Indole-3-Carbinol Alleviates Allergic Skin inflammation via Periostin/Thymic Stromal Lymphopoietin Suppression in Atopic Dermatitis

Indole-3-Carbinol Alleviates Allergic Skin inflammation via Periostin/Thymic Stromal Lymphopoietin Suppression in Atopic Dermatitis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Indole-3-Carbinol Alleviates Allergic Skin inflammation via Periostin/Thymic Stromal Lymphopoietin Suppression in Atopic Dermatitis Yun-Mi Kang, Hye-Min Kim, Minho Lee, Hyo-Jin An This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4073342/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Dec, 2024 Read the published version in Chinese Medicine → Version 1 posted 4 You are reading this latest preprint version Abstract Background Atopic dermatitis (AD) is a chronic multifactorial inflammatory skin disorder with a complex etiology. Despite its increasing prevalence, treatment of AD is still limited. Indole-3-carbinol (I3C) is found in cruciferous vegetables and is formed when these vegetables are cut, chewed, or cooked; it exerts diverse pharmacological activities. Methods HaCaT keratinocytes stimulated with tumor necrosis factor-α and interferon-γ mixture and NC/Nga mice stimulated with 2,4-dinitrochlorobenzen (DNCB) were used for AD models, in vitro and in vivo , respectively. Results The results showed that I3C reduced the expression of pro-inflammatory cytokines, thymic stromal lymphopoietin (TSLP), and periostin in in vitro model. Oral administration of I3C alleviated AD-like skin inflammatory symptoms, including serum IgE levels, epidermal thickening, inflammatory cell infiltration, transepidermal water loss, and scratching behavior. Moreover, I3C decreased the expression of TSLP and periostin and recovered the expression of skin barrier proteins by inhibiting the mitogen-activated protein kinase and nuclear factor-κB pathways in the skin of DNCB-induced AD mice. Conclusions I3C is suggested as a potential therapeutic alternative for the treatment of AD by repressing allergic inflammatory pathways. atopic dermatitis indole-3-carbinol TSLP periostin DNCB keratinocyte Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Atopic dermatitis (AD) is a long-standing and periodically recurring inflammatory disorder of the skin, which has seen a notable rise in incidence over recent years [ 1 ]. The pathogenesis of AD involves both impairment of the skin barrier and allergic inflammation of the skin, with the T-helper (Th) 2-type immune response playing a predominant role in the dermatological manifestations of the condition [ 2 ]. A wide array of allergens have been implicated in triggering AD, encompassing dietary allergens, airborne allergens, infectious agents, and physical irritants [ 3 ]. The immune response to these allergens involves both the innate and adaptive immune systems. It is widely acknowledged that repeated or continuous exposure to these allergens can lead to persistent chronic allergic inflammatory conditions [ 4 ]. Formerly regarded solely as constituents of the skin's protective barrier, keratinocytes are now recognized as integral components of the innate immune system, actively engaging in the inflammatory processes of AD by secreting pro-inflammatory cytokines and chemokines [ 5 ]. Notably, cytokines produced by activated keratinocytes, especially thymic stromal lymphopoietin (TSLP), are pivotal in triggering or enhancing Th2 immune responses, as well as driving the inflammatory processes in AD by mediating interactions between keratinocytes and dendritic cells (DCs) [ 6 ]. TSLP, akin to interleukin-7 (IL-7), is synthesized by epithelial cells residing at the barrier interfaces of the skin, lungs, and gastrointestinal tract [ 7 ] emerging as a key regulator of AD, notably abundant in keratinocytes within AD-afflicted human skin [ 8 ]. Furthermore, the induction of AD-like dermatitis in TSLP transgenic mice underscores its significance [ 9 ], while the absence of TSLP receptors in mice leads to a failure in developing allergic skin inflammation following epicutaneous sensitization to allergens [ 10 ]. Additionally, periostin, an extracellular matrix protein belonging to the fasciclin family, exhibits heightened expression in the skin of AD patients, with its levels significantly correlated to disease severity. It plays a crucial role in sustaining and exacerbating allergic skin inflammation by facilitating the production of TSLP and promoting Th2-driven immune reactions [ 11 ]. Periostin not only stimulates keratinocyte proliferation and viability but also directly triggers TSLP production [ 12 ], thereby creating a feedback loop that reinforces the Th2 immune response and keratinocyte activation within the AD pathology. Indole-3-carbinol (I3C) is abundant in cruciferous vegetables; broccoli, brussels sprouts, cabbage, collards, cauliflower, kale, mustard greens, turnips, and rutabagas. I3C is formed when these vegetables are cut, crushed, or cooked by the breakdown of glucosinolate glucobrassicin [ 13 ]. Within the gastrointestinal tract, I3C undergoes conversion into a biologically active dimer known as 3,3′-diindolylmethane (DIM) when ingested. Several studies have indicated that I3C has therapeutic potential for both the prevention and treatment of cancer as a chemopreventive agent in preclinical cancer models [ 14 ]. I3C and its metabolic derivatives suppress the proliferation of various cancer cell lines by targeting a wide spectrum of signaling pathways governing cell cycle progression, hormonal homeostasis, angiogenesis, cell cycle progression, cell survival, and proliferation[ 15 ]. Moreover, accumulating evidence has shown that I3C has multiple therapeutic activities, including anti-ulcer[ 16 ], anti-adipogenic[ 17 ], anti-inflammatory[ 18 , 19 ], anti-oxidant[ 20 ], cardioprotective[ 21 ], and cancer chemopreventive activities. Despite the fact that I3C possesses many potential therapeutic activities and advances in preclinical research, it has never been studied in experimental AD models. In the current study, we investigated the effect of I3C on AD pathogenesis using a 1-chloro-2,4-dinitrochlorobenzene (DNCB)-induced AD model and human keratinocytes. Materials and Methods Chemicals and reagents Sigma; EMD Millipore (Billerica, MA, USA) supplied I3C (I7256, ≥ 96%), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and all other chemicals for this study. Bio-Techne Ltd. (Abingdon, OX, UK) provided recombinant human tumor necrosis factor (TNF)-α and recombinant human IFN-γ. Life Technologies, Inc. (Grand Island, NY, USA) supplied Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin. Primary antibodies against p-p38 (cat no. 9211), p38 (cat no. 9212), c-Jun N-terminal kinase (JNK) (cat no. 9252), p-JNK (cat no. 9251), and p-TAK1 (cat no. 4508) were acquired from Cell Signaling Technology Inc. (Danvers, MA, USA). Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) provided primary antibodies against periostin (cat. sc-398631), TAK1 (cat. sc-7967), p-IκB-α (cat. sc-8404), IκB-α (cat. sc-203), involucrin (cat. sc-21748), loricrin (cat. sc-9542), and β-actin (Cat. sc-81178). Abcam (Cambridge, UK) and Novus Biologicals (Centennial, CO, USA) were the sources of TSLP (ab188766) and loricrin, respectively. Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA) supplied horseradish peroxidase-conjugated secondary antibodies. R&D Systems Inc. (Minneapolis, MN, USA) provided ELISA kits for TNF-α, IL-1β, IL-6, and IgE. Takara Bio, Inc. (Shiga, Japan) supplied SYBR Premix Ex Taq, and Bioneer Corporation (Daejeon, South Korea) provided oligonucleotide primers. Cell viability and sample treatment Provided by Professor Jae-Young Um (Kyung Hee University, Republic of Korea), HaCaT keratinocytes underwent cultivation at 37°C in DMEM supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL) within a humidified atmosphere of 5% CO 2 . To determine the toxicity of I3C on HaCaT keratinocytes, an MTT assay was performed. Cells were seeded in 96-well culture plates at a density of 5 × 10 4 cells/mL in culture medium and allowed to attach for 24 h. Cells were treated with medium containing various concentrations of I3C. After incubation for 24 h, the cells were treated with 50 µL of MTT (5 µg/mL) for 4 h. For investigation purposes, cells were seeded at a density of 1×10 5 cells per well, incubated for 24 h, and subjected to treatment with I3C at concentrations of 25, 50, and 100 µM for 1 h at 37°C in a humidified environment with 5% CO 2 . Subsequently, the cells were stimulated with 10 ng/mL of TNF-α/IFN-γ at 37°C, and the formazan precipitate was dissolved in DMSO. Absorbance was measured at 540 nm using a microplate reader. DNCB-induced AD model Acquired from Charles River Laboratories (Harlan Laboratories, Inc., Wilmington, MA, USA), thirty NC/Nga male mice (6 weeks old; 20–25 g body weight) were meticulously maintained under consistent conditions: a temperature range of 20–25 ˚C, humidity between 40–60%, and a 12-hour light/dark cycle. Randomly assigned to one of the five groups (n = 6 per group), the mice underwent induction of AD-like symptoms and skin lesions using DNCB. For the induction of AD-like symptoms and skin lesions, DNCB was employed. In a concise overview, the dorsal skin of the mice underwent stripping with cellophane tape. Subsequently, it was topically sensitized with 100 µL of 1% DNCB dissolved in a 4:1 v/v mixture of acetone and olive oil, applied to the shaved area of the dorsal surface for three consecutive days, followed by a four-day period of no treatment. Following the initial challenge that induced AD-like symptoms, the treatment regimen involved repeated stripping and application of 100 µL of 0.5% DNCB for a duration of 28 days. Mice received topical administration of either vehicle (acetone/olive oil), dexamethasone (5 mg/kg admixed in Vaseline), or I3C (50 or 100 mg/kg oral administration) 4 hours after each DNCB treatment, once a day over a span of 4 weeks. At the experiment's conclusion, lymph nodes and spleens were obtained for body weight comparison, and skin tissues were subjected to histological and western blot analyses. All procedures adhered to university guidelines and received approval from the Ethical Committee for Animal Care and the Use of Laboratory Animals, Korean Medicine, Sangji University (Wonju, Korea; approval no. 2021-05). Evaluation of dermatitis severity The severity of clinical dermatitis, evaluated at the experiment's onset and conclusion, utilized the method outlined by Yamamoto and colleagues. The scoring system, encompassing the development of erythema/hemorrhage, scarring/dryness, edema, and excoriation/erosion, employed a scale of 0 for none, 1 for mild ( 60%). The cumulative sum of individual scores was employed as the dermatitis score. Cytokine and IgE assay Culture media were collected post-treatment with I3C and stored at − 70°C. The levels of TNF-α, IL-1β, and IL-6 were measured using ELISA kits, according to the manufacturer’s instructions. At the conclusion of the experiment, blood samples were procured from each mouse. Serum was acquired through centrifugation at 1700 × g for 30 min and stored at − 80 ℃ until analysis. IgE release was measured using an ELISA kit, according to the manufacturer’s protocol. Measurement of transepidermal water loss (TEWL) The assessment of TEWL on the dorsal skin of NC/Nga mice was conducted at the 8-week mark using GPskin Barrier Light (Gpskin, Seoul, Republic of Korea), adhering to established protocols. TEWL in the mouse dorsal skin was measured under specific conditions at 24°C and 50–55% humidity. Placing the probe at the center of the shaved dorsum area of each mouse recorded the TEWL value in g/m2/h. Statistical values were expressed as a fold change compared to the control group. Histopathological analysis Dorsal skin samples were collected from the mice at the end of the study period. To detect epidermal thickness and inflammatory cells, the samples were fixed in 10% buffered formalin, embedded in paraffin, and sectioned into 4-µm-thick slices. Staining with hematoxylin and eosin (H&E) and toluidine blue was performed. Pathological changes in all stained skin sections were observed through a DM IL LED microscope (Leica, Wetzlar, Germany) and documented using a DFC295 camera (Leica, Wetzlar, Germany). Digital images were captured from each slide (three per group) and measured using the Leica Application Suite (Leica, Wetzlar, Germany). For immunohistochemistry (IHC), skin tissue slides were deparaffinized in xylene, rehydrated at different concentrations of ethanol (100%, 95%, 90%, 80%, and 70%), and hydrated with water. To quench endogenous peroxidase activity, slides were incubated with 0.6% H 2 O 2 in 50% MeOH. The slides were permeabilized with 0.3% Triton in PBS, pre-blocked with 10% NGS for 1 h, and then incubated overnight with a specific primary antibody at 4°C. The sections were then washed three times and incubated for 1 h with HRP-labeled secondary antibodies at room temperature. The antibody–antigen interaction was visualized using chromogenic DAB with hematoxylin and eosin counterstaining. For immunofluorescence analysis, the skin sections were incubated with primary antibodies overnight at 4°C. After washing, the slides were incubated with the secondary antibody Alexa Fluor 594-conjugated goat anti-mouse Invitrogen. Glass slides with mounted coverslips were utilized, and images were captured on a Leica TCS SP5 (LAS AF) microscope (Leica Microsystems) connected to a light microscope. Western blot analysis For the suspension of segments from cells or dorsal tissues, PRO-PREP™ protein extraction solution (Intron Biotechnology, Inc., Seoul, Korea) was employed, followed by a 20-minute incubation at 4°C. Cell debris elimination involved microcentrifugation at 11,000 × g for 30 minutes at 4°C, succeeded by the swift freezing of the supernatant. Protein concentration determination utilized Bio-Rad protein assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA) as per the manufacturer’s protocol. Cellular proteins from treated and untreated cell extracts (10–30 µL) underwent electroblotting onto a polyvinylidene fluoride membrane after separation with 8–12% SDS-PAGE. The membrane was subject to a 1-hour incubation with blocking solution (5% skim milk) at room temperature, followed by an overnight incubation with primary antibodies (1: 1,000) at 4°C. Subsequent washing three times with Tween 20/Tris-buffered saline (T/TBS) preceded a 2-hour incubation with a horseradish peroxidase-conjugated secondary antibody (1:2,000) at room temperature. Final steps involved three washes with T/TBS and development using enhanced chemiluminescence (GE Healthcare Life Sciences, Chalfont, UK). Densitometric analysis was executed with the Bio-Rad Quantity One software version 4.3.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Statistical analysis Data are expressed as the mean ± standard deviation of triplicate experiments. Statistically significant differences were compared using one-way analysis of variance and Dunnett’s post-hoc test. P < 0.05 was considered a statistically significant difference. Statistical analysis was performed using the SPSS statistical analysis software (version 19.0, IBM SPSS, Armonk, NY, USA). Results I3C inhibited the pro-inflammatory response and expression of TSLP and periostin in HaCaT keratinocytes Conducting an MTT assay enabled the evaluation of I3C's cytotoxic impact on human HaCaT keratinocytes. Notably, I3C exhibited no cytotoxic effects at concentrations up to 100 µM (Fig. 1 A), prompting further in vitro investigations at concentrations of 25, 50, and 100 µM. The study proceeded to analyze I3C's influence on the production of pro-inflammatory cytokines, namely TNF-α, IL-1β, and IL-6, in keratinocytes stimulated with TNF-α and IFN-γ. Results indicated a significant elevation in cytokine secretion following stimulation compared to baseline conditions, yet pre-treatment with I3C led to a decrease in TNF-α, IL-1β, and IL-6 levels in HaCaT keratinocytes (Fig. 1 B–D), with a more pronounced inhibitory effect observed on TNF-α and IL-1β. Given the recognized involvement of TSLP and periostin in AD pathogenesis [ 12 ], the study subsequently explored the impact of I3C on their expression. Western blot analysis revealed heightened TSLP and periostin expression in response to TNF-α and IFN-γ stimulation, which were attenuated upon treatment with I3C in HaCaT keratinocytes (Fig. 1 E). These findings suggest a potential role for I3C in mitigating skin inflammation through the inhibition of TSLP and periostin expression. I3C alleviated DNCB-induced AD-like skin symptoms in mice For the purpose of validating the effects of I3C in an in vivo setting, an experimental model of AD induced by DNCB was devised. Sensitization of mouse dorsal skin was achieved through the application of 1% DNCB, subsequently leading to the development of AD-like skin inflammation. Evaluation of the results revealed pronounced clinical manifestations on the dorsal skin of DNCB-challenged mice, characterized by erythema, edema, scarring, dryness, excoriation, and hemorrhage (Fig. 2 A), accompanied by elevated dermatitis scores and serum levels of IgE (Fig. 2 B and C). Remarkably, oral administration of I3C significantly mitigated AD-like symptoms and reduced IgE serum levels in the mice (Fig. 2 A–C). Furthermore, DNCB-induced enlargement of axillary lymph nodes and spleens was observed, which was notably reversed by I3C treatment, leading to decreased lymph node and spleen size and weight (Fig. 3 D and E). Additionally, trans-epidermal water loss (TEWL) and the frequency of scratching behaviors in I3C-treated mice were significantly diminished compared to DNCB-treated mice, with levels even lower than those observed in the positive control DEX group (Fig. 3 F and G). I3C attenuated histological alterations in skin of DNCB-induced AD mice The histopathological assessment of dorsal skin tissue, conducted through H&E and toluidine blue staining, provided additional insights into the impact of I3C on the histological characteristics of AD in mice. DNCB-challenged mice exhibited notable epidermal thickening and infiltration of inflammatory cells (Fig. 3 A and B). Additionally, toluidine blue staining revealed notable mast cell infiltration within the dermis of DNCB-induced AD mice (Fig. 3 C and D). Nevertheless, oral administration of I3C significantly mitigated epidermal thickening and reduced the abundance of mast cells within the skin tissue of AD mice (Fig. 3 A–D). I3C decreased the periostin and TSLP expression and recovered skin barrier proteins in the skin of DNCB-induced AD mice To explore the contribution of periostin to the pathogenesis of AD, we initially conducted a comparative analysis of periostin expression in skin lesions obtained from AD-afflicted mice. Utilizing immunohistochemical and immunofluorescent staining techniques, we observed a significant upregulation of periostin expression in the lesional skin of mice induced with DNCB-induced AD in contrast to skin samples from normal mice. Notably, periostin accumulation within the dermal region of the skin was particularly evident. However, upon treatment with I3C, there was a noticeable downregulation of periostin expression in the skin (Fig. 4 A and B). Furthermore, as depicted in Fig. 4 C, a substantial reduction in both TSLP and periostin expression was observed in the skin of mice administered with I3C compared to those in the DNCB group (Fig. 4 C). Subsequently, we examined alterations in the expression levels of epidermal proteins, specifically involucrin and loricrin, crucial constituents involved in forming the skin barrier. In the skin of DNCB-induced AD mice, diminished levels of involucrin and loricrin were observed compared to the normal group, whereas a pronounced increase was evident in the skin of mice treated with I3C (Fig. 4 C). I3C regulated p38, JNK MAPKs and NF-κB pathway in skin of DNCB-induced AD mice To expand our understanding of the underlying mechanisms in vivo, we investigated the potential molecular pathways through which I3C exerts its inhibitory effects on AD-like skin inflammation in mice induced with DNCB-induced AD. Our focus shifted towards examining the impact of I3C on components of the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways. As illustrated in Fig. 5 A, DNCB treatment led to heightened phosphorylation levels of p38 and JNK MAPK compared to those observed in the normal group, whereas these levels markedly decreased in the skin of mice treated with I3C. Moreover, DNCB exposure resulted in increased phosphorylation levels of TAK1 and IκBα, critical regulators of NF-κB activation; however, treatment with I3C led to a notable reduction in phosphorylation levels in DNCB-induced AD mice (Fig. 5 B). Discussion AD poses a formidable challenge in the realm of healthcare, lacking effective preventive measures or definitive treatments. Its onset stems from a multifaceted interplay of environmental and genetic factors [ 22 ], with immune-inflammatory complexities often heralding the development of asthma and other allergic conditions [ 23 ]. Periostin has recently emerged as a promising biomarker of type 2 inflammation in allergic diseases [ 24 ], notably induced by signature type 2 cytokines like IL-4 and IL-13 or exacerbating their effects, thereby propagating allergic skin inflammation [ 25 ]. Moreover, periostin assumes a critical role in AD pathogenesis by orchestrating the release of pro-inflammatory cytokines and chemokines, such as TSLP, IL-25, and IL-33, from activated keratinocytes [ 11 ]. In light of this, our study sought to investigate the therapeutic potential of targeting periostin and TSLP in AD-like skin inflammation. To establish an AD-like skin inflammation model, we devised a mouse model involving sensitization and challenge with DNCB, a commonly employed skin irritant. Our findings revealed that repeated DNCB applications upregulated TSLP and periostin expression concurrent with scratching-induced responses, epidermal hyperplasia, and compromised skin barrier integrity. Notably, our observations align with previous reports demonstrating that recurrent sensitization with house dust mite induces periostin accumulation in the dermis of mice [ 11 ]. This investigation marks the inaugural study to propose the anti-inflammatory effects of I3C on skin keratinocytes and in the AD animal model. Our observations revealed that I3C markedly reduced the production of pro-inflammatory cytokines, notably TNF-α and IL-1β, emanating from activated keratinocytes (referenced in Fig. 1 B–D). The stimulation of cytokines in keratinocytes and the application of DNCB on mouse dorsal skin significantly elevated the expression levels of TSLP and periostin, which were effectively diminished by I3C intervention (as depicted in Figs. 1 E and 4 ). Notably, we detected elevated serum IgE levels in DNCB-treated mice, a response instigated by the Th2 cell-mediated activation of B cells [ 26 ]; however, these levels were substantially reduced following I3C treatment (illustrated in Fig. 2 C). Th2 cytokines are known to suppress the expression of critical skin barrier proteins such as filaggrin, involucrin, and loricrin, leading to allergic skin inflammation and barrier impairment [ 27 ]. Our findings align with this understanding, showing an escalation in TSLP and periostin levels concomitant with the reduction of the skin barrier proteins involucrin and loricrin in DNCB-induced AD-like skin conditions, which were subsequently ameliorated by I3C treatment (as shown in Fig. 4 C). Nonetheless, significant expression of filaggrin in skin lesions was not detected in our study, either in keratinocytes or in DNCB-treated mice (data not presented). Through this study, we have elucidated the anti-inflammatory properties of I3C on AD-like skin, highlighting its potential to inhibit TSLP and periostin expression and modulate Th2 responses and skin barrier functionality. Periostin serves as a mediator of epithelial–mesenchymal transition via the MAPK signaling pathway [ 28 ] and it activates the NF-κB pathway through integrins, thereby contributing to the activation of epithelial/mesenchymal interactions within the skin [ 29 ]. Its significance as a matricellular protein in allergic diseases lies in its ability to bind to various integrin molecules on cell surfaces, thereby providing signals for tissue development and remodeling [ 30 ]. Accumulated periostin directly influences keratinocytes to produce cytokines such as TSLP by binding to αv integrin receptors on their surface. Notably, among these integrins, αvβ3 is recognized for its ability to activate NF-κB, a process critical for TSLP expression [ 31 ]. Intriguingly, several studies have investigated the suppressive effect of I3C on NF-κB activation [ 15 , 32 ], underscoring the significance of NF-κB inhibition within keratinocytes in the context of allergic skin inflammation. Furthermore, our study suggests that I3C mitigates AD-related skin inflammation by downregulating TSLP/periostin expression and inhibiting the signaling pathways associated with TSLP/periostin, including NF-κB and MAPK (depicted in Fig. 5 ). I3C is abundant in Cruciferae or Brassicaceae family plants, and medicinal herbs like Raphanus sativus Linné, Brassica campestris L., and Isatis indigotica Fort. are emblematic of this family in traditional Chinese medicine [ 33 , 34 ]. Since I3C is primarily found in Cruciferae vegetables and has various therapeutic benefits [ 35 , 36 ], there's a need for research on its content in Cruciferae herbs, including those mentioned. Additional in-depth studies derived from this study could clarify the composition and therapeutic efficacy of these herbal medicines, providing valuable insights into their pharmacological properties. Conclusions This study underscores the relevance of TSLP/periostin in AD-like skin and keratinocytes, hinting at the therapeutic potential of I3C in addressing TSLP/periostin-associated pathophysiology. However, elucidating the impact of I3C on skin-constituting cells beyond keratinocytes is imperative, necessitating further exploration of the molecular mechanisms underlying I3C's actions within the skin. Given the emerging roles of TSLP and periostin as diagnostic markers in conditions like asthma and obstructive lung disease [ 37 ], it is anticipated that I3C may exert multifaceted therapeutic effects against various diseases, including allergic skin inflammation. Our investigation reveals the collaborative involvement of the MAPK/NF-κB pathways in DNCB-induced AD-like skin inflammation and barrier dysfunction orchestrated by TSLP and periostin, effects substantially mitigated by I3C treatment. These findings underscore the therapeutic potential of targeting TSLP/periostin in AD skin pathogenesis and advocate for the utility of I3C in managing skin inflammation, particularly in AD. Abbreviations Atopic dermatitis (AD); Indole-3-carbinol (I3C); 2,4-dinitrochlorobenzen (DNCB); transepidermal water loss (TEWL); thymic stromal lymphopoietin (TSLP); mitogen-activated protein kinase (MAPK); nuclear factor-κB (NF-κB) Declarations Consent for publication Not applicable. Availability of data and materials The data of this study are available from the corresponding author upon reasonable request. Conflicts of interest The authors declare no conflicts of interest. Acknowledgements This research was supported by Korea Institute for Advancement of Technology (KIAT) (Project no. P0017805, HRD Program for Industrial Innovation) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea). Authors’ contributions Yun-Mi Kang: Investigation, Writing- Original draft preparation. Hye-Min Kim: Methodology, Visualization, Investigation, Writing- Reviewing and editing. Minho Lee: Validation, Supervision, Funding acquisition. Hyo-Jin An: Conceptualization, Data curation, Validation, Supervision. All authors have read and accept this as the final version of the manuscript. References Nutten S: Atopic dermatitis: global epidemiology and risk factors. Annals of nutrition & metabolism 2015, 66 Suppl 1: 8-16. Werfel T, Allam JP, Biedermann T, Eyerich K, Gilles S, Guttman-Yassky E, Hoetzenecker W, Knol E, Simon HU, Wollenberg A, et al: Cellular and molecular immunologic mechanisms in patients with atopic dermatitis. 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Boguniewicz M, Leung DY: Atopic dermatitis: a disease of altered skin barrier and immune dysregulation. Immunological reviews 2011, 242: 233-246. Izuhara K, Nunomura S, Nanri Y, Ono J, Takai M, Kawaguchi A: Periostin: An emerging biomarker for allergic diseases. Allergy 2019, 74: 2116-2128. Ozceker D, Yucel E, Sipahi S, Dilek F, Ozkaya E, Guler EM, Kocyigit A, Guler N, Tamay Z: Evaluation of periostin level for predicting severity and chronicity of childhood atopic dermatitis. Postepy dermatologii i alergologii 2019, 36: 616-619. Deo SS, Mistry KJ, Kakade AM, Niphadkar PV: Role played by Th2 type cytokines in IgE mediated allergy and asthma. Lung India : official organ of Indian Chest Society 2010, 27: 66-71. Mitamura Y, Nunomura S, Nanri Y, Ogawa M, Yoshihara T, Masuoka M, Tsuji G, Nakahara T, Hashimoto-Hachiya A, Conway SJ, et al: The IL-13/periostin/IL-24 pathway causes epidermal barrier dysfunction in allergic skin inflammation. Allergy 2018, 73: 1881-1891. Chen L, Tian X, Gong W, Sun B, Li G, Liu D, Guo P, He Y, Chen Z, Xia Y, et al: Periostin mediates epithelial-mesenchymal transition through the MAPK/ERK pathway in hepatoblastoma. Cancer biology & medicine 2019, 16: 89-100. Taniguchi K, Arima K, Masuoka M, Ohta S, Shiraishi H, Ontsuka K, Suzuki S, Inamitsu M, Yamamoto KI, Simmons O, et al: Periostin controls keratinocyte proliferation and differentiation by interacting with the paracrine IL-1alpha/IL-6 loop. The Journal of investigative dermatology 2014, 134: 1295-1304. Kuhn B, del Monte F, Hajjar RJ, Chang YS, Lebeche D, Arab S, Keating MT: Periostin induces proliferation of differentiated cardiomyocytes and promotes cardiac repair. Nature medicine 2007, 13: 962-969. Scatena M, Almeida M, Chaisson ML, Fausto N, Nicosia RF, Giachelli CM: NF-kappaB mediates alphavbeta3 integrin-induced endothelial cell survival. The Journal of cell biology 1998, 141: 1083-1093. Takada Y, Andreeff M, Aggarwal BB: Indole-3-carbinol suppresses NF-kappaB and IkappaBalpha kinase activation, causing inhibition of expression of NF-kappaB-regulated antiapoptotic and metastatic gene products and enhancement of apoptosis in myeloid and leukemia cells. Blood 2005, 106: 641-649. Pena M, Guzman A, Martinez R, Mesas C, Prados J, Porres JM, Melguizo C: Preventive effects of Brassicaceae family for colon cancer prevention: A focus on in vitro studies. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 2022, 151: 113145. Zhang N, Jing P: Anthocyanins in Brassicaceae: composition, stability, bioavailability, and potential health benefits. Critical reviews in food science and nutrition 2022, 62: 2205-2220. Centofanti F, Buono A, Verboni M, Tomino C, Lucarini S, Duranti A, Pandolfi PP, Novelli G: Synthetic Methodologies and Therapeutic Potential of Indole-3-Carbinol (I3C) and Its Derivatives. Pharmaceuticals 2023, 16 . Bacil GP, Romualdo GR, Rodrigues J, Barbisan LF: Indole-3-carbinol and chlorogenic acid combination modulates gut microbiome and attenuates nonalcoholic steatohepatitis in a murine model. Food research international 2023, 174: 113513. Nejman-Gryz P, Gorska K, Paplinska-Goryca M, Proboszcz M, Krenke R: Periostin and Thymic Stromal Lymphopoietin-Potential Crosstalk in Obstructive Airway Diseases. Journal of clinical medicine 2020, 9 . Cite Share Download PDF Status: Published Journal Publication published 26 Dec, 2024 Read the published version in Chinese Medicine → Version 1 posted Reviewers agreed at journal 25 Apr, 2024 Reviewers invited by journal 24 Mar, 2024 Editor assigned by journal 18 Mar, 2024 First submitted to journal 11 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4073342","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":283355624,"identity":"b18ee508-850a-4c95-9dc4-25ad206b3790","order_by":0,"name":"Yun-Mi Kang","email":"","orcid":"","institution":"Sangji University","correspondingAuthor":false,"prefix":"","firstName":"Yun-Mi","middleName":"","lastName":"Kang","suffix":""},{"id":283355625,"identity":"b22f4759-f319-4113-9615-27b98450d693","order_by":1,"name":"Hye-Min Kim","email":"","orcid":"","institution":"Sangji University","correspondingAuthor":false,"prefix":"","firstName":"Hye-Min","middleName":"","lastName":"Kim","suffix":""},{"id":283355626,"identity":"e8cb9578-7be9-4cb0-aaa9-4666eda68b44","order_by":2,"name":"Minho Lee","email":"","orcid":"","institution":"Dongguk University","correspondingAuthor":false,"prefix":"","firstName":"Minho","middleName":"","lastName":"Lee","suffix":""},{"id":283355627,"identity":"8ac8aa74-9b73-4ae7-9620-dce63f88f7fb","order_by":3,"name":"Hyo-Jin An","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAs0lEQVRIiWNgGAWjYBACA4YDDAwfDhwAcxgbiNXCOINELQwMzDwkaTFnPPzssc2ZO4kN7IcfMM7cQ4QWy4Zj5sY5N54lNvCkGTBueEaMww4cMJPO+XA4sYEhh4HxwQGitBz/Jm0B0sL/hmgtZ8ykGW4AtUgAbdlApJYyyZ4zh43bJJ4ZHJxBlJYbx7dJ/Dh2WLafP/nhwx5itDBIQFWxATFRGhgY+BuIUzcKRsEoGAUjGAAAnBtC+w7krwgAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-2937-874X","institution":"Kyung Hee University","correspondingAuthor":true,"prefix":"","firstName":"Hyo-Jin","middleName":"","lastName":"An","suffix":""}],"badges":[],"createdAt":"2024-03-11 11:31:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4073342/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4073342/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13020-024-01042-5","type":"published","date":"2024-12-26T15:57:39+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53562046,"identity":"96ee8e3c-c3fe-414c-a9bb-e2161c806641","added_by":"auto","created_at":"2024-03-27 13:46:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":143721,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of I3C on the pro-inflammatory cytokines, TSLP, and periostin in TNF-α/IFN-γ-stimulated HaCaT keratinocytes.\u003c/strong\u003e(A) Cell viability was measured using MTT assay in HaCaT keratinocytes. (B-D) Pro-inflammatory cytokines productions were measured using ELISA kit. (E) TSLP and periostin were western-blotted from total proteins. Internal control featured β-actin. Densitometric analysis was facilitated using Bio-Rad Quantity One® Software. The data presented represent the mean ± S.D. of three independent experiments. ### p \u0026lt; 0.001 vs. the control group; *p \u0026lt; 0.05, and ***p \u0026lt; 0.001 vs. the TNF-α/IFN-γ-stimulated group.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4073342/v1/b1875188d3c730efe5bc2510.png"},{"id":53562041,"identity":"1c573057-92b3-4b2e-b158-b13e824be8c0","added_by":"auto","created_at":"2024-03-27 13:46:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":258972,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of I3C on the clinical features of DNCB-induced AD skin in NC/Nga mice. \u003c/strong\u003e(A) Clinical features of AD-skin symptoms of mice. (B) Measurement of dermatitis scores occurred once a week over 5 weeks. The dermatitis score, defined as the sum of scores graded for each symptom, was recorded. (C) Serum level of IgE was measured using an ELISA kit. (D) Lymph nodes and (E) spleen weights of mice were recorded and shown as weight/body weight ratio (%). (F) Transepidermal water loss (TEWL) and (G) scratching number of mice were measured at end of 5\u0026nbsp;weeks. ###\u003csup\u003e \u003c/sup\u003ep \u0026lt; 0.001 vs. the control group;\u003csup\u003e \u003c/sup\u003e*p \u0026lt; 0.05, **p \u0026lt; 0.01, and ***p \u0026lt; 0.001 vs. DNCB-stimulated group.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4073342/v1/dc378c2a8a5a8884deb79542.png"},{"id":53562029,"identity":"97448f3b-800b-43d7-adbe-8526cefcdfbb","added_by":"auto","created_at":"2024-03-27 13:45:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1245122,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of I3C on histological alterations in DNCB-induced AD skin in NC/Nga mice. \u003c/strong\u003e(A) AD mouse skin lesions stained with H\u0026amp;E, showcasing a scale bar of 200 μm. (B) Measurement of epidermal thickness involved assessing H\u0026amp;E stained sections under a microscope. (C) Presentation of AD mouse skin lesions stained with toluidine blue, featuring a scale bar of 200 μm. (D) Mast cell infiltration in toluidine blue stained sections is quantified as the average total count across five fields. The presented data signify the mean ± S.D. of three independent experiments. ### p \u0026lt; 0.001 vs. the control group; ***p \u0026lt; 0.001 vs. the DNCB-stimulated group.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4073342/v1/6e8652a7966467a163864d2a.png"},{"id":53562039,"identity":"8ae1f2d2-bc34-47d8-ac9d-4209753c091d","added_by":"auto","created_at":"2024-03-27 13:46:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1079536,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of I3C on periostin, TSLP and skin barrier proteins in DNCB-induced AD skin in NC/Nga mice.\u003c/strong\u003ePeriostin expressions in mouse dorsal skin. Skin sections stained by (A) immunohistochemistry (scale bar = 50 μm) or (B) immunofluorescence with anti-periostin antibody. Red: periostin staining, blue: DAPI nuclei staining (scale bar = 10 μm). (C) TSLP, periostin, involucrin, and loricrin were the focus of western blotting in total protein preparations. Specific antibodies facilitated this analysis, with β-actin serving as the internal control. Densitometric assessment was conducted using Bio-Rad Quantity One® Software. The data depicted convey the mean ± S.D. of three independent experiments. ### p \u0026lt; 0.001 vs. the control group; ***p \u0026lt; 0.001 vs. the DNCB-stimulated group.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4073342/v1/1a1b11956e0308ea3cf34936.png"},{"id":53562030,"identity":"58bf693f-910a-445d-90a7-1c1bce1a8395","added_by":"auto","created_at":"2024-03-27 13:45:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":238912,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of I3C on the activation of MAPKs and NF-κB pathway in DNCB-induced AD skin in NC/Nga mice.\u003c/strong\u003eTotal proteins were prepared and western blotted for (A) p-JNK, JNK, p-p38, p38, (B) p-TAK1, TAK1, p-IκBα, and IκBα using specific antibodies. Internal control β-actin guided the western blotting for this segment. Densitometric analysis, facilitated by Bio-Rad Quantity One® Software, was applied to the data, illustrating the mean ± S.D. of three independent experiments. ### p \u0026lt; 0.001 vs. the control group; **p \u0026lt; 0.01, and ***p \u0026lt; 0.001 vs. the DNCB-stimulated group.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4073342/v1/0d4a2cb9dddbe48155e45748.png"},{"id":73056708,"identity":"09dd123e-b937-4b01-8fba-9dffcc3c4181","added_by":"auto","created_at":"2025-01-06 10:17:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5089663,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4073342/v1/1b1a4cdd-dbbf-48f2-bd7b-285ad0eee14b.pdf"}],"financialInterests":"","formattedTitle":"Indole-3-Carbinol Alleviates Allergic Skin inflammation via Periostin/Thymic Stromal Lymphopoietin Suppression in Atopic Dermatitis","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAtopic dermatitis (AD) is a long-standing and periodically recurring inflammatory disorder of the skin, which has seen a notable rise in incidence over recent years [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The pathogenesis of AD involves both impairment of the skin barrier and allergic inflammation of the skin, with the T-helper (Th) 2-type immune response playing a predominant role in the dermatological manifestations of the condition [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. A wide array of allergens have been implicated in triggering AD, encompassing dietary allergens, airborne allergens, infectious agents, and physical irritants [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The immune response to these allergens involves both the innate and adaptive immune systems. It is widely acknowledged that repeated or continuous exposure to these allergens can lead to persistent chronic allergic inflammatory conditions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFormerly regarded solely as constituents of the skin's protective barrier, keratinocytes are now recognized as integral components of the innate immune system, actively engaging in the inflammatory processes of AD by secreting pro-inflammatory cytokines and chemokines [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Notably, cytokines produced by activated keratinocytes, especially thymic stromal lymphopoietin (TSLP), are pivotal in triggering or enhancing Th2 immune responses, as well as driving the inflammatory processes in AD by mediating interactions between keratinocytes and dendritic cells (DCs) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. TSLP, akin to interleukin-7 (IL-7), is synthesized by epithelial cells residing at the barrier interfaces of the skin, lungs, and gastrointestinal tract [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] emerging as a key regulator of AD, notably abundant in keratinocytes within AD-afflicted human skin [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Furthermore, the induction of AD-like dermatitis in TSLP transgenic mice underscores its significance [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], while the absence of TSLP receptors in mice leads to a failure in developing allergic skin inflammation following epicutaneous sensitization to allergens [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Additionally, periostin, an extracellular matrix protein belonging to the fasciclin family, exhibits heightened expression in the skin of AD patients, with its levels significantly correlated to disease severity. It plays a crucial role in sustaining and exacerbating allergic skin inflammation by facilitating the production of TSLP and promoting Th2-driven immune reactions [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Periostin not only stimulates keratinocyte proliferation and viability but also directly triggers TSLP production [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], thereby creating a feedback loop that reinforces the Th2 immune response and keratinocyte activation within the AD pathology.\u003c/p\u003e \u003cp\u003eIndole-3-carbinol (I3C) is abundant in cruciferous vegetables; broccoli, brussels sprouts, cabbage, collards, cauliflower, kale, mustard greens, turnips, and rutabagas. I3C is formed when these vegetables are cut, crushed, or cooked by the breakdown of glucosinolate glucobrassicin [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Within the gastrointestinal tract, I3C undergoes conversion into a biologically active dimer known as 3,3\u0026prime;-diindolylmethane (DIM) when ingested. Several studies have indicated that I3C has therapeutic potential for both the prevention and treatment of cancer as a chemopreventive agent in preclinical cancer models [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. I3C and its metabolic derivatives suppress the proliferation of various cancer cell lines by targeting a wide spectrum of signaling pathways governing cell cycle progression, hormonal homeostasis, angiogenesis, cell cycle progression, cell survival, and proliferation[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Moreover, accumulating evidence has shown that I3C has multiple therapeutic activities, including anti-ulcer[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], anti-adipogenic[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], anti-inflammatory[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], anti-oxidant[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], cardioprotective[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and cancer chemopreventive activities. Despite the fact that I3C possesses many potential therapeutic activities and advances in preclinical research, it has never been studied in experimental AD models. In the current study, we investigated the effect of I3C on AD pathogenesis using a 1-chloro-2,4-dinitrochlorobenzene (DNCB)-induced AD model and human keratinocytes.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and reagents\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSigma; EMD Millipore (Billerica, MA, USA) supplied I3C (I7256, \u0026ge;\u0026thinsp;96%), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and all other chemicals for this study. Bio-Techne Ltd. (Abingdon, OX, UK) provided recombinant human tumor necrosis factor (TNF)-α and recombinant human IFN-γ. Life Technologies, Inc. (Grand Island, NY, USA) supplied Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin. Primary antibodies against p-p38 (cat no. 9211), p38 (cat no. 9212), c-Jun N-terminal kinase (JNK) (cat no. 9252), p-JNK (cat no. 9251), and p-TAK1 (cat no. 4508) were acquired from Cell Signaling Technology Inc. (Danvers, MA, USA). Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) provided primary antibodies against periostin (cat. sc-398631), TAK1 (cat. sc-7967), p-IκB-α (cat. sc-8404), IκB-α (cat. sc-203), involucrin (cat. sc-21748), loricrin (cat. sc-9542), and β-actin (Cat. sc-81178). Abcam (Cambridge, UK) and Novus Biologicals (Centennial, CO, USA) were the sources of TSLP (ab188766) and loricrin, respectively. Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA) supplied horseradish peroxidase-conjugated secondary antibodies. R\u0026amp;D Systems Inc. (Minneapolis, MN, USA) provided ELISA kits for TNF-α, IL-1β, IL-6, and IgE. Takara Bio, Inc. (Shiga, Japan) supplied SYBR Premix Ex Taq, and Bioneer Corporation (Daejeon, South Korea) provided oligonucleotide primers.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell viability and sample treatment\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eProvided by Professor Jae-Young Um (Kyung Hee University, Republic of Korea), HaCaT keratinocytes underwent cultivation at 37\u0026deg;C in DMEM supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 \u0026micro;g/mL) within a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e. To determine the toxicity of I3C on HaCaT keratinocytes, an MTT assay was performed. Cells were seeded in 96-well culture plates at a density of 5\u003cem\u003e\u0026times;\u003c/em\u003e10\u003csup\u003e4\u003c/sup\u003e cells/mL in culture medium and allowed to attach for 24 h. Cells were treated with medium containing various concentrations of I3C. After incubation for 24 h, the cells were treated with 50 \u0026micro;L of MTT (5 \u0026micro;g/mL) for 4 h. For investigation purposes, cells were seeded at a density of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well, incubated for 24 h, and subjected to treatment with I3C at concentrations of 25, 50, and 100 \u0026micro;M for 1 h at 37\u0026deg;C in a humidified environment with 5% CO\u003csub\u003e2\u003c/sub\u003e. Subsequently, the cells were stimulated with 10 ng/mL of TNF-α/IFN-γ at 37\u0026deg;C, and the formazan precipitate was dissolved in DMSO. Absorbance was measured at 540 nm using a microplate reader.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eDNCB-induced AD model\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAcquired from Charles River Laboratories (Harlan Laboratories, Inc., Wilmington, MA, USA), thirty NC/Nga male mice (6 weeks old; 20\u0026ndash;25 g body weight) were meticulously maintained under consistent conditions: a temperature range of 20\u0026ndash;25 ˚C, humidity between 40\u0026ndash;60%, and a 12-hour light/dark cycle. Randomly assigned to one of the five groups (n\u0026thinsp;=\u0026thinsp;6 per group), the mice underwent induction of AD-like symptoms and skin lesions using DNCB. For the induction of AD-like symptoms and skin lesions, DNCB was employed. In a concise overview, the dorsal skin of the mice underwent stripping with cellophane tape. Subsequently, it was topically sensitized with 100 \u0026micro;L of 1% DNCB dissolved in a 4:1 v/v mixture of acetone and olive oil, applied to the shaved area of the dorsal surface for three consecutive days, followed by a four-day period of no treatment. Following the initial challenge that induced AD-like symptoms, the treatment regimen involved repeated stripping and application of 100 \u0026micro;L of 0.5% DNCB for a duration of 28 days. Mice received topical administration of either vehicle (acetone/olive oil), dexamethasone (5 mg/kg admixed in Vaseline), or I3C (50 or 100 mg/kg oral administration) 4 hours after each DNCB treatment, once a day over a span of 4 weeks. At the experiment's conclusion, lymph nodes and spleens were obtained for body weight comparison, and skin tissues were subjected to histological and western blot analyses. All procedures adhered to university guidelines and received approval from the Ethical Committee for Animal Care and the Use of Laboratory Animals, Korean Medicine, Sangji University (Wonju, Korea; approval no. 2021-05).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of dermatitis severity\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe severity of clinical dermatitis, evaluated at the experiment's onset and conclusion, utilized the method outlined by Yamamoto and colleagues. The scoring system, encompassing the development of erythema/hemorrhage, scarring/dryness, edema, and excoriation/erosion, employed a scale of 0 for none, 1 for mild (\u0026lt;\u0026thinsp;20%), 2 for moderate (20\u0026ndash;60%), and 3 for severe (\u0026gt;\u0026thinsp;60%). The cumulative sum of individual scores was employed as the dermatitis score.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCytokine and IgE assay\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCulture media were collected post-treatment with I3C and stored at \u0026minus;\u0026thinsp;70\u0026deg;C. The levels of TNF-α, IL-1β, and IL-6 were measured using ELISA kits, according to the manufacturer\u0026rsquo;s instructions. At the conclusion of the experiment, blood samples were procured from each mouse. Serum was acquired through centrifugation at 1700 \u0026times; g for 30 min and stored at \u0026minus;\u0026thinsp;80 ℃ until analysis. IgE release was measured using an ELISA kit, according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of transepidermal water loss (TEWL)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe assessment of TEWL on the dorsal skin of NC/Nga mice was conducted at the 8-week mark using GPskin Barrier Light (Gpskin, Seoul, Republic of Korea), adhering to established protocols. TEWL in the mouse dorsal skin was measured under specific conditions at 24\u0026deg;C and 50\u0026ndash;55% humidity. Placing the probe at the center of the shaved dorsum area of each mouse recorded the TEWL value in g/m2/h. Statistical values were expressed as a fold change compared to the control group.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eDorsal skin samples were collected from the mice at the end of the study period. To detect epidermal thickness and inflammatory cells, the samples were fixed in 10% buffered formalin, embedded in paraffin, and sectioned into 4-\u0026micro;m-thick slices. Staining with hematoxylin and eosin (H\u0026amp;E) and toluidine blue was performed. Pathological changes in all stained skin sections were observed through a DM IL LED microscope (Leica, Wetzlar, Germany) and documented using a DFC295 camera (Leica, Wetzlar, Germany). Digital images were captured from each slide (three per group) and measured using the Leica Application Suite (Leica, Wetzlar, Germany).\u003c/p\u003e \u003cp\u003eFor immunohistochemistry (IHC), skin tissue slides were deparaffinized in xylene, rehydrated at different concentrations of ethanol (100%, 95%, 90%, 80%, and 70%), and hydrated with water. To quench endogenous peroxidase activity, slides were incubated with 0.6% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in 50% MeOH. The slides were permeabilized with 0.3% Triton in PBS, pre-blocked with 10% NGS for 1 h, and then incubated overnight with a specific primary antibody at 4\u0026deg;C. The sections were then washed three times and incubated for 1 h with HRP-labeled secondary antibodies at room temperature. The antibody\u0026ndash;antigen interaction was visualized using chromogenic DAB with hematoxylin and eosin counterstaining. For immunofluorescence analysis, the skin sections were incubated with primary antibodies overnight at 4\u0026deg;C. After washing, the slides were incubated with the secondary antibody Alexa Fluor 594-conjugated goat anti-mouse Invitrogen. Glass slides with mounted coverslips were utilized, and images were captured on a Leica TCS SP5 (LAS AF) microscope (Leica Microsystems) connected to a light microscope.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFor the suspension of segments from cells or dorsal tissues, PRO-PREP\u0026trade; protein extraction solution (Intron Biotechnology, Inc., Seoul, Korea) was employed, followed by a 20-minute incubation at 4\u0026deg;C. Cell debris elimination involved microcentrifugation at 11,000 \u0026times; g for 30 minutes at 4\u0026deg;C, succeeded by the swift freezing of the supernatant. Protein concentration determination utilized Bio-Rad protein assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA) as per the manufacturer\u0026rsquo;s protocol. Cellular proteins from treated and untreated cell extracts (10\u0026ndash;30 \u0026micro;L) underwent electroblotting onto a polyvinylidene fluoride membrane after separation with 8\u0026ndash;12% SDS-PAGE. The membrane was subject to a 1-hour incubation with blocking solution (5% skim milk) at room temperature, followed by an overnight incubation with primary antibodies (1: 1,000) at 4\u0026deg;C. Subsequent washing three times with Tween 20/Tris-buffered saline (T/TBS) preceded a 2-hour incubation with a horseradish peroxidase-conjugated secondary antibody (1:2,000) at room temperature. Final steps involved three washes with T/TBS and development using enhanced chemiluminescence (GE Healthcare Life Sciences, Chalfont, UK). Densitometric analysis was executed with the Bio-Rad Quantity One software version 4.3.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eData are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of triplicate experiments. Statistically significant differences were compared using one-way analysis of variance and Dunnett\u0026rsquo;s post-hoc test. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered a statistically significant difference. Statistical analysis was performed using the SPSS statistical analysis software (version 19.0, IBM SPSS, Armonk, NY, USA).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eI3C inhibited the pro-inflammatory response and expression of TSLP and periostin in HaCaT keratinocytes\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eConducting an MTT assay enabled the evaluation of I3C's cytotoxic impact on human HaCaT keratinocytes. Notably, I3C exhibited no cytotoxic effects at concentrations up to 100 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), prompting further in vitro investigations at concentrations of 25, 50, and 100 \u0026micro;M. The study proceeded to analyze I3C's influence on the production of pro-inflammatory cytokines, namely TNF-α, IL-1β, and IL-6, in keratinocytes stimulated with TNF-α and IFN-γ. Results indicated a significant elevation in cytokine secretion following stimulation compared to baseline conditions, yet pre-treatment with I3C led to a decrease in TNF-α, IL-1β, and IL-6 levels in HaCaT keratinocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u0026ndash;D), with a more pronounced inhibitory effect observed on TNF-α and IL-1β. Given the recognized involvement of TSLP and periostin in AD pathogenesis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], the study subsequently explored the impact of I3C on their expression. Western blot analysis revealed heightened TSLP and periostin expression in response to TNF-α and IFN-γ stimulation, which were attenuated upon treatment with I3C in HaCaT keratinocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). These findings suggest a potential role for I3C in mitigating skin inflammation through the inhibition of TSLP and periostin expression.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eI3C alleviated DNCB-induced AD-like skin symptoms in mice\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFor the purpose of validating the effects of I3C in an in vivo setting, an experimental model of AD induced by DNCB was devised. Sensitization of mouse dorsal skin was achieved through the application of 1% DNCB, subsequently leading to the development of AD-like skin inflammation. Evaluation of the results revealed pronounced clinical manifestations on the dorsal skin of DNCB-challenged mice, characterized by erythema, edema, scarring, dryness, excoriation, and hemorrhage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), accompanied by elevated dermatitis scores and serum levels of IgE (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and C). Remarkably, oral administration of I3C significantly mitigated AD-like symptoms and reduced IgE serum levels in the mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;C). Furthermore, DNCB-induced enlargement of axillary lymph nodes and spleens was observed, which was notably reversed by I3C treatment, leading to decreased lymph node and spleen size and weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and E). Additionally, trans-epidermal water loss (TEWL) and the frequency of scratching behaviors in I3C-treated mice were significantly diminished compared to DNCB-treated mice, with levels even lower than those observed in the positive control DEX group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF and G).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eI3C attenuated histological alterations in skin of DNCB-induced AD mice\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe histopathological assessment of dorsal skin tissue, conducted through H\u0026amp;E and toluidine blue staining, provided additional insights into the impact of I3C on the histological characteristics of AD in mice. DNCB-challenged mice exhibited notable epidermal thickening and infiltration of inflammatory cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). Additionally, toluidine blue staining revealed notable mast cell infiltration within the dermis of DNCB-induced AD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D). Nevertheless, oral administration of I3C significantly mitigated epidermal thickening and reduced the abundance of mast cells within the skin tissue of AD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;D).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eI3C decreased the periostin and TSLP expression and recovered skin barrier proteins in the skin of DNCB-induced AD mice\u003c/b\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo explore the contribution of periostin to the pathogenesis of AD, we initially conducted a comparative analysis of periostin expression in skin lesions obtained from AD-afflicted mice. Utilizing immunohistochemical and immunofluorescent staining techniques, we observed a significant upregulation of periostin expression in the lesional skin of mice induced with DNCB-induced AD in contrast to skin samples from normal mice. Notably, periostin accumulation within the dermal region of the skin was particularly evident. However, upon treatment with I3C, there was a noticeable downregulation of periostin expression in the skin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B). Furthermore, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, a substantial reduction in both TSLP and periostin expression was observed in the skin of mice administered with I3C compared to those in the DNCB group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Subsequently, we examined alterations in the expression levels of epidermal proteins, specifically involucrin and loricrin, crucial constituents involved in forming the skin barrier. In the skin of DNCB-induced AD mice, diminished levels of involucrin and loricrin were observed compared to the normal group, whereas a pronounced increase was evident in the skin of mice treated with I3C (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eI3C regulated p38, JNK MAPKs and NF-κB pathway in skin of DNCB-induced AD mice\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo expand our understanding of the underlying mechanisms in vivo, we investigated the potential molecular pathways through which I3C exerts its inhibitory effects on AD-like skin inflammation in mice induced with DNCB-induced AD. Our focus shifted towards examining the impact of I3C on components of the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, DNCB treatment led to heightened phosphorylation levels of p38 and JNK MAPK compared to those observed in the normal group, whereas these levels markedly decreased in the skin of mice treated with I3C. Moreover, DNCB exposure resulted in increased phosphorylation levels of TAK1 and IκBα, critical regulators of NF-κB activation; however, treatment with I3C led to a notable reduction in phosphorylation levels in DNCB-induced AD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAD poses a formidable challenge in the realm of healthcare, lacking effective preventive measures or definitive treatments. Its onset stems from a multifaceted interplay of environmental and genetic factors [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], with immune-inflammatory complexities often heralding the development of asthma and other allergic conditions [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Periostin has recently emerged as a promising biomarker of type 2 inflammation in allergic diseases [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], notably induced by signature type 2 cytokines like IL-4 and IL-13 or exacerbating their effects, thereby propagating allergic skin inflammation [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Moreover, periostin assumes a critical role in AD pathogenesis by orchestrating the release of pro-inflammatory cytokines and chemokines, such as TSLP, IL-25, and IL-33, from activated keratinocytes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In light of this, our study sought to investigate the therapeutic potential of targeting periostin and TSLP in AD-like skin inflammation. To establish an AD-like skin inflammation model, we devised a mouse model involving sensitization and challenge with DNCB, a commonly employed skin irritant. Our findings revealed that repeated DNCB applications upregulated TSLP and periostin expression concurrent with scratching-induced responses, epidermal hyperplasia, and compromised skin barrier integrity. Notably, our observations align with previous reports demonstrating that recurrent sensitization with house dust mite induces periostin accumulation in the dermis of mice [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis investigation marks the inaugural study to propose the anti-inflammatory effects of I3C on skin keratinocytes and in the AD animal model. Our observations revealed that I3C markedly reduced the production of pro-inflammatory cytokines, notably TNF-α and IL-1β, emanating from activated keratinocytes (referenced in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u0026ndash;D). The stimulation of cytokines in keratinocytes and the application of DNCB on mouse dorsal skin significantly elevated the expression levels of TSLP and periostin, which were effectively diminished by I3C intervention (as depicted in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Notably, we detected elevated serum IgE levels in DNCB-treated mice, a response instigated by the Th2 cell-mediated activation of B cells [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]; however, these levels were substantially reduced following I3C treatment (illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Th2 cytokines are known to suppress the expression of critical skin barrier proteins such as filaggrin, involucrin, and loricrin, leading to allergic skin inflammation and barrier impairment [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Our findings align with this understanding, showing an escalation in TSLP and periostin levels concomitant with the reduction of the skin barrier proteins involucrin and loricrin in DNCB-induced AD-like skin conditions, which were subsequently ameliorated by I3C treatment (as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Nonetheless, significant expression of filaggrin in skin lesions was not detected in our study, either in keratinocytes or in DNCB-treated mice (data not presented). Through this study, we have elucidated the anti-inflammatory properties of I3C on AD-like skin, highlighting its potential to inhibit TSLP and periostin expression and modulate Th2 responses and skin barrier functionality.\u003c/p\u003e \u003cp\u003ePeriostin serves as a mediator of epithelial\u0026ndash;mesenchymal transition via the MAPK signaling pathway [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and it activates the NF-κB pathway through integrins, thereby contributing to the activation of epithelial/mesenchymal interactions within the skin [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Its significance as a matricellular protein in allergic diseases lies in its ability to bind to various integrin molecules on cell surfaces, thereby providing signals for tissue development and remodeling [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Accumulated periostin directly influences keratinocytes to produce cytokines such as TSLP by binding to αv integrin receptors on their surface. Notably, among these integrins, αvβ3 is recognized for its ability to activate NF-κB, a process critical for TSLP expression [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Intriguingly, several studies have investigated the suppressive effect of I3C on NF-κB activation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], underscoring the significance of NF-κB inhibition within keratinocytes in the context of allergic skin inflammation. Furthermore, our study suggests that I3C mitigates AD-related skin inflammation by downregulating TSLP/periostin expression and inhibiting the signaling pathways associated with TSLP/periostin, including NF-κB and MAPK (depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eI3C is abundant in Cruciferae or Brassicaceae family plants, and medicinal herbs like Raphanus sativus Linn\u0026eacute;, Brassica campestris L., and Isatis indigotica Fort. are emblematic of this family in traditional Chinese medicine [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Since I3C is primarily found in Cruciferae vegetables and has various therapeutic benefits [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], there's a need for research on its content in Cruciferae herbs, including those mentioned. Additional in-depth studies derived from this study could clarify the composition and therapeutic efficacy of these herbal medicines, providing valuable insights into their pharmacological properties.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study underscores the relevance of TSLP/periostin in AD-like skin and keratinocytes, hinting at the therapeutic potential of I3C in addressing TSLP/periostin-associated pathophysiology. However, elucidating the impact of I3C on skin-constituting cells beyond keratinocytes is imperative, necessitating further exploration of the molecular mechanisms underlying I3C's actions within the skin. Given the emerging roles of TSLP and periostin as diagnostic markers in conditions like asthma and obstructive lung disease [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], it is anticipated that I3C may exert multifaceted therapeutic effects against various diseases, including allergic skin inflammation. Our investigation reveals the collaborative involvement of the MAPK/NF-κB pathways in DNCB-induced AD-like skin inflammation and barrier dysfunction orchestrated by TSLP and periostin, effects substantially mitigated by I3C treatment. These findings underscore the therapeutic potential of targeting TSLP/periostin in AD skin pathogenesis and advocate for the utility of I3C in managing skin inflammation, particularly in AD.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAtopic dermatitis (AD); Indole-3-carbinol (I3C); 2,4-dinitrochlorobenzen (DNCB); transepidermal water loss (TEWL); thymic stromal lymphopoietin (TSLP); mitogen-activated protein kinase (MAPK); nuclear factor-\u0026kappa;B (NF-\u0026kappa;B)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;This research was supported by Korea Institute for Advancement of Technology (KIAT) (Project no. P0017805, HRD Program for Industrial Innovation) funded by the Ministry of Trade, Industry \u0026amp; Energy (MOTIE, Korea).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYun-Mi Kang: Investigation, Writing- Original draft preparation. Hye-Min Kim: Methodology, Visualization, Investigation, Writing- Reviewing and editing. Minho Lee: Validation, Supervision, Funding acquisition. Hyo-Jin An: Conceptualization, Data curation, Validation, Supervision. All authors have read and accept this as the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNutten S: \u003cstrong\u003eAtopic dermatitis: global epidemiology and risk factors.\u003c/strong\u003e \u003cem\u003eAnnals of nutrition \u0026amp; metabolism \u003c/em\u003e2015, \u003cstrong\u003e66 Suppl 1:\u003c/strong\u003e8-16.\u003c/li\u003e\n\u003cli\u003eWerfel T, Allam JP, Biedermann T, Eyerich K, Gilles S, Guttman-Yassky E, Hoetzenecker W, Knol E, Simon HU, Wollenberg A, et al: \u003cstrong\u003eCellular and molecular immunologic mechanisms in patients with atopic dermatitis.\u003c/strong\u003e \u003cem\u003eThe Journal of allergy and clinical immunology \u003c/em\u003e2016, \u003cstrong\u003e138:\u003c/strong\u003e336-349.\u003c/li\u003e\n\u003cli\u003eWerfel T: \u003cstrong\u003eThe role of leukocytes, keratinocytes, and allergen-specific IgE in the development of atopic dermatitis.\u003c/strong\u003e 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\u003cstrong\u003e9\u003c/strong\u003e.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"atopic dermatitis, indole-3-carbinol, TSLP, periostin, DNCB, keratinocyte","lastPublishedDoi":"10.21203/rs.3.rs-4073342/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4073342/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAtopic dermatitis (AD) is a chronic multifactorial inflammatory skin disorder with a complex etiology. Despite its increasing prevalence, treatment of AD is still limited. Indole-3-carbinol (I3C) is found in cruciferous vegetables and is formed when these vegetables are cut, chewed, or cooked; it exerts diverse pharmacological activities.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eHaCaT keratinocytes stimulated with tumor necrosis factor-α and interferon-γ mixture and NC/Nga mice stimulated with 2,4-dinitrochlorobenzen (DNCB) were used for AD models, \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, respectively.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe results showed that I3C reduced the expression of pro-inflammatory cytokines, thymic stromal lymphopoietin (TSLP), and periostin in \u003cem\u003ein vitro\u003c/em\u003e model. Oral administration of I3C alleviated AD-like skin inflammatory symptoms, including serum IgE levels, epidermal thickening, inflammatory cell infiltration, transepidermal water loss, and scratching behavior. Moreover, I3C decreased the expression of TSLP and periostin and recovered the expression of skin barrier proteins by inhibiting the mitogen-activated protein kinase and nuclear factor-κB pathways in the skin of DNCB-induced AD mice.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eI3C is suggested as a potential therapeutic alternative for the treatment of AD by repressing allergic inflammatory pathways.\u003c/p\u003e","manuscriptTitle":"Indole-3-Carbinol Alleviates Allergic Skin inflammation via Periostin/Thymic Stromal Lymphopoietin Suppression in Atopic Dermatitis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-27 13:45:44","doi":"10.21203/rs.3.rs-4073342/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-04-26T03:55:59+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-25T01:41:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-18T17:43:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Chinese Medicine","date":"2024-03-11T07:31:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a2050274-6e4b-4f70-91b8-4542a5f5107d","owner":[],"postedDate":"March 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-30T16:05:16+00:00","versionOfRecord":{"articleIdentity":"rs-4073342","link":"https://doi.org/10.1186/s13020-024-01042-5","journal":{"identity":"chinese-medicine","isVorOnly":false,"title":"Chinese Medicine"},"publishedOn":"2024-12-26 15:57:39","publishedOnDateReadable":"December 26th, 2024"},"versionCreatedAt":"2024-03-27 13:45:44","video":"","vorDoi":"10.1186/s13020-024-01042-5","vorDoiUrl":"https://doi.org/10.1186/s13020-024-01042-5","workflowStages":[]},"version":"v1","identity":"rs-4073342","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4073342","identity":"rs-4073342","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}