant beneficial effect by modulating the inflammatory effects promoted by LPS-G through activation of the TLR4/NALP3/NFkB/IL-1β signaling pathways and ROS production.