Anti-Inflammatory and Antifungal Effects of Steroidal Compounds from Thymus vulgaris Against Malassezia-Induced Inflammation in Keratinocytes

The phytosteroids β-sitosterol, stigmasterol, and campesterol have been identified through the use of high-performance liquid chromatography (HPLC) from an extract of Thymus vulgaris. The separation was performed on a C18 reversed-phase column using a standard gradient for elution. The retention times (RT) of individual sterol compounds were compared to those of authentic reference standards for identification. The purity of all three compounds was determined using HPLC peak area normalization. The chemical structures of β-sitosterol, stigmasterol, and campesterol were assigned according to chromatographic patterns along with published reference data, as shown in Table 1.

Table 1. Molecular formula, molecular weight, and chemical structures of steroidal compounds isolated from Thymus vulgaris

Compound	Molecular Formula	Molecular Weight (g/mol)	Chemical Structure
β-Sitosterol	C₂₉H₅₀O	414.7	t1.jpg
Stigmasterol	C₂₉H₄₈O	412.7	t2.jpg
Campesterol	C₂₈H₄₈O	400.7	t3.jpg

Human keratinocyte cell lines, HaCaT, were used because they are very appropriate for simulating the skin environment and have already been mentioned to elicit inflammatory responses to external stimuli. The experiments were carried out in a successive three-tier manner: treatment of Malassezia-induced keratinocytes with steroidal compounds from Thymus vulgaris [15], assessment of inflammatory gene expression using qRT-PCR [16], study on the pathway of NF-κΒ concerned with inflammation [17].

The HaCaT keratinocytes have been supplied by CLS Cell Lines Service at Eppelheim, Germany. A culture of HaCaT keratinocytes was maintained in a complete medium containing DMEM (High Glucose) with 10% FBS, 1% Penicillin-Streptomycin, and incubated in a 37℃, humidified incubator with 5% CO₂, as described previously. Malassezia globosa (ATCC 96807) and Malassezia restricta (ATCC 33555) were obtained from the American Type Culture Collection (ATCC), USA. Both fungal strains were cultured on Dixon’s agar and incubated at 32℃ for 48–72 h under aerobic conditions. Yeasts were harvested, washed in sterile PBS, and the concentration of yeast cells was adjusted to the appropriate dilution for further use.

2.1 HPLC analysis of steroidal compounds

By means of HPLC, the purity and identity of steroidal compounds obtained from Thymus vulgaris were evaluated. The assessment employed a Reversed-Phase C18 column (250 mmlong × 4.6 mm) having a particle size of 5 microns. The mobile-phase solvent consisted of 90% methanol and 10% water (v/v), delivered at 1.0 mL/min under Isocratic conditions. Chromatographic separation took place at room temperature, while the UV detector was set at 210 nm for the detection of sterols. For each sample, 20 µL was injected into the column. The individual sterols were identified through the use of RT in comparison with reference standards (β-sitosterol, stigmasterol, and campesterol). The purity of each peak was assessed by the resolution of the peaks obtained on the chromatogram.

2.2 Malassezia cells and HaCaT cells co-culturing

An in vitro inflammation model was created by directly co-culturing HaCaT keratinocytes with Malassezia cells. HaCaT cells were cultured in plates until they reached approximately 70-80% confluent. Malassezia globosa and Malassezia restricta were obtained from Dixon's agar media, washed 2 times using sterile phosphate-buffered saline (PBS), and heat-inactivated at 65℃ for 30 minutes to inhibit fungal overgrowth while maintaining any immunostimulatory components. The heat-inactivated yeast cells were then suspended in serum-free DMEM and added to the HaCaT cultures at a multiplicity of infection (MOI) of 10 fungal units. The co-cultures were incubated under a humidified atmosphere of 37℃ and 5% CO₂ for 24 hours prior to the addition of steroids and subsequent studies.

2.3 The MTT assay for HaCaT cells viability

The MTT assay was performed on HaCaT keratinocytes to evaluate the cytotoxicity of the steroidal compounds before any functional experiments. The HaCaT keratinocytes were seeded at a density of 1 × 10⁴ cells per well into 96-well plates and incubated for 24 hours to allow for cell adhesion. After incubation, the steroidal compounds were added at 10, 25, 50, and 100 µM and incubated for 24 hours under normal culture conditions (37℃ and 5% CO₂). After the 24 hour incubation, the cells were treated with MTT Solution at a concentration of 0.5 mg/mL for 4 hours. The formazan crystals formed from the conversion of the MTT by the viable cells were dissolved in DMSO, and the absorbance was measured at 570 nm using a microplate reader. The cell viability was reported as a percentage of the untreated control cells. After that, the cells were exposed to Thymus vulgaris steroidal compounds at various concentrations ranging from 10 to 100 µM in order to assess the dose-dependent effects. The controls included keratinocytes that were not treated and those that were treated with the solvent only. At the end of the treatment period, RNA was extracted using TRIzol reagent. The NanoDrop spectrophotometer was used to evaluate the purity and concentration of RNA. Only the highest quality of RNA was turned into cDNA for qRT-PCR analysis, which was intended for the quantification of cytokines IL-1β, TNF-α, IL-6, and COX-2—these are the ones that typically become more active during inflammation induced by Malassezia.

2.4 Gene expression and NF-κB pathway analysis

Quantitative real-time PCR (qRT-PCR) was used to measure the expression of the pro-inflammatory cytokines and genes related to the NFκB pathway. RNA was purified from the treated HaCaT keratinocyte cell lines and the untreated control cell lines and then converted to cDNA via reverse transcription using standard methods. Specific primers targeting IL-1β, TNF-α, IL-6, COX-2 (PTGS2), and GAPDH (a reference gene) were designed for amplification with the qRT-PCR system and validated prior to their use in the experiments. The expected size of each amplicon and the primer sequences used for amplification can be found in Table 2. The expression of each gene relative to GAPDH, utilized for internal normalisation, was calculated using the 2^⁻ΔΔCt method.

Table 2. Primer sequences used for qRT-PCR analysis

Western blot analysis was used to determine whether or not there was a modulation of the NF-kB signaling pathway at the protein level. Total protein was extracted from the cells, which was then separated via SDS-PAGE, before being transferred to a PVDF membrane. The membranes were then immunoblotted using primary antibodies against total NF-kB p65, phosphorylated NF-kB p65 (Ser536), and IκBα, all of which were obtained from Cell Signaling Technology (Danvers, MA). The antibodies were diluted according to the manufacturer's recommendations, and detailed information about the antibodies, including catalog numbers and working dilution can be found in Table 3. The bands on the membrane were visualized using enhanced chemiluminescence, and densitometric analysis of the bands was completed using ImageJ Software. In order to normalize the intensity of each band to that of β-actin, the band intensities were averaged over three independent trials.

In order to further explore the role of NF-κB signaling in mediating the effects of glucocorticoids (steroid-like compounds) derived from Thymus vulgaris, small interfering RNA (siRNA) technology was applied to eliminate specific genes that encode for proteins that make up the NF-κB pathway. Specifically, the subunit p65 (RelA) was eliminated from HaCaT keratinocytes through transfection with Lipofectamine RNAiMAX, a method recommended by the manufacturer. A control group of non-transfected, wild-type keratinocytes was also maintained. Details regarding the siRNA sequences, catalog numbers, suppliers, and methods of transfection are shown in Table 4. The efficiency of the knockdown for the NF-κB p65 (RelA) subunit was confirmed to be approximately 80% by use of Western blotting, at 48 h following transfection. Following elimination of the p65 (RelA) subunit gene in HaCaT keratinocytes, the keratinocytes were then treated with T. vulgaris compound extracts to determine if the reduction in expression of proinflammatory cytokines was dependent upon inhibition of the NF-κB pathway.

Table 3. Primary antibodies used for Western blotting analysis

Table 4. siRNA sequences and transfection conditions used for NF-κB pathway silencing

2.5 Antimicrobial assays

Antimicrobial assays and fungal viability testing using compounds derived from Thymus vulgaris, the antifungal capacity of these products will be determined against two clinically significant species of the genus Malassezia (Malassezia globosa and Malassezia restricta). Fungal samples will be isolated on Dixon’s agar and subsequently placed in optimal growth conditions through incubation at 32 degrees Celsius for a period of 72 hours. At the end of this period, the culture will be in a period of logarithmic growth; therefore, the fungus will be prepared as a suspension and placed into increasing concentrations of steroidal compound (5-100 µM).

2.5.1 Growth inhibition assay (OD at 600 nm)

To evaluate the ability of steroidal products to inhibit fungal growth, treated and untreated fungal samples will be subjected to standard incubation conditions. Once incubated, the total number of colonies on the treated and untreated samples will be measured using a spectrophotometer at 600 nm (OD600). Measurements of OD600 will provide information regarding the growth and multiplication of the fungal samples in question. All tests will be executed in triplicate to increase the reliability of the data obtained.

2.5.2 Viability testing of fungi (Live/dead staining)

Fungal viability will also be evaluated using a dual fluorescent staining method in addition to the OD600 reading. After treatment with the steroidal compounds, Malassezia cells will be collected and rinsed twice with PBS before being stained with SYTO9 and propidium iodide (PI) following the protocol provided by the manufacturer. SYTO9 stains live fungal cells green, with SYTO9 penetrating unbroken cell membranes. Propidium Iodide (PI) stains dead fungal cells red due to its ability to only penetrate cells with damaged membranes. Once the samples were prepared, they were then photographed with a fluorescent microscope. The images contained a minimum of five random microscopic views per sample to count live and dead cells. To determine the percentages of live and dead fungal cells, the following two equations were used: 1. Lives (%) = (Green Stained Cells / Total Cells) × 100; 2. Dying (%) = (Red Stained Cells / Total Cells) × 100. The data obtained from the viability tests were analysed along with OD600 measurements to provide an overall assessment of how effective the Thymus vulgaris steroidal compounds had been against fungal cells.

2.6 Statistical analysis

Triplicates would be demonstrated by each of the experiments, and the data would be analyzed according to the standard deviation. Statistical significance was assessed using one-way ANOVA followed by Tukey’s post-hoc test. A p-value of < 0.05 was considered significant. Relative mRNA levels were calculated using the 2^-ΔΔCt method, with housekeeping genes GAPDH and β-actin as controls.

The aim of the present work was to investigate the anti-inflammatory action and the antimicrobial properties of steroidal compounds isolated from Thymus vulgaris on Malassezia-stimulated keratinocytes. These data show that the investigated compounds represent potent anti-inflammatory agents, since they lower the proinflammatory gene expression at subtoxic concentrations, inducing downregulation of up to 80±5%, partial inhibition of the NF-κB signaling pathway, and antibacterial activity. These findings largely align with previous studies and provide the initial evidence on the dual therapeutic nature of Thymus vulgaris for Malassezia-associated skin diseases like seborrheic dermatitis and dandruff. Promoted mRNA levels of pro-inflammatory cytokines (IL-1β, TNF-α, and COX-2) were reduced by Thymus vulgaris steroidal compounds, displaying a reduced inflammation [18]. Cytokines are crucial immune response elements associated with Malassezia-induced skin aggravating inflammatory processes. The suppression of these mediators dose-dependently demonstrated that Thymus vulgaris compounds can give specific anti-inflammatory effects without affecting the immune response ultimately [19]. This is particularly relevant in chronic skin diseases where a balance between suppression of inflammation to avoid tissue damage and impairment of an intact defense mechanism, such as the skin barrier, needs to be achieved. Natural compounds, such as those derived from Thymus vulgaris extracts, have previously shown the ability to affect inflammatory pathways [20]. One study, for example, demonstrated that the essential oil of Thymus vulgaris could downregulate the production of TNF-α and IL-6 upon lipopolysaccharide stimulation of macrophages [21]. In this research, we demonstrated that the inflammatory cytokines produced by Malassezia-stimulated keratinocytes were reduced in the same manner, thus proving the pharmacological steroid compounds to be effective in the reduction of inflammation during the fungal skin infection process. The research conducted in this study has shed more light on the association between the NF-κB pathway and inflammation management. The transcription factor NF-κB is involved in that signal and regulates the expression of pro-inflammatory genes like IL-1β, TNF-α, and COX-2; thus, it is a vital component of the whole process. It is usually upregulated in microbial infections, including those caused by Malassezia [22]. The results of treating the cells with steroidal-like compounds obtained from Thymus vulgaris (thyme) indicate that one of the main effects of these compounds on the cellular signaling is through the phosphorylation of IκBα suppression and thus, NF-κB pathway suppression. The decline of the phosphorylated variants results in a lowered cytosolic-to-nuclear translocation of NF-κB and, thus, a suppression of the pro-inflammatory gene transcripts’ generation [23]. The phosphorylated-IκBα levels underwent a significant 65% reduction, and at the same time, a decline in p-NF-κB was observed, suggesting that the substances produced from Thymus vulgaris are working directly against crucial mediators that are associated with the NF-κB signaling pathway. This supports findings from other research on the regulation of inflammatory pathways by plant-based steroids. For example, it has been proven that the Steroidal saponins of various plants can stop NF-κB activation by hindering IκBα phosphorylation [24].

NF-κB repression by Thymus vulgaris reveals molecular antinflammatory links [25]. The inhibition of the NF-κB subunits results in a reduction of the inflammatory response of keratinocytes, even in the absence of steroid treatment. Such findings provide evidence that the anti-inflammatory effect of Thymus vulgaris steroids is primarily the result of NF-κB regulation; thus, the pathway's pivotal role in Malassezia-induced inflammation has been basically asserted.

This study shows that Thymus vulgaris's steroidal compounds inhibit NFκB pathway activation induced by Malassezia in cultured keratinocytes. Interpreting how these compounds work requires that multiple signaling pathways be examined and combined. The results of our study found that treatment with steroidal compounds significantly increased levels of total IκB, while decreasing levels of phosphorylated IκBα and NFκB p65, and decreased NFκB's nuclear localization. Hence, our data indicate that various phytosterols inhibit the canonical activation cascade of NFκB. These results are supported by other researchers who found that curcumin inhibits the function of IKK [26], while both resveratrol and curcumin directly act to decrease IKK activity and prevent NFκB from binding to its DNA binding site [27, 28].

Interestingly, the phytosterol, beta-sitosterol, along with other phytosterols, has shown to reduce NF-kB activity in models of inflammation, showing that Sterol structure may share affinity for kinases/adaptor proteins acting upstream from NF-kB signal activations [29]. Although our results do not directly measure IKK activity, we do show decreases in phospho I-kappaB alpha and phospho NF-kappaB, which indicate that the steroidal compounds interrupt the signalling pathway that activates NF-kappaB prior to translocating into the nucleus, so we can't definitively make statements about being an inhibitor of IKK Kinases or altering the activity of receptor and adaptor complexes, such as toll-like receptors. Further work is needed to demonstrate that the compounds do directly inhibit IKK Kinases or Alter receptors and adaptor complexes upstream from IKK Kinases. Combining structural studies with mechanistic assays will allow us to put T.vulgaris sterols into perspective with other naturally occurring NF-kB modulators.

The extracts of Thymus vulgaris have a remarkable ability to kill Malassezia species (M. globosa and M. restricta), mostly associated with dermatological manifestations- such as dandruff and seborrheic dermatitis-largely from being an active agent toward inflammation. The minimum inhibitory concentrations (MICs) determined at 75 µM for M. globosa and 100 µM for M. restricta show that these compounds can effectively prevent fungal growth even at very low dosages. The outcomes of the use of live/dead dyes and the absorbance (OD) decrease at 600 nm showed concentration-dependent antifungal activity, where the 100 µM concentration completely stopped the growth of the fungi [26]. The cell death dye confirmed this, showing that all the fungal cells were dead at this particular dose scale. These findings agree with other research on the antifungal properties of Thymus vulgaris [30].

The antibacterial as well as the antifungal activities that different thyme essential oils exhibited; the latter were active against fungi like Candida albicans. This may also be furthered by developing the results of such studies because it has been shown in the current study that steroidal compounds from Thymus vulgaris are active against the Malassezia species. The two roles of the steroidal compounds produced from Thymus vulgaris indicate a therapeutic advantage in the treatment of inflammatory conditions and the infectious organism itself [31]. These agents will be directed against both the inflammatory response and the microbes directly responsible for skin diseases; hence, a more holistic approach toward treating these conditions. This may be of further benefit in seborrheic dermatitis, where overpopulation of Malassezia drives an inflammatory response [32]. The anti-inflammatory action of many of the compounds here, through inhibition of NF-κB, may complement their fungicidal action and provide a fuller complement to conventional anti-inflammatories, lacking in activity against the microbial element associated with these diseases.

The common conventional treatments for Malassezia-associated skin diseases are antifungal agents, steroids, and anti-inflammatory medications. Inhibiting the growth of the fungus with antifungals has no effect on the inflammation produced [33]. On the other hand, topical corticosteroids and NSAIDs exert anti-inflammatory activities but may induce skin atrophy, immunosuppression, or irritation. A safe alternative could be steroidal compounds of Thymus vulgaris, which address the etiopathogenesis of the disease with fewer side effects [34].

As well as having both antifungal and anti-inflammatory activity, the potential therapeutic use of steroidal compounds from Thymus vulgaris will be influenced by (i) the ability of the steroidal compounds to penetrate through the skin and into the underlying tissues, (ii) the ability of the final formulation to remain stable, and (iii) the degree of compatibility with existing therapies. The use of natural sources of phytosterols, including β-sitosterol, stigmasterol, and campesterol, as lipophilic compounds, may support transdermal uptake via topically applied lipids (formulations that are creams, liposomes, and nano-emulsions) [35, 36]. In addition, the relative chemical stability of steroidal compounds compared to polyphenolic antioxidants may be considered to support the use of steroidal compounds as active ingredients in topical pharmaceutical formulations [37]. The presence of both antifungal and anti-inflammatory activities of steroidal compounds raises the possibility that these may be used together with azole antifungal agents in a synergistic manner; evidence suggests that both fungal terpenoids and natural sterols can enhance the antifungal efficacy of azoles via disruption of fungal membrane integrity with enhancement of drug delivery through the fungal membrane [38]. On the contrary, further research is necessary to establish whether or not steroidal compounds from T. vulgaris and topical corticosteroids may interact antagonistically or additively to increase the risk of persistent fungal colonisation due to concurrent suppression of pro-inflammatory signals [39]. Therefore, future studies should include skin permeation studies, stability testing, and combination index analyses to define the appropriate treatment setting for T. vulgaris steroidal compounds pertaining to Malassezia-related inflammatory dermatoses.

Finally, these compounds are derived from nature, and natural products are generally considered to provide safer modes of treatment compared to synthetic drugs. A study highlighted the ability of natural compounds to reduce adverse effects associated with conventional treatment regimens [40].