Therapeutic potential of phytochemical luteolin in restoring skin barrier by inhibiting antimicrobial peptides associated with TRAF6/TAK1/IKK/IκB in psoriasis-like HaCaT models

Abstract

Autoimmune skin diseases, such as psoriasis are primarily characterized by excessive inflammation and skin barrier damage. The disrupted skin barrier is directly related to skin dryness. Luteolin, a flavonoid compound, has various biological activities such as antioxidant, anticancer, anti-aging, and anti-inflammatory. However, little has been known about the anti-psoriatic therapeutic function of luteolin. This study focused on the recovery of the skin barrier by luteolin through the regulation of antimicrobial peptides (AMPs) as well as anti-inflammatory activity, using TNF-α/IL-17A/IFN-γ-stimulated HaCaT cells. First, luteolin significantly inhibited the mRNA expression of antimicrobial peptides LL-37, human β-defensin-2 (hBD-2), S100A7, and S100A8 by regulating IκB/NF-κB signaling pathway through TRAF6/IκB. Moreover, luteolin increased the expression of filaggrin, loricrin and involucrin, which are essential of cornified envelope (CE) formation in epidermis. These results suggest anti-inflammatory luteolin has the function of restoring the disrupted skin barrier by regulating antimicrobial peptides and cornified envelope proteins.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10068-025-01894-z.

Introduction

Psoriasis is one of the intractable skin diseases related to autoimmune responses (Griffiths et al., 2021). Small DNA/RNA nucleic acid fragments produced from skin cells by exposure to ultraviolet (UV) rays, invasion of pathogens such as viruses and bacteria, and by other physical damages, can act as new auto-antigens in the human body, and auto-antibodies are produced against these, leading to persistent chronic inflammation as an autoimmune diseases (Ben Abdallah et al., 2021; Lande et al., 2007). Recently, a direct evidences between psoriasis and antimicrobial peptides (AMPs) have been reported (Büchau and Gallo, 2007; Takahashi and Yamasaki, 2020). AMPs, known to be secreted by leukocytes or epithelial cells to remove external pathogens as one of the innate immune responses, but are secreted excessively in psoriasis patients, maintaining high levels abnormally (Ma et al., 2020). Usually, in the presence of infection by viruses/bacteria, keratinocytes in the epidermis immediately release AMPs such as LL-37 and effectively control pathogens (Lai and Gallo, 2009; Lande et al., 2014). Then, AMPs directly induce the cell wall or membrane destruction of pathogens by creating pore (Ma et al., 2020). AMPs form complexes through binding with self-DNA or RNA fragments released from damaged or killed cells, which eventually become auto-antigens and activate dendritic cells (pDC) to differentiate into mDC (Gilliet and Lande, 2008; Lande et al., 2007). On the other side, abnormal and excessive secretion of AMPs repeatedly induces inflammatory responses in psoriasis, so it is very important to effectively control them from the perspective of psoriasis treatment (Peric et al., 2009; Takahashi and Yamasaki, 2020).

When inflammatory cytokines, such as TNF-α, IL-17 A, IL-22, and IFN-γ, secreted by differentiated Th1, Th2, and Th17 cells, are released into the dermis, they also have a significant impact on keratinocytes in the adjacent epidermal layer (Grän et al., 2020; Sieminska et al., 2024). IL-17 A is secreted by Th17 cells and binds to the receptor (IL-17 A receptor) in keratinocytes, thereby interfering with epidermal differentiation and promoting cell proliferation, thereby causing thickened epidermis (Brembilla et al., 2018). In addition, IL-17 A abnormally activates the innate immune system by excessively inducing the production of AMPs such as β-defensin (hBD-2), S100 A7 and LL37 (Takahashi and Yamasaki, 2020; Liang et al., 2006). IFN-γ, secreted from differentiated Th1 cells, is a type II interferon involved in both innate and adaptive immunity. It stimulates macrophages or dendritic cells to produce CCL20 chemokine and activates Th17 cells to promote their migration to the psoriasis environment. Co-stimulation with IFN-γ and IL-17 A induces overexpression of human β-defensin-2 (hBD-2) in keratinocytes, which is further increased by IFN-γ after being induced by IL-17 A (Kryczek et al., 2008; Schroder et al., 2004). TNF-α, IL-17 A, IFN-γ, and IL-22 cytokines stimulate keratinocytes in the epidermal layer to induce epidermal hyper-proliferation, hyper-secretion of antimicrobial peptides, and excessive inflammatory responses in a chain reaction, which not only causes psoriasis but also continuously repeats a vicious cycle (Zhou et al., 2022). This explains why psoriasis often relapses after treatment or is ultimately difficult to cure, despite the development of various treatments and therapies (Lee et al., 2024). Treatment of psoriasis involves the combined use of biological agents (monoclonal antibody, bio-drugs) along with topical medications such as steroids, retinoids, calcineurin inhibitors, and sunlight therapy (Sugumaran et al., 2024). Nevertheless, when treatment is discontinued, there are realistic limitation to treatment because they cause side effects such as long-term toxicity, decreased immunity, infection, and carcinogenesis along with relapse. In particular, it is important to develop complementary treatments for the deterioration of quality of life and mental stress due to long-term use (Lee and Kim, 2023). In this study, we focused on polyphenol compounds including phenolic acids, flavonoids, alkaloids, terpenoids derived from natural products (Lee et al., 2024; Le et al., 2024). These natural compounds have pharmacological properties such as anti-inflammatory, anti-proliferative, and antioxidant properties, and have been considered as candidates for inhibitors of key inflammatory signaling pathways in psoriasis (Fernandes et al., 2023). In particular, natural products are considered promising therapeutic strategies for the treatment of psoriasis because it has the potential to inhibit inflammatory signaling pathways and regulate the proliferation and differentiation of keratinocytes (Huang et al., 2019; Lee et al., 2024; Zhou et al., 2022). Luteolin, a flavonoid compound extracted from various plants such as carrots, broccoli, and celery, is a yellow pigment that has strong health benefits that far outweigh other phytochemicals, including antioxidant, anti-inflammatory, wound healing, cardioprotective, antidiabetic, and anticancer effects (Huang et al., 2023). Various studies have shown that luteolin effectively inhibits the expression of inflammatory cytokines such as TNF-α, IL-1β, IL-6, IL-8, and IL-17 in keratinocytes, fibroblasts, and various immune cell models. It has also been reported to regulate signaling molecules such as NF-κB, JAK–STAT, and TLR (Gendrisch et al., 2021; Huang et al., 2023;). Luteolin exhibited potent anti-inflammatory effects in a TNF-α-induced keratinocyte model, inhibiting the production of inflammatory mediators such as IL-6, IL-8, and VEGF (Weng et al., 2014). In addition, it has shown potential as a psoriasis treatment through its effects of inhibiting IFN-γ expression (Lv et al., 2020). However, little is known about whether luteolin has the potential to treat psoriasis by modulating antimicrobial peptides and improving the skin barrier, in addition to its anti-inflammatory activity. Ultimately, this study aimed to demonstrate that luteolin improves the skin barrier by regulating AMPs and inflammatory cytokine/chemokine in a HaCaT cell model stimulated with TNF-α, IL-17 A, and IFN-γ, which are major cytokines in psoriasis lesions, and to confirm its potential as a new candidate for the treatment of psoriasis.

Material and methods

Chemicals and reagents

Luteolin, brazilin, dimethylsulfoxide (DMSO), tumor necrosis factor-α (TNF-α), chloroform, isopropanol used in this experiment were purchased directly from sigma-aldrich (St. Louis, MO, USA). IL-17 A and IFN-γ were purchased from R&D systems (Minneapolis, MN, USA). DMEM (Dulbecco's modified eagle's medium), DPBS (Dulbecco's phosphate buffered saline), and FBS (Fetal bovine serum) were purchased from Welgene (Kyungsan, Kyungbuk, Korea), and the cell counting kit-8 (CCK-8) was purchased from Dogen Bio (Seoul, Korea). Penicillin–Streptomycin (10,000 U/mL) and 0.05% Trypsin–EDTA were purchased from Gibco (Grand Island, NY, USA). Luteolin was dissolved in DMSO, and a 20 mg/mL stock was prepared, diluted in FBS-free DMEM on the experiment of cell treatment.

Cell culture

HaCaT (human keratinocytes) cell was purchased from CLS (Cell lines service GmbH, Eppelheim, Baden-Wurttemberg, Germany). HaCaT were cultured in DMEM medium containing 10% inactivated FBS (Welgene, Gyeongbuk, Korea) and 1% penicillin-streptomysin (Gibco, Grand Island, NY, USA). They were maintained in an incubator at 5% CO₂ and 37 °C. The medium was replaced with fresh medium every day, and the cells were subcultured when they reached more than 70% confluent.

Cell viability assay

Cell cytotoxicity was observed using the CCK-8 (Dogen Bio, Seoul, Korea). HaCaT cells were seeded at 5.0 × 10⁴ cells/well in a 24-well plate containing culture medium supplemented with 10% FBS. After stabilization for 24 h, TNF-α/IL-17 A/IFN-ɤ (each 0.02 μg/mL) and luteolin (1, 3, 5, 7, 10, and 20 μg/mL) were simultaneously treated and cultured for 24 h. The medium was removed, and the CCK-8 reagent was diluted 1:10 ratio with phenol-red free medium and then reacted with the cells for 30 min. Afterwards, 100 μL of the supernatant was dispensed into a 96-well plate, and the absorbance was measured at 450 nm using a microplate spectrophotometer (BioTek, Winooski, VT, USA).

$$\text{Cell viability}\left(\%\right)=\left(\frac{{\text{OD}}_{sampl{e}^a}-{\text{OD}}_{{\text{blank}}^{\text{b}}}}{{\text{OD}}_{sampl{e}^c}-{\text{OD}}_{{\text{blank}}^{\text{b}}}}\right)$$

a: Absorbance of the group treated with sample.

b: Absorbance of CCK-8 solution.

c: Absorbance of the control group.

RNA preparation and RT-qPCR

HaCaT keratinocytes were seeded at 4.0 × 105 cells/well in 6-well plates and cultured for 24 h for stablization. Then, TNF-α/IL-17 A/IFN-ɤ (0.02 μg/mL each) and 1, 3, and 5 μg/mL luteolin were co-treated and incubated for 24 h. After treatment, the cells were washed with DPBS buffer. Total RNA was extracted from cells using TRIzol Reagent (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions. Then, 200 μL of chloroform was added, mixed using a vortex, and centrifuged at 14,000 rpm for 15 min at 4 °C. The supernatant (about 380 μL) of the separated mixture was separated, mixed with an equal volume (about 380 μL) of isopropanol, and left to stand for 10 min at 4 °C to precipitate the RNA. The mixture was centrifuged again at 14,000 rpm for 15 min at 4 °C to obtain an RNA pellet, which was washed with 1 mL of cold 75% EtOH. After centrifugation under the same conditions (14,000 rpm, 15 min, 4 °C), 75% EtOH was removed and the pellet was dried at room temperature. Finally, total RNA was obtained by dissolving it in DEPC-treated RNase-free water. Reverse transcription was performed using the Revertra Ace-α-® kit (TOYOBO, Osaka, Japan) according to the manufacturer's protocol. Total RNA (1.5 μg/mL), DEPC-water, 5 × RT Buffer, dNTPs mixture, Oligo (dT)20, RNase inhibitor, and ReverTra Ace were added to a PCR tube, and cDNA was synthesized under the conditions of 42 °C for 20 min, 99 °C for 5 min, and 4 °C for 5 min. The synthesized cDNA (20 μL) was diluted with 80 μL of 1 × Tris/EDTA buffer (Sigma Aldrich, St. Louis, MO, USA). The StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA, USA) was used to amplify specific genes. Each TaqMan probe (Applied Biosystems, Foster City, CA, USA), TaqMan™ Universal Master Mix II, with UNG (Applied Biosystems, Foster City, CA, USA), DEPC-water, and synthesized cDNA were sequentially dispensed into MicroAmp™ Fast Reaction 8-Tubes Strip (Applied Biosystems, Foster City, CA, USA). PCR conditions were as follows. After the UNG activation step, polymerase activation was performed at 50 °C for 2 min, and an initial denaturation process was performed at 95 °C for 10 min. Thereafter, the PCR cycle including denaturation process at 95 °C for 15 s and annealing process at 60 °C for 1 min was repeated a total of 45 times. All samples were repeated three times in total. The TaqMan probes information used in this experiment were presented separately in Table 1.

Table 1.

Gene symbol, name, and assay ID of TaqMan probes used in RT-qPCR analysis

Symbol	Gene name	Assay ID
GAPDH	Glyceraldehyde-3-phosphate dehydrogenase	Hs02786624_g1
IL-1β	Interleukin 1 beta	Hs01555410_m1
IL-6	Interleukin 6	Hs00174131_m1
CXCL8	Chemokine (C–X–C motif) ligand 8	Hs00174103_m1
CCL20	Chemokine (C–C motif) ligand 20	Hs01011368_m1
CAMP	Cathelicidin antimicrobial peptide (LL-37)	Hs00189038_m1
DEFB4 A	Defensin beta 4 A (hBD-2)	Hs00175474_m1
S100 A7	S100 calcium binding protein A7	Hs00161488_m1
S100 A8	S100 calcium binding protein A8	Hs00374263_m1
FLG	Filaggrin	Hs00856927_g1
LOR	Loricrin	Hs01894962_s1
IVL	Involucrin	Hs00846307_s1

Western blotting

HaCaT cells were seeded at 6.0 × 10⁵ or 7.0 × 10⁵ cells/well in a 6-well plate and cultured for 24 h. TNF-α/IL-17 A/IFN-ɤ (0.02 μg/mL each) and 1, 3, and 5 μg/mL Luteolin were simultaneously treated and cultured for 24 h. HaCaT cells were washed twice with DPBS at 4 °C, and 200 μL of RIPA lysis buffer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) containing Halt™ Protease and Phosphatase Inhibitor Cocktail (100X) (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and 0.5 M EDTA Solution (100X) (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was added, and the cells were lysed using a shaker for 15 min. Cells were collected using a Cell Scraper and centrifuged at 14,000 rpm and 4 °C for 15 min. The precipitated cell debris in the extracted cell lysate was removed, and only the cell lysate containing proteins was collected. The extracted proteins were mixed with diluted 4 × Laemmli Sample Buffer (62.5 mM Tris–HCl, pH 6.8, 10% glycerol, 1% LDS, 0.005% Bromophenol Blue) (Bio-Rad Laboratories, Hercules, CA, USA) and 10% β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA), followed by denaturation at 95 °C for 5 min. The proteins were loaded onto a 10% SDS-PAGE Gel. The proteins were separated by size through an electrophoresis process under the conditions of 110 V and 100 min. Trans-Blot® Turbo™ Transfer System (Bio-Rad, California, USA) was used to transfer to PVDF membrane (polyvinylidene fluoride, Invitrogen, Waltham, Massachusetts, USA) and blocked with 5% skim milk for 1 h at room temperature. The membrane was washed at least 4 times for 5 min with 1 × TBST containing 0.1% Tween-20, and then reacted with primary antibody overnight at 4 °C. After washing with 1 × TBST, secondary antibody was treated and stirred and reacted at room temperature for 1 h. After washing 3 times for 10 min with 1 × TBST, ECL (FastGene, Western ECL kit) was dispensed onto PVDF membrane, and target proteins were detected and visualized using an image processing device (Microchemi-DNR, Neve Yamin, Israel). Protein quantitative analysis was performed using Image J (N.I.H, Bethesda, MD, USA). The antibodies used in this experiment are presented in Table 2.

Table 2.

Detailed antibody information for western blot analysis

Symbol	Protein name	Catalog No	Dilution
GAPDH	Glyceraldehyde 3 phosphate dehydrogenase	MA5-15,738a	1:1000
IκBα	Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha	4812b	1:1000
p-IκBα	Phospho-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha	2859b	1:1000
IKKα/β	IκB kinase, alpha/beta	MA5-32,274a	1:1000
p-IKKα/β	Phospho-IκB kinase, alpha/beta	2697b	1:1000
TAK1	Transforming growth factor beta-activated kinase 1	5206b	1:1000
TRAF6	TNF receptor associated factor 6	702,286a	1:500
Filaggrin	Filament aggregating protein	PA5-115,235a	1:1000
Secondary Antibody	Goat anti-Mouse IgG (H + L) Secondary Antibody, HRP	31,430a	1:1000
	Goat anti-Rabbit IgG (H + L) Secondary Antibody, HRP	31,460a	1:1000

^aInvitrogen™

^bCell Signaling Technology™

Immunocytochemistry staining

HaCaT cells were seeded at 4.0 × 10⁵ cells/well on coverslips in 6-well plates and stabilized for 24 h. Then, the cells were simultaneously treated with TNF-α/IL-17 A/IFN-ɤ (0.02 μg/mL each) and luteolin (1, 3, 5 μg/mL) and cultured for 24 h. After stimulation, the cells were fixed with methanol (Honeywell, Charlotte, NC, USA) at −20 °C for 10 min and washed three times with cold DPBS. The cells were incubated with DPBS containing 0.1% Triton X-100 for 10 min at room temperature and then washed three times with DPBS. To block nonspecific binding sites, 3% bovine serum albumin (BSA; Rockland Immunochemicals, Inc., Limerick, PA, USA) was used for blocking at room temperature for 1 h. Primary antibodies against NFκB p65 (Cat. # PA5-16,545, Invitrogen, Waltham, MA, USA) was added and incubated overnight at 4 °C. After washing the cells three times with DPBS, AlexaFluor 594 conjugated goat anti-rabbit IgG (Invitrogen, Waltham, MA, USA) secondary antibody was added and reacted at room temperature for 1 h. After washing three times with DPBS, the cells were stained with DAPI (Sigma-Aldrich, St. Louis, MO, USA) for 10 min. Fluorescence images were visualized using a Nikon Eclipse Ti-S/L100 microscope (Nikon, Tokyo, Japan). Images were further processed using NIS-Elements software (Version 4.0, Nikon, Tokyo, Japan) and ImageJ software.

Trans-epithelial electrical resistance (TEER) assay

To evaluate trans-epithelial electrical resistance (TEER), HaCaT cells (2.0 × 10⁴ cells/well) were seeded into 0.4 µm insert wells of a 12-well plate and allowed to stabilize for 24 h. Following stabilization, the cells were treated with TNF-α, IL-17 A, and IFN-γ (20 ng/mL each) in combination with luteolin at concentrations of 1, 3, and 5 μg/mL in serum-free medium. TEER measurements were conducted at 0, 6, 12, and 24 h using an EVOM™ Epithelial Volt/Ohm Meter 3 (World Precision Instruments, Sarasota, FL, USA). Final TEER values were calculated by multiplying the measured resistance (Ω) by the insert surface area (1.13 cm²), with results expressed in Ω·cm². Prior to measurement, STX4 electrodes (World Precision Instruments, Sarasota, FL, USA) were sterilized with 75% ethanol and equilibrated in serum-free medium for 5 min. All experiments were performed in triplicate, and the recorded data were statistically analyzed.

Statistical processing

All experiments were repeated more than three times, and the results (mean and standard deviation) obtained through Student's t-test were analyzed. If the p value was less than 0.05, the experimental results were considered statistically significant.

Results and discussion

RNA Seq analysis in psoriasis like HaCaT keratinocyte model showed specific increase in antimicrobial peptides (AMPs) expression

Psoriasis is one of the intractable skin diseases caused by autoimmune disorders. We performed mechanistic studies at the cellular and molecular levels using a cell model optimized for psoriasis research. To validate this model, we observed changes in mRNA expression levels of psoriasis marker genes using RNA-Seq analysis (Macrogen, Seoul, Korea). Heatmap analysis of changes in mRNA expression levels of key genes is shown in complementary data. For the psoriasis-like cell model, HaCaT cells were stimulated with three major cytokines, such as TNF-α/IL-17 A/INF-γ, and then compared with normal cells. Compared with the normal control, HaCaT cells stimulated with TNF-α/IL-17 A/INF-γ showed increased expression of inflammatory cytokines (IL-6, IL-23 A, TNF-α), inflammatory chemokines (CXCL8, CCL2, CCL20), and AMPs (DEFB4 A, S100 A7). However, it was observed that cornified envelope (CE) genes related to the skin barrier, such as filaggrin, ivolucrin were decreased significantly. From above results, it was confirmed that the expression of AMPs and inflammatory cytokine genes significantly increased compared to the normal group in the condition of psoriasis, but skin barrier related CE genes were actually decreased.

Luteolin significantly suppressed the expression of inflammatory cytokines and chemokines mRNA in a psoriasis-like cell model

Luteolin is a yellow flavonoid compound found in the leaves of various plants, including chamomile, celery, rosemary, and peppermint, and has a 3′,4′,5,7-tetrahydroxyflavone structure (Fig. 1A). First, the cytotoxic concentration of luteolin was determined in HaCaT cells by performing a CCK-8 assay. First, luteolin was co-treated with TNF-α/IL-17 A/IFN-γ (0.02 μg/mL each) at concentrations up to 20 μg/mL in a HaCaT psoriasis-like model. As a result, no toxicity was observed at up to 10 μg/mL, but cell viability was not observed at the 20 μg/mL condition. Therefore, the maximum concentration for HaCaT cell treatment was set to 5 μg/mL, which is relatively safe considering toxicity, and was applied to all future experiments (Fig. 1B). Here, brazilin (3 μg/mL), a polyphenol compound, was used as a positive control (Choi and Hwang, 2019). Second, RT-PCR was performed to observe the expression level of IL-1β mRNA, an inflammatory cytokine, in a HaCaT psoriasis-like model stimulated with TNF-α/IL-17 A/IFN-γ. As a result, the TNF-α/IL-17 A/IFN-γ treatment group showed a 4.22 ± 0.22-fold increase compared to the untreated group, and decreased to 2.30 ± 0.12-fold when brazilin was used as the positive control group. In the luteolin treatment group, the decrease was 2.53 ± 0.12, 2.80 ± 0.12, and 2.55 ± 0.12-fold at concentrations of 1, 3, and 5 μg/mL. Therefore, luteolin inhibited by 40.10 ± 2.32%, 33.69 ± 2.32%, and 39.69 ± 1.67% compared to the TNF-α/IL-17 A/IFN-γ treatment group, respectively (Fig. 1C). Next, the mRNA expression of IL-6, a cytokine that induces psoriasis, was observed to increase 5.70 ± 0.71-fold in the TNF-α/IL-17 A/IFN-γ treatment group, and the expression was suppressed 1.52 ± 0.23, 1.13 ± 0.09, and 0.92 ± 0.05-fold at concentrations of 1, 3, and 5 ug/mL, respectively, showing that luteolin also suppressed IL-6 expression (Fig. 1D). In addition, the anti-inflammatory activity of luteolin was similarly observed in the regulation of chemokines that induce psoriasis (Fig. 1E, F). In HaCaT cells stimulated with TNF-α/IL-17 A/IFN-ɤ, the mRNA expressions of CXCL8 and CCL20 were increased by 18.59 ± 1.51 folds and 34.77 ± 2.81 folds, respectively, compared to the untreated group. The mRNA expression of CXCL8 was down-regulated by 6.41 ± 1.35, 4.21 ± 0.29, and 4.75 ± 0.84-fold in the luteolin 1, 3, and 5 μg/mL compared to the TNF-α/IL-17 A/IFN-ɤ treatment group (Fig. 1E), and the mRNA expression of CCL20 was also decreased by 8.28 ± 0.55, 4.28 ± 0.23, and 4.44 ± 0.46-fold, respectively (Fig. 1F).

Fig. 1.

Luteolin significantly inhibited the mRNA expression of psoriasis related anti-inflammatory cytokines and chemokines in TNF-α, IL-17 A, IFN-γ induced HaCaT cells. (A) Chemical structure of luteolin, a flavonoid family. (B) CCK-8 assay was performed to evaluate the cytotoxic concentration of luteolin. HaCaT cells were treated with (0.02 μg/mL each) and luteolin (1, 3, 5, 7, 10, and 20 μg/mL). The cytotoxicity of luteolin was observed from a concentration of 20 μg/mL, but the maximum concentration was set at 5 μg/mL to compare with brazilin (3 μg/mL) as positive control. (C, D) After stimulation of HaCaT cells with TNF-α/IL-17 A/IFN-ɤ (0.02 μg/mL each), the mRNA expression of psoriasis-inducing cytokines (IL-1β, IL-6) were observed using RT-qPCR, and significant inhibition of IL-1β and IL-6 were observed by luteolin. In addition, psoriasis-inducing chemokines (E) CXCL8 and (F) CCL20 were observed to be dose-dependently inhibited by luteolin

Luteolin significantly inhibited the mRNA expression of antimicrobial peptides (AMPs) such as LL-37, Human β-defensin-2, S100 A7, and S100 A8 through IKK/IκB/NFκB pathway

RT-PCR was performed to observe the mRNA expression level of the antimicrobial peptide LL-37 in a HaCaT psoriasis-like model stimulated with TNF-α/IL-17 A/IFN-γ. As a result, the TNF-α/IL-17 A/IFN-γ treatment group showed a 2.70 ± 0.45-fold increase compared to the untreated group, and brazilin-treated group as used the positive control, showed no significant change with a 2.56 ± 0.44-fold change. However, LL-37 was significantly reduced by 3.25, 1.50, and 1.18-fold at 1, 3, and 5 μg/mL luteolin concentrations, respectively, and significance was observed only at 5 ug/mL (Fig. 2A). Next, we observed the mRNA expression of human hBD-2, another antimicrobial peptide known as a psoriasis-inducing antimicrobial peptide, and confirmed that luteolin significantly inhibited it in a concentration-dependent manner (Fig. 2B). In HaCaT cells stimulated with TNF-α/IL-17 A/IFN-γ, the hBD-2 mRNA level increased 21.21 ± 2.53-fold, and decreased 3.19 ± 0.38-fold in the presence of brazilin, suggesting that there were no problems in all experimental conditions. When treated with 1, 3, and 5 ug/mL of luteolin, the hBD-2 mRNA expression levels significantly decreased by 18.41 ± 1.34, 5.70 ± 0.46, and 3.87 ± 0.39-fold, respectively. The efficacy of luteolin was similarly observed for S100 A7 (psoriasin), an antimicrobial peptide that causes psoriasis. The mRNA expression of the S100 A7 gene was increased by 22.46 ± 2.41-fold, and when luteolin was treated at concentrations of 1, 3, and 5 μg/mL, the mRNA expression was observed to be 12.50 ± 0.51, 3.35 ± 0.22, and 0.02, respectively (Fig. 2C). Finally, luteolin was also found to significantly inhibit the S100 calcium-binding protein A8 (S100 A8) gene (Fig. 2D). In summary, the above results suggest that luteolin, a polyphenol-type flavonoid compound, significantly inhibited the expression of AMPs in a HaCaT cell model similar to psoriasis under non-cytotoxic conditions. In fact, the excessive expression of AMPs have been confirmed in the skin of psoriasis patients through actual clinical studies. Therefore, the regulatory function of antimicrobial peptides such as LL-37 by luteolin can be seen as a very meaningful result in terms of therapeutic potential as development of luteolin as a psoriasis-treat biomaterial. The expression mechanism of AMPs is related to the NF-κB pathway, an inflammatory signaling pathway. NF-κB or AP1 binds to the DEFB4 promoter and regulates the expression of hBD2, which is directly involved in the progression of the psoriatic inflammatory response (Johansen et al., 2016; Scheenstra et al., 2020). Therefore, in this study, Western blot was performed to confirm the antimicrobial peptide inhibitory signaling pathway by luteolin at the cellular and molecular levels. First, we confirmed whether luteolin was involved in the phosphorylation of IκB protein, an NF-κB inhibitor. Phosphorylation of IκB protein was increased by TNF-α/IL-17 A/IFN-γ stimulation and significantly inhibited by brazilin, and luteolin not only reduced phosphorylation but also promoted the degradation of IκB protein (Fig. 2E). Second, we observed IKK protein, which phosphorylates IκB, and confirmed that IKK phosphorylation was also significantly inhibited by luteolin (Fig. 2G). These results suggest that luteolin regulates IκB/NF-κB by controlling intracellular IKK protein phosphorylation. Therefore, it appears that the antimicrobial peptide-regulated signaling pathway by luteolin directly regulates the NF-κB signaling pathway via IKK.

Fig. 2.

Luteolin significantly inhibited the mRNA expression of antimicrobial peptides (AMPs) such as LL-37, Human β-defensin-2, S100 A7, and S100 A8 throughout IκB/NFκB/IKK signaling pathway. (A) To confirm the function of luteolin, RT-PCR was used focusing on the mRNA expression level of LL-37, a representative antimicrobial peptide. In addition, luteolin (5 μg/mL) significantly inhibited the mRNA expression of other AMPs, (B) hBD-2, (C) S100 A7, and (D) S100 A8 in the presence of TNF-α/IL-17 A/IFN-ɤ. (E, F) AMPs expression is known to be regulated by the NF-kB pathway. Therefore, the phosphorylation regulatory function of IkB and IKK, which are NF-kB pathway proteins that regulate antimicrobial peptide expression, was observed through Western blot, and it was confirmed phosphorylational regulation was significantly inhibited by luteolin as concentration dependent manner

Luteolin affects NF-κB nuclear translocation by regulating TAK1/TRAF6 located upstream of NF-kB

We aimed to confirm the subcellular localization of NF-κB protein by luteolin under TNF-α/IL-17 A/IFN-γ signaling conditions through immunostaining assay. We found that most of the NF-κB transcription factors were translocated from the cytoplasm to the nucleus under the condition of treating HaCaT cells with TNF-α/IL-17 A/IFN-γ. However, in the presence of brazilin, a positive control, most of the NF-κB transcription factors remained in the cytoplasm. As expected, we confirmed that luteolin also effectively blocks NF-κB activation by inhibiting the translocation of NF-κB (Fig. 3A). Moreover, luteolin also regulates TAK1 and TRAF6 proteins, which are IL-17 A signaling pathway proteins (Fig. 3B, D). Here, luteolin negatively regulates TAK1 and TRAF6 protein, which is located upstream of IKK. Thus, this suggests that luteolin has role in the antimicrobial peptide gene expression derived from the effective suppression of both upstream and downstream in TNF-α/IL-17 A/IFN-γ stimulation signals. Finally, we also observed changes in TRAF6, which were significantly suppressed by luteolin. As is known, TRAF6 is an IL-17 A signaling protein and is also linked to TAK1. Therefore, luteolin has a high potential to be developed as an anti-psoriatic agent because it effectively suppresses not only the inflammatory signal of TNF-α but also the major IL-17 A signaling protein. Taken together, these results suggest that luteolin can effectively regulate the expression of antimicrobial peptides by directly regulating the transcription factor NF-kB.

Fig. 3.

Luteolin affects NFκB nuclear translocation by regulating TAK1/TRAF6 located upstream of NFkB. (A) Immunocytochemistry was performed to observe that luteolin inhibits the nuclear translocation of NF-κB p65. NF-κB p65 was visualized as red fluorescence, and nuclei were visualized as blue fluorescence using DAPI. (B, C) Phosphorylation of TAK1, a key upstream pathway, was assessed by Western blotting in HaCaT cells stimulated with TNF-α/IL-17 A/IFN-γ. TAK1 protein, located upstream of IKK, was slightly increased by TNF-α/IL-17 A/IFN-γ stimulation and markedly decreased by luteolin. This suggests that luteolin is involved in regulating TAK1 protein expression. (D, E) TRAF6 protein was also significantly downregulated by luteolin. From the above results, we could see that luteolin regulates the NFkB pathway by regulating the IKK/ikB/NFkB pathway through TRAF6/TAK1 regulation. Protein phosphorylation levels were quantified using ImageJ software, and the relative expression levels of each protein were analyzed compared with GAPDH

Luteolin increased the expression of filaggrin, loricrin, involucrin, which are essential of cornified envelope (CE) formation in epidermis

The main pathogenesis of psoriasis is damage to the skin barrier due to excessive inflammation, increased antimicrobial peptides, and decreased cornified envelop (CE) proteins that form the stratum corneum. CE include filaggrin, involucrin, loricrin, and keratin proteins. They play an important role in the skin barrier of the stratum corneum. Recently, it has been reported that filaggrin and loricrin are decreased in keratinocytes stimulated by TNF-α and IFN-γ. Therefore, from the perspective of psoriasis treatment, it is very important to increase the expression level of CE components and improve the skin barrier. Therefore, we observed changes in the expression level of CE component gene mRNA in TNF-α/IL-17 A/IFN-γ-stimulated HaCaT cells. As a result, luteolin significantly increased the filaggrin gene (Fig. 4A), and the expression levels of loricrin and involucrin were also significantly increased (Fig. 4B, C). In addition, when we observed the protein expression level of filaggrin by Western blot, we found that the protein expression level increased in a concentration-dependent manner, similar to the mRNA results (Fig. 4D). Here, we identified a novel regulatory function of luteolin, as restoring the expression of filaggrin, loricrin, and involucrin essential proteins for CE formation—is considered an important strategy for treating psoriasis. In this study, we found that the expression of filaggrin, loricrin, and involucrin was significantly reduced in HaCaT cells stimulated with TNF-α/IL-17 A/IFN-γ. Luteolin treatment, however, significantly increased the gene expression of these proteins, suggesting that luteolin may be highly effective in restoring damaged skin barrier function in psoriasis lesions.

Fig. 4.

Luteolin affects skin barrier function through overexpression of filaggrin, loricrin, and involucrin, which are essential for the formation of the keratinocyte enhanced layer (CE) of the epidermis. The skin barrier recovery by luteolin was confirmed in HaCaT cells induced with TNF-α/IL-17 A/IFN-ɤ, which is similar to psoriasis. As a result, (A) filaggrin, (B) loricrin, and (C) involucrin mRNA expression levels were down-regulated under TNF-α/IL-17 A/IFN-ɤ stimulation conditions, but significantly up-regulated by luteolin. This suggests that the skin barrier can be restored by increasing CE protein expression by luteolin. (D) In ​​addition, when we observed filaggrin protein expression by luteolin through Western blot, filaggrin, which was reduced in a psoriasis-like environment, was significantly up-regulated by luteolin in a dose-dependent manner. (E) Skin barrier function of luteolin through TEER analysis. In the TNF-α/IL-17 A/IFN-γ treatment group, the TEER value significantly decreased, but when treated with 3 μg/mL and 5 μg/mL of luteolin, the TEER resistance value increased, indicating the restoring of skin barrier. (F) Schematic diagram of the psoriasis improvement of luteolin through anti-inflammatory, AMPs inhibition, and CE protein induction in human epidermal keratinocytes

TEER assay indicates luteolin restores the skin barrier weakened by excessive inflammatory response

To evaluate the skin barrier recovery of luteolin, TEER (Trans-epithelial electrical resistance) analysis was performed. In the TNF-α/IL-17 A/IFN-γ treatment group, TEER values ​​decreased continuously from 0 to 24 h, indicating that the skin barrier was damaged due to inflammation. In the positive control brazilin-treatment group, TEER values was increased after 8 h, indicating that the skin barrier was recovered. Finally, in the test groups treated with luteolin at each concentration (1, 3, and 5 μg/mL), TEER values was increased similarly to brazilin after 8 h. The skin barrier improvement effect of luteolin was confirmed by TEER analysis (Fig. 4E). In summary, the skin barrier regulation function of luteolin was proven by increasing the expression level of filaggrin protein, a skin barrier component protein, inducing an increase in the expression level of another cornified envelope protein, and finally increasing the TEER resistance value.

Finally, this study focused on the inflammation-modulating mechanism of another polyphenolic compound, luteolin, and examined its various psoriasis-modulating activities. Specifically, we demonstrated that luteolin can contribute to regulating AMPs and promote skin barrier recovery. Our findings showed that luteolin strongly inhibits the expression of AMPs (LL-37, hBD2, S100 A7, and S100 A8), which are overexpressed in psoriasis. Additionally, to confirm the regulation of the NF-κB signaling pathway, we analyzed the expression of IκB, IKK, TAK1, and TRAF6 proteins. It was found that luteolin effectively regulates IκB and the upstream signaling pathway to suppress the expression of inflammatory cytokines and AMPs. Moreover, we identified a novel regulatory function of luteolin, as restoring the expression of filaggrin, loricrin, and involucrin essential proteins for CE formation—is considered an important strategy for treating psoriasis. In this study, we found that the expression of filaggrin, loricrin, and involucrin was significantly reduced in HaCaT cells stimulated with TNF-α/IL-17 A/IFN-γ. In conclusion, this study has clarified the psoriasis control efficacy and potential of luteolin as a therapeutic candidate at the cellular level (Fig. 4F). Based on these findings, we expect to see more meaningful results in future drug development research. Previous studies have reported that activator protein 1 (AP-1) and c-JUN bind to histone deacetylase 1 (HDAC1) in keratinocytes stimulated with TNF-α and IFN-γ, forming a complex that inhibits filaggrin (FLG) promoter activity (Ahn et al., 2022). This mechanism has been proposed as a key factor in the decreased expression of filaggrin in psoriasis. It is possible that luteolin regulates this pathway to restore filaggrin expression. Therefore, further studies to explore this pathway seem necessary. Second, since this study was conducted using a cell model (HaCaT), additional research using animal models is needed to confirm the antipsoriatic effects of luteolin more clearly. Luteolin has the disadvantage of low absorption and limited bioavailability due to its lipophilic properties. Therefore, studies using Imiquimod (IMQ)-induced psoriasis mouse models are needed to improve luteolin's absorption and stability using drug delivery systems such as liposomes, nanocapsules, and microemulsions (Taheri et al., 2021; Wu et al., 2018; Miyashita et al., 2022). If the mechanism of action of luteolin is more clearly elucidated through future animal and clinical studies, it is expected to be developed as a new therapeutic candidate for psoriasis.

Supplementary Information

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Author contributions

Data preparation, data collection and analysis, and initial manuscript writing were led by Hui Su Chung. Formal analysis was performed by Hui Su Chung. Design of conceptualization, supervision, paper writing, editing and reviewing were performed by Hyung Seo Hwang. All authors contributed to the study design and approved the final manuscript.

Data availability

All data in the study are included in this published article.

Declarations

Conflict of interest

The authors have no competing interests to declare that are relevant to the content of this article. All authors have approved the manuscript and agree with its submission.

Ethical approval

This study does not include any experiments involving animals and human participants.