A tryptophol-containing emulgel ameliorates imiquimod-induced psoriasis in mice by decreasing the expression of JAK/STAT pathway members

Arnon Pudgerd 1

Kamonwan Jongsomchai 1

Watcharamat Muangkaew 2

Laorrat Phuapittayalert 1

Sataporn Jamsuwan 1

Teera Chanmanee 1

Rapeepun Vanichviriyakit 3

Passanesh Sukphopetch 2✉ Emailnatthanej.lup@mahidol.ac.th

1 Division of Anatomy, School of Medical Sciences University of Phayao 56000 Phayao Thailand

2 Department of Microbiology and Immunology, Faculty of Tropical Medicine Mahidol University 10400 Bangkok Thailand

3 Department of Anatomy, Faculty of Science Mahidol University Rama VI Rd 10400 Bangkok Thailand

4 Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science Mahidol University Rama VI Rd 10400 Bangkok Thailand

Arnon Pudgerd¹

Kamonwan Jongsomchai¹

Watcharamat Muangkaew ²

Laorrat Phuapittayalert¹

Sataporn Jamsuwan¹

Teera Chanmanee ¹

Rapeepun Vanichviriyakit^3,4

Passanesh Sukphopetch^2,*

¹ Division of Anatomy, School of Medical Sciences, University of Phayao, Phayao, Thailand 56000

² Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand 10400

³ Department of Anatomy, Faculty of Science, Mahidol University, Rama VI Rd, Bangkok 10400, Thailand

⁴ Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, Rama VI Rd, Bangkok, Thailand 10400

Corresponding author: natthanej.lup@mahidol.ac.th

Abstract

Psoriasis is a chronic skin inflammatory disease that involves the IL-23/IL-17 axis. The disease is characterized by severe inflammation, prominent epidermal hyperplasia, scaling and erythema. Tryptophol (TOH) is a quorum-sensing molecule that has anti-inflammatory properties. In this study, we demonstrated the ability of a TOH-containing emulgel to alleviate imiquimod (IMQ)-induced psoriasis in mice. The results revealed that the TOH-containing emulgel decreased the pathological score of the psoriasis area and severity index (PASI). Together with the suppression of keratinocyte proliferation and differentiation, the TOH-containing emulgel alleviated hyperkeratosis and downregulated the expression of K10, K6, K16, K17 and involucrin. Moreover, reduced mast cell infiltration was observed, along with the downregulation of ST2 and IL-33 expression in the mouse skin lesions. CD4⁺ cell infiltration was reduced, and decreased levels of IL-17A and IL-23 in both the serum and psoriatic skin lesions were associated with reduced activation of the JAK2/STAT3 signalling pathway. Additionally, the TOH-containing emulgel suppressed systemic inflammation in the axillary lymph nodes; alleviated IMQ-induced splenomegaly; downregulated the expression of TNFα, IL-1 and IL-6 in the splenic and lymph node tissues; and reduced the serum levels of TNFα and IL-1β. This study revealed that a TOH-containing emulgel attenuates IMQ-induced inflammation in psoriatic mice and presents a novel therapeutic approach for this condition.

Keywords:

Quorum-sensing Molecule

Tryptophol

Psoriasis

Inflammation

Emulgel

Introduction

Psoriasis is a chronic inflammatory skin disease resulting from an uncontrolled immune response that causes keratinocyte hyperproliferation and the recruitment of inflammatory cells, including CD4 + T cells and mast cells, to the skin^{1 2,3,4,5,6}. Although the molecular mechanisms driving the pathogenesis of psoriasis are unclear, several studies have proposed that T helper 17 (Th17) cells play a critical role in the development of psoriasis because they are the main source of the cytokine IL-17^3,7,8. Th17 cells differentiate from naïve CD4⁺ cells primarily through the activation of IL-23 in the JAK2/STAT3 signalling pathway^3,7,9. Thus, the IL-23/IL-17 axis is essential in psoriasis¹⁰.

During the onset of psoriasis, the proinflammatory cytokines TNFα and IL-1β affect keratinocyte proliferation and differentiation^11,12. They decrease the expression of keratin (K)10^13,14 and increase the expression of involucrin^15,16 as well as K6, K16 and K17^11,17. The upregulation of these genes reflects keratinocyte activation and hyperproliferation ¹⁸, which results in the development of histopathological features such as epidermal thickening and parakeratosis^13,16. Moreover, the response of keratinocytes to IL-17 increases the expression of antimicrobial peptides, including S100A8 and S100A9, which are hallmarks of psoriasis^8,19.

Although treatments for psoriasis, including methotrexate, cyclosporine, retinoic acid, vitamin D3 derivatives, and biologic agents such as alefacept, have been widely applied, a complete cure for psoriasis remains elusive^1,20. Furthermore, certain medications, such as cyclosporine and retinoic acid, are associated with adverse side effects¹. Consequently, combination therapies integrating traditional treatments and biologic agents have been explored as alternative options for managing psoriasis. However, these approaches may pose challenges, including cumulative toxicity and compromised safety profiles²¹. Thus, the development of new treatments for psoriasis has become a focus of interest.

Quorum sensing is a process that enables microbes to communicate through hormone-like molecules and regulates vital processes such as biofilm formation, virulence, and metabolic adaptation, highlighting its importance in microbial behaviour and therapeutic research²². Tryptophol (indol-3-ethanol, TOH) is a quorum-sensing molecule isolated from bacteria and fungi. Tryptophol is a metabolite of tryptophan and tryptamine²³. It exhibits antimicrobial activity against the food contaminant Campylobacter jejuni²³. TOH also inhibits the growth of Candida albicans and Scedosporium apiospermum²⁴. In aquaculture, TOH increases the survival rate of Marsupenaeus japonicus after white spot syndrome virus infection by inhibiting viral replication²⁵. Low concentrations of TOH have little effect on normal lymphocytes but induce extensive apoptosis and inhibit the proliferation of leukaemia U937 cells through the activation of protease enzymes (caspase-8 and 3), leading to the cleavage of poly (ADP‒ribose) polymerase^26,27. Additionally, TOH reduces excessive inflammation and inhibits the effects of TNFα²⁸. Although TOH has potential therapeutic effects, it is typically dissolved in organic solvents such as ethanol, and repeated topical application can induce skin irritation and contact dermatitis. Therefore, an TOH-containing emulgel was formulated to avoid these adverse effects and effectively deliver TOH to the skin, where it can be absorbed. The TOH-containing emulgel did not cause skin irritation, increase skin hydration or reduce transepidermal water loss²⁴. Therefore, this study aimed to investigate the potential effect of this TOH-containing emulgel on IMQ-induced psoriasis in mice. The results revealed that the TOH-containing emulgel reduced skin inflammation, providing evidence for its potential as a future strategy to treat psoriasis.

Results

The TOH-containing emulgel ameliorated psoriasis-like symptoms in IMQ-induced mice

To investigate the potential effect of TOH-containing emulgel on psoriasis, an animal model was established by applying IMQ to BALB/c mice for 7 consecutive days. Compared with those in the cream base and tryptophol groups, the IMQ + Emulgel group presented typical symptoms of psoriasis, such as erythema, scaling and skin thickening (Fig. 1a). Compared with IMQ + Emulgel, the TOH-containing emulgel alleviated the symptoms of IMQ-induced psoriasis (Fig. 1a), reduced the psoriasis area and severity index (PASI) score in the mice (Fig. 1b-d) and resulted in a lower cumulative score (Fig. 1e). Although IMQ caused weight loss in psoriatic mice from the 2nd to the 7th days, the weight loss in the IMQ + Tryptophol group was not significantly different from that in the cream base and tryptophol groups. Moreover, the IMQ + Tryptophol group gained significantly more weight than the IMQ + Emulgel group on days 2 (-0.67 ± 0.57 versus − 1.80 ± 0.41, p < 0.01), 3 (-0.762 ± 0.92 versus − 4.12 ± 0.55, p < 0.001) and 4 (-0.62 ± 1.03 versus − 4.55 ± 1.07, p < 0.001). Although there was no significant difference in body weight from days 5 to 7, the mice in the IMQ + Tryptophol group had greater body weights than those in the IMQ + Emulgel group did (Fig. 1f).

Fig. 1

TOH-containing emulgel ameliorates psoriasis symptoms in IMQ-induced psoriatic mice. (a) Comparison of dorsal skin lesions on the 7th day among different groups of mice. (b-e) PASI scores, including erythema (b), scaling (c), skin thickness (d) and total PASI score, of mouse dorsal skin in all groups of mice were evaluated daily (n = 8). (f) The weights of the mice in all groups were measured daily (n = 8). The data are presented as the means ± SDs. **p < 0.01, ***p < 0.001.

The TOH-containing emulgel abrogated the morphological changes and reduced keratinocyte proliferation and differentiation in IMQ-induced psoriatic mice

To investigate the pathology of the mouse dorsal skin, H&E and immunohistochemical staining were performed. Compared with the IMQ + Emulgel group, the IMQ + Tryptophol group presented alleviated epidermal parakeratosis and acanthosis (Fig. 2a). The epidermal thickness was significantly lower in the IMQ + Tryptophol group than in the IMQ + Emulgel group (77.43 ± 12.80 versus 160.71 ± 33.59, p < 0.001), whereas the epidermal thicknesses in the cream base (14.35 ± 1.29) and tryptophol groups (14.67 ± 1.29) appeared normal (Fig. 2a and b). Similarly, Ki67 expression in proliferative cells (Fig. 2c) was significantly lower in keratinocytes, especially in the stratum basale, stratum spinosum and hair follicle, in the IMQ + Tryptophol group (191.05 ± 48.20) than in the IMQ + Emulgel group (419.05 ± 66.56) (Fig. 2d, p < 0.01). The expression of Ki67 was not significantly different between the cream base (71.55 ± 20.58) and tryptophol groups (74.35 ± 22.46) (Fig. 2c and d). Additionally, keratinocyte differentiation was assessed by evaluating K10 and involucrin expression. K10 is associated with the terminal differentiation of keratinocytes. Compared with that in the IMQ + Emulgel group (1.50 ± 0.46), the K10 mRNA expression in the IMQ + Emulgel group was significantly greater (2.57 ± 0.53) (Fig. 2e, p < 0.05). Although K10 expression was upregulated in the IMQ + Tryptophol group, it was not significantly different from that in the cream base (1.01 ± 0.19) and tryptophol groups (1.03 ± 0.17) (Fig. 2e). The mRNA expression of involucrin, a keratinocyte differentiation marker, was significantly greater in the IMQ + Emulgel group (4.33 ± 1.44) than in the IMQ + Tryptophol group (2.39 ± 0.33) (Fig. 2f, p < 0.05). However, involucrin expression was not different between and the cream base (1.00 ± 0.13) and the tryptophol groups (1.21 ± 0.25) (Fig. 2f).

Fig. 2

TOH-containing emulgel alleviated epidermal hyperplasia in IMQ-induced psoriatic mice. (a) Representative H&E sections of the dorsal skin of the mice in all the groups. Histological analysis revealed greater severity of parakeratosis and acanthosis in the IMQ + Emulgel group than in the IMQ + Tryptophol group. Representative images of skin sections were taken at 10X magnification, with a scale bar of 100 µm. (b) Quantification of epidermal thickness in all groups (n = 8). (c) Representative Ki67 immunostaining of the dorsal skin of all groups of mice. Ki67-positive cells are indicated by black arrows. Representative images of skin sections were taken at 10X magnification (scale bar: 100 µm), and the inset images were taken at 40X magnification (scale bar: 50 µm). (d) Quantification of Ki67-positive cells (n = 4). (e and f) qRT‒PCR analysis of the keratinocyte differentiation markers K10 and involucrin (n = 4), respectively. The data are expressed as fold changes relative to the cream base and tryptophol groups. All the data are presented as the means ± SDs; * p < 0.05, ** p < 0.01 and *** p < 0.001.

Additionally, K6, K16 and K17 are considered hallmarks of psoriasis, and their expression is associated with hyperproliferation of keratinocytes. qRT‒PCR revealed significantly decreased mRNA expression of K6 (IMQ + Tryptophol group: 15.54 ± 3.84 versus the IMQ + Emulgel group: 35.22 ± 10.41, p < 0.01; Fig. 3a), K16 (IMQ + Tryptophol group: 73.61 ± 14.64 versus the IMQ + Emulgel group: 271.95 ± 148.52, p < 0.05; Fig. 3b) and K17 (IMQ + Tryptophol group: 3.34 ± 0.69 versus the IMQ + Emulgel group: 7.20 ± 3.03, p < 0.01; Fig. 3c) in the skin of the IMQ + Tryptophol group compared with the IMQ + Emulgel group. To investigate whether the cytokines TNFα and IL-1β increase K6, K16 and K17 expression, psoriatic skin was homogenized, and ELISA was performed. Compared with those in the cream base group, the levels of TNFα (15.63 ± 0.00) in the cream base group (19.54 ± 0.00) were lower (Fig. 3d, p < 0.01). Moreover, IMQ-induced psoriasis in mice resulted in significantly lower levels of TNFα in the IMQ + Tryptophol group (22.46 ± 1.81) than in the IMQ + Emulgel group (31.74 ± 4.87) (Fig. 3d, p < 0.001). IL-1β cytokine production was significantly greater in the psoriatic skin of the IMQ + Emulgel group than in that of the IMQ + Tryptophol group (Fig. 3e, p < 0.001). These results indicate that the TOH-containing emulgel reduced IMQ-induced keratinocyte proliferation and differentiation in IMQ-induced psoriasis in mice.

Fig. 3

TOH-containing emulgel reduced keratinocyte proliferation by downregulating K6, K16, and K17. (a-c) qRT‒PCR results showing downregulated mRNA expression of K6, K16, and K17 in all groups of mice (n = 4). (d-e) TNFα and IL-1β levels in the dorsal skin of the mice in all the groups were measured using ELISA (n = 5). All the data are presented as the means ± SDs; * p < 0.05, ** p < 0.01 and *** p < 0.001.

The TOH-containing emulgel reduced mast cell infiltration by downregulating the expression of IL33/ST2

To evaluate mast cell activation in psoriatic skin lesions, histological sections were stained with Giemsa. The number of mast cells that infiltrated the dermal layer of psoriatic lesions (Fig. 4a) was significantly lower in the IMQ + Tryptophol group (24.25 ± 3.10) than in the IMQ + Emulgel group (32.99 ± 3.32) (Fig. 4b, p < 0.001). To further investigate the mRNA expression of ST2 and IL-33, which are associated with mast cell infiltration, qRT‒PCR was performed. ST2 mRNA expression in psoriatic skin lesions was lower in the IMQ + Tryptophol group (1.74 ± 0.26) than in the IMQ + Emulgel group (5.04 ± 0.73) (Fig. 4c, p < 0.01). Additionally, ST2 gene expression was not significantly different between the IMQ + Tryptophol group and the cream base (1.05 ± 0.41) and tryptophol groups (1.17 ± 0.59). IL-33 mRNA expression was downregulated in the IMQ + Tryptophol group (2.23 ± 0.95) compared with the IMQ + Emulgel group (6.82 ± 2.82) (Fig. 4d, p < 0.05), which corresponded with a decrease in IL-33 cytokine levels detected in psoriatic skin lesions of the IMQ + Tryptophol group (1103.12 ± 3.50) compared with the IMQ + Emulgel group (1198.44 ± 3.49) (Fig. 4e, p < 0.001). However, the expression of the cytokine IL-33 did not significantly differ between the psoriatic skin lesions of the IMQ + Tryptophol group and those of the cream base (1154.69 ± 12.83) and tryptophol groups (1154.93 ± 10.47) (Fig. 4e). These results suggest that the TOH-containing emulgel reduced mast cell infiltration and skin inflammation.

Fig. 4

TOH-containing emulgel reduced mast cell infiltration in IMQ-induced psoriatic mice. (a) Representative images of Giemsa-stained dorsal skin from all groups of mice. Representative images of skin sections were taken at 10X magnification (scale bars: 100 µm) and inset images were taken at 40X magnification (scale bars: 50 µm). (b) Quantification of mast cell accumulation in the dorsal skin of the mice in all the groups (n = 8). (c-d) Gene expression levels of ST2 and IL-33 were measured via qRT‒PCR (n = 4). (e) Cytokine levels of IL-33 in the dorsal skin of mice in all groups were measured using ELISA (n = 5). All the data are presented as the means ± SDs; * p < 0.05, ** p < 0.01, and *** p < 0.001.

The TOH-containing emulgel reduced CD4⁺ T-cell infiltration and alleviated IL-17A/IL-23

To assess the effect of the TOH-containing emulgel on CD4⁺ T-cell accumulation in the skin of IMQ-induced psoriatic mice, immunohistochemical staining of CD4⁺ T cells was performed, and the results are shown in Fig. 5a. The results revealed a significant decrease in the number of CD4⁺ cells in the dermis of the IMQ + Tryptophol group (22.20 ± 4.27) compared with the IMQ + Emulgel group (53.47 ± 2.44) (Fig. 5b, p < 0.001). Furthermore, the levels of inflammatory cytokines related to psoriasis were investigated by ELISA. The results revealed significantly lower IL-17A cytokine levels in the psoriatic skin and serum of the IMQ + Tryptophol group (skin: 712.89 ± 139.94 and serum: 702.34 ± 57.11) than in the IMQ + Emulgel group (skin: 934.25 ± 44.98 and serum: 999.22 ± 1.75) (Fig. 5c, p < 0.01 and Fig. 5d, p < 0.001). Additionally, the IL-17A cytokine level was lower in the skin of the tryptophol group (523.44 ± 16.75) than in that of the cream base group (722.66 ± 62.25) (Fig. 5c, p < 0.001), and there was no significant difference in the IL-17A level between the cream base and IMQ + Tryptophol groups (Fig. 5c). However, the IL-17A cytokine levels in the serum of the IMQ-Tryptophol group were not significantly different from those in the cream base (625.78 ± 68.30) and tryptophol groups (678.13 ± 116.55) (Fig. 5d). The IL-17A cytokine level corresponded with the IL-23 cytokine level in both the skin and serum. The results revealed a significant decrease in IL-23 cytokine production in both psoriatic skin and serum in the IMQ + Tryptophol group (skin: 337.24 ± 6.38 and serum: 337.89 ± 5.38) compared with the IMQ + Emulgel group (skin: 442.06 ± 10.89 and serum: 453.78 ± 6.26) (Fig. 5e and f, p < 0.001). Additionally, IL-23 levels were lower in both the skin (208.99 ± 8.82) and serum (212.24 ± 11.76) samples than in the cream base samples (skin: 273.44 ± 48.54 and serum: 356.12 ± 13.40) (Fig. 5e and f, p < 0.001), and the IMQ + Tryptophol samples were not significantly different from the cream base samples (Fig. 5e and f). These results suggested that the TOH-containing emulgel reduced CD4⁺ T-cell infiltration and skin inflammation.

Fig. 5

TOH-containing emulgel reduced CD4⁺ T-cell infiltration and the release of inflammatory cytokines in IMQ-induced psoriatic mice. (a) Representative images of immunohistochemical staining of CD4⁺ cells in the dorsal skin of the mice in all the groups. Representative images of skin sections were taken at 10X and 40X magnification (scale bars: 100 µm and 50 µm, respectively). The CD4⁺ cells are indicated by a black arrow. (b) Quantification of CD4⁺ cells in all groups (n = 5). (c-f) Inflammatory cytokine production in the skin and serum (n = 5). The data are presented as the means ± SDs; * p < 0.05, ** p < 0.01, and *** p < 0.001.

The TOH-containing emulgel downregulated the antimicrobial peptide genes S100A8 and S100A9

To determine the expression of S100A8 and S100A9, dorsal skin samples were harvested, and qRT‒PCR was performed; the results are shown in Fig. 6. The expression of the S100A8 mRNA was significantly lower in the IMQ + Tryptophol group (17.79 ± 5.49) than in the IMQ + Emulgel group (36.79 ± 12.86) (Fig. 6a, p < 0.05). However, S100A9 expression was downregulated in the IMQ + Tryptophol group (29.21 ± 8.58), but it was not significantly different from that in the IMQ + Emulgel group (48.11 ± 20.10) (Fig. 6b).

Fig. 6

TOH-containing emulgel downregulated the expression of the S100A8 and S100A9 genes. (a-b) qRT‒PCR results showing downregulated mRNA expression of S100A8 and S100A9 in all groups of mice. All the data are presented as the means ± SDs (n = 4), * p < 0.05, ** p < 0.01 and *** p < 0.001.

The TOH-containing emulgel reduced the expression of JAK/STAT pathway members

To evaluate the effects of the TOH-containing emulgel on skin inflammation, the JAK2/STAT3 pathway was analysed using immunohistochemical staining, qRT‒PCR, and western blotting. Immunohistochemical staining revealed significantly decreased pJAK2 expression in keratinocytes and dermal cells in the IMQ + Tryptophol group (1021.17 ± 207.69) compared with the IMQ + Emulgel group (1656 ± 150.57) (Fig. 7a and b, p < 0.01). Similarly, JAK2 mRNA expression was markedly lower in the IMQ + Tryptophol group (2.03 ± 1.11) than in the IMQ + Emulgel group (6.85 ± 1.71) (Fig. 7c, p < 0.001). Although JAK2 protein levels tended to decrease in the IMQ + Tryptophol group, the difference was not statistically significant (Supplementary Figures S1a, S1b, and S2). Additionally, downregulated mRNA expression of JAK1 (0.91 ± 0.31) and JAK3 (0.93 ± 0.33) was observed in the IMQ + Tryptophol group compared with the IMQ + Emulgel group (JAK1: 2.65 ± 0.99; JAK3: 2.22 ± 0.99) (Supplementary Figure S4a and b).

Fig. 7

TOH-containing emulgel reduced the expression of JAK2 in IMQ-induced psoriatic mice. (a) Representative images of immunohistochemical staining for JAK2 in the dorsal skin of the mice in all the groups. Representative images of skin sections were taken at 10X and 40X magnification (scale bars: 100 µm and 50 µm, respectively). JAK2-positive cells are indicated by black arrows. (b) Quantification of JAK2-positive cells in all groups (n = 4). (c) mRNA expression of JAK2 (n = 4). All the data are presented as the means ± SDs (n = 4), * p < 0.05, ** p < 0.01 and *** p < 0.001.

Immunostaining of STAT3 revealed a significant reduction in the number of positive cells in the IMQ + Tryptophol group (878.50 ± 266.35) compared with the IMQ + Emulgel group (1508.00 ± 328.06) (p < 0.05, Fig. 8a and b). Although STAT3 mRNA expression tended to decrease in the IMQ + Tryptophol group (2.81 ± 0.34) compared with the IMQ + Emulgel group (3.95 ± 0.66) (Fig. 8c), this difference was not statistically significant. Similarly, STAT3 protein levels also tended to decrease in the IMQ + Tryptophol group, but the difference was not statistically significant (Supplementary Figures S1c, S1d, and S3). In addition, the mRNA expression of STAT1 demonstrated a similar decreasing trend in the IMQ + Tryptophol group (1.29 ± 0.41) compared with the IMQ + Emulgel group (1.86 ± 0.48) (Supplementary Figure S4c).

Fig. 8

TOH-containing emulgel reduced the expression of STAT3 in IMQ-induced psoriatic mice. (a) Representative images of immunohistochemical staining for STAT3 in the dorsal skin of the mice in all the groups. Representative images of skin sections were taken at 10X and 40X magnification (scale bars: 100 µm and 50 µm, respectively). STAT3-positive cells are indicated by black arrows. (b) Quantification of STAT3-positive cells in all groups. (c) mRNA expression of STAT3. (d-e) mRNA expression of genes downstream of the JAK/STAT pathway, including BCL2 (d) and CCND1 (e). All the data are presented as the means ± SDs (n = 4), * p < 0.05, and *** p < 0.001.

To explore the downstream signalling pathways of the JAK/STAT pathway, the mRNA expression levels of BCL2 and CCND1 were analysed. The results revealed a significant reduction in BCL2 mRNA expression in the IMQ + Tryptophol group (1.12 ± 0.37) compared with the IMQ + Emulgel group (4.90 ± 0.80) (p < 0.001, Fig. 8d). Furthermore, CCND1 mRNA expression tended to decrease in the IMQ + Tryptophol group (1.21 ± 0.62) compared with the IMQ + Emulgel group (2.22 ± 0.51) (Fig. 8e). These findings suggest that the TOH-containing emulgel effectively reduces the expression of JAKs and STATs in IMQ-induced psoriatic mice. This reduction indicates its potential to alleviate inflammation by suppressing the JAK/STAT signalling pathway.

The TOH-containing emulgel reduced systemic inflammation in IMQ-induced psoriatic mice

To investigate whether the TOH-containing emulgel alleviated systemic inflammation induced by IMQ, spleens and axillary lymph nodes were removed, and the mRNA expression of inflammation-related genes was quantified. The results revealed that the TOH-containing emulgel inhibited IMQ-induced splenomegaly in the IMQ + Emulgel group (Fig. 9a) and significantly decreased the relative spleen weight in the IMQ + Tryptophol group (9.13 ± 0.44) compared with the IMQ + Emulgel group (10.92 ± 0.74) (Fig. 9b, p < 0.01). The mRNA expression of inflammatory-related genes, including TNFα, IL-1 and IL-6, was significantly downregulated in the IMQ + Tryptophol group (TNFα; 2.13 ± 0.43, IL-1; 2.82 ± 1.09 and IL-6; 57.21 ± 19.99) compared with the IMQ + Emulgel group (TNFα; 4.66 ± 1.68, IL-1; 12.59 ± 2.64 and IL-6; 479.49 ± 93.30) (Fig. 9c-e, p < 0.05, p < 0.001 and p < 0.001, respectively). However, although there was no difference in relative lymph node weight between the IMQ + Tryptophol group (0.31 ± 0.06) and the IMQ + Emulgel group (0.39 ± 0.08) (Fig. 9f and g), the mRNA expression of TNFα, IL-1 and IL-6 was significantly lower in the IMQ + Tryptophol group (TNFα; 2.89 ± 0.52, IL-1; 8.62 ± 0.95 and IL-6; 1.30 ± 0.63) than in the IMQ + Emulgel group (TNFα; 7.84 ± 2.59, IL-1; 130.31 ± 48.98 and IL-6; 2.71 ± 0.79) (Fig. 9h‒j, p < 0.05, p < 0.01 and p < 0.05, respectively). These results corresponded with the serum levels of TNFα and IL-1β, which were significantly lower in the IMQ + Tryptophol group (TNFα; 8.20 ± 0.87 and IL-1β; 617.19 ± 13.53) than in the IMQ + Emulgel group (TNFα; 12.11 ± 0.87 and IL-1β; 693.75 ± 16.94) (Fig. 9k and l, p < 0.001). Notably, the mRNA expression of these genes in the spleen and axillary lymph nodes did not differ between the tryptophol and cream base groups (Fig. 9c-e and h-j). Additionally, the cytokine level of TNFα was notably lower in the tryptophol group than in the cream base group (Fig. 9k), with no difference in the IL-1β level between the groups (Fig. 9l). These results revealed that the TOH-containing emulgel reduced systemic inflammation in IMQ-induced psoriatic mice.

Fig. 9

The TOH-containing emulgel suppressed IMQ-induced systemic inflammation. (a) Representative image of the spleen at the end of the experiment. (b) Relative spleen weights of the mice in all groups (n = 5). (c-e) The mRNA expression of TNFα, IL-1 and IL-6 in the spleens of all groups of mice (n = 4). (f) Representative image of right axillary lymph nodes at the end of the experiment. (g) Relative right axillary lymph node weights of the mice in all groups (n = 5). (h-j) The mRNA expression of TNFα, IL-1 and IL-6 in the lymph nodes of all groups of mice (n = 4). (k-l) Serum levels of TNF-α and IL-1β in all groups of mice (n = 5). All the data are presented as the means ± SDs (n = 4), * p < 0.05, ** p < 0.01 and *** p < 0.001.

Discussion

Psoriasis is a chronic inflammatory skin disease that currently has no cure and lowers patients’ quality of life¹. Cytokines and immune networks are being investigated as potential targets in the research and development of new therapeutic options^5,29,30. TOH is a quorum-sensing molecule isolated from bacteria and fungi with potential effects in treating various pathogenic fungi²⁴. A previous study revealed that TOH can counteract excessive inflammation and suppress the effects of TNFα on the immune response²⁸. Treatment with a TOH-containing emulgel improved skin hydration and reduced transepidermal water loss in human skin²⁴. This study demonstrated that the TOH-containing emulgel attenuated IMQ-induced psoriasis in mice. In IMQ-induced psoriatic mice, TOH-containing emulgel treatment resulted in a decreased cumulative PASI score, which indicated that erythema, scaling and skin thickening were all alleviated after treatment. Additionally, the application of IMQ is frequently associated with severe weight loss, as observed in this study. Previous research suggests that the weight loss associated with IMQ application is not a direct result of reduced food and water intake but is rather attributed to the activation of the solute carrier family 15 member 4 (SLC15A4) gene and the subsequent accumulation of CD4⁺ T lymphocytes and plasmacytoid dendritic cells³³. In this study, the IMQ + Emulgel group exhibited weight loss, despite the nontoxic nature of the pure emulgel. These findings suggest that the weight reduction was primarily attributed to psoriasis development, which likely caused stress-induced reductions in food intake and metabolic disruptions associated with inflammation. The observed increase in body weight may be a result of the decreased severity of psoriasis in response to the emulgel treatment.

Moreover, the most common clinical characteristics of IMQ-induced psoriatic mice include increased keratinocyte hyperproliferation and differentiation^3,16,29. Ki67 was used to evaluate keratinocyte proliferation because it is a nucleoprotein associated with cell proliferation. Both IMQ-induced psoriatic mice and patients with psoriasis exhibit increased Ki67 expression in keratinocytes in the basal layer of the dermis^16,34. This cell proliferation marker was also upregulated in the IMQ + emulgel group before treatment in this study. Notably, the number of Ki67-positive cells was reduced when the TOH-containing emulgel was applied to IMQ-induced psoriatic mice. Therefore, it is possible that the TOH-containing emulgel has an effect on abnormal keratinocyte proliferation. Additionally, the expression of K10 and involucrin, which are involved in keratinocyte differentiation, was investigated¹⁶. K10 is a differentiation-related protein expressed in all layers of the epidermis except the stratum basale³⁵. Although there is increased epidermal thickness in psoriasis, the expression of K10 is decreased^13,14. This demonstrated that there is a loss of control over the division of keratinocytes because K10 plays an important role in regulating cell proliferation and the expression of filaggrin and cornified envelope proteins³⁵. This study revealed significantly greater K10 expression in the IMQ + Emulgel group than in the control and IMQ + Tryptophol groups. These results were similar to those of Chen and colleagues, who reported that IMQ increased the mRNA expression of K10¹⁶. Additionally, high K10 expression was also detected in patients with mild psoriasis³⁵. Thereby, the increased K10 expression observed in this study may be a result of IMQ treatment. Mutations in the K10 protein increase the proliferation of suprabasal keratinocytes, leading to hyperkeratosis, as observed in epidermolytic ichthyosis patients. Therefore, K10 mutations may lead to abnormal formation of keratin intermediate filaments in suprabasal keratinocytes, which disrupts their structural stability. Additionally, this mutation in the K10 protein elevates the levels of cyclin D, cyclin E, and phospho-Akt, all of which contribute to keratinocyte hyperproliferation in psoriasis³⁵. However, the mechanism through which IMQ increases K10 expression must be investigated further. Moreover, involucrin is expressed mainly in keratinocytes in the upper stratum spinosum and stratum granulosum of healthy skin, where it helps stabilize the cornified envelope^15,16,36. In psoriatic patients, this expression pattern extends into the middle part of the stratum spinosum^37,38, with increased expression also observed in IMQ-induced psoriatic mice^16,39. Increased involucrin expression results in altered keratinocyte proliferation, leading to the formation of psoriatic plaques³⁶. Interestingly, the application of the TOH-containing emulgel to IMQ-induced psoriatic mice ameliorated keratinocyte hyperproliferation and differentiation by downregulating K10 and involucrin expression. These findings suggest that the TOH-containing emulgel effectively mitigates abnormal keratinocyte proliferation.

In addition to K10 and involucrin, increased expression of K6, K16 and K17 is associated with increased epidermal thickness¹⁷. These keratins are considered markers of psoriasis because they regulate keratinocyte proliferation and the inflammatory response during the disease¹⁹. Both patients with psoriasis and IMQ-induced psoriatic mice exhibit highly upregulated expression of these keratins^11,17. The underlying mechanism involves IL-1β-mediated Nrf2 nuclear translocation in psoriasis keratinocytes, resulting in increased expression of K6, K16, and K17¹¹. Another proinflammatory cytokine, TNFα, also upregulates K6 expression through the NF-κB signalling pathway⁴⁰. Therefore, both Nrf2 and NF-κB are important regulators of the inflammatory response, and they communicate and influence each other⁴¹. Therefore, IL-1β and TNFα activate keratinocytes by increasing the expression of K6, K16 and K17, as observed in the IMQ-Emulgel group. However, the expression of these cytokines and genes was reduced in the mice after treatment with the TOH-containing emulgel.

In addition to the dysregulation of the innate and adaptive immune responses in the skin tissue, the infiltration of mast cells, CD4⁺ cells and other immune cells is considered a hallmark of psoriasis⁴. An increased number and activation of mast cells have been reported in both patients with psoriasis and IMQ-induced psoriatic mice⁵. Mast cells are activated in response to infection or other forms of stress-induced inflammation by IL-33 by binding to its receptor IL-33R (T1/ST2 or ST2). The activated mast cells subsequently produce inflammatory cytokines, including TNFα, IL-1β and IL-6⁴². A previous study revealed that IL-33 is released from keratinocytes and subsequently initiates inflammation in psoriasis⁴³. This study revealed that IL-33 cytokine levels and mRNA expression were increased in psoriatic skin, along with increased mast cell accumulation and the upregulation of ST2. Interestingly, the TOH-containing emulgel was able to alleviate this pathological condition. These findings might be related to our finding that TOH-containing emulgel did not cause skin irritation and improved skin hydration by decreasing transepidermal water loss²⁴. Previous studies have shown that improving skin hydration has the potential to alleviate psoriasis symptoms⁴⁴. Additionally, TOH may reduce the abnormal proliferation of keratinocytes, similar to the inhibition of leukaemia U937 cell proliferation^26, and subsequently reduce the autocrine production of IL-33 from keratinocytes and reduce inflammation^43,45. In this case, TOH-containing emulgel may improve skin hydration and relieve skin inflammation from IMQ-induced psoriasis.

In this study, in addition to mast cell infiltration, the infiltration of CD4⁺ lymphocytes into dermal tissue was observed as an initial phase of psoriasis⁴⁶. Our study also revealed a decrease in CD4⁺ cell accumulation following TOH-containing emulgel treatment. Moreover, JAK2/STAT3 signalling is considered an important pathway in psoriasis development because it regulates Th cell differentiation, and JAK2/STAT3 pathway members have been found to be upregulated in IMQ-induced psoriatic mice⁴⁷. STAT3 plays an important role in the immune system and is activated by IL-6 and IL-23. After JAKs (JAK1, JAK2, JAK3 and TYK2) are activated, they undergo phosphorylation and subsequently activate STATs. In particular, STAT3 has been reported to promote Th cell development through downstream signalling^7,9. Additionally, the activation of STAT3 results in the upregulation of the BCL2 and cyclin D1 (CCND1) genes, which are involved in cell survival and proliferation^7,48. Increased STAT3 upregulation leads to disease initiation and maintenance, mediating cytokine signalling pathways involved in psoriasis pathogenesis⁴⁹, especially the IL23/IL17 axis^7,8. Inhibiting the phosphorylation of STAT3 reduces IMQ-induced psoriasis in mice and reduces K17 expression in keratinocytes⁴⁹. This study revealed a significant decrease in the number of STAT3-positive cells and a decreasing trend in STAT3 gene and protein expression after TOH-containing emulgel treatment. Additionally, upstream JAKs, especially JAK2, which has been associated with keratinocyte activation and proliferation⁵⁰, have been found to be involved in psoriasis. The activation of JAK2/STAT3 signalling in keratinocytes promoted IL-1β secretion⁵¹. This finding is consistent with our study, which revealed numerous keratinocytes positively stained for JAK2, along with a significant increase in JAK2 mRNA expression and a trend towards increased protein expression after TOH-containing emulgel treatment. Conversely, mice treated with the TOH-containing emulgel presented a decreased number of keratinocytes positively stained for JAK2, reduced JAK2 gene and protein expression, and decreased expression of the downstream signalling proteins Bcl2 and CCND1. These data suggest that TOH-containing emulgel treatment reduces IMQ-induced psoriasis in mice by suppressing the JAK2/STAT3 pathway. A previous study revealed that inhibiting the expression of JAK2/STAT3 prevents the development of psoriasis⁴⁷.

In addition, activated Th17 cells secrete IL-17 and TNFα, and IL-17 also stimulates keratinocytes to produce the antimicrobial peptides S100A8 and S100A9⁸. S100A8/S100A9 dimers (calprotectin) bind to Toll-like receptor 4 (TLR4) and trigger an inflammatory response in target cells¹⁹. This process occurs not only in immune cells but also in keratinocytes. Additionally, this study revealed that the TOH-containing emulgel reduced IL-17A and IL23 cytokine levels in both the dorsal skin and serum of mice and decreased the levels of S100A8 and S100A9. Thus, it alleviates the inflammatory microenvironment of the skin, resulting in a decrease in CD4⁺ cell accumulation and antimicrobial peptide gene expression. These results suggest that this TOH-containing emulgel may alleviate skin inflammation by modulating the IL-17/IL-23 axis and the JAK2/STAT3 pathway. However, the mechanism by which TOH regulates the JAK2/STAT3 pathway must be investigated in depth.

Finally, IMQ-induced psoriasis in mice has been reported to cause systemic inflammation characterized by splenomegaly along with increased serum levels of TNFα and IL-6²⁹. This study revealed that a TOH-containing emulgel reduced systemic inflammation, not only by decreasing the spleen size but also by reducing the axillary lymph node size. Proinflammatory cytokine mRNA and serum levels were also decreased after TOH-containing emulgel treatment.

Conclusion

This study demonstrated that a TOH-containing emulgel alleviates IMQ-induced psoriasis in mice by reducing inflammatory cell accumulation and cytokine secretion, primarily by suppressing the immune responses mediated by IL-17 and IL-23 through the regulation of JAK/STAT signalling pathway. Given its effectiveness in treating psoriasis and its nonirritating properties in healthy mice, the TOH-containing emulgel is a promising therapeutic agent for alleviating psoriasis. However, further in-depth investigations, including investigations of the levels of phosphorylated and unphosphorylated forms of JAK/STAT pathway members and other signalling pathway members, are needed. Additionally, the toxicity of the TOH-containing emulgel was not assessed in this study. Therefore, it is essential to evaluate its acute and chronic toxicity in the future to provide a comprehensive safety evaluation. Thus, additional experiments are needed for future application and clinical research.

Materials and methods

Materials

Tryptophol (TOH; C10H11NO) and bovine serum albumin (BSA) (Cat no. 9048-46-8) were purchased from Sigma‒Aldrich (St. Louis, MO, USA). Mayer’s haematoxylin (Cat no. 05-06002/L) and eosin Y plus alcoholic solution (Cat no. 05-11007/L) were purchased from Bio-Optica Milano Spa (San Faustino, Milano, Italy). Absolute ethanol (Cas no.64-17-5), xylene (Cas no. 1330-20-7) and formaldehyde 35–40% (Cas no. 50-00-0) were purchased from RCI Labscan (Pathumwan, Bangkok, Thailand). Giemsa stain solution (RA-002-05) was purchased from Biotech Reagent (Bangkok, Thailand). Toluene mounting media (Cas no. 108-88-3) was purchased from Fisher Chemical (Geel, Belgium, UK). An ELISA MAX™ Deluxe Set Mouse IL-1β Kit (Cat no. 432604), ELISA MAX™ Deluxe Set Mouse TNFα Kit (Cat no. 430904), ELISA MAX™ Deluxe Set Mouse IL-17A Kit (Cat no. 432504), ELISA MAX™ Deluxe Set Mouse IL-23 Kit (Cat no. 433704) and LEGEND MAX™ Mouse IL-33 ELISA Kit (Cat no. 436407) were purchased from BioLegend (San Diego, California). An anti-β-actin (13E5) rabbit monoclonal antibody (#4970S) was purchased from Cell Signaling Technology (Danvers, Massachusetts, USA). A rabbit polyclonal anti-STAT3 antibody (AB31370), rabbit polyclonal anti-Ki67 antibody (ab15580), rabbit monoclonal anti-Jak2 (phosphoY1007 + Y1008) antibody (AB32101), rabbit monoclonal anti-CD4 antibody (EPR19514) and protein blocking solution (AB64226) were purchased from Abcam (Cambridge, UK). The secondary antibody peroxidase AffiniPure goat anti-rabbit IgG (H&L) (111-035-003) was purchased from Jackson ImmunoResearch (West Grove, Pennsylvania, USA). The peroxidase (HRP) substrate Vector NovaRed (SK-4800) was purchased from Vector Laboratories (Newark, California). TRI Reagent® (Cat no. TR118) was purchased from Molecular Research Center, Inc. (Montgomery Rd., Cincinnati, OH, USA). ReverTra Ace™ qPCR RT Master Mix with gDNA Remover was purchased from TOYOBO Research Reagents (Osaka, Japan). A SensiFAST™ SYBR® No-ROX kit (BIO-98050, Bioline) was purchased from Meridian Bioscience (River Hills Drive, Cincinnati, OH, USA). T-PER™ tissue protein extraction reagent (REF 78510) was purchased from Pierce Biotechnology (Rockford, USA). Protease inhibitors (Roche cOmplete™, Mini Protease Inhibitor Cocktail, REF 04 693 159 001), chloroform (Cas no. 67-66-3) and 2-propanol (MilliporeSigma™, 1.09634) were purchased from Merck (Darmstadt, Germany). QubitTM protein assay kit (Invitrogen, REF Q33211) was purchased from Life Technologies Corporation (Eugene, Oregon, USA). BioTrace hydrophobic polyvinylidene fluoride (PVDF) membranes with 0.45 µm pores (Pall no.66543) were purchased from Pall Corporation (Pensacola, USA). BD Difco™ skim milk (Ref 232100) was purchased from Becton Dickinson and Company (Sparks, Maryland, USA). The Colorcode prestain protein marker (Abbkine, Cat no. BMM3001) and superKine™ West Pico PLUS chemiluminescent substrate (Abbkine, Cat no. BMU101-EN) were purchased from CliniSciences (Nanterre, France). Vaseline (petroleum jelly) was purchased from Unilever (London, UK). Five percent imiquimod cream (Aldara™) was purchased from Ensign Laboratories Pty Ltd. (Australia).

Preparation of the TOH-containing emulgel

The emulgel formulation and emulgel containing TOL (100 µM) were prepared as previously described²⁴. The concentration of TOL used was chosen on the basis of in vitro cell viability assays. The HaCat cell culture method is described in Supplementary Data 1, and the results for cell viability are shown in Supplementary Figure S5.

Animal experiments

Healthy male BALB/c mice (6–8 weeks of age, weight 27 ± 1.37 g) were purchased from Nomura International Siam (Thailand). All the mice were housed under specific pathogen-free (SPF) conditions with the appropriate humidity and temperature and a 12-hour light/dark cycle. The mice were allowed access to a standard pellet diet and drinking water ad libitum throughout the experiment.

All experimental protocols and animal protocols used were approved by the ethical committee of the University of Phayao, Thailand (Approval Number 1-020-65).

Establishment of IMQ-induced psoriasis-like lesions in mice

Thirty-two mice were randomly divided into four groups (n = 8 mice per group): (1) the control cream base group (cream base), (2) the control TOH-containing emulgel group (tryptophol), (3) the psoriasis nontreatment group (IMQ + Emulgel), and (4) the psoriasis treatment with the TOH-containing emulgel group (IMQ + tryptophol). Before starting the experimental treatment for one day, the dorsal skin of the mice was shaved.

Experimental treatment

1. Vaseline (100 µl) was applied to the shaved areas of mice in the cream base group at 7:00 am, followed by application of the emulgel without TOH (100 µl) at 1:00 pm and 7:00 pm for 7 consecutive days.

2. Vaseline (100 µl) was applied to the shaved areas of mice in in the tryptophol group, at 7.00 am, followed by application of the TOH-containing emulgel (100 µl) at 1.00 pm and 7.00 pm for 7 consecutive days.

3. The 5% IMQ cream (topical dose 62.5 mg) was applied to the shaved areas of mice in the IMQ-Emulgel group at 7.00 am), followed by application of the emulgel without TOH (100 µl) at 1.00 pm and 7.00 pm for 7 consecutive days.

4. The 5% IMQ cream (topical dose 62.5 mg) was applied to the shaved areas of mice in the IMQ + Tryptophol group at 7.00 am, followed by application of the TOH-containing emulgel (100 µl) at 1.00 pm and 7.00 pm for 7 consecutive days.

After 7 days of treatment, all the mice were anaesthetized with isoflurane and sacrificed by cervical dislocation. Blood collection was performed via cardiac puncture. Skin lesions, lymph nodes and spleens were collected for further analysis.

Assessment of skin inflammation severity

The severity of the skin lesions was investigated by a modified human scoring system, the PASI. The mice were assessed daily and individually for erythema, scaling, and skin thickening. Each parameter was scored independently on a scale from 0 to 4 (0, none; 1, mild; 2, moderate; 3, marked; and 4, obvious). The cumulative score, which combines erythema, scaling, and skin thickening, was used to evaluate the severity of skin inflammation^52,53.

Histological analysis

H&E staining

The skin lesions were collected and preserved in 4% paraformaldehyde, embedded in paraffin and then cut into 4 µm-thick sections with a rotary microtome. In accordance with standard H&E staining procedures, the tissue sections were rehydrated, stained with H&E, dehydrated, cleared, mounted on slides and coverslipped for pathological observation by light microscopy. Epidermal thickness was accurately measured with ImageJ software.

Giemsa staining

The skin sections were deparaffinized and rehydrated with a decreasing series of ethanol and distilled water. These sections were subsequently stained with Giemsa solution for 45 minutes, after which the excess dye was washed away with distilled water. The sections were dehydrated in an ethanol series and cleared with xylene. Finally, the sections were mounted on slides and coverslipped. These skin sections were observed and photographed under a microscope. The mast cells were counted in 5 areas per 10x field.

Immunohistochemistry

After nonspecific antigens were blocked with protein blocking solution, the tissue sections were incubated with anti-CD4 (dilution 1:50), anti-Ki67 (dilution 1:100), anti-pJAK2 (dilution 1:50), and anti-STAT3 (dilution 1:50) antibodies at room temperature for 3 hours. After washing, the tissue sections were incubated with the appropriate secondary antibody (dilution 1:500) for 1 hour at room temperature. The slides were washed, and positive signals were subsequently visualized with NOVA Red, followed by counterstaining with Mayer’s haematoxylin. For quantitative analysis, the slides were imaged at a magnification of 10x. The CD4⁺, Ki67, pJAK2 and STAT3-positive cells were counted in 4 areas per 10X field.

Body weight and lymphatic organ weight

The body weights of the mice were recorded on days 1, 3, 5 and 7. The right axillary lymph nodes and spleens of the mice were then removed, cleaned and weighed after sacrifice on the 8th day. The right axillary lymph node weight and spleen weight were normalized to the body weight to obtain the organ index.

Cytokine analysis by ELISA

Serum and skin tissue preparation

The dorsal skin of the control or experimental mice was collected, the attached connective tissue was removed, and 0.1 cm³ of tissue was added to 1 ml of normal saline. The tissue was subsequently cut and ground thoroughly in a homogenizer to obtain a tissue suspension. The homogenized tissue was centrifuged at 4°C at 3,000 rpm for 15 min. The supernatant was removed and stored at -20 ºC for the ELISA experiments²⁹.

The blood samples were allowed to clot at room temperature for 30 min and subsequently centrifuged at 2,500 × g at 4 ºC for 15 min to collect the serum. The serum was collected and stored at -20 ºC for the ELISA experiments.

The levels of IL-1β, TNF-α, IL17A, IL-23 and IL-33 in the dorsal skin and serum samples from the control and experimental groups were measured using commercial ELISA kits following the manufacturer’s instructions. The absorbance was measured at 450 nm using a spectrophotometer (VersaMax™ Microplate Reader, Molecular Devices, San Jose, CA, USA). The calculation and analyses of the data were conducted using the SoftMax Pro software version 6 (Molecular Devices).

RNA isolation and cDNA synthesis

Total RNA from the spleens, lymph nodes and dorsal skin of control and experimental mice was isolated using TRIzol Reagent® according to the manufacturer's protocol. The amount and quality of RNA were measured with a Nanodrop One (Thermo Fisher Scientific). From each sample, 1 µg of RNA was reverse transcribed into cDNA with cDNA synthesis kits according to the manufacturer's instructions. The cDNA mixture was stored at − 20°C for further analysis.

Analysis of gene expression by quantitative real-time PCR

The relative expression levels of the genes encoding TNFα, IL-1, IL-6, IL-33, ST2, K10, K6, K16, K17, involucrin, S100A8, S100A9, JAK1, JAK2, JAK3, BCL2, CCND1, STAT1 and STAT3, as well as TNFα, IL-1 and IL-6, in the dorsal skin of control and experimental mice as well as TNFα, IL-1 and IL-6 in the spleens and lymph nodes were measured via quantitative real-time PCR (qRT‒PCR) via a QIAquant Real-Time PCR Thermal Cycler (Qiagen, USA). The amplification was performed on a 96-well plate with a 20 µL reaction mixture consisting of 1 µL of cDNA from each tissue, 10 µL of SYBR, 0.4 µL of specific primers (10 µM/µL), and 8.2 µL of sterile distilled water. The thermal cycle profile for qRT‒PCR was as follows: one cycle at 95°C for 3 min; 40 cycles at 95°C for 3 s for denaturation and 60°C for 20 s for annealing/extension; and one cycle at 95°C for 15 s, 60°C for 1 min, 95°C for 30 s, and 60°C for 15 s, respectively. All reactions were analysed in triplicate, and β-actin was also amplified as an internal control. The details of the primers used in this experiment are presented in Supplementary Table 1. The comparative CT method (2^−ΔΔCT method) was used to analyse the relative expression levels of inflammatory cytokine genes according to Livak and to Schmittgen⁵⁴. Target gene expressed was normalized to β-actin expression.

Western blot analysis

The dorsal skin of the mice (50 mg/sample) was homogenized and extracted with tissue protein extraction reagent supplemented with a protease inhibitor. The protein concentration was determined using a Qubit™ protein assay kit and Qubit™ 4 Fluorometer (Invitrogen, REF Q3326, Life Technologies Holding Pte Ltd, Singapore).. Protein samples (100 µg/each) were separated by 7.5% and 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis and then transferred to PVDF membranes (0.45 µm). The membranes were incubated with protein blocking buffer (3% skim milk and 2% BSA in 0.05% PBST) overnight at 4°C and subjected to incubation with primary antibodies for 3 hours at room temperature. The antibodies used to detect specific proteins included anti-pJAK2 (dilution 1:1,000), anti-STAT3 (dilution 1:500) and anti-β-actin (dilution 1:2,000) antibodies. All the antibodies were diluted with blocking buffer. Afterwards, the blots were washed with PBST 3 times (5 min each) and subsequently incubated with secondary antibody (dilution 1:5000). Then, the blots were visualized using an enhanced chemiluminescence detection system (S1060; Azure Biosystems). Finally, the protein bands were quantified using ImageJ software (National Institutes of Health, USA).

Statistical analysis

The results of this study are presented as the means ± standard deviations (SDs). Differences were assessed by one-way analysis of variance (ANOVA) or Student's t test. All the statistical analyses and graphs were generated with GraphPad software version 9.1.0. Statistical significance was considered at p values less than 0.05.

Data availability

The datasets used and/or analysed in this study are available from the corresponding author upon reasonable request.

Electronic Supplementary Material

Below is the link to the electronic supplementary material

Supplementary Material 1

Data Availability

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

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Acknowledgement

We are very grateful to the Laboratory Animal Research Center, University of Phayao for the animal facility as well as School of Medical Sciences, University of Phayao, Department of Microbiology and Immunology, Faculty of Tropical Medicine, Centex Shrimp and Department of Anatomy, Faculty of Science, Mahidol University for laboratory facility.

Funding

This project is funded by a grant from the National Research Council of Thailand (NRCT): Contract number N42A660825 awarded to Arnon Pudgerd and a grant from the Health Systems Research Institute (Grant number: HSRI 67 − 027) awarded to Passanesh Sukphopetch.

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Author Contribution

A.P. conceptualized, designed, and performed the study; participated in data collection and analysis; validated the data; performed formal analyses; and wrote the original draft of the manuscript. K.J. performed the study; collected the data; and performed the data analysis. L.P., S.J. and T.C. contributed to the data collection. W.M. and P.S. performed the cell culture and TOH cytotoxicity tests and prepared the emulgel and TOH-containing emulgel. R.V performed the study; validated the results; and interpreted the data. P.S. conceptualized and performed the study; interpreted the data; supervised the study; and reviewed and edited the manuscript. All the authors reviewed the manuscript and approved the final version.

Accordance and ARRIVE guidelines

Accordance: We confirmed that all the experiments in this study were performed in accordance with the relevant guidelines and regulations.

All the procedures of the study followed the ARRIVE guidelines.

Yes