New use of psoralen isoflavones

Abstract

The application provides a new use of psoralen isoflavone, the psoralen isoflavone can significantly inhibit the protein expression of PDE4B in the skin of AD mice induced by dinitrochlorobenzene and in the keratinocytes stimulated by TNF-alpha / IFN-gamma, after the treatment of the psoralen isoflavone, the dermatitis characteristics such as redness, scaling, ulceration and the like of the skin of the AD mice can be effectively improved; the psoralen isoflavone can significantly reduce the scratching frequency of the AD mice, and effectively relieve itching; the psoralen isoflavone reduces the transdermal water loss of the skin of the AD mice; H&E staining shows that the psoralen isoflavone obviously reduces the epidermal thickness of the skin of the AD mice; toluidine blue staining shows that the psoralen isoflavone significantly reduces the number of mast cells in the skin tissue of the AD mice and improves the infiltration state of the mast cells, and has important application value in the preparation of PDE4B inhibitors.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to novel uses of psoralen isoflavones. Background Technology

[0002] Atopic dermatitis (AD), also known as eczema, is a chronic, relapsing, inflammatory skin disease. The prevalence of atopic dermatitis is gradually increasing worldwide, affecting 15%-30% of children and 2-10% of adults globally. AD is often accompanied by recurrent itching, eczematous lesions, bleeding, and erosion, leading to complications such as depression, anxiety, and sleep disorders, severely impacting the physical and mental health of AD patients. The significant medical and social burden of AD constitutes a global health problem.

[0003] Treatment of Alzheimer's disease (AD) aims to improve clinical symptoms, eliminate triggering and / or aggravating factors, and prevent complications and recurrence. Currently, clinical treatment of AD mainly relies on the application of topical medications, such as topical corticosteroids (TCS), topical calcineurin inhibitors (TCI), and phosphodiesterase 4 (PDE4) inhibitors. Long-term use of TCS may lead to adverse reactions such as telangiectasia, skin atrophy, striae, acne, and purpura. The use of TCI may cause burning sensations at the application site and the risk of skin infection. The use of PDE4 inhibitors may cause adverse reactions such as nausea and vomiting.

[0004] PDE4B is a member of the PDE4 family of cyclic adenosine monophosphate (cAMP)-specific cyclic nucleotides. It is highly expressed in keratinocytes and is a major driver of inflammation. Increased PDE4B expression is observed in patients with chronic inflammatory skin diseases such as Alzheimer's disease (AD). The PDE4B inhibitor difamilast inhibits PDE4B expression, promotes intracellular cAMP expression, and leads to CREB phosphorylation. CREB phosphorylation upregulates KPRP expression, restoring skin barrier function and thus playing a therapeutic role in AD. Currently, difamilast is the only marketed drug that directly targets PDE4B, with a low incidence of adverse reactions, avoiding the nausea and vomiting associated with topical PDE4 inhibitors.

[0005] According to the "Guidelines for the Diagnosis and Treatment of Atopic Dermatitis in Traditional Chinese Medicine," the main TCM syndrome differentiation includes spleen and kidney yang deficiency syndrome, spleen deficiency and blood dryness syndrome, and heart and spleen heat accumulation syndrome. Treatment focuses on strengthening the spleen, consolidating the kidneys, and clearing the heart. Exploring further TCM treatments for atopic dermatitis (AD) based on these principles has significant clinical application value. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a new use for psoralen isoflavones.

[0007] The technical solution adopted in this invention is:

[0008] The use of corylin in the preparation of PDE4B inhibitors, the chemical structural formula of which is as follows:

[0009]

[0010] The molecular formula of the above-mentioned psoralen isoflavones is C 20 H 16 O4, molecular weight 320.3, CAS number: 53947-92-5.

[0011] Use of psoralen isoflavones in the preparation of drugs or skin care products for the prevention and treatment of atopic dermatitis.

[0012] Uses of psoralen isoflavones in the preparation of skin barrier repair drugs or skin care products.

[0013] Preferably, the use of the above-mentioned psoralen isoflavones is to improve skin barrier repair by improving skin redness, scaling, and ulceration.

[0014] Preferably, in the use of the above-mentioned psoralen isoflavones, the skin barrier repair is for relieving itching.

[0015] Preferably, in the use of the above-mentioned psoralen isoflavones, the skin barrier repair is to reduce skin moisture loss.

[0016] Preferably, in the use of the above-mentioned psoralen isoflavones, the skin barrier repair is to reduce the thickness of the skin epidermis.

[0017] Preferably, in the use of the above-mentioned psoralen isoflavones, the medicine is a topical medicine.

[0018] A drug / skincare product for the prevention and treatment of atopic dermatitis, the main active ingredient of which includes the aforementioned psoralen isoflavones.

[0019] A skin barrier repair drug / skincare product, whose main active ingredient includes the aforementioned psoralen isoflavones.

[0020] The beneficial effects of this invention are:

[0021] In a mouse model of atopic dermatitis induced by dinitrochlorobenzene (DNCB), the skin showed significant thickening, accompanied by redness, scaling, and ulceration. The study found that psoralen isoflavones significantly inhibited the protein expression of PDE4B in the skin and TNF-α / IFN-γ-stimulated keratinocytes of DNCB-induced AD mice. Treatment with psoralen isoflavones effectively improved the dermatitis characteristics of redness, scaling, and ulceration in AD mice; psoralen isoflavones also significantly reduced the frequency of scratching in AD mice, effectively... Psoralen isoflavones relieve itching; they reduce transepidermal water loss in AD mice; H&E staining shows that psoralen isoflavones significantly reduce epidermal thickness in AD mice; toluidine blue staining shows that psoralen isoflavones significantly reduce the number of mast cells in the skin tissue of AD mice and improve mast cell infiltration; psoralen isoflavones significantly reduce splenomegaly and decrease serum IgE levels in AD mice, while also reducing the mRNA expression of cytokines IL-4, IL-5, IL-6, IL-13, and TSLP in the skin of AD mice. Psoralen isoflavones effectively increase the protein expression of filaggrin, loricrin, and involucrin in the skin tissue of AD mice, and have a role in repairing the skin barrier in AD mice, showing promising application potential in the treatment of atopic dermatitis. Attached Figure Description

[0022] Figure 1 A simplified flowchart for model making.

[0023] Figure 2 Figure showing the effect of corylin on the appearance of AD mice.

[0024] Figure 3 Effects of corylin on skin lesion scores in DNCB-induced AD mice (n=6), ### P<0.001 vs Control ## P<0.01 vs Control *** P<0.001 vsModel ** P<0.01 vsModel * P<0.05vsModel.

[0025] Figure 4 The effect of corylin on scratching behavior in AD mice (n=6), ### P<0.001 vs Control ** P<0.01 vsModel * P<0.05 vsModel

[0026] Figure 5 Effects of corylin on skin TEWL in AD mice (n=6), ### P<0.001vns Control *** P<0.001 vsModel ** P<0.01vsModel.

[0027] Figure 6 H&E stained skin pathology section and skin epidermal thickness (n=3), ### P<0.001vsControl *** P<0.001vsModel.

[0028] Figure 7 Toluidine blue stained skin pathological sections and the number of skin mast cells (n=3), ### P<0.001vsControl *** P<0.001 vsModel ** P<0.01vsModel.

[0029] Figure 8 Effects of corylin on spleen index in DNCB-induced AD mice (n=6), ### P<0.001 VS Control ** P<0.01 vsModel * P<0.05vsModel.

[0030] Figure 9 Effects of corylin on serum IgE in AD mice (n=3), ## P<0.01 VSControl ** P<0.01 VS Model.

[0031] Figure 10 Effects of corylin on the expression of skin cytokines mRNA in Alzheimer's disease (AD) mice (n=3), ### P<0.001 VS Control ## P<0.01 VS Control # P<0.05VS Control ***P<0.00VSModel ** P<0.05VS Model.

[0032] Figure 11 The effect of corylin on filaggrin protein expression in AD mice (n=3), ### P<0.001 VS Control *** P<0.001 VS Model.

[0033] Figure 12 The effect of corylin on involucrin protein expression in AD mice. (n=3), ## P<0.01 VS Control ** P<0.01 VS Model * P<0.05VS Model.

[0034] Figure 13 The effect of corylin on loricrin protein expression in AD mice. (n=3), ### P<0.001 VS Control *** P<0.001 VS Model.

[0035] Figure 14 Screening for safe concentrations of corylin in HaCaT cells. (n=18), ** P<0.01 vs Control.

[0036] Figure 15 Screening for safe concentrations of corylin in HEK cells (n=12), ** P<0.01vsControl.

[0037] Figure 16 Effects of different TNF-α / IFN-γ stimulation times on cytokine mRNA levels in HaCaT cells (n=3), ### P<0.001 VS Control ## P<0.01 VS Control.

[0038] Figure 17Effects of TNF-α / IFN-γ stimulation for 24 h on cytokine mRNA levels in HEK cells (n=3)

[0039] ### P<0.001 VS Control ## P<0.01 VS Control

[0040] Figure 18 Effects of corylin on the viability of TNF-α / IFN-γ stimulated HaCaT cells (n=18), ### P<0.001 vs Control *** P<0.001 vs Model ** P<0.01 vs Model * P<0.05 vs Model.

[0041] Figure 19 Effects of corylin on LDH release in TNF-α / IFN-γ stimulated HaCaT cells (n=18), ### P<0.001 vs Control *** P<0.001 vs Model ** P<0.01 vs Model.

[0042] Figure 20 Effects of corylin on the viability of TNF-α / IFN-γ stimulated HEK cells. (n=12), ### P<0.001 vs Control *** P<0.001 vs Model ** P<0.01 vs Model * P<0.05vsModel.

[0043] Figure 21 Effects of corylin on LDH release in HEK cells stimulated by TNF-α / IFN-γ (n=12), ### P<0.001 vs Control *** P<0.001 vs Model ** P<0.01 vs Model.

[0044] Figure 22 The effect of corylin on cytokines in HaCaT cells stimulated by TNF-α / IFN-γ. (n=3), ### P<0.001 VS Control ## P<0.01 VS Control # P<0.05VS Control *** P<0.001 VS Model ** P<0.01 VS Model * P<0.05VS Model.

[0045] Figure 23 The effect of corylin on related cytokines in HEK cells after TNF-α / IFN-γ stimulation. (n=3), ### P<0.001 VS Control ## P<0.01 VS Control # P<0.05VSControl *** P<0.001 VS Model ** P<0.01 VS Model * P<0.05VS Model.

[0046] Figure 24 The effect of corylin on the mRNA levels of skin barrier function-related indicators in HaCaT cells after TNF-α / IFN-γ stimulation. (n=3), ## P<0.01 VS Control # P<0.05VSControl *** P<0.001 VS Model ** P<0.01 VS Model * P<0.05VS Model.

[0047] Figure 25 The effect of corylin on filaggrin protein expression in HaCaT cells after TNF-α / IFN-γ stimulation. (n=3), ### P<0.001 VS Control *** P<0.001 VS Model **P<0.01 VSModel.

[0048] Figure 26 The effect of corylin on the expression of Loricrin protein in HaCaT cells after TNF-α / IFN-γ stimulation. (n=3), ### P<0.001 VS Contrl *** P<0.001 VS Model ** P<0.01 VSModel.

[0049] Figure 27 The effect of corylin on the expression of involucrin protein in HaCaT cells after TNF-α / IFN-γ stimulation. (n=3), ### P<0.001 VS Control *** P<0.001 VS Model ** P<0.01VS Model.

[0050] Figure 28 The effect of corylin on the mRNA levels of skin barrier function-related indicators in HEK cells after TNF-α / IFN-γ stimulation. (n=3), ## P<0.01 VS Control # P<0.05VS Control ** P<0.01 VS Model * P<0.05VS Model.

[0051] Figure 29 The effect of corylin on filaggrin protein expression in HEK cells after TNF-α / IFN-γ stimulation. (n=3), ### P<0.001 VS Control *** P<0.001 VS Model ** P<0.01 VSModel.

[0052] Figure 30 The effect of corylin on the expression of involucrin protein in HEK cells after TNF-α / IFN-γ stimulation. (n=3), ### P<0.001 VS Control ***P<0.00VS Model ** P<0.0VSModel * P<0.05VS Model.

[0053] Figure 31 The effect of corylin on the expression of Loricrin protein in HEK cells after TNF-α / IFN-γ stimulation. (n=3), ## P<0.01 VS Control ** P<0.01 VS Model * P<0.05VS Model.

[0054] Figure 32 The effect of corylin on PDE4B protein expression in AD mice. (n=3), ## p<0.01 VS Control # p<0.05VS Control * p<0.05VS Model.

[0055] Figure 33 Effects of corylin on PDE4B protein expression in HaCaT cells (n=3), ## P<0.01 VS Control ** P<0.01 VS Model * P<0.05VS Model.

[0056] Figure 34 Effects of corylin on PDE4B protein expression in HEK cells (n=3), ### P<0.001 VS Control ** P<0.001 VS Model ** P<0.01 VS Model.

[0057] Figure 35 To verify PDE4B overexpression in HEK cells using qRT-PCR. (n=3), # P<0.05VSControl.

[0058] Figure 36 To verify PDE4B overexpression in HEK cells using Western blot. (n=3), # P<0.05 vs Control.

[0059] Figure 37 The effect of corylin on the expression of cytokine mRNA in HEK cells after transfection with an overexpression plasmid. (n=3), ### P<0.001 VS Control ## P<0.01 VS Control # P<0.05VSControl *** P<0.001 VS Model ** P<0.01 VS Model * P<0.05VS Model &&& P<0.001 VS Corylin+TNF-α / IFN-γ && P<0.01 VS Corylin+TNF-α / IFN-γ & P<0.05VS Corylin+TNF-α / IFN-γ.

[0060] Figure 38 The effect of corylin on the mRNA of skin barrier function-related indicators in HEK cells after transfection with an overexpression plasmid. (n=3), ## P<0.01 VS Control # P<0.05VS Control ** P<0.01VS Model * P<0.05VS Model & P<0.05VS Corylin+TNF-α / I FN-γ.

[0061] Figure 39 The effect of corylin on the protein expression of filaggrin in HEK cells after transfection with an overexpression plasmid. (n=3), ## P<0.01 VS Control ** P<0.01 VS Model && P<0.05VS Corylin+TNF-α / IFN-γ.

[0062] Figure 40The effect of corylin on the protein expression of involucrin in HEK cells after being transfected with an overexpression plasmid. (n=3), ### P<0.001 VS Control *** P<0.001 VS Model && P<0.05VS Corylin+TNF-α / IFN-γ.

[0063] Figure 41 The effect of corylin on the protein expression of loricrin in HEK cells after being transfected with an overexpression plasmid. (n=3), ### P<0.001 VS Control ** P<0.01 VS Model && P<0.05VS Corylin+TNF-α / IFN-γ. Detailed Implementation

[0064] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0065] Example 1

[0066] Study on the effects of corylin on DNCB-induced AD mouse model

[0067] A DNCB-induced AD mouse model was established. Using crisaborole as a positive control, the effects of psoralen isoflavones on skin lesion scores, scratching behavior, transepidermal water loss (TEWL), spleen index, and serum IgE in AD mice were investigated. Skin tissue pathological sections were examined to investigate the effects of psoralen isoflavones on epidermal thickness and mast cells. qRT-PCR was used to detect the expression of cytokines and skin barrier-related indicators in skin tissue. Western blot was used to detect the expression of skin barrier-related proteins in skin tissue to explore the ameliorative effect of psoralen isoflavones on the AD mouse model.

[0068] 1. Materials

[0069] 1.1 Reagents and Consumables

[0070] 4% tissue cell fixative (Beijing Solarbio Company)

[0071] Crizoborole Ointment (2%, 1g ointment contains 20mg crisaborole) Pharmacia and Upjohn Company LLC

[0072] Corylin (CAS No.: 53947-92-5, purity >98%), Shanghai Yuanye Biotechnology Co., Ltd.

[0073] RNAprepPure Animal Tissue Total RNA Extraction Kit (DP431) Tiangen Biotech (Beijing) Co., Ltd.

[0074] DNCB Alfaisa Chemicals Ltd.

[0075] PDE4B antibody (72096s), Cell Signaling Technology, USA

[0076] Corylin (CAS No.: 53947-92-5, purity >98%), Shanghai Yuanye Biotechnology Co., Ltd.

[0077] Six-well white transparent cell culture plate (3516) Costar, USA

[0078] StarScript III Reverse Transcription Kit (Beijing Kangrun Chengye Biotechnology Co., Ltd.)

[0079] 2X Universal SYBR Green Fast qPCR Mix, Wuhan Aiboteke Biotechnology Co., Ltd.

[0080] RNApure Tissure&Cell Kit CW0584S

[0081] cDNA reverse transcription kit, manufactured by Abi (USA).

[0082] FastStart Universal SYBR Green Master (Roche, Switzerland)

[0083] Primers Shanghai Sangon Biotech

[0084] Involucrin antibody (sc-21748), Santa Cruz Pharmaceuticals, USA

[0085] Loricrin antibody (55439-1-AP) Wuhan Sanying Biotechnology Co., Ltd.

[0086] Filaggrin antibody (A20011) Wuhan Aibote Biotechnology Co., Ltd.

[0087] β-actin antibody (3700S), Cell Signaling Technology, USA

[0088] Goat anti-rabbit horseradish peroxidase (HPR) labeled antibody, Cell Signaling Technology, USA

[0089] Goat anti-mouse horseradish peroxidase (HPR) labeled antibody, Cell Signaling Technology, USA

[0090] Horseradish peroxidase chemiluminescent reagent (ECL luminescence solution), Millipore Corporation, USA

[0091] 1.2 Instruments

[0092] Real-time fluorescent quantitative PCR instrument (LightCycler480), ROCHE, Switzerland

[0093] Multi-probe skin testing system, CK, Germany

[0094] Mini centrifuge, Thermo Fisher, USA

[0095] Gel imaging system, Bio-Rad, USA

[0096] 1.3 Experimental animals

[0097] SPF-grade female BALB / C mice aged 6-8 weeks, weighing about 18g, SPF grade, were purchased from Sibio Biotechnology Co., Ltd. (license No. SCK (Jing) 2019-0010). The mice were housed in the Animal Center of Tianjin University of Traditional Chinese Medicine, at room temperature of 20-26°C and relative humidity of 40-70%. The mice were housed in separate cages with conventional food and water, and experiments were carried out after 1 week of adaptive feeding.

[0098] 2 Methods

[0099] 2.1 Drug preparation

[0100] (1) Blank matrix solution: olive oil and acetone are mixed at a ratio of 1:3.

[0101] (2) 2% DNCB: weigh 160 mg of DNCB powder and dissolve it in 8 mL of blank matrix solution.

[0102] (3) 0.6% DNCB: weigh 36 mg of DNCB powder and dissolve it in 12 mL of blank matrix solution.

[0103] (4) Blank preparation: Carbomer 940 was weighed and dispersed in 10 ml of deionized water. The mixture was stirred at 500 rpm for 3 h at room temperature until no obvious lumps were observed, followed by swelling at 4 °C for 12 h. Ethylparaben (0.2%) and glycerol (10%) were added to Carbomer 940 and stirred until homogeneous to obtain the blank gel. Methylparaben was dissolved in PBS at 75 °C to form the aqueous phase. Phospholipids were dissolved in ethanol at 75 °C, and behenyl glycerol and medium-chain glycerols were dissolved in chloroform at 75 °C. The two were mixed to form the oil phase. The oil phase was slowly added to the aqueous phase. The mixture was rapidly stirred at 75 °C (500 rpm) until the solution became clear, followed by slow stirring (300 rpm) to remove the organic phase. The solution was cooled to room temperature and filtered through a 0.22 μm filter to obtain the blank NLC, which was stored at 4 °C for later use. The blank gel and blank NLC were mixed at a 1:2 ratio to obtain the blank preparation.

[0104] (5) Corylin preparation: Carbomer 940 was weighed and dispersed in 10 ml of deionized water. The mixture was stirred at 500 rpm for 3 h at room temperature until no obvious lumps were observed, followed by swelling at 4 °C for 12 h. Ethylparaben (0.2%) and glycerol (10%) were added to Carbomer 940 and stirred until homogeneous to obtain a blank gel. Methylparaben was dissolved in PBS at 75 °C to form the aqueous phase. Phospholipids and psoralen isoflavone standards were dissolved in ethanol at 75 °C, and behenicol and medium-chain glycerides were dissolved in chloroform at 75 °C. The two were mixed to form the oil phase. The oil phase was slowly added to the aqueous phase. The mixture was rapidly stirred at 75 °C (500 rpm) until the solution became clear, followed by slow stirring (300 rpm) to remove the organic phase. The solution was cooled to room temperature and filtered through a 0.22 μm filter membrane to obtain Corylin-NLC, which was stored at 4 °C for later use. Corylin formulation was obtained by mixing blank gel and Corylin-NLC at a ratio of 1:2.

[0105] 2.2 Animal Grouping

[0106] (1) Pre-experimental treatment

[0107] One day before DNCB induction, most of the hair on the back of the mice was removed with an electric shaver, and then depilatory cream was applied to completely remove the hair on the back and fully expose the skin (2×2cm).

[0108] (2) Experimental grouping and drug administration

[0109] On days 1 and 4, the blank control group received 100 μL of blank matrix solution, while the rest received 100 μL of 2% DNCB. On day 7, the mice were randomly divided into 5 groups of 6 mice each using a random number table: (1) blank control group (Control), (2) model group (Mode1), (3) Base group, (4) Corylin treatment group, and (5) Crisaborole ointment group. Treatment began, and the therapeutic drugs were administered daily during the treatment period. Simultaneously, 100 μL of 0.6% DNCB was applied to the skin on the back every 3 days. A simplified procedure is described below. Figure 1 .

[0110] Mice were randomly divided into 5 groups of 6 mice each:

[0111] Control group: 100 μL of blank matrix solution was applied to the skin of mice;

[0112] Model group: For initial sensitization, 100 μL of 2% DNCB was applied to the skin on the back of the mice every 2 days. Seven days after the first induction, 100 μL of 0.6% DNCB was applied to the back of the mice every 2 days, and drug administration was initiated.

[0113] Base group: 2 hours after DNCB drug stimulation, the blank preparation was applied to the skin of mice twice a day, with an 8-hour interval between the two applications;

[0114] Corylin group: Two hours after DNCB stimulation, mice were given Corylin preparation at a concentration of 0.6 mg / g applied to their skin twice a day, with an 8-hour interval between applications.

[0115] Crisaborole group: 2 hours after DNCB stimulation, 90 mg of Crisaborole ointment was applied to the skin of mice twice a day, with an 8-hour interval between applications.

[0116] 2.3. Mouse skin lesion scoring determination

[0117] The severity of skin lesions in mice was scored on days 0, 5, 8, 11, 14, 17, 20 and 22, based on the following four aspects: edema / papules; erythema / hemoptysis; epidermal peeling; scaling / dryness. Each score was divided into four levels: asymptomatic (0), mild symptoms (1), moderate symptoms (2) and severe symptoms (3). The scores were determined by the three observers other than the experimenter.

[0118] 2.4 Measurement of Scratching Behavior in Mice

[0119] Scratching behavior in mice was assessed by counting the number of scratches. Ten minutes after the last sensitization, each mouse was placed in a separate cage, and the number of times the mouse scratched the stimulated area with its hind paws within 10 minutes was recorded. Each scratch of the skin at the DNCB-stimulated area was counted as one valid scratch. If scratching continued for more than 3 seconds, it was counted as two scratches, and the mouse was manually intervened to stop the scratching behavior after 3 seconds.

[0120] 2.5. TEWL Measurement in Mice

[0121] Transdermal water loss in the skin lesions of mice in each group was measured using the Tewameter TM300 probe of a multi-probe skin analyzer. The standard measurement method was used, with the measurement time set to 20 seconds per measurement, and the average value was read 15 seconds later.

[0122] 2.6. Histopathological examination of mouse skin

[0123] 2.6.1 H&E staining to observe mouse epidermal thickness

[0124] Samples from each group of mice were fixed in 4% formaldehyde, dehydrated, and permeabilized before being embedded in paraffin and sectioned using a microtome. The sections were then dewaxed and rehydrated. Hematoxylin and 0.5% eosin staining was performed. The sections were then dehydrated, permeabilized, and mounted with neutral resin. Five fields of view were randomly selected under a microscope, and the average epidermal thickness was calculated for each field.

[0125] 2.6.2 Toluidine blue staining to observe the number of mast cells

[0126] The specific steps for toluidine blue staining are as follows.

[0127] (1) Dewaxing paraffin sections to anhydrous: The sections were placed in environmentally friendly dewaxing solution I for 20 min, environmentally friendly dewaxing solution II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min in sequence, and then washed with water.

[0128] (2) Toluidine blue staining: Animal tissue sections are immersed in the staining solution for 2-5 minutes, washed with water, slightly differentiated with 0.1% glacial acetic acid, and the reaction is terminated by washing with ultrapure water. The degree of differentiation is controlled under a microscope. After washing with ultrapure water, the sections are placed in an oven to dry.

[0129] (3) Clearing and mounting: Place the section in clean xylene for 10 min to clear, then mount with neutral resin.

[0130] Then, randomly select 5 fields of view under the microscope and calculate the average value of mast cells.

[0131] 2.7 Evaluation of Spleen Index in Mice

[0132] Mice were euthanized by cervical dislocation, with their left abdomen exposed. The skin was cut open to reveal a long, dark strip of spleen tissue. The spleen was removed, rinsed in physiological saline, and blotted dry with filter paper. Its weight was measured to calculate the spleen index, and a photograph was taken for recording. The spleen index formula is: Spleen Index = Spleen weight (g) / Mouse body weight (g) × 100%.

[0133] 2.8 ELISA detection of IgE levels in mouse serum samples

[0134] On day 20, mice were anesthetized with isoflurane, and blood was collected by removing the eyeballs. The collected blood was placed in a 1.5 mL centrifuge tube and allowed to stand at room temperature for 30 min. It was then centrifuged at 2000 rpm for 20 min, and the supernatant was collected and stored at -80℃. Serum IgE levels were measured using an ELISA kit.

[0135] 2.9 qRT-PCR detection of cytokines in mouse skin tissue

[0136] 2.9.1 Preparation of cDNA from mouse skin tissue

[0137] (1) Weigh 30-50mg of sample and place it in a glass tissue homogenizer. Add 1mL of Trizol and grind thoroughly to lyse. Centrifuge at 12000rpm at 4℃ for 10min.

[0138] (2) Take the supernatant, being careful not to aspirate the precipitate. The pipette tip should move down with the liquid level. Let stand at room temperature for 2 minutes, then add 0.2 times the volume of the supernatant in chloroform. Shake vigorously for 15 seconds, let stand at room temperature for 3 minutes, and centrifuge at 12000 rpm at 4°C for 15 minutes.

[0139] (3) Transfer the aqueous phase to a new 2mL centrifuge tube, with about 600μL of supernatant (if the supernatant is less than 600μL, add enzyme-free water to 600μL), and add 300μL RL and 450μL isopropanol.

[0140] (4) Take 700 μL of aqueous phase onto the CR3 column, centrifuge at 12000 rpm for 30 s at room temperature, discard the downstream liquid, and reassemble the column.

[0141] (5) Add 350 μL of RW1 protein removal solution to the CR3 column, centrifuge at 12000 rpm at room temperature for 30-60 s, discard the downstream liquid, and put the adsorption column back into the collection tube.

[0142] (6) Mix 10 μL DNase I and 70 μL LRDD by gently inverting to prepare DNase I working solution. Add 80 μL of DNase I working solution to the center of the CR3 adsorption column and let stand at room temperature for 15 min.

[0143] (7) Add 350 μL of protein removal solution RW1 to the adsorption column CR3, centrifuge at 12000 rpm at room temperature for 30-60 s, discard the downstream liquid, and put the adsorption column back into the collection tube.

[0144] (8) Add 500 μL of washing buffer RW to the adsorption column CR3, centrifuge at 12000 rpm for 30-60 s at room temperature, discard the downstream waste liquid, put the adsorption column CR3 back into the collection tube, and repeat this step once. Centrifuge at 12000 rpm for 2 min at room temperature to remove as much excess liquid as possible from the centrifuge column.

[0145] (9) Transfer the adsorption column CR3 to a new 1.5 mL centrifuge tube, add 30-100 μL of RNase-Free Water to the middle of the adsorption membrane, let it stand at room temperature for 2 min, and centrifuge at 12000 rpm for 2 min at room temperature to obtain the RNA solution.

[0146] (10) Preparation of cDNA by reverse transcription

[0147] Prepare the reverse transcription system by adding enzyme-free water to 10 μL, then add the RNA sample to bring the total volume to 20 μL. Perform the reaction. After the reaction is complete, briefly centrifuge and store the cDNA sample at -20°C for later use.

[0148] 2.9.2 qRT-PCR reaction

[0149] Each group was configured with 3 replicates for subsequent experiments. The qRT-PCR system was prepared by adding 1 μL of PCR Forward Primer, 10 μL of FastStart Universal SYBR Green Master, and 1 μL of PCR Reverse Primer to each well. The cDNA loading volume was 1 μL, and DEPC water was added to bring the total volume to 20 μL. Amplification was performed using GAPDH as an internal control on a Roche 480 instrument. The qRT-PCR primer sequences are shown in Table 1.

[0150] Table 1 Primer sequences for GAPDH and cytokines

[0151]

[0152] 2.9.3 Data Analysis

[0153] The amplification reaction is complete. Ct values ​​for each sample are obtained, and a single-peak melting curve is observed. The mRNA expression level of the target gene is calculated using the following formula.

[0154] ΔCt=Ct 待测基因 -Ct 内参基因 (2-1)

[0155]

[0156] Gene expression = 2 -ΔΔCt (2-3)

[0157] 2.10. Western blot detection of skin barrier-related proteins in mouse skin tissue

[0158] 2.10.1 Preparation of total protein from mouse skin tissue

[0159] (1) Take 20 mg of mouse skin tissue from each group and place it in a 1.5 mL centrifuge tube. Cut it into small pieces. Add 400 μL of physiological saline, mix well, centrifuge and discard the supernatant. Repeat the washing step with physiological saline twice.

[0160] (2) Add 200 μL of RIPA tissue lysis buffer to each tube, place on ice, and sonicate to disrupt the animal skin tissue. After sonication, place on ice for 15 min, centrifuge at 4°C, and the supernatant is the protein to be extracted.

[0161] (3) Preparation of standard curve samples: Protein standards are serially diluted to prepare standard curve samples of different concentrations.

[0162] (4) Protein sample preparation: Dilute the protein sample in (2) with PBS.

[0163] (5) Preparation of BCA working solution: Prepare BCA working solution according to the instructions and store at room temperature.

[0164] (6) Take 20 μL of protein sample and standard curve sample and add them to the well plate in sequence. Add 200 μL of BCA working solution to each well, mix well, incubate at 37℃ for 30 min, and detect the absorbance at 560 nm.

[0165] (7) Plot a standard curve using the concentration of the standard and the absorbance value corresponding to the well, and calculate the protein concentration of the sample.

[0166] 2.10.2 Western blot detection

[0167] (1) Preparation of separating and stacking gels: Prepare separating and stacking gels of the corresponding concentrations according to the molecular weight of the target protein.

[0168] (2) Adding sample proteins: According to the experimental grouping order, add the pre-stained protein Maker and protein samples to the corresponding lanes of the gel in sequence;

[0169] (3) Electrophoresis: When performing electrophoresis, first connect the electrophoresis tank to the power supply of the electrophoresis apparatus and set the constant voltage to 80V. After electrophoresis for 30 minutes, readjust the electrophoresis conditions to a constant voltage of 110V and continue electrophoresis for about 90 minutes until the protein sample migrates to the bottom of the separating gel. After completing the electrophoresis, turn off the power to terminate the electrophoresis process.

[0170] (4) Transfer: After electrophoresis, remove the gel and place the sponge, filter paper, separating gel, polyvinylidene fluoride (PVDF) membrane, filter paper and sponge pre-wetted with transfer solution into the wet transfer clamp in a "sandwich" manner. Remove air bubbles, clamp the wet transfer clamp tightly and place it into the wet transfer apparatus filled with transfer solution. Place the wet transfer apparatus in an ice bath to maintain a low temperature environment and transfer the membrane for 110 min under a constant current of 200mA.

[0171] (5) Sealing: After the transfer is completed, remove the wet transfer clamp, cut the PVDF membrane according to the gel size, then soak the PVDF membrane in 1×TBST solution to remove excess transfer solution, and place the PVDF membrane in 5% milk powder sealing solution prepared with 1×TBST solution to seal at room temperature for 2 hours.

[0172] (6) Washing the membrane: Discard the 5% milk powder sealing solution and soak the PVDF membrane multiple times with 1×TBST solution to wash away the milk powder sealing solution on the surface of the PVDF membrane;

[0173] (7) Primary antibody incubation overnight: Place the PVDF membrane in the corresponding diluted target protein primary antibody dilution solution and incubate overnight at 4°C on a shaker;

[0174] (8) Washing the membrane: The primary antibody was recovered the next day, and the membrane was washed multiple times with 1×TBST to remove the primary antibody from the surface of the PVDF membrane.

[0175] (9) Secondary antibody incubation: Place the PVDF membrane in the corresponding diluted secondary antibody solution and incubate it on a shaker at room temperature for 90 min;

[0176] (10) Washing the membrane: recover the secondary antibody, wash the membrane multiple times with 1×TBST to remove the secondary antibody from the surface of the PVDF membrane, and immerse the PVDF membrane in 1×TBST.

[0177] (11) Development and imaging: Prepare and mix solutions A and B of the enhanced chemiluminescence (ECL) reagent in equal proportions according to experimental requirements. Prepare fresh for each use. Add ELC reagent to the PVDF membrane to completely wet it. Expose the membrane in a multi-functional imager and analyze the electrophoretic bands using ImageJ software.

[0178] 3. Statistical processing

[0179] All data were analyzed using SPSS 25 statistical software. Results are expressed as mean ± standard deviation. To indicate that the differences between groups were statistically significant using one-way ANOVA, P < 0.05 was considered statistically significant.

[0180] 4. Results

[0181] 4.1 Comparison of skin appearance in AD mice

[0182] This experiment investigated the effect of Corylin on skin lesions and dermatitis scores in a DNCB-induced AD mouse model. The results showed that the control group mice had smooth, rosy skin; the model group exhibited dermatitis symptoms such as edema, scarring, dry erythema, epidermal scaling, and erosion, indicating the successful establishment of the DNCB-induced AD mouse model. Both the Corylin and Crisaborole treatment groups significantly improved the DNCB-induced dermatitis symptoms in mice (P<0.001, P<0.01). See [link to relevant documentation]. Figure 2 , Figure 3 .

[0183] 4.2 Effects of Corylin on Scratching Behavior in AD Mice

[0184] One of the characteristics of AD is itching, and the number of scratches is used as one of the indicators for evaluating the efficacy of Corylin in a DNCB-induced AD mouse model. Figure 4 As shown, compared with the control group, the number of scratches in the model group mice was significantly increased (P<0.001). The number of scratches in the Crisaborole and Corylin administration groups was significantly reduced (P<0.001).

[0185] 4.3 Effects of Corylin on TEWL in AD Mice

[0186] Scratching caused by itching damages the skin's barrier function, and the persistent "itch-scratching" cycle further exacerbates the damage to the skin barrier function in Alzheimer's disease (AD). The skin barrier maintains skin hydration and prevents transepidermal water loss. Damage to the skin barrier function increases TEWL (transepidermal water loss). Figure 5 It was found that, compared with the Control group, DNCB-induced TEWL in AD mice was significantly increased (P<0.001); compared with the Model group, the positive control drug Cresaborole significantly reduced TEWL in mouse skin (P<0.001). TEWL in mice in the Corylin-treated group was also significantly downregulated (P<0.001, P<0.01).

[0187] 4.4 Effects of Corylin on the Skin Tissue Structure of AD Mice

[0188] To better clarify whether Corylin could improve skin lesions in an AD mouse model, H&E staining and toluidine blue staining were performed on the dorsal skin of mice. H&E staining showed that, compared with the control group, the epidermal thickness in the model group was significantly increased (P<0.001); administration of Corylin and Crisaborole significantly reduced the epidermal thickness of mice (P<0.001, P<0.01). Toluidine blue staining was used to observe the effect of Corylin on mast cells in the skin of AD mice. Toluidine blue staining showed that, compared with the control group, the number of mast cells and increased infiltration were significantly increased in the skin of the model group (P<0.001); compared with the model group, the number of mast cells and infiltration were significantly reduced in the skin of mice treated with Corylin and Crisaborole (P<0.001, P<0.01). See [link to article]. Figure 6 , Figure 7 .

[0189] 4.5 Effect of Corylin on spleen index in AD mice

[0190] Alzheimer's disease (AD) not only manifests as skin lesions but also affects immune organs. The spleen in DNCB-induced mice was significantly larger than in normal mice. Changes in the spleen index can reflect the level of inflammation in mice. Compared with the control group, the spleen index of mice in the model group was significantly increased (P<0.001). Crisaborole and Corylin administration significantly reduced the spleen index in mice (P<0.01, P<0.05). See [link / reference needed]. Figure 8 .

[0191] 4.6 Effect of Corylin on serum IgE in AD mice

[0192] Elevated serum IgE levels are an important clinical feature of Alzheimer's disease (AD). The expression levels of IgE in the serum of mice in each group were detected using ELISA. The results showed that the serum IgE level in the Model group was significantly higher than that in the Control group (P<0.001). After drug administration, the serum IgE levels in the Crisaborole and Corylin groups were significantly lower (P<0.001), see [link to relevant documentation]. Figure 9 .

[0193] 4.7 Effects of Corylin on the expression of cytokine mRNA in the skin of AD mice

[0194] Impaired skin barrier function leads to the production of various pro-inflammatory mediators, inflammatory cytokines, and chemokines by epithelial cells in response to external stimuli, initiating the inflammatory response in Alzheimer's disease (AD). This experiment examined cytokines such as IL-4, IL-5, IL-6, IL-13, and TSLP to explore whether Corylin could downregulate the expression of IL-4, IL-5, IL-6, IL-13, and TSLP mRNA. Results showed that compared with the control group, the mRNA levels of IL-4, IL-5, IL-6, IL-13, and TSLP in the model skin were significantly increased (P<0.001). After administration of crisaborole and Corylin, the levels of IL-4, IL-5, IL-6, IL-13, and TSLP cytokines in mouse skin were significantly decreased (P<0.001). Figure 10 .

[0195] 4.8 Effects of Corylin on the expression of skin barrier-related proteins in AD mice

[0196] In BALB / c mice, compared with the Control group, the expression levels of skin barrier function-related proteins such as filaggrin, loricrin, and involucrin were significantly decreased in the Model group (P<0.001, P<0.01). Compared with the Model group, both the Corylin-treated and Crisaborole-treated groups significantly increased the expression levels of skin barrier function-related proteins such as filaggrin, loricrin, and involucrin (P<0.001, P<0.01, P<0.05). See Figure 11 , Figure 12 , Figure 13 .

[0197] 5. Summary

[0198] Corylin effectively improved dermatitis characteristics such as redness, scaling, and ulceration in AD mice; it significantly reduced the frequency of scratching in AD mice, effectively relieving itching; it reduced transepidermal water loss in AD mouse skin; H&E staining showed that Corylin significantly reduced epidermal thickness in AD mice; toluidine blue staining showed that Corylin significantly reduced the number of mast cells in AD mouse skin tissue and improved mast cell infiltration; Corylin significantly reduced spleen swelling, decreased serum IgE levels in AD mice, and reduced mRNA expression of cytokines IL-4, IL-5, IL-6, IL-13, and TSLP in AD mouse skin. Corylin effectively increased the protein expression of Filaggrin, Loricrin, and Involucrin in AD mouse skin tissue, indicating a role in repairing the skin barrier in AD mice.

[0199] Example 2

[0200] Study on the effect of Corylin on a TNF-α / IFN-γ-stimulated cellular inflammation model

[0201] A cellular inflammation model was established by inducing keratinocytes with TNF-α and IFN-γ. After administration of Corylin, cell viability of TNF-α and IFN-γ-induced keratinocytes was detected using a CCK-8 assay, and LDH release from TNF-α and IFN-γ-induced keratinocytes was detected using a lactate dehydrogenase assay. qRT-PCR was used to detect the mRNA expression of cytokines IL-6, IL-8, IL-1β, MDC, and TSLP, as well as filaggrin, loricrin, and involucrin in TNF-α and IFN-γ-induced keratinocytes. Western blot was used to detect the protein expression of filaggrin, loricrin, and involucrin in TNF-α and IFN-γ-induced keratinocytes to examine the ameliorative effect of Corylin on the cellular inflammation model. The effect of Corylin on Alzheimer's disease (AD) was investigated at the cellular level.

[0202] 1. Materials

[0203] 1.1 Reagents and Consumables

[0204]

[0205]

[0206] 1.2 Instruments

[0207] Real-time quantitative PCR instrument (LightCycler 480), Roche, Switzerland

[0208] Gel imaging systems from Bio-Rad, USA

[0209] 1.3 Cell lines

[0210] HaCaT and HEK were both purchased from the ATCC cell bank.

[0211] 2. Method

[0212] 2.1 Solution Preparation

[0213] (1) Preparation of Corylin solution: Accurately weigh 3.20 mg of Corylin standard, dissolve it in 100 μL of DMSO to obtain 1×10 5The Corylin standard stock solution of μM was diluted with the corresponding complete culture medium to obtain the Corylin sample solution of the corresponding concentration.

[0214] (2) Preparation of 1×CCK-8 working solution: Dilute 10×CCK-8 working solution 10 times with MEM or DMEM basal medium according to the culture medium used for the cells being tested, and mix well.

[0215] (3) Preparation of TNF-α working solution: Dissolve TNF-α powder into a standard stock solution of 2 μg / mL according to the instructions, and then dilute it stepwise as needed to the corresponding concentration of TNF-α working solution.

[0216] (4) Preparation of IFN-γ working solution: Dissolve IFN-γ powder into a standard stock solution of 2μg / mL according to the instructions, and then dilute it stepwise as needed to the corresponding concentration of IFN-γ working solution.

[0217] 2.2. Culture and treatment of keratinocytes

[0218] 2.2.1. Recovery of keratinocytes

[0219] Remove frozen HaCaT and HEK cells from the -80°C freezer and immediately place them in a 37°C water bath to thaw rapidly. After sterilization, immediately transfer them to a biosafety cabinet. Transfer the cell suspension to centrifuge tubes containing fresh complete culture medium, centrifuge, discard the supernatant, add an appropriate amount of the corresponding complete culture medium, resuspend and mix well, and continue culturing in T25 culture flasks. Incubate at 37°C in a 5% CO2 incubator.

[0220] 2.2.2 Keratinocyte passage and seed plate

[0221] Under a microscope, observe the cells until they reach approximately 80% confluence, then passage them. Discard the complete culture medium in the flask, add 2 mL of PBS solution, gently shake, wash twice, discard the PBS solution, add 1 mL of 1X 0.25% trypsin-EDTA, and incubate at 37°C, 5% CO2 for 5 minutes. After the cells become rounded and detach, stop the digestion with an appropriate amount of the corresponding complete culture medium, transfer the digested cell suspension to a 15 mL centrifuge tube, and centrifuge moderately. Discard the cell supernatant, add an appropriate amount of MEM or DMEM complete culture medium, mix well, and transfer at a 1:4 ratio to a new cell culture flask. Incubate at 37°C, 5% CO2 for further experiments.

[0222] 2.3 Screening of Corylin for Safe Concentrations in Keratinocytes

[0223] The CCK-8 assay kit was used to evaluate the cytotoxicity of Corylin against keratinocytes. HaCaT cells / HEK cells were cultured at a ratio of 1 × 10⁻⁶ cells / cells. 5 HaCaT / HEK cells were seeded at a density of 80% / mL in 96-well plates. Corylin was added at the appropriate concentration. After 24 hours, 1×CCK-8 working solution was added, and the cells were incubated at 37°C for 0.5 hours. The absorbance was then measured using a microplate reader to calculate cell viability.

[0224] 2.4 Establishment of a TNF-α / IFN-γ-stimulated human keratinocyte inflammation model

[0225] 2.4.1 Establishment of a TNF-α / IFN-γ-stimulated HaCaT cell inflammation model

[0226] HaCaT cells were divided into 5×10 5 Cells were seeded at a density of 10 cells / well in 6-well plates. Once the cells had adhered and grown to 80%, 2 mL of MEM complete medium with a final concentration of 10 ng / mL TNF-α and 10 ng / mL IFN-γ was added to each well and the cells were incubated for 6 h or 24 h, respectively. Cells were then collected and cytokine expression levels were detected using qRT-PCR.

[0227] 2.4.1.1 Preparation of cDNA from TNF-α / IFN-γ-stimulated HaCaT cells

[0228] (1) Discard the culture medium, wash twice with pre-cooled PBS, add 1 mL of Trizol reagent to lyse the cells, collect the cells into a 1.5 mL centrifuge tube, and let stand on ice for 5 min.

[0229] (2) Add 200 μL of chloroform to each tube, mix by inverting for 15 seconds, let stand on ice for 15 minutes, and then centrifuge at 4℃ and 12000 rpm for 5 minutes.

[0230] (3) Gently aspirate the colorless and transparent supernatant into a 1.5 mL centrifuge tube, being careful not to touch the precipitate.

[0231] (4) Add an equal volume of isopropanone to each tube, mix well, and let stand on ice for 5 minutes. Then centrifuge at 4°C and 12,000 rpm for 10 minutes.

[0232] (5) Discard the supernatant. A small amount of white precipitate can be seen at the bottom of the tube. Add 1 mL of 75% ethanol to resuspend the precipitate and centrifuge at 4℃ and 12000 rpm for 5 min.

[0233] (6) Discard the supernatant and repeat step (5).

[0234] (7) Carefully aspirate the supernatant, place the centrifuge tube in a ventilated place to dry for about 2 hours until the edge of the white precipitate becomes transparent, and then add 20 μL of enzyme-free water to dissolve and obtain the RNA solution.

[0235] (8) Reverse transcription to prepare cDNA: The steps are the same as (10) in 1.2.9.1.

[0236] 2.4.1.2 qRT-PCR reaction

[0237] The steps are the same as in Example 1, and the qRT-PCR primer sequences are shown in Table 2.

[0238] Table 2 Primer sequences for GAPDH and cytokines

[0239]

[0240] 2.4.1.3 Data Analysis

[0241] The steps are the same as in Example 1.

[0242] 2.4.2 Establishment of a TNF-α / IFN-γ-stimulated HEK cell inflammation model

[0243] HEK cells were divided into 5×10 5 Cells were seeded at a density of 1 cell / well in 6-well plates. Once the cells had adhered and grown to 80%, the appropriate concentration of Corylin was added, and the cells were cultured for another 2 hours. The supernatant was discarded, and 2 mL of MEM complete medium with a final concentration of 10 ng / mL TNF-α and 10 ng / mL IFN-γ was added to each well. The cells were then incubated for 24 hours. Cells were collected and cytokine expression levels were detected using qRT-PCR.

[0244] 2.4.2.1 Preparation of cDNA from HEK cells stimulated by TNF-α / IFN-γ

[0245] Replace the cells with HEK cells, and follow the same steps as in 2.2.3.1.1.

[0246] 2.4.2.2 qRT-PCR reaction

[0247] The steps are the same as in 2.2.3.1.2.

[0248] 2.4.2.3 Data Analysis

[0249] The steps are the same as in 2.2.3.1.3.

[0250] 2.5 Effects of Corylin on TNF-α / IFN-γ-stimulated keratinocyte cell viability and LDH release

[0251] 2.5.1 Effects of Corylin on cell viability and LDH release in HaCaT cells stimulated by TNF-α / IFN-γ

[0252] HaCaT cells were used at a rate of 1×10 5 Cells were seeded at a concentration of [number] cells / mL in 96-well plates, followed by the addition of Corylin solution at the appropriate concentration. After 2 hours, medium containing 10 ng / mL TNF-α and 10 ng / mL IFN-γ was added to each well and incubated for 24 hours. Then, 1×CCK-8 working solution was added, and the plates were incubated at 37°C for 0.5 hours. The absorbance was measured using a microplate reader to calculate cell viability. Approximately 50 μL of cell culture medium was then transferred from each well to a new 96-well plate, and 50 μL of Assay Buffer was added to each well. The plates were incubated at 37°C in the dark for 15 minutes.

[0253] 2.5.2 Effects of Corylin on TNF-α / IFN-γ-stimulated HEK cell viability and LDH release

[0254] Replace the cells with HEK cells, and follow the same steps as in 2.2.5.1.

[0255] 2.6 Effects of Corylin on Cytokines in Keratinocytes After TNF-α / IFN-γ Stimulation

[0256] 2.6.1 Effects of Corylin on Cytokines in HaCaT Cells After TNF-α / IFN-γ Stimulation

[0257] HaCaT cells / HEK cells were arranged at a ratio of 5 × 10 5 Cells were seeded at a density of 10 cells / well in 6-well plates. After 80% cell adhesion and growth, Corylin solution of the appropriate concentration was added. Two hours later, 2 mL of MEM / DMEM complete medium with a final concentration of 10 ng / mL TNF-α and 10 ng / mL IFN-γ was added to each well and the cells were incubated for 24 hours. Cells were then collected and cytokine expression levels were detected using qRT-PCR.

[0258] 2.6.1.1 Preparation of cDNA

[0259] The steps are the same as in 2.2.3.1.1.

[0260] 2.6.1.2, qRT-PCR

[0261] The steps are the same as in 2.2.3.1.2.

[0262] 2.6.1.3 Data Analysis

[0263] The steps are the same as in 2.2.3.1.3.

[0264] 2.6.2 Effects of Corylin on Cytokines in HEK Cells After TNF-α / IFN-γ Stimulation

[0265] 2.6.2.1 Preparation of cDNA

[0266] The steps are the same as in 2.2.3.1.1.

[0267] 2.6.2.2, qRT-PCR

[0268] The steps are the same as in 2.2.3.1.2.

[0269] 2.6.2.3 Data Analysis

[0270] The steps are the same as in 2.2.3.1.3.

[0271] 2.7 Effects of Corylin on Skin Barrier Function-Related Indicators in Keratinocytes After TNF-α / IFN-γ Stimulation

[0272] HaCaT cells / HEK cells were arranged at a ratio of 5 × 10 5 Cells were seeded at a density of 1 cell / well in 6-well plates, and the appropriate concentration of Corylin was added. After culturing for 2 hours, the supernatant was discarded. When the cells adhered and grew to 80%, 2 mL of MEM complete medium with a final concentration of 10 ng / mL TNF-α and 10 ng / mL IFN-γ was added to each well and incubated for 24 hours. Cells were collected and the expression levels of skin barrier function-related indicators were detected by qRT-PCR and Western blot.

[0273] 2.7.1 qRT-PCR

[0274] 2.7.1.1 Preparation of cDNA

[0275] The steps are the same as in 2.2.3.1.1.

[0276] 2.7.1.2, qRT-PCR

[0277] The steps are the same as in 2.2.3.1.2.

[0278] 2.7.1.3 Data Analysis

[0279] The steps are the same as in 2.2.3.1.3.

[0280] 2.7.2 Western blot

[0281] 2.7.2.1 Preparation of protein samples

[0282] (1) Add 100 μL of RIPA lysis buffer to the cell pellets collected above, resuspend them and place them on ice for 15 min.

[0283] (2) Centrifuge at 4℃ and 12000rpm for 10min, and the supernatant is the extracted protein.

[0284] The remaining steps are the same as in 1.2.10.1.

[0285] 2.7.2.2, Western blot

[0286] The steps are the same as in 1.2.10.2.

[0287] 3. Statistical processing

[0288] The steps are the same as in 1.3.

[0289] 4. Results

[0290] 4.1 Screening of Corylin for Safe Concentrations in Keratinocytes

[0291] 4.1.1 Screening of safe concentrations of Corylin for HaCaT cells

[0292] Figure 14 The results showed that the cell viability of the 100 μM Corylin solution group was 109.42 ± 10.57%, which was significantly higher than that of the Control group (P < 0.01). The cell viability of the 1 and 10 μM Corylin solution groups did not show significant changes compared with the Control group (P > 0.05). The 1 and 10 μM Corylin concentrations were selected for subsequent experiments.

[0293] 4.1.2 Screening of safe concentrations of Corylin in HEK cells

[0294] Figure 15 The results showed that the cell viability of the 100 μM Corylin solution group was 114.55 ± 11.50%, which was significantly higher than that of the Control group (P < 0.01). The cell viability of the 1 and 10 μM Corylin solution groups did not show significant changes compared with the Control group (P > 0.05). The 1 and 10 μM Corylin concentrations were selected for subsequent experiments.

[0295] 4.2 Establishment of a TNF-α / IFN-γ-stimulated human keratinocyte cellular inflammation model

[0296] 4.2.1 Establishment of a TNF-α / IFN-γ-stimulated HaCaT cell inflammation model

[0297] Figure 16The results showed that, compared with the Control group, after 6 h of TNF-α / IFN-γ stimulation of HaCaT cells, the mRNA levels of cytokines such as IL-6, IL-8, MDC, and TSLP did not change significantly. However, after 24 h of TNF-α / IFN-γ stimulation of HaCaT cells, the mRNA levels of cytokines such as IL-4, IL-1β, IL-6, IL-8, MDC, and TSLP increased significantly (P<0.001, P<0.01). Therefore, TNF-α / IFN-γ stimulation of HaCaT cells for 24 h was selected as the modeling condition for subsequent experiments on HaCaT cells.

[0298] 4.2.2 Establishment of a TNF-α / IFN-γ-stimulated HEK cell inflammation model

[0299] Figure 17 The results showed that, compared with the Control group, the mRNA levels of cytokines such as IL-4, IL-1β, IL-6, IL-8, MDC, and TSLP were significantly increased after TNF-α / IFN-γ stimulation of HEK cells for 24 h (P<0.001, P<0.01, P<0.05). Therefore, TNF-α / IFN-γ stimulation of HEK cells for 24 h was selected as the modeling condition for subsequent HEK cell experiments.

[0300] 4.3 Effects of Corylin on TNF-α / IFN-γ-stimulated keratinocyte cell viability and LDH release

[0301] 4.3.1 Effects of Corylin on cell viability and LDH release in HaCaT cells stimulated by TNF-α / IFN-γ

[0302] The results showed that the cell viability in the Model group was 84.30±6.11%, and the LDH release was 157.88±6.82%. Compared with the Control group, the cell viability was significantly decreased (P<0.001), and the LDH release was significantly increased (P<0.001), indicating that the TNF-α / IFN-γ-stimulated HaCaT cell inflammation model was successfully established. Compared with the Model group, 10 -3 The -1 μM methyl orylin solution group significantly improved cell viability (P<0.001, P<0.01, P<0.05) and reduced LDH release (P<0.001, P<0.01, P<0.05). Figure 18 , Figure 19 .

[0303] 4.3.2 Effects of Corylin on cell viability and LDH release in HEK cells stimulated by TNF-α / IFN-γ

[0304] The results showed that the cell viability in the Model group was 84.91±6.70%, and the LDH release was 157.82±6.41%. Compared with the Control group, the cell viability was significantly decreased (P<0.001), and the LDH release was significantly increased (P<0.001), indicating that the TNF-α / IFN-γ-stimulated HaCaT cell inflammation model was successfully established. Compared with the Model group, 10 -3 The -1 μM methyl orylin solution group significantly improved cell viability (P<0.001, P<0.01) and reduced LDH release (P<0.05). Figure 20 , Figure 21 .

[0305] 4.4 Effects of Corylin on Cytokines in Keratinocytes After TNF-α / IFN-γ Stimulation

[0306] 4.4.1 Effects of Corylin on related cytokines in HaCaT cells after TNF-α / IFN-γ stimulation

[0307] like Figure 22 As shown, compared with the Control group, the Model group had significantly increased mRNA levels of cytokines such as IL-4, IL-1β, IL-6, IL-8, MDC, and TSLP (P<0.001, P<0.01). After receiving 10 [units of something], -3 Following administration of 1 μM orylin, cytokine mRNA levels were significantly downregulated (P<0.001, P<0.01, P<0.05).

[0308] 4.4.2 Effects of Corylin on Related Cytokines in HEK Cells After TNF-α / IFN-γ Stimulation

[0309] like Figure 23 As shown, compared with the Control group, the Model group had significantly increased mRNA levels of cytokines such as IL-4, IL-1β, IL-6, IL-8, MDC, and TSLP (P<0.001, P<0.01, P<0.05). -3 After administration of 1 μM orylin, the levels of cytokine mRNA were significantly reduced (P<0.001, P<0.01, P<0.05).

[0310] 4.5 Effects of Corylin on Skin Barrier Function-Related Indicators in Keratinocytes After TNF-α / IFN-γ Stimulation

[0311] 4.5.1 Effects of Corylin on Skin Barrier Function-Related Indicators in HaCaT Cells After TNF-α / IFN-γ Stimulation

[0312] In HaCaT cells, compared with the Control group, the Model group showed significantly lower mRNA and protein expression levels of skin barrier function-related indicators such as Filagrin, Involucrin, and Loricrin (P<0.001). Compared with the Model group, cells treated with 10... -3 -10 -1 μM of Corylin significantly increased the mRNA and protein expression levels of skin barrier function-related indicators such as Filaggrin, Involucrin, and Loricrin (P<0.001, P<0.01). Figure 24 , Figure 25 , Figure 26 , Figure 27 As shown.

[0313] 4.5.2 Effects of Corylin on Skin Barrier Function-Related Indicators in HEK Cells After TNF-α / IFN-γ Stimulation

[0314] In HEK cells, compared with the Control group, the Model group showed significantly lower mRNA and protein expression levels of skin barrier function-related indicators such as Filagrin, Involucrin, and Loricrin (P<0.001, P<0.01). Compared with the Model group, cells treated with 10... -1 -10 -3 μM of Corylin significantly increased the mRNA and protein expression levels of skin barrier function-related indicators such as Filaggrin, Involucrin, and Loricrin (P<0.001, P<0.01, P<0.05). Figure 28 , Figure 29 , Figure 30 , Figure 31 As shown.

[0315] 5. Summary

[0316] A cellular inflammation model was successfully constructed by stimulating keratinocytes with TNF-α and IFN-γ. Corylin significantly increased the cell viability of TNF-α and IFN-γ-stimulated keratinocytes and inhibited LDH release from TNF-α and IFN-γ-stimulated keratinocytes. Corylin effectively reduced the mRNA expression of cytokines IL-6, IL-8, IL-1β, MDC, and TSLP in TNF-α and IFN-γ-stimulated keratinocytes, and significantly increased the mRNA and protein expression of filaggrin, loricrin, and involucrin.

[0317] Example 3

[0318] Corylin's study on the ameliorative effect of PDE4B on AD models

[0319] This embodiment first used Western blot experiments to investigate the changes in PDE4B expression in AD mice and TNF-α / IFN-γ-stimulated keratinocytes by Corylin. To clarify the key role of PDE4B in AD, PDE4B was overexpressed using plasmid transfection technology to explore the effect of PDE4B expression changes on inflammation and barrier function-related biomarkers in AD. qRT-PCR and Western blot were then used to clarify the regulatory effect of Corylin on PDE4B in HEK cells, and further investigation was conducted to determine whether the protective effect of Corylin on the TNF-α / IFN-γ-stimulated HEK inflammation model was achieved through the regulation of PDE4B molecules.

[0320] 1. Materials

[0321] 1.1 Reagents and Consumables

[0322] Overexpression plasmids from China Gemma Gene Co., Ltd.

[0323] GPtransmate transfection reagent, China Gemma Gene Co., Ltd.

[0324] Primers Shanghai Sangon Biotech

[0325] The remaining reagents and consumables are the same as in Example 2.

[0326] 1.2 Instruments

[0327] Same as Example 2.

[0328] 1.3 Cell lines

[0329] Both HaCaT cells and HEK cells were purchased from the ATCC cell bank.

[0330] 2. Method

[0331] 2.1 The solution preparation steps are the same as in Example 2.

[0332] 2.2. Culture and treatment of keratinocytes

[0333] 2.2.1. Keratinocyte resuscitation, the steps are the same as in Example 2.

[0334] 2.2.2 Passaging of keratinocytes, the steps are the same as in Example 2.

[0335] 2.2.3. Keratinocyte administration treatment, the steps are the same as in Example 2.

[0336] 2.3 Effect of Corylin on PDE4B expression in AD models

[0337] 2.3.1 Western blot detection of PDE4B protein expression, the procedure is the same as in Example 2.

[0338] 2.4 Effects of PDE4B overexpression of Corylin on a TNF-α / IFN-γ-stimulated HEK cell inflammation model

[0339] 2.4.1 Verification of PDE4B overexpression efficiency in HEK cells

[0340] Take HEK cells in good growth condition, and administer at a ratio of 4 × 10⁻⁶. 5 Cells were seeded at a density of 10 cells / mL in 60 mm culture dishes. When the cells reached approximately 60-80% confluency, PDE4B overexpression plasmid transfection was performed. A control group, a mock transfection group, and a model group (TNF-α / IFN-γ co-stimulation) were set up. For transfection, 500 μL of serum-free medium and 15 μL of GP transfection reagent were added to tube A and mixed well. 500 μL of serum-free medium and 15 μL of PDE4B-OE were added to tube B and mixed well. After standing for 5 min, the solution from tube A was directly added to tube B, gently mixed, and incubated at room temperature for 15-20 min. Then, 4 mL of LDM complete medium was added and the mixture was transferred to a culture dish. Cells were placed in an incubator for transfection for 5-7 h. After transfection, the transfection solution was discarded, and 5 mL of LDM complete medium was added to terminate the transfection. qRT-PCR and Western blot were used to detect cell transfection.

[0341] The steps for cDNA preparation and qRT-PCR reaction are the same as in 2.2.4.1.1-2.2.4.1.3. Primer sequences are shown in Table 3.

[0342] Table 3 Primer sequences

[0343]

[0344] The steps for protein sample preparation and Western blot detection are the same as in Example 2.

[0345] 2.4.2 Effect of Corylin on Cytokine Expression in HEK Cells after PDE4B Overexpression

[0346] Take HEK cells in good growth condition, and administer at a ratio of 4 × 10⁻⁶. 5Cells were seeded at a density of 10 cells / mL in 60 mm culture dishes. When the cells reached approximately 60-80% confluency, PDE4B overexpression plasmid transfection experiments were performed. The following groups were set up: Control group, transfection reagent group (Mock group), model group (TNF-α / IFN-γ co-stimulation), TNF-α / IFN-γ co-stimulation + Corylin group, and TNF-α / IFN-γ co-stimulation + Corylin + PDE4B-OE group. For transfection, 500 μL of serum-free medium and 15 μL of GPtransmate transfection reagent were added to tube A and mixed well. 500 μL of serum-free medium and 15 μL of PDE4B-OE were added to tube B and mixed well. After standing for 5 min, the solution from tube A was directly added to tube B, gently mixed, and incubated at room temperature for 15-20 min. Then, 4 mL of LDM complete medium was added to the culture dish, and the cells were placed in an incubator for 5-7 h for transfection. After transfection, the transfection solution was discarded, and 5 ml of LDM complete medium was added to terminate the transfection. qRT-PCR and Western blot were used to detect cell transfection status. Subsequent experiments were performed after 48 hours of culture. qRT-PCR was then used to detect the expression of PDE4B and cytokines.

[0347] The cDNA preparation and qRT-PCR reaction steps are the same as in Example 2.

[0348] 2.4.3 Effect of PDE4B overexpression on the expression of skin barrier-related proteins in TNF-α / IFN-γ stimulated HEK cells by Corylin

[0349] The transfection procedure for overexpressing PDE4B plasmid was the same as in 3.2.5.1. Subsequently, Western blot was used to detect skin barrier-related proteins.

[0350] The steps for protein sample preparation and Western blot detection are the same as in Example 2.

[0351] 3. Statistical processing

[0352] The steps are the same as in 1.3.

[0353] 4. Results

[0354] 4.1 Effect of Corylin on PDE4B expression in AD models

[0355] like Figure 32 As shown in the figure. In vivo results showed that, compared with the control group, the expression level of PDE4B protein in the model group was significantly increased (P<0.05). Compared with the model group, both the Corylin and Crisaborole administration groups significantly reduced the expression level of PDE4B protein (P<0.05).

[0356] Depend on Figure 33 , Figure 34 The results showed that, in vitro experiments, the expression level of PDE4B protein in the Model group was significantly increased compared with the Control group in HaCaT cells and HEK cells (P<0.001). Compared with the Model group, the expression level of PDE4B protein in the Corylin group was significantly inhibited in a dose-dependent manner (P<0.001, P<0.01, P<0.05).

[0357] 4.2 Effects of PDE4B overexpression of Corylin on a TNF-α / IFN-γ-stimulated HEK cell inflammation model

[0358] 4.2.1 Verification of PDE4B overexpression efficiency in HEK cells

[0359] like Figure 35 , Figure 36 As shown, compared with the Control group, the expression levels of PDE4B mRNA and protein in the MOCK group were not significantly increased (P>0.05); after transfection of the PDE4B overexpression plasmid into HEK cells, compared with the Control group, the expression levels of PDE4B mRNA and protein in the PDE4B overexpression plasmid transfection group were significantly increased (P<0.001, P<0.05), indicating that the PDE4B overexpression plasmid was successfully transfected into HEK cells and can be used for subsequent experiments.

[0360] 4.2.2 Effect of PDE4B overexpression of Corylin on cytokine expression in HEK cells

[0361] like Figure 37 As shown, in HEK cells, compared with the Control group, cytokine expression was significantly increased in the Model group (P<0.001, P<0.01), confirming the successful establishment of the model. Compared with the Model group, administration of Corylin significantly reduced cytokine expression levels (P<0.001, P<0.01), indicating that Corylin has an ameliorative effect on the TNF-α / IFN-γ-stimulated HEK cell inflammation model. Compared with the group receiving TNF-α / IFN-γ stimulation and Corylin, the cytokine release was significantly increased in the groups receiving PDE4B overexpression plasmid and Corylin (P<0.001, P<0.01), indicating that the ameliorative effect of Corylin on the TNF-α / IFN-γ-stimulated HEK cell inflammation model was eliminated by the PDE4B overexpression plasmid. These results suggest that Corylin may exert its ameliorative effect on the TNF-α / IFN-γ-stimulated HEK cell inflammation model through the potential target of PDE4B.

[0362] 4.2.3 Effects of Corylin on Skin Barrier Function-Related Indicators in HEK Cells After PDE4B Overexpression

[0363] like Figure 38 , Figure 39 , Figure 40 , Figure 41 As shown, in HEK cells, compared with the Control group, the expression of skin barrier-related indicators in the Model group was significantly reduced (P<0.001, P<0.01), proving the successful construction of the model. Compared with the Model group, administration of Corylin significantly upregulated the expression of skin barrier-related indicators (P<0.001, P<0.01), indicating that Corylin has an ameliorative effect on the TNF-α / IFN-γ-stimulated HEK cell inflammation model. Compared with the group receiving TNF-α / IFN-γ stimulation and Corylin, the expression of skin barrier-related indicators in the groups receiving PDE4B overexpression plasmid and Corylin was significantly reduced (P<0.001, P<0.01, P<0.05), indicating that the ameliorative effect of Corylin on the TNF-α / IFN-γ-stimulated HEK cell inflammation model was eliminated by the PDE4B overexpression plasmid. These results suggest that Corylin exerts an ameliorative effect on the inflammatory response induced by TNF-α / IFN-γ stimulation of cells through the potential target PDE4B.

[0364] 5. Summary

[0365] Corylin significantly inhibited PDE4B protein expression in DNCB-induced AD mice and TNF-α / IFN-γ-stimulated keratinocytes. Overexpression of PDE4B reversed the trend of Corylin in reducing cytokine expression in TNF-α / IFN-γ-stimulated keratinocytes, and also reversed the trend of Corylin in increasing the expression of filaggrin, loricrin, and involucrin. The ameliorative effect of Corylin on the TNF-α / IFN-γ-stimulated keratinocyte inflammation model was reversed by PDE4B overexpression.

[0366] The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. Application of psoralen isoflavones in the preparation of drugs for the prevention and treatment of atopic dermatitis.

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