Aqueous surfactant preparation for relieving atopic dermatitis and application

Abstract

The invention provides a water-based surfactant preparation for relieving atopic dermatitis and application, and belongs to the field of external preparation development. The active ingredient of the aqueous surfactant preparation is an anionic surfactant for inhibiting an STAT3 signal. NF-kappa B and MAPK signal channels are indirectly inhibited by inhibiting STAT3 activation, and expression of inflammatory factors is remarkably reduced. Compared with a traditional surfactant SLS, the water-based surfactant preparation does not affect cell activity, shows good anti-inflammatory activity in vivo and in vitro, has the advantages of relieving atopic dermatitis skin lesion, reducing serum IgE level, relieving mast cell infiltration and the like, and provides a safer cleaning or external anti-inflammatory choice for AD patients. Therefore, the traditional surfactant is utilized to relieve the inflammation of atopic dermatitis, and meanwhile, the problem that the traditional surfactant irritates skin and aggravates inflammatory response is avoided.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of external preparation development, and particularly relates to a water-based surfactant preparation for relieving atopic dermatitis and application. BACKGROUND

[0002] Atopic dermatitis (AD) is a common chronic inflammatory skin disease, and its main features include persistent inflammation, skin barrier dysfunction and immunoglobulin E (IgE)-mediated sensitization. The prevalence of children is as high as 25%, and the prevalence of adults is about 6% to 10%. The excessive activation of Th2 type inflammatory response and the dysfunction of skin barrier are the core mechanisms of AD.

[0003] Skin cleaning is a key link in the management of AD, and appropriate cleaning can effectively remove irritants and allergens on the skin surface. Surfactants are the main components of cleaning agents, which can reduce the skin surface tension, emulsify and remove dirt, sebum and sweat. However, the hygiene hypothesis suggests that the improvement of family economic level and cleaning conditions may be related to the increase of AD incidence, which indicates that the overuse of cleaning products may cause skin irritation and aggravate AD symptoms. SUMMARY

[0004] The present application provides a water-based surfactant preparation for relieving atopic dermatitis and application, so as to solve the technical problem of how to use traditional surfactants to relieve inflammation of atopic dermatitis while avoiding the problem of traditional surfactants stimulating the skin and aggravating the inflammatory response.

[0005] In a first aspect, the present application provides a water-based surfactant preparation for relieving atopic dermatitis, wherein the effective component of the water-based surfactant preparation is an anionic surfactant for inhibiting STAT3 signal, and the STAT signal pathway has the following performance: indirectly inhibiting the signal pathways of NF-κB and MAPK.

[0006] Optionally, the anionic surfactant is sodium dodecyl benzene sulfonate or a structural analogue of sodium dodecyl benzene sulfonate.

[0007] Optionally, the mass concentration of the anionic surfactant is 0.1% to 1.0%.

[0008] Optionally, the mass concentration of the anionic surfactant is 0.5% to 1.0%.

[0009] Optionally, the dosage form of the water-based surfactant preparation includes a patch, a paste, an ointment, a cream, a gel, an oil, a microneedle, a film, a cataplasm, a spray and a dressing agent.

[0010] Optionally, the aqueous surfactant preparation further comprises a pharmaceutically acceptable carrier.

[0011] Optionally, the pharmaceutically acceptable carrier comprises one or more of ethanol, an aqueous carrier, glycerol, an aqueous gel, propylene glycol, and a vegetable oil.

[0012] In a second aspect, the present application provides the use of the aqueous surfactant preparation of any one of the embodiments of the first aspect in the manufacture of a medicament for treating atopic dermatitis.

[0013] Optionally, the aqueous surfactant preparation is administered topically to the skin lesion area once or multiple times per day.

[0014] Optionally, the aqueous surfactant preparation has any one of the pharmacological effects of down-regulating the expression of TSLP and IL-6 key inflammatory factors, relieving atopic dermatitis lesions, reducing the epidermal thickness of the lesion site, reducing serum IgE levels, and reducing mast cell infiltration, and is used in the manufacture of a medicament for treating atopic dermatitis.

[0015] Compared with the prior art, the above technical solutions provided by the embodiments of the present application have the following advantages:

[0016] The present application provides an aqueous surfactant preparation for relieving atopic dermatitis, which uses an anionic surfactant that inhibits STAT3 signaling as an effective ingredient, indirectly inhibits the NF-κB and MAPK signaling pathways by inhibiting STAT3 activation, and significantly reduces the expression of inflammatory factors. Compared with the traditional surfactant SLS, the aqueous surfactant preparation of the present application does not affect cell viability, and shows good anti-inflammatory activity in vivo and in vitro, has advantages such as relieving atopic dermatitis lesions, reducing serum IgE levels, and reducing mast cell infiltration, and provides a safer cleaning or external anti-inflammatory option for AD patients. Thus, the traditional surfactant is used to achieve inflammation relief for atopic dermatitis, while avoiding the problem of stimulating the skin and aggravating the inflammatory response of the traditional surfactant. BRIEF DESCRIPTION OF DRAWINGS

[0017] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and serve to explain the principles of the present application together with the description.

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings needed to be used in the embodiments or prior art description will be briefly introduced as follows. Obviously, for those skilled in the art, other drawings can also be obtained based on these drawings without any creative effort.

[0019] Figure 1A molecular structure of SDBS provided for the embodiments of the present application;

[0020] Figure 2 A time axis diagram of a mouse AD-like model induced by MC903 provided for the embodiments of the present application;

[0021] Figure 3 Representative images of clinical manifestations of MC903-induced AD-like mice after topical treatment with ethanol, SDBS, desonide or tacrolimus on day 21 provided for the embodiments of the present application;

[0022] Figure 4 Comparison of ear lesion thickness (left, mm) and dermatitis severity score (right) of mice on day 21 provided for the embodiments of the present application;

[0023] Figure 5 Serum total immunoglobulin E (IgE) levels of MC903-induced AD-like mice provided for the embodiments of the present application;

[0024] Figure 6 Representative histological images of hematoxylin-eosin (H&E) staining (400x) of skin tissue (left) and epidermis thickness (right, pm) of mice on day 21 provided for the embodiments of the present application;

[0025] Figure 7 Toluidine blue staining of ear skin tissue of MC903-induced AD-like mice to show mast cell infiltration. The right panel shows the number of mast cells counted in 5 randomly selected high power fields (HPF, 400x);

[0026] Figure 8 Immunohistochemical analysis showing TSLP expression (brown staining, 400x) in the epidermis of the ears of MC903-induced AD-like mice. The right panel shows the number of TSLP-positive cells counted in 5 randomly selected high power fields (HPF, 400x) (n = 8);

[0027] Figure 9 Serum TSLP (left) and IL-6 (right) levels of MC903-induced AD-like mice provided for the embodiments of the present application;

[0028] Figure 10 Tslp, Il4, Il6 and Il13 mRNA expression levels in the ears of MC903-induced AD-like mice relative to the expression of the β-actin gene provided for the embodiments of the present application;

[0029] Figure 11Cell viability (n=3) of HaCaT cells pretreated with different concentrations of SDBS (0, 5, 10, 15, 20, 25 and 30 pg / ml) provided for the examples of the present application;

[0030] Figure 12 TSLP and IL-6 protein levels and mRNA levels in T / I-stimulated HaCaT cells treated with different concentrations of SDBS (0, 2, 5, 10, 20 pg / ml) determined by enzyme-linked immunosorbent assay (ELISA) (n=6) provided for the examples of the present application;

[0031] Figure 13 Western blot analysis of p-STAT3, STAT3, p-NF-κB p65, NF-κB p65, p-IκB-α, IκB-α, p-p38 and p38 protein levels in MC903-induced AD-like mouse ears (n=5) provided for the examples of the present application;

[0032] Figure 14 Band density quantification of p-STAT3, STAT3, p-NF-κB p65, NF-κB p65, p-IκB-α, IκB-α, p-p38 and p38 proteins in MC903-induced AD-like mouse ears normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (n=5) provided for the examples of the present application;

[0033] Figure 15 p-STAT3, STAT3, p-p38, p38, p-ERK1 / 2, ERK1 / 2, p-cytoplasmic NF-κB p65, cytoplasmic NF-κB p65, p-nuclear NF-κB p65, nuclear NF-κB p65, p-IκB-α, IκB-α protein levels in T / I-stimulated HaCaT cells treated with different concentrations of SDBS (0, 2, 5, 10, 20 pg / ml) (n=3) provided for the examples of the present application;

[0034] Figure 16Band density quantification of p-STAT3, STAT3, p-p38, p38, p-ERK1 / 2, ERK1 / 2, p-cytoplasmic NF-κB p65, cytoplasmic NF-κB p65, p-IκB-α and IκB-α in T / I-stimulated HaCaT cells treated with different concentrations of SDBS (0, 2, 5, 10, 20 μg / ml) and normalized to β-actin (n=3);

[0035] Figure 17 TSLP and IL-6 protein levels in MC903-induced AD-like mice treated with 10 μg / ml SDBS and / or colivelin (n=6) provided for the embodiments of the present application;

[0036] Figure 18 Timeline of SDBS treatment and intraperitoneal injection of colivelin in MC903-induced AD-like mice provided for the embodiments of the present application;

[0037] Figure 19 Representative images of clinical scores and histological H&E staining (200x) of MC903-induced AD-like mice treated with ethanol, SDBS, desonide and / or colivelin on day 21 (n=8) provided for the embodiments of the present application;

[0038] Figure 20 Dermatitis scores of mouse skin lesions (n=8) provided for the embodiments of the present application;

[0039] Figure 21 Epidermal thickness (μm) of mouse skin lesions (n=8) provided for the embodiments of the present application;

[0040] Figure 22 p-STAT3, STAT3, p-IκB-α, IκB-α, p-ERK1 / 2 and ERK1 / 2 protein levels in the ears of MC903-induced AD-like mice by Western immunoblotting analysis (n=5) provided for the embodiments of the present application;

[0041] Figure 23 Band density quantification of p-STAT3, STAT3, p-IκB-α, IκB-α, p-ERK1 / 2 and ERK1 / 2 protein in the ears of MC903-induced AD-like mice and normalized to β-actin (n=5) provided for the embodiments of the present application. DETAILED DESCRIPTION

[0042] The technical solutions and advantages of the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0043] Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or prepared by existing methods.

[0044] Atopic dermatitis (AD) is a common chronic inflammatory skin disease often accompanied by epidermal barrier impairment and Th2-dominated immune imbalance. Anionic surfactants widely used in cleansing products, such as SLS (sodium lauryl sulfate), are considered to potentially disrupt the skin barrier and induce proinflammatory cytokine expression, thereby exacerbating AD symptoms.

[0045] Sodium dodecyl benzene sulfonate (SDBS) is a widely used anionic surfactant, commonly used as a cleaning agent and emulsifier in household, industrial and cosmetic products. In cosmetics, SDBS is used as a cleansing ingredient, and its degree of skin absorption is related to pH value. Toxicology studies have shown that SDBS has low acute toxicity by oral or dermal exposure, and no systemic toxicity has been observed with repeated dermal exposure. However, at high concentrations (15%), SDBS, like other cleansing agents, can cause severe irritation reactions in animal skin. The Cosmetic Ingredient Review Expert Panel (CIR) recommends optimizing SDBS formulations to reduce their irritancy. Studies have found that SDBS can cause direct cytotoxicity to human keratinocytes (HaCaT cells) at certain concentrations (≥20 μg / ml) and cause morphological changes. However, unlike the more studied anionic surfactant sodium lauryl sulfate (SLS), SDBS does not significantly up-regulate the expression of key pro-inflammatory cytokines such as IL-1α, IL-6, IL-8 and TNF-α, suggesting that its mechanism of action in skin inflammation may be different. Studies have pointed out that the difference in carbon chain length of anionic surfactants may lead to differences in their biological effects, further highlighting the need for in-depth study of the mechanism of action of SDBS. However, the mechanism of action of sodium dodecyl benzene sulfonate as a surfactant in AD inflammation has not been clearly defined, and it has long been suspected of potentially exacerbating inflammation.

[0046] The existing cleansing products for AD patients tend to lack specificity, and conventional surfactants such as SLS have strong irritation and obvious pro-inflammatory effects. In addition, there is no research on the development of SDBS for anti-inflammatory purposes, especially the lack of reasonable dosage form design and application methods based on its ability to regulate inflammatory signaling pathways.

[0047] Therefore, based on the above-mentioned problem of how to use traditional surfactants to achieve inflammation relief for atopic dermatitis while avoiding the irritation of traditional surfactants to the skin and exacerbating inflammatory reactions, the embodiments of the present application provide the following content:

[0048] The present application provides an aqueous surfactant preparation for relieving atopic dermatitis, the effective component of which is an anionic surfactant that inhibits STAT3 signaling, which has the following performance: indirectly inhibits the signaling pathways of NF-κB and MAPK.

[0049] It should be noted that STAT3 (Signal Transducer and Activator of Transcription 3) is a transcription factor activated by cytokines (such as IL-6), which forms a dimer through phosphorylation and enters the nucleus to regulate gene expression.

[0050] NF-κB (Nuclear Factor-kappaB) is a core regulator of inflammatory response, involved in TLRs, TNF-α, and other signal transduction. STAT3 inhibition can reduce the activation of NF-κB through downstream regulation, thereby reducing the expression of inflammatory factors such as IL-6 and TNF-α.

[0051] MAPK (Mitogen-Activated Protein Kinase) includes subtypes such as ERK, JNK, and p38, involved in cell proliferation, differentiation, and inflammatory response.

[0052] In some embodiments, the anionic surfactant is sodium dodecyl benzene sulfonate or a structural analogue of sodium dodecyl benzene sulfonate.

[0053] In some embodiments, the mass concentration of the anionic surfactant is 0.1% to 1.0%.

[0054] In some embodiments, the mass concentration of the anionic surfactant is 0.5% to 1.0%.

[0055] The technical scheme provided by the embodiments of the present application uses sodium dodecyl benzene sulfonate (SDBS) as an effective component, indirectly inhibits NF-κB and MAPK signaling pathways by inhibiting STAT3 activation, and significantly reduces the expression of inflammatory factors. Compared with the traditional surfactant SLS, the preparation of the present application does not affect cell viability, and shows good anti-inflammatory activity in vivo and in vitro. It has the advantages of relieving atopic dermatitis lesions, reducing serum IgE levels, reducing mast cell infiltration, etc., and provides a safer cleaning or external anti-inflammatory option for AD patients. For example, the mass concentration of the anionic surfactant can be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.7%, 0.9%, 1.0%, etc.

[0056] In some embodiments, the dosage form of the aqueous surfactant preparation includes a patch, a paste, an ointment, a cream, a gel, an oil, a microneedle, a film, a cataplasm, a spray, and a dressing agent.

[0057] In some embodiments, the aqueous surfactant preparation further comprises a pharmaceutically acceptable carrier.

[0058] In some embodiments, the pharmaceutically acceptable carrier comprises one or more of ethanol, an aqueous carrier, glycerol, an aqueous gel, propylene glycol, and a vegetable oil.

[0059] In addition to using SDBS as the main component, the embodiments of the present application can also attempt to use anionic surfactants with similar structures or mechanisms of action to replace it, such as structural analogues of sodium dodecyl benzene sulfonate or the addition of other STAT3 signal inhibiting synergistic components. The carrier part can also be replaced by glycerol, aqueous gel or other skin-friendly matrix to meet the needs of different groups of people.

[0060] In some embodiments, the preparation method of the aqueous surfactant preparation is to uniformly mix sodium dodecyl benzene sulfonate and a pharmaceutically acceptable carrier according to a set mass ratio to obtain the aqueous surfactant preparation.

[0061] Based on one overall inventive concept, the present application provides the use of the aqueous surfactant preparation of any one of the above embodiments in the treatment of atopic dermatitis.

[0062] In some embodiments, the administration method of the aqueous surfactant preparation is to apply it topically and externally to the skin lesion area once or multiple times a day.

[0063] In some embodiments, the aqueous surfactant formulation has any one of the pharmacodynamic effects of down-regulating TSLP, IL-6 key inflammatory factor expression, alleviating atopic dermatitis lesions, reducing epidermal thickness at the lesion site, reducing serum IgE levels, and reducing mast cell infiltration in the treatment of atopic dermatitis.

[0064] It should be noted that TSLP is a key initiator of Th2-type immune response, which is secreted by keratinocytes, can activate dendritic cells and promote the release of inflammatory factors such as IL-4, IL-5, IL-13.

[0065] IL-6 is an upstream activator of the STAT3 pathway, which promotes Th17 differentiation and exacerbates inflammation.

[0066] IgE is a key antibody of allergic reactions, and its level is positively correlated with the severity of atopic dermatitis.

[0067] Mast cells infiltrate in large quantities in AD lesions, releasing histamine, tryptase and other pruritogenic mediators.

[0068] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples are not specified, which are generally determined according to the industry standard. If there is no corresponding industry standard, it is carried out according to the general international standard, the conventional condition, or according to the condition suggested by the manufacturer.

[0069] In this example, MC903-induced AD-like mouse models and TNF-α / IFN-γ (T / I)-stimulated keratinocyte models were used to systematically investigate the effects of SDBS on AD and its potential molecular mechanisms, and to evaluate its effects on the clinical manifestations, histopathological changes, immune responses and related signaling pathways of AD.

[0070] 1. Materials and methods

[0071] 1.1 Animal experiments

[0072] 6-week-old male BALB / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were raised in a specific pathogen-free (SPF) environment. All animal experiments were conducted in accordance with the ARRIVE guidelines to ensure animal welfare and ethical standards, and the research protocol was approved by the Ethics Committee of the Medical Department of Peking University and implemented in accordance with its guidelines.

[0073] The AD-like mouse model was established by applying 20 μL (2 nmol) of MC903 topically to the skin behind the ears of both ears daily for 20 consecutive days; the ethanol-treated group served as the control. From day 15 to day 20, the experimental mice were topically applied with 20 μL of vehicle (ethanol), 0.1% SDBS (Sigma-Aldrich, USA, dissolved in ethanol), 1% SDBS, desonide cream (Huabang Pharmaceutical, China), or tacrolimus ointment (Astellas Pharma, Japan) daily as the positive control group ( Figure 2 Starting from the 15th day, STAT3 agonist colivelin was injected intraperitoneally 2 hours before local treatment every day at a dose of 7 nmol / mouse ( Figure 18 On days 15, 17, 19, and 21, mice were anesthetized with 2% isoflurane and photographed (Canon EOS1200D, Japan). Dermatitis severity was assessed by erythema / hemorrhage, edema, and scaling / dryness, and each score was assigned a numerical value (0: none; 1: mild; 2: moderate; 3: severe). Ear thickness was also measured using a vernier caliper (Satatool, USA).

[0074] 1.2 Histological analysis

[0075] After the experiment, bilateral ear skin samples were collected from mice and fixed in 4% paraformaldehyde for 24 hours. After dehydration and paraffin embedding, the samples were stained with hematoxylin and eosin (H&E) and toluidine blue to detect mast cells. Five high-power fields (HPF) were randomly selected from each section, and the number of mast cells was counted by three pathologists. Images were acquired using a light microscope (Leica DM4000B, Germany).

[0076] 1.3 Immunohistochemical staining

[0077] Paraffin sections were treated with xylene and graded ethanol, rinsed in PBS containing 0.025% Triton X-100, and subjected to high-temperature antigen retrieval. Sections were blocked with normal goat serum for 20 minutes at room temperature and incubated with rabbit polyclonal anti-TSLP antibody (1:500, Abcam, UK) overnight at 4°C. The sections were then incubated with horseradish peroxidase-conjugated anti-rabbit IgG for 30 minutes and developed with diabetic retinopathy (DAB). Five high-power fields (HPF) were randomly selected from each section, and the number of TSLP-positive cells was counted by three pathologists.

[0078] 1.4 Cell culture

[0079] Human keratinocyte cell line HaCaT (Sigma-Aldrich, USA) was cultured in high-glucose DMEM medium (Sigma-Aldrich, USA) containing 10% fetal bovine serum and maintained in a 37°C, 5% CO2 incubator.

[0080] 1.5 Cell viability assay

[0081] HaCaT cells were cultured at 5×10 4 Cells were seeded in 96-well plates and treated with different concentrations of SDBS or SLS. After 24 hours of treatment, 300 μL of MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to each well and incubated at 37°C for 2–4 hours. The crystals were then dissolved in DMSO and the absorbance at 550 nm was measured using a microplate reader (Perkin-Elmer, USA).

[0082] 1.6 Enzyme-linked immunosorbent assay (ELISA)

[0083] On the day of the experiment, whole blood was collected from mice and centrifuged at 3000 rpm for 10 minutes to obtain serum. Serum IgE, TSLP, and IL-6 levels were measured using ELISA kits (Abcam, UK). To detect the expression of proinflammatory cytokines, HaCaT cells were pretreated with varying concentrations of SDBS (0.5–30 μg / ml) for 24 hours and then stimulated with T / I for 24 hours. To evaluate the effect of the STAT3 agonist colivelin, cells were pretreated with colivelin (2 μg / ml) for 1 hour, post-treated with 10 μg / ml SDBS for 24 hours, and then stimulated with T / I for 24 hours. Cell supernatants were collected and IL-6 and TSLP levels were measured using ELISA kits (Abgent, USA).

[0084] 1.7 Real-time fluorescence quantitative PCR

[0085] Total RNA was extracted using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. 500 ng of RNA was extracted from each sample and reverse transcribed into cDNA using a cDNA synthesis kit (Invitrogen, USA) (total reaction volume: 20 μL). Transcripts were detected using the SYBR Green assay (Applied Biosystems, Sweden), with replicate wells set up for each sample.

[0086] The primer sequences used were as follows: Tslp (forward: 5′-TGAAGCCTATGACACCACTGA-3′, reverse: 5′-TCCCCTACGCTTTCTTGTCCT-3′);

[0087] Il4(forward:5'-GGTCTCAACCCCCAGCTAGT-3', reverse:5'-GCCGATGATCTCCTCAAGTGAT-3');

[0088] Il6 (forward: 5'-TAGTCCTTCCTACCCCAATTTCC-3', reverse: 5'-TTGGTCCTTAGCCACTCCTTC-3');

[0089] Il13 (forward: 5'-CCTGGCTCTTGCTTGCCTT-3', reverse: 5'-GGTCTTGTGTGATGTTGCTCA-3');

[0090] β-actin (forward: 5'-CATGTACGTTGCTATCCAGGC-3', reverse: 5'-CTCCTTAATGTCACGCACGAT-3');

[0091] TSLP (forward: 5'-ATGTTCGCCATGAAAACTAAGGC-3', reverse: 5'-GCGACGCCACAATCCTTGTA-3');

[0092] IL6 (forward: 5'-ACTCACCTCTTCAGAACGAATTG-3', reverse: 5'-CCATCTTTGGAAGGTTCAGGTTG-3');

[0093] IL33 (forward: 5'-GTGACGGTGTTGATGGTAAGAT-3', reverse: 5'-AGCTCCACAGAGTGTTCCTTG-3');

[0094] CCL17 (forward: 5'-TTCTCTGCAGCACATCCACGCA-3', reverse: 5'-CTGGAGCAGTCCTCAGATGTCT-3');

[0095] GAPDH (forward: 5'-GGAGCGAGATCCCTCCAAAAT-3', reverse: 5'-GGCTGTTGTCATACTTCTCATGG-3').

[0096] 1.8 Western blot

[0097] 1.8 Western blotHaCaT cells and mouse ear tissues were lysed with RIPA lysis buffer (Beyotime, China) to extract total proteins, and the protein concentration was determined by BCA method. Equal amounts of proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes (PALL, USA). The membranes were blocked with 5% bovine serum albumin for 1 h and incubated with primary antibodies at 4°C overnight. The antibodies used included p-STAT3, STAT3, p-p38, p38, p-IκB-a, IκB-a, p-NF-κB p65 (cytoplasm / nucleus), ERK1 / 2 (Cell Signaling Technology, USA; Abeam, UK), b-actin (cytoplasmic internal reference), and Lamin B (nuclear internal reference) (Connoisseur, China). Mouse samples used corresponding source antibodies. After three TBST washes, the membranes were incubated with horseradish peroxidase-labeled secondary antibodies (Cell Signaling Technology, USA) at room temperature for 1 h. ECL was used for development, and image analysis was performed using Fiji / ImageJ software (version 2.16.0 / 1.54g).

[0098] 1.9 Statistical analysis

[0099] Statistical analysis was performed using GraphPad Prism software (version 10.3.1, GraphPad Software, USA). Independent sample t-test was used for comparison between two groups, and one-way ANOVA was used for comparison among multiple groups. P<0.05 was considered statistically significant.

[0100] 2 Results

[0101] 2.1 Topical application of SDBS alleviates MC903-induced AD-like dermatitis

[0102] To determine the role of SDBS in the pathogenesis of AD, we used MC903 to induce AD-like skin lesions by topical application on the ears of mice, followed by topical SDBS treatment Figure 2 ). The results showed that MC903 successfully induced AD-like dermatitis. Topical application of 0.1% and 1% SDBS significantly reduced dermatitis symptoms, as evidenced by a significant decrease in dermatitis scores (p<0.001) and ear thickness (p<0.001) Figure 3 , Figure 4 ). Histopathological analysis showed that MC903-induced skin lesions had obvious features such as hyperkeratosis, parakeratosis, acanthosis, and lymphocyte infiltration, while 0.1% and 1% SDBS treatment significantly improved these pathological changes and significantly reduced epidermal thickness (p<0.001) Figure 6 ). In addition, SDBS treatment also reduced mast cell infiltration (p<0.001) Figure 7), and reduced serum IgE levels (p<0.0001) Figure 5 ) The remission effects of 0.1% and 1% SDBS were comparable to the positive control drugs, desonide cream and tacrolimus ointment, in terms of dermatitis scores, ear thickness, epidermal thickness, mast cell infiltration, and serum IgE levels Figures 3-7

[0103] 2.2 SDBS can inhibit the expression of inflammatory factors in AD-like skin lesions and T / I-stimulated HaCaT cells

[0104] Further investigate the effects of SDBS on the expression of AD-related cytokines at the protein and mRNA levels. Immunohistochemical results showed that TSLP was highly expressed in the cytoplasm of epidermal cells in the MC903-induced AD-like mouse ear skin Figure 8 ) ELISA detection showed that the levels of TSLP and IL-6 in the serum were significantly higher than those in the control group Figure 9 ) Real-time fluorescent quantitative PCR results showed that the mRNA levels of Tslp, Il4, Il6, and Il13 were significantly increased Figure 10 ) After treatment with 0.1% or 1% SDBS, the staining intensity of TSLP was significantly reduced (p<0.001), the serum levels of TSLP (p<0.0001) and IL-6 (p<0.0001) were decreased, and the mRNA levels of Tslp (p<0.01), Il4 (p<0.0001), Il6 (p<0.001), and Il13 (p<0.0001) were significantly down-regulated, with effects comparable to those of desonide and tacrolimus Figures 8-10

[0105] In in vitro experiments, ELISA detection showed that different concentrations of SDBS (2, 5, and 10 μg / ml) could inhibit the expression of TSLP (p<0.01) and IL-6 (p<0.01) induced by T / I co-stimulation Figure 12 ), while real-time PCR also showed that it could down-regulate the mRNA levels of TSLP, IL6, IL33, and CCL17 (p<0.05). In contrast, SLS at the same concentrations failed to inhibit the expression of the above-mentioned cytokines induced by T / I, but instead observed a significant increase in the mRNA expression of TSLP, IL6, and IL33 (p<0.05). Notably, SDBS at concentrations below 20 μg / ml had no significant effect on the viability of HaCaT cells Figure 11 ), while SLS at concentrations ≥5 μg / ml significantly inhibited cell viability.

[0106] 2.3 SDBS inhibits the activation of STAT3, NF-κB, and MAPK signaling pathways

[0107] ​​Further explore the regulation of SDBS on signaling pathways in in vitro and in vivo models. Western blot results showed that in MC903-induced AD-like mouse ear skin, the phosphorylation levels of STAT3, NF-κB P65, IκB-α and P38 MAPK were significantly increased, while 0.1% and 1% SDBS treatment could significantly inhibit the phosphorylation expression of the above molecules( Figures 13-14 ). In the HaCaT cell model, SDBS also inhibited T / I-induced STAT3, P38 MAPK and ERK1 / 2 phosphorylation expression. Although the total protein level of cytoplasmic NF-κB P65 did not change significantly, the nuclear NF-κB P65 expression in the SDBS treatment group was significantly lower than that in the T / I stimulation group. In addition, 20 μg / ml and 10 μg / ml SDBS could significantly reduce p-IκB-α expression( Figures 15-16 ).

[0108] 2.4 SDBS inhibits MC903-induced AD-like dermatitis and related signaling pathways through STAT3-dependent mechanism

[0109] To further verify whether the inhibitory effect of SDBS on T / I-stimulated HaCaT cells is dependent on the STAT3 pathway, we used STAT3-specific agonist colivelin for intervention. The results showed that the levels of TSLP and IL-6 in the T / I treatment group were increased, and significantly decreased after SDBS treatment. After the addition of colivelin, the levels of TSLP and IL-6 in the T / I+SDBS treatment group were significantly increased(p<0.0001)( Figure 17 ), suggesting that colivelin can weaken the inhibitory effect of SDBS on TSLP and IL-6 expression. Subsequently, colivelin was also given in the MC903-induced AD-like mouse model( Figure 18 ), and the results showed that SDBS could significantly improve the clinical and histological manifestations of dermatitis, but in the colivelin intervention group, the dermatitis score(p<0.05) and epidermal thickness(p<0.01) were significantly higher than those in the SDBS treatment group alone( Figures 19-21 ), suggesting that colivelin can partially reverse the remission effect of SDBS on AD-like dermatitis.

[0110] Further analysis of the changes in signaling pathways found that the levels of p-STAT3, p-IκB-α and p-ERK1 / 2 were significantly increased in MC903-induced lesions, and SDBS treatment could inhibit their expression. Compared with the T / I+SDBS group, the p-STAT3 / STAT3(p<0.05), p-IκB-α / IκB-α(p<0.0001) and p-ERK1 / 2 / ERK1 / 2(p<0.0001) ratios in the colivelin intervention group were significantly increasedFigures 22-23 ), further indicating that STAT3 activation could interfere with the inhibitory effect of SDBS on NF-κB and MAPK signaling pathways.

[0111] In summary, sodium dodecyl benzene sulfonate (SDBS) is a widely used anionic surfactant. Contrary to the possible inflammatory effects speculated by the "hygiene hypothesis", previous studies have shown that SDBS exposure does not significantly upregulate the expression of key proinflammatory factors (such as IL-1α, IL-6, IL-8 and TNF-α). This example systematically evaluated the effect of SDBS on the pathogenesis of atopic dermatitis (AD) through in vivo and in vitro experiments. The results showed that topical application of 0.1% and 1% SDBS can alleviate the clinical symptoms of MC903-induced AD-like mouse dermatitis, including dermatitis scores, lesion thickness, mast cell infiltration and serum IgE levels. In addition, SDBS significantly reduced the concentrations of TSLP and IL-6 in serum, and downregulated the mRNA expression levels of Tslp, Il6, Il4 and Il13 in skin tissue. In vitro experiments further confirmed that SDBS could concentration-dependently inhibit the expression of TSLP and IL-6 proteins and their related mRNAs (TSLP, IL6, IL33, and CCL17) after T / I stimulation without affecting the activity of HaCaT cells. Further mechanistic studies showed that SDBS could significantly inhibit the activation of STAT3, NF-κB, and MAPK signaling pathways, and this effect was verified both in vitro and in vivo models. When the STAT3 agonist colivelin was administered, the alleviating effect of SDBS on MC903-induced AD-like dermatitis (including clinical scores, histopathological improvement, and inhibition of STAT3, IκB-α, and ERK1 / 2 phosphorylation) was significantly weakened, suggesting that its mechanism of action depends on the regulation of the STAT3 signaling pathway.

[0112] AD is one of the most common chronic inflammatory skin diseases, with typical features of recurrent inflammation, epidermal barrier dysfunction, and IgE-mediated immune response. MC903-induced AD-like mouse model is widely used in the study of AD pathogenesis and therapeutic targets. In this application, MC903-treated mice showed obvious erythema, edema, scab and capillary dilation in the ear, and SDBS intervention significantly alleviated the above symptoms, and 1% SDBS had a stronger effect than 0.1% SDBS. Histological observation also showed that the MC903 group had typical AD pathological features (epidermal hyperplasia, spongiotic changes and perivascular lymphocyte infiltration), and SDBS treatment significantly improved the above symptoms, and the epidermal thickness of the 1% SDBS group was reduced more obviously. The key mechanisms of AD include type 2 immune imbalance and IgE-mediated hyperresponsiveness. Elevated serum total IgE is an important marker of AD. In this application, SDBS reduced the serum IgE level of MC903-induced AD-like mice. Mast cells are the main target cells of IgE activation and are involved in the early inflammatory response of AD, and SDBS can also reduce mast cell infiltration in the skin lesions. TSLP is an important cytokine for the initiation of type 2 immune response, and this example demonstrates that SDBS can significantly inhibit the protein and mRNA expression of TSLP in skin lesions and reduce its serum level.

[0113] TNF-α and IFN-γ combined stimulation of keratinocytes can induce the expression of various inflammatory factors, and the T / I stimulated HaCaT cell model is constructed in this application to further demonstrate that SDBS can significantly reduce the expression of key factors such as TSLP, IL6, IL33 and CCL17. IL-4 and IL-13 are core factors that induce IgE class switching and promote inflammation, and SDBS treatment can down-regulate the expression of Il4 and Il13 in mouse skin. IL-6 not only promotes Th2 differentiation but also inhibits Treg function, promotes disease progression in AD, and SDBS can significantly inhibit its expression in vivo and in vitro. IL-33 is an epithelial-derived alarm, which drives type 2 immunity, and CCL17 is a chemotactic factor related to disease severity, and this application found that SDBS can inhibit the mRNA expression of both. In contrast, SLS, as a widely used anionic surfactant, did not show the above inhibitory effect under the same conditions, but instead up-regulated the expression of TSLP, IL6 and IL33.

[0114] STAT3 is a member of the signal transducer and activator of transcription family, and is a key regulator of AD inflammation. The present application found that SDBS can significantly inhibit the phosphorylation expression of STAT3 in MC903 mouse ear and T / I stimulated HaCaT cells. NF-κB and MAPK pathway cooperatively regulate a variety of pro-inflammatory factors, and is a key pathway of AD inflammation. SDBS treatment significantly inhibits the phosphorylation activation of p65, IκB-α and P38 / ERK1 / 2, and blocks the nuclear translocation process of NF-κB, suggesting that it has a multi-target anti-inflammatory mechanism. Further intervention using STAT3 agonist colivelin was found to significantly reverse the inhibitory effect of SDBS on TSLP and IL-6, and also weaken the remission effect of SDBS on dermatitis score and epidermal thickness in vivo, and reactivated IκB-α and ERK1 / 2 signals, suggesting that the inhibitory effect of SDBS on NF-κB and MAPK depends on STAT3 activity.

[0115] In summary, SDBS, as a widely used anionic surfactant, its biological effects on the skin have not been fully recognized. The present application first confirmed that 0.1% and 1% SDBS can significantly alleviate the clinical and histological manifestations of AD-like dermatitis in vivo and in vitro, inhibit the expression of key inflammatory factors of AD, and play a protective role through the STAT3-dependent NF-κB / MAPK signaling pathway. The present application provides a new theoretical basis and research idea for the further development of AD suitable cleaning products.

[0116] Various embodiments of the present application can exist in the form of a range; it should be understood that the description in the form of a range is only for the convenience and brevity, and should not be understood as a hard limit on the scope of the present application; therefore, it should be considered that the described range has been specifically disclosed all possible sub-ranges and single values within the range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the described range, such as 1, 2, 3, 4, 5 and 6, regardless of the range. In addition, whenever a numerical range is indicated in the present application, it refers to any cited number (fraction or integer) within the indicated range.

[0117] In addition, in the description of the specification of the present application, the terms "include", "contain" and the like mean "include but not limited to". In this text, relationship terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between the entities or operations.

[0118] The foregoing detailed description of the application has been presented for purposes of illustration and description. Various modifications and changes can be made to these embodiments without departing from the spirit and scope of the application. It is intended that the scope of the application should not be limited by the particular representative embodiments described above.

Claims

1. An aqueous surfactant preparation for alleviating atopic dermatitis, characterized in that: The active ingredient of the aqueous surfactant preparation is an anionic surfactant that inhibits STAT3 signaling. The inhibition of the STAT signaling pathway has the following manifestations: indirectly inhibiting the signaling pathways of NF-κB and MAPK.

2. The aqueous surfactant preparation according to claim 1, characterized in that The anionic surfactant is sodium dodecylbenzenesulfonate or a structural analogue of sodium dodecylbenzenesulfonate.

3. The aqueous surfactant preparation according to claim 1, characterized in that The mass concentration of the anionic surfactant is 0.1% to 1.0%.

4. The aqueous surfactant preparation according to claim 3, characterized in that The mass concentration of the anionic surfactant is 0.5% to 1.0%.

5. The aqueous surfactant preparation according to claim 1, characterized in that The dosage forms of the aqueous surfactant preparation include patches, pastes, ointments, creams, gels, oils, microneedles, film coatings, papules, sprays and dressings.

6. The aqueous surfactant preparation according to claim 1, characterized in that The aqueous surfactant formulation further includes a pharmaceutically acceptable carrier.

7. The aqueous surfactant preparation according to claim 6, characterized in that The pharmaceutically acceptable carrier includes one or more of ethanol, aqueous carrier, glycerol, aqueous gel, propylene glycol and vegetable oil.

8. Use of the aqueous surfactant preparation according to any one of claims 1 to 7 in a drug for treating atopic dermatitis.

9. The use according to claim 8, characterized in that The aqueous surfactant preparation is administered by topical application to the skin lesion area once or more per day.

10. The use according to claim 8, characterized in that The aqueous surfactant preparation has any one of the following medicinal effects: down-regulating the expression of key inflammatory factors TSLP and IL-6, alleviating atopic dermatitis lesions, reducing the epidermal thickness of the lesion site, reducing serum IgE levels, and reducing mast cell infiltration, and is used in drugs for treating atopic dermatitis.

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