New application of psoralen isoflavone

Abstract

The invention provides a new application of psoralen isoflavone, the psoralen isoflavone can significantly inhibit protein expression of PDE4B in dinitrochlorobenzene induced AD mouse skin and TNF-alpha / I FN-gamma stimulated keratinocytes, and after treatment with the psoralen isoflavone, dermatitis characteristics such as skin redness and swelling, scales and ulceration of AD mice can be effectively improved; the psoralen isoflavone can be used for remarkably reducing the scratching frequency of AD mice and effectively relieving pruritus; the psoralen isoflavone can reduce the skin percutaneous moisture loss of AD mice; hamp, Hamp; e dyeing shows that the psoralen obviously reduces the skin epidermis thickness of AD mice; toluidine blue staining shows that the psoralen isoflavone remarkably reduces the number of mast cells in AD mouse skin tissue and improves the infiltration state of the mast cells, and the psoralen isoflavone has important application value in preparation of a PDE4B inhibitor.

Description

Technical Field

[0001] The invention relates to the technical field of medicine, in particular to new use of psoralea corylifolia isoflavones. Background Art

[0002] Atopic dermatitis (AD), also known as eczema, is a chronic, recurrent, inflammatory skin disease. The prevalence of atopic dermatitis is gradually increasing worldwide, with 15%-30% of children and 2-10% of adults suffering from the disease. AD is often accompanied by clinical symptoms such as recurrent itching, eczematoid lesions, bleeding and erosion, causing complications such as depression, anxiety, and sleep disorders, which seriously affect the physical and mental health of AD patients. The significant medical and social burden of AD constitutes a global health problem.

[0003] The treatment of AD aims to improve clinical symptoms, eliminate inducing and / or aggravating factors, and prevent complications and recurrences. Currently, the treatment of AD in clinical practice mainly relies on the use of topical medications, such as topical corticosteroids (TCS), topical calcineurin inhibitors (TCIs), and phosphodiesterase 4 (PDE4) inhibitors. Long-term use of TCS may cause adverse reactions such as telangiectasia, skin atrophy, striae, acne, and purpura. The use of TCI can cause a burning sensation at the application site and the risk of skin infection. The use of PDE4 inhibitors can cause adverse reactions such as nausea and vomiting.

[0004] PDE4B is a member of the cyclic adenosine monophosphate (cAMP)-specific cyclic nucleotide PDE4 family. It is highly expressed in keratinocytes and is one of the main factors driving inflammation. PDE4B expression is increased in patients with chronic inflammatory skin diseases such as AD. The PDE4B inhibitor Difamilast inhibits the expression of PDE4B, promotes the expression of intracellular cAMP and leads to the phosphorylation of CREB. CREB phosphorylation upregulates the expression of KPRP, restores the skin barrier function, and plays a role in treating AD. Currently, Difamilast is the only marketed drug that directly targets PDE4B. It has a low incidence of adverse reactions and can avoid adverse reactions such as nausea and vomiting caused by topical PDE4 inhibitors.

[0005] According to the TCM Diagnosis and Treatment Guidelines for Atopic Dermatitis, TCM syndrome differentiation mainly includes spleen and kidney yang deficiency syndrome, spleen deficiency and blood dryness syndrome, heart and spleen heat accumulation syndrome, etc. The treatment is mainly to strengthen the spleen, strengthen the kidney and clear the heart. On this basis, exploring more therapeutic effects of traditional Chinese medicine on AD has important clinical application value. Summary of the invention

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

[0007] The technical solution adopted by the present invention is:

[0008] The use of corylin in the preparation of a PDE4B inhibitor, wherein the chemical structural formula of corylin is as follows:

[0009]

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

[0011] Use of psoralea corylifolia isoflavones in preparing medicines or skin care products for preventing and treating atopic dermatitis.

[0012] Use of psoralea corylifolia isoflavones in preparing skin barrier repair medicines or skin care products.

[0013] Preferably, in the above-mentioned use of psoralea corylifolia isoflavones, the skin barrier repair is to improve skin redness, scaling and ulceration.

[0014] Preferably, in the above-mentioned use of psoralea corylifolia isoflavones, the skin barrier repair is to relieve itching.

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

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

[0017] Preferably, in the above-mentioned use of psoralea corylifolia isoflavones, the medicine is an external-use medicine.

[0018] A medicine / skin care product for preventing and treating atopic dermatitis, wherein the main active ingredients include the above-mentioned psoralen isoflavones.

[0019] A skin barrier repair medicine / skin care product, the main active ingredients of which include the above-mentioned psoralea corylifolia isoflavones.

[0020] The beneficial effects of the present invention are:

[0021] The skin of atopic dermatitis model mice induced by dinitrochlorobenzene (DNCB) showed obvious thickening, accompanied by redness, swelling, scaling, ulceration and other phenomena. The study found that psoralen isoflavones can significantly inhibit the protein expression of PDE4B in the skin of AD mice induced by dinitrochlorobenzene (DNCB) and in keratinocytes stimulated by TNF-α / IFN-γ. After treatment with psoralen isoflavones, the dermatitis characteristics of AD mice, such as redness, swelling, scaling, ulceration, etc., can be effectively improved; psoralen isoflavones can significantly reduce the number of scratching times of AD mice, effectively Relieve itching; Psoralen isoflavones reduce transepidermal water loss in the skin of AD mice; H&E staining shows that Psoralen isoflavones significantly reduce the thickness of the epidermis of 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 the infiltration of mast cells; Psoralen isoflavones significantly reduce the degree of spleen swelling and reduce the level of IgE in the serum of AD mice, while 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, have the effect of repairing the skin barrier of AD mice, and also have a good application prospect in the treatment of atopic dermatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a simple flow chart for modeling.

[0023] Figure 2 This is a diagram showing the effect of corylin on the appearance of AD mice.

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

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

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

[0027] Figure 6 H&E stained skin pathology sections 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<0.001vsModel ** P<0.01vsModel.

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

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

[0031] Fig.10 Effects of corylin on the expression of cytokine mRNA in the skin of 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] Fig.11 Effect of corylin on the expression of Filaggrin protein in AD mice (n=3), ### P<0.001 VS Control *** P<0.001 VS Model.

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

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

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

[0036] Fig.15 To screen the safe concentration of corylin for HEK cells (n=12), ** P<0.01vsControl.

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

[0038] Fig.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] Fig.18 Effects of corylin on the viability of HaCaT cells stimulated by TNF-α / IFN-γ (n=18), ### P<0.001 vs Control *** P<0.001 vs Model ** P<0.01 vs Model * P<0.05vsModel.

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

[0042] Fig. 20 Effects of corylin on the viability of 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 * P<0.05vsModel.

[0043] Fig.21 Effect 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<0.01vs Model.

[0044] Fig. 22 Effects 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] Fig.23 Effects of corylin on related cytokines in HEK 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.

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

[0047] Fig.25 Effect of corylin on the expression of Filaggrin protein in HaCaT cells stimulated by TNF-α / IFN-γ (n=3), ### P<0.001 VS Control *** P<0.001 VS Model **P<0.01 VSModel.

[0048] Fig.26 Effect of corylin on the expression of loricrin protein in HaCaT cells stimulated by TNF-α / IFN-γ (n=3), ### P<0.001 VS Control *** P<0.001 VS Model ** P<0.01 VSModel.

[0049] Fig. 27 Effect of corylin on the expression of involucrin protein in HaCaT cells stimulated by TNF-α / IFN-γ (n=3), ### P<0.001 VS Control *** P<0.001 VS Model ** P<0.01 VSModel.

[0050] Fig.28 Effects 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] Fig.29 Effect of corylin on the expression of Filaggrin protein in HEK cells stimulated by TNF-α / IFN-γ (n=3), ### P<0.001 VS Control *** P<0.001 VS Model ** P<0.01 VSModel.

[0052] Fig.30 Effect of corylin on the expression of involucrin protein in HEK cells stimulated by TNF-α / IFN-γ (n=3), ### P<0.001 VS Control ***P<0.00VS Model ** P<0.0VS Model * P<0.05VS Model.

[0053] Fig.31 Effect of corylin on the expression of loricrin protein in HEK cells stimulated by TNF-α / IFN-γ (n=3), ## P<0.01 VS Control ** P<0.01 VS Model * P<0.05VS Model.

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

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

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

[0057] Fig.35 qRT-PCR validation of PDE4B overexpression in HEK cells (n=3), # P<0.05VSControl.

[0058] Fig.36 Western blot verification of PDE4B overexpression in HEK cells (n=3), # P<0.05VS Control.

[0059] Fig.37 Effects of corylin on cytokine mRNA expression in HEK cells after overexpression plasmid transfer (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] Fig.38 Effects of corylin on mRNA expression of skin barrier function-related indicators in HEK cells after overexpression plasmid transfer (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] Fig.39 Effect of corylin on protein expression of filagrin in HEK cells after overexpression plasmid transfer (n=3), ## P<0.01 VS Control ** P<0.01 VS Model && P<0.05VS Corylin+TNF-α / IFN-γ.

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

[0063] Fig.41 Effect of corylin on the protein expression of loricrin in HEK cells after overexpression plasmid transfer (n=3), ### P<0.001 VS Control ** P<0.01 VS Model && P<0.05VS Corylin+TNF-α / IFN-γ. DETAILED DESCRIPTION

[0064] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0065] Example 1

[0066] Effect of corylin on DNCB-induced AD mouse model

[0067] A DNCB-induced AD mouse model was established, and crisaborole was used as a positive drug to investigate the effects of psoralen isoflavones on skin lesion scores, scratching behavior, transepidermal water loss (TEWL), spleen index and serum IgE in AD mice. Skin tissue pathological sections were used to investigate the effects of psoralen isoflavones on skin 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 improvement 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] Crisaborole 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 Biochemical Technology (Beijing) Co., Ltd.

[0074] DNCB Alfa Aesar Chemicals Ltd.

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

[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] StarScriptⅢ Reverse Transcription Kit Beijing Kangrun Chengye Biotechnology Co., Ltd. manage

[0079] 2X Universal SYBR Green Fast qPCR Mix Wuhan Abotek Biotech Co., Ltd. company

[0080] RNApure Tissure&Cell Kit CW0584S

[0081] cDNA reverse transcription kit Abi Company, USA

[0082] FastStart Universal SYBR Green Master ROCHE, Switzerland

[0083] Shanghai Bioengineering

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

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

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

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

[0088] Goat anti-Immuno-Hotradine Peroxidase (HPR) antibody Cell Signaling Technology Company

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

[0090] Horseradish peroxidase chemiluminescent agent (ELC luminescent liquid) Millipore, USA

[0091] 1.2 Instruments

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

[0093] Multi-probe skin testing system German CK company

[0094] Mini centrifuge Thermo Fisher USA

[0095] Gel imaging system Bio-Rad, USA

[0096] 1.3 Experimental Animals

[0097] SPF grade 6-8 week old BALB / C female mice, weighing about 18 g, SPF grade, were purchased from Sibeifu Biotechnology Co., Ltd. (license number SCK (Beijing) 2019-0010). The mice were raised in the Animal Center of Tianjin University of Traditional Chinese Medicine, with a room temperature of 20-26 °C and a relative humidity of 40-70%. The mice were raised in separate cages and fed with regular food and water. The experiment was conducted after 1 week of adaptive feeding.

[0098] 2. Methods

[0099] 2.1 Drug preparation

[0100] (1) Blank matrix solution: Mix olive oil and acetone in 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: Weigh Carbomer 940 and disperse it in 10 ml of deionized water. Stir at 500 rpm for 3 h at room temperature until there are no obvious lumps, and then swell at 4 °C for 12 h. Add ethyl paraben (0.2%) and glycerol (10%) to Carbomer 940 and stir evenly to obtain blank gel. Dissolve in PBS in a 75 °C water bath to form the water phase. Dissolve phospholipids in ethanol in a 75 °C water bath, dissolve behenic acid glyceride and medium-chain glyceride in chloroform in a 75 °C water bath, and mix the two to form the oil phase. Slowly inject the oil phase into the water phase. Stir rapidly at 75 °C (500 r / min) until the solution is clear, then stir slowly (300 r / min), evaporate the organic phase, cool to room temperature, filter through a 0.22 μm filter membrane, obtain blank NLC, and store in a refrigerator at 4 °C for later use. Mix blank gel and blank NLC in a ratio of 1:2 to obtain a blank preparation.

[0104] (5) Corylin preparation: Weigh Carbomer 940 and disperse it in 10 ml of deionized water. Stir at 500 rpm for 3 h at room temperature until there are no obvious lumps, and then swell at 4 °C for 12 h. Add ethyl paraben (0.2%) and glycerol (10%) to Carbomer 940 and stir evenly to obtain a blank gel. Dissolve it in PBS in a 75 °C water bath to obtain the aqueous phase. Dissolve phospholipids and psoralen isoflavone standards in ethanol in a 75 °C water bath, and dissolve behenic acid glyceride and medium-chain glycerides in chloroform in a 75 °C water bath. Mix the two to obtain the oil phase. Slowly inject the oil phase into the aqueous phase. Stir rapidly at 75 °C (500 r / min) until the solution is clear, then stir slowly (300 r / min), evaporate the organic phase, cool to room temperature, filter through a 0.22 μm filter membrane, and obtain Corylin-NLC. Store in a refrigerator at 4 °C for later use. Blank gel and Corylin-NLC were mixed at a ratio of 1:2 to obtain Corylin preparation.

[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 shaved with an electric shaver, and then a depilatory cream was applied to completely remove the hair on the back and fully expose the skin (2×2 cm).

[0108] (2) Experimental groups and drug administration

[0109] On D1 and D4, the blank group was given 100 μL of blank matrix solution, and the rest were given 100 μL of 2% DNCB. On D7, the mice were randomly divided into 5 groups with 6 mice in each group using a random number table, namely (1) blank control group (Control), (2) model group (Mode1), (3) Base group, (4) Corylin group, and (5) Crisaborole ointment group. The drug was started and the treatment drug was given every day during the drug administration period. At the same time, 100 μL of 0.6% DNCB was applied to the back skin once every 3 days. The simple process is shown in the figure. Figure 1 .

[0110] The mice were randomly divided into 5 groups, 6 mice in each group, respectively:

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

[0112] Model group: At the first sensitization, 2% DNCB 100 μL was applied to the back skin once every 2 days. 7 days after the first induction, 0.6% DNCB 100 μL was applied to the back of the mice once every 2 days and drug administration began;

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

[0114] Corylin group: 2 hours after DNCB drug stimulation, Corylin preparation with a concentration of 0.6 mg / g was applied to the mouse skin twice a day, with an interval of 8 hours between the two applications.

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

[0116] 2.3. Determination of skin lesion scores in mice

[0117] On day 0, day 5, day 8, day 11, day 14, day 17, day 20, and day 22, the degree of skin lesions of mice was scored based on the following four aspects, including: edema / papule; erythema / hemorrhage; epidermal exfoliation; scaling / dryness. Each score was divided into four levels: no symptoms (0), mild symptoms (1), moderate symptoms (2), and severe symptoms (3). The scores were determined unanimously by three observers other than the experimenter.

[0118] 2.4. Mouse scratching behavior measurement

[0119] The scratching behavior of mice was evaluated by counting the number of scratches. 10 minutes after the last sensitization, each mouse was placed in a cage and the number of scratches of the mouse with its hind paws at the stimulation site was recorded within 10 minutes. The scratching of the skin at the DNCB stimulation site by the mouse was counted as one effective scratch. If the scratching lasted for more than 3 seconds, it was counted as 2 scratches, and the scratching behavior of the mouse was stopped by manual intervention after 3 seconds.

[0120] 2.5. Determination of TEWL in mice

[0121] The transepidermal water loss of the skin lesion area of ​​each group of mice was measured by the Tewameter TM300 probe of the skin multi-probe tester. The standard measurement method was used, the measurement time was set to 20 s / time, and the average value was read after 15 s.

[0122] 2.6. Mouse skin histopathological examination

[0123] 2.6.1. Observation of mouse epidermal thickness by H&E staining

[0124] The samples of mice in each group were fixed with 4% formaldehyde, dehydrated, and permeabilized before being embedded in paraffin and sliced ​​using a microtome. The slices were dewaxed and rehydrated. They were stained with hematoxylin staining solution and 0.5% eosin. They were then dehydrated, permeabilized, and sealed with neutral gum. Five fields of view were randomly selected under the microscope, and the average epidermal thickness in the five fields of view was calculated.

[0125] 2.6.2. Observation of mast cell number by toluidine blue staining

[0126] Toluidine blue staining, the specific steps are as follows.

[0127] (1) Dewaxing the paraffin sections until they are free of water: sequentially place the sections 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% alcohol for 5 min, and then wash with water.

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

[0129] (3) Transparent sealing: Put the slices into clean xylene for 10 minutes and seal them with neutral gum.

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

[0131] 2.7 Evaluation of mouse spleen index

[0132] The mice were killed by dislocating the neck, and the left side of the abdomen was exposed with the abdomen facing upward. The skin was cut open, and long dark spleen tissue was visible. The spleen was removed and washed in physiological saline. The surface moisture was absorbed with filter paper, and its weight was calculated to calculate the spleen index and photographed for record. 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 D20, the mice were anesthetized with isoflurane, and the eyeballs were removed to collect blood. The collected blood was placed in a 1.5 mL centrifuge tube at room temperature for 30 min, centrifuged at 2000 rpm for 20 min, and the supernatant was stored at -80°C. The serum IgE level was measured with 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 30-50 mg of sample and place in a glass tissue grinder. Add 1 mL of Trizol and grind thoroughly. Centrifuge at 12,000 rpm for 10 min at 4°C.

[0138] (2) Take the supernatant, being careful not to absorb the precipitate; the pipette tip should move downward as the liquid level moves downward; let it stand at room temperature for 2 min, add 0.2 times the volume of supernatant chloroform; shake vigorously for 15 s, let it stand at room temperature for 3 min, and centrifuge at 12,000 rpm for 15 min at 4°C.

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

[0140] (4) Take 700 μL of the aqueous phase and transfer it to the CR3 column. Centrifuge at 12,000 rpm at room temperature for 30 s. Discard the downstream liquid and reassemble the column.

[0141] (5) Add 350 μL of RW1 deproteinization solution to the CR3 column, centrifuge at 12,000 rpm at room temperature for 30-60 seconds, discard the downstream liquid, and return the adsorption column to the collection tube.

[0142] (6) 10 μL DNase I, 70 μL RDD, gently invert to mix and prepare DNase I working solution, add 80 μL DNase I working solution to the center of the CR3 adsorption column, and leave at room temperature for 15 min.

[0143] (7) Add 350 μL of deproteinized solution RW1 to the adsorption column CR3, centrifuge at 12,000 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 rinse solution RW to the adsorption column CR3, centrifuge at room temperature and 12,000 rpm for 30-60 seconds, discard the downstream waste liquid, put the adsorption column CR3 back into the collection tube, and repeat this step once. Centrifuge at room temperature and 12,000 rpm for 2 minutes 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 RNase-Free Water to the middle part of the adsorption film, let it stand at room temperature for 2 min, and centrifuge it at 12,000 rpm at room temperature for 2 min to obtain the RNA solution.

[0146] (10) Reverse transcription to prepare cDNA

[0147] Prepare the reverse transcription system, add enzyme-free water to 10 μL, and add RNA sample to make the total volume 20 μL. Operate on the machine. After the reaction is completed, centrifuge briefly and store the cDNA sample at -20℃ for later use.

[0148] 2.9.2 qRT-PCR reaction

[0149] Three replicate wells were set up in each group for subsequent experiments. The qRT-PCR system was prepared by adding 1 μL PCR Forward Primer, 10 μL FastStart Universal SYBR Green Master, and 1 μL PCR Reverse Primer to each well. The cDNA loading volume was 1 μL, and DEPC water was added to make the total volume reach 20 μL. GAPDH was used as the internal reference, and the amplification reaction was performed in the Roche480 instrument. The qRT-PCR primer sequences are shown in Table 1.

[0150] Table 1 GAPDH and cytokine primer sequences

[0151]

[0152] 2.9.3 Data Analysis

[0153] The amplification reaction is completed. The Ct value of each sample is obtained, and a single-peak melting curve reaction is observed. The mRNA expression level of the target gene is calculated according to 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) From each group, 20 mg of mouse skin tissue was cut into small pieces in a 1.5 mL centrifuge tube. 400 μL of saline was added, mixed, centrifuged and the supernatant was discarded. The washing step with saline was repeated twice.

[0160] (2) Add 200 μL RIPA tissue lysis buffer to each tube, place on ice, and ultrasonically disrupt the animal skin tissue. Place on ice for 15 minutes after ultrasonication, centrifuge at 4°C, and the supernatant is the protein to be extracted.

[0161] (3) Preparation of standard curve samples: The protein standard is gradiently diluted to produce standard curve samples of different concentrations.

[0162] (4) Preparation of protein sample: 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) Pipette 20 μL of protein sample and standard curve sample and add them to the well plate in turn. Add 200 μL of BCA working solution to each well, mix well, incubate at 37°C for 30 min, and detect the absorbance at 560 nm.

[0165] (7) Draw a standard curve based on the concentration of the standard and the corresponding absorbance value of the well, and calculate the protein concentration of the sample.

[0166] 2.10.2 Western blot detection

[0167] (1) Preparation of separation gel and stacking gel: Prepare separation gel and stacking gel of corresponding concentration according to the molecular weight of the target protein;

[0168] (2) Adding sample protein: Add the prestained protein Maker and protein samples to the corresponding lanes of the gel in the order of experimental grouping;

[0169] (3) Electrophoresis: When conducting an electrophoresis experiment, first connect the electrophoresis tank to the electrophoresis instrument power supply and set the constant voltage to 80 V. After electrophoresis for 30 min, readjust the electrophoresis conditions to a constant voltage of 110 V and continue electrophoresis for about 90 min until the protein sample migrates to the bottom of the separation gel. After the electrophoresis is completed, turn off the power supply to terminate the electrophoresis process.

[0170] (4) Transfer: After the electrophoresis is completed, the gel is removed and the sponge, filter paper, separation gel, polyvinylidene fluoride (PVDF) membrane, filter paper, and sponge pre-soaked with transfer solution are placed in a wet transfer clip in the form of a "sandwich". The bubbles are removed and the wet transfer clip is clamped and placed in a wet transfer apparatus filled with transfer solution. The wet transfer apparatus is placed in an ice bath to maintain a low temperature environment. The membrane is transferred for 110 min at a constant current of 200 mA.

[0171] (5) Blocking: After the transfer is completed, remove the wet transfer clip, cut the PVDF membrane according to the gel size, then soak the PVDF membrane in 1×TBST solution, remove the excess electrotransfer solution, and place the PVDF membrane in 5% milk powder blocking solution prepared from 1×TBST solution for blocking at room temperature for 2 h;

[0172] (6) Washing the membrane: discard the 5% milk powder blocking solution and soak the membrane in 1×TBST solution several times to wash away the milk powder blocking solution on the surface of the PVDF membrane;

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

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

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

[0176] (10) Washing the membrane: Recover the secondary antibody and wash the membrane several times with 1× TBST to remove the secondary antibody on the surface of the PVDF membrane. Then, soak the PVDF membrane in 1× TBST.

[0177] (11) Development and photography: Prepare solution A and solution B of the enhanced chemiluminescence (ECL) reagent in equal proportions according to experimental requirements and mix them evenly. Add ELC reagent on the PVDF membrane to completely soak it. Place the membrane in a multifunctional imager for exposure and use Image J software to analyze the electrophoresis bands.

[0178] 3. Statistical processing

[0179] All data were analyzed using SPSS25 statistical analysis software, and the results were expressed as mean ± standard deviation. One-way analysis of variance was used to indicate the differences among the groups, and P < 0.05 was considered statistically significant.

[0180] 4. Results

[0181] 4.1 Comparison of the appearance of AD mouse skin

[0182] This experiment investigated the effect of Corylin on the back skin lesions and dermatitis scores of the DNCB-induced AD mouse model. The results showed that the skin of the mice in the Control group was ruddy and smooth; the Model group showed dermatitis-like features such as edema, scars, erythema and dryness, epidermal scaling and erosion, indicating that the DNCB-induced AD mouse model was successfully constructed. Both the Corylin-administered group and the Crisaborole-administered group significantly improved the DNCB-induced dermatitis-like features of the mouse skin (P<0.001, P<0.01). Figure 2 , Figure 3 .

[0183] 4.2 Effect of Corylin on the scratching behavior of AD mice

[0184] One of the characteristics of AD is pruritus, and the number of scratching is used as one of the indicators to observe the efficacy of Corylin on DNCB-induced AD mouse model. Figure 4 As shown in the figure, compared with the Control group, the scratching frequency of mice in the Model group increased significantly (P<0.001). Crisaborole and Corylin administration groups significantly reduced the scratching frequency of mice (P<0.001).

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

[0186] Scratching caused by itching can damage the skin's barrier function. The persistent "itch-scratching" cycle further aggravates the damage to the skin barrier function of AD. The skin barrier can maintain the skin's moisture content and prevent the body's water from being lost through the skin. Damage to the skin barrier function will increase TEWL. Figure 5 It can be seen that compared with the Control group, the TEWL of DNCB-induced AD mice was significantly increased (P<0.001); compared with the Model group, the positive drug Crisaborole significantly reduced the TEWL of mouse skin (P<0.001). The TEWL of mice in the Corylin 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] In order to better determine whether Corylin can improve the skin lesions in the AD mouse model, H&E staining and toluidine blue staining were performed on the back skin of mice. H&E staining showed that compared with the Control group, the epidermal thickness of the Model group was significantly increased (P<0.001); the epidermal thickness of mice was significantly reduced after administration of Corylin and Crisaborole (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 in the skin of the Model group increased significantly, and the infiltration increased (P<0.001); compared with the Model group, the number and infiltration of mast cells in the skin of mice given Corylin and Crisaborole were significantly reduced (P<0.001, P<0.01). See Figure 6 , Figure 7 .

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

[0190] AD not only manifests as skin damage, but also has certain effects on immune organs. The spleen of DNCB-induced mice is significantly larger than that of normal mice. Changes in the spleen index can reflect the inflammation in the mouse body. Compared with the Control group, the spleen index of the Model group mice was significantly increased (P<0.001). Crisaborole and Corylin administration groups significantly reduced the spleen index of mice (P<0.01, P<0.05). Figure 8 .

[0191] 4.6 Effect of Corylin on Serum IgE in AD Mice

[0192] Increased serum IgE levels are one of the important clinical features of AD patients. ELISA was used to detect the expression level of IgE in the serum of mice in each group. The results showed that compared with the Control group, the IgE level in the serum of the Model group mice was significantly increased (P<0.001). After drug administration, the IgE levels in the serum of mice in the Crisaborole and Corylin groups were significantly decreased (P<0.001). Fig. 9 .

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

[0194] Damage to the skin barrier can cause epithelial cells to produce various pro-inflammatory mediators, inflammatory cytokines and chemokines under external stimulation, initiating inflammatory responses in AD. This experiment detected cytokines such as IL-4, IL-5, IL-6, IL-13, and TSLP, aiming to explore whether Corylin can downregulate the expression of IL-4, IL-5, IL-6, IL-13, and TSLP mRNA. The 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 IL-4, IL-5, IL-6, IL-13, and TSLP cytokines in the mouse skin were significantly reduced (P<0.001), see. Fig.10 .

[0195] 4.8 Effect of Corylin on the Expression of Skin Barrier-Related Proteins in the Skin of 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 in the Model group were significantly decreased (P<0.001, P<0.01). Compared with the Model group, the Corylin group and the Crisaborole group could significantly increase the expression levels of skin barrier function-related proteins such as Filaggrin, Loricrin and Involucrin (P<0.001, P<0.01, P<0.05). Fig.11 , Fig.12 , Fig.13 .

[0197] 5. Summary

[0198] Corylin effectively improves the dermatitis characteristics of AD mice, such as redness, scaling, and ulceration; Corylin significantly reduces the number of scratching times in AD mice, effectively relieving itching; Corylin reduces transepidermal water loss in AD mice; H&E staining shows that Corylin significantly reduces the thickness of the epidermis in AD mice; Toluidine blue staining shows that Corylin significantly reduces the number of mast cells in the skin tissue of AD mice and improves the infiltration of mast cells; Corylin significantly reduces the degree of spleen swelling, reduces the level of IgE in the serum of AD mice, and reduces the mRNA expression of cytokines IL-4, IL-5, IL-6, IL-13, and TSLP in the skin of AD mice. Corylin effectively increases the protein expression of Filaggrin, Loricrin, and Involucrin in the skin tissue of AD mice, and has the effect of repairing the skin barrier of AD mice.

[0199] Example 2

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

[0201] The keratinocytes were induced by TNF-α and IFN-γ to establish a cellular inflammation model. After administration of Corylin, the cell viability of keratinocytes induced by TNF-α and IFN-γ was detected by CCK-8 kit, and the LDH release of keratinocytes induced by TNF-α and IFN-γ was detected by lactate dehydrogenase kit. The mRNA expression of cytokines IL-6, IL-8, IL-1β, MDC and TSLP and Filaggrin, Loricrin and Involucrin in keratinocytes induced by TNF-α and IFN-γ was detected by qRT-PCR; the protein expression of Filaggrin, Loricrin and Involucrin in keratinocytes induced by TNF-α and IFN-γ was detected by Western blot to investigate the improvement effect of Corylin on the cellular inflammation model. The effect of Corylin on AD was studied at the cellular level.

[0202] 1. Materials

[0203] 1.1 Reagents and consumables

[0204]

[0205]

[0206] 1.2 Instruments

[0207] Real-time fluorescence quantitative PCR instrument (LightCycler480) ROCHE, Switzerland

[0208] Gel imaging system Bio-Rad, USA

[0209] 1.3 Cell lines

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

[0211] 2. Methods

[0212] 2.1. Solution preparation

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

[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 to be tested, and mix well.

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

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

[0217] 2.2. Culture and treatment of keratinocytes

[0218] 2.2.1. Recovery of keratinocytes

[0219] Take out the frozen HaCaT cells and HEK cells from the -80℃ refrigerator and immediately put them in a water bath at 37℃ to thaw the cells quickly. After disinfection, transfer them to the biosafety cabinet immediately, transfer the cell suspension to a centrifuge tube with fresh complete medium, discard the supernatant after centrifugation, add the corresponding complete medium, resuspend and mix, and continue to culture in a T25 culture bottle. Culture in a 37℃, 5% CO2 incubator.

[0220] 2.2.2. Keratinocyte passaging and plating

[0221] Observe the cells under the microscope and perform subculture when the growth reaches about 80%. Discard the complete medium in the bottle, add 2mL PBS solution, shake gently, wash twice, aspirate and discard the PBS solution, add 1mL 1X 0.25% trypsin-EDTA, and place in a 37°C, 5% CO2 cell culture incubator for 5 minutes. After the cells become round and fall off, use an appropriate amount of the corresponding complete medium to terminate the digestion, transfer the digested cell suspension to a 15mL centrifuge tube, and centrifuge moderately. Discard the cell supernatant, add an appropriate amount of MEM or DMEM complete medium, mix well, and transfer to a new cell culture bottle at a ratio of 1:4, and place it in a 37°C, 5% CO2 cell culture incubator for continued culture in preparation for subsequent experiments.

[0222] 2.3. Screening of safe concentration of Corylin for keratinocytes

[0223] CCK-8 kit was used to evaluate the cytotoxicity of corylin on keratinocytes. HaCaT cells / HEK cells were cultured at a rate of 1×10 5 The cells were inoculated in a 96-well plate at a density of 100 / mL. When the HaCaT cells / HEK cells reached 80% of the adherent growth, the corresponding concentration of Corylin was added. 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 measured by a microplate reader to calculate the cell survival rate.

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

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

[0226] HaCaT cells were cultured at a rate of 5 × 10 5 The cells were seeded at a density of 100 cells / well in a 6-well plate. When the cell adhesion growth reached 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 6 h or 24 h, respectively. The cells were collected and the cytokine expression levels were detected by qRT-PCR.

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

[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 place 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°C and 12,000 rpm for 5 minutes.

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

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

[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°C and 12,000 rpm for 5 min.

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

[0234] (7) Carefully discard the supernatant and place the centrifuge tube in a ventilated place to dry for about 2 h until the edge of the white precipitate becomes transparent. Then add 20 μL of enzyme-free water to dissolve 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 were the same as in Example 1, and the qRT-PCR primer sequences were shown in Table 2.

[0238] Table 2 GAPDH and cytokine primer sequences

[0239]

[0240] 2.4.1.3 Data Analysis

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

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

[0243] HEK cells were cultured at 5×10 5 The cells were seeded at a density of 100 cells / well in a 6-well plate. When the cell adhesion growth reached 80%, the corresponding concentration of Corylin was added and the culture was continued for 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 and incubated for 24 hours. The cells were collected and the cytokine expression levels were detected by qRT-PCR.

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

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

[0246] 2.4.2.2 qRT-PCR reaction

[0247] The steps are the same as 2.2.3.1.2.

[0248] 2.4.2.3 Data Analysis

[0249] The steps are the same as 2.2.3.1.3.

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

[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 cultured at 1×10 5 / mL concentration was inoculated in a 96-well plate, and then the corresponding concentration of Corylin solution was added. After 2 hours, a medium containing 10ng / mL TNF-α and 10ng / mL IFN-γ was added to each well and incubated for 24 hours. Then, 1×CCK-8 working solution was added and incubated at 37℃ for 0.5h. The absorbance was measured by a microplate reader to calculate the cell viability. Then, about 50μL of cell culture medium was aspirated from the well and transferred to a new 96-well plate. 50μL of Assay Buffer was added to each well and incubated for 15min at 37℃ in the dark.

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

[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 cultured at 5×10 5 Cells were seeded at a density of 100 cells / well in a 6-well plate, and the corresponding concentration of Corylin solution was added after the cells adhered to the wall and grew to 80%. After 2 hours, 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 incubated for 24 hours. The cells were collected and the cytokine expression levels were detected by qRT-PCR.

[0258] 2.6.1.1. Preparation of cDNA

[0259] The steps are the same as 2.2.3.1.1.

[0260] 2.6.1.2 qRT-PCR

[0261] The steps are the same as 2.2.3.1.2.

[0262] 2.6.1.3 Data Analysis

[0263] The steps are the same as 2.2.3.1.3.

[0264] 2.6.2 Effect 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 2.2.3.1.1.

[0267] 2.6.2.2 qRT-PCR

[0268] The steps are the same as 2.2.3.1.2.

[0269] 2.6.2.3 Data Analysis

[0270] The steps are the same as 2.2.3.1.3.

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

[0272] HaCaT cells / HEK cells were cultured at 5×10 5 Cells were seeded at a density of 100 cells / well in a 6-well plate, and the corresponding concentration of Corylin was added. The culture was continued for 2 hours, and the supernatant was discarded. When the cell adhesion growth reached 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. The cells were collected and the expression levels of skin barrier function-related indicators were detected by qRT-PCR and Western blot methods, respectively.

[0273] 2.7.1 qRT-PCR

[0274] 2.7.1.1. Preparation of cDNA

[0275] The steps are the same as 2.2.3.1.1.

[0276] 2.7.1.2 qRT-PCR

[0277] The steps are the same as 2.2.3.1.2.

[0278] 2.7.1.3 Data Analysis

[0279] The steps are the same as 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 each of the cell pellets collected above, resuspend and place on ice for 15 min.

[0283] (2) Centrifuge at 4°C, 12,000 rpm for 10 min. The supernatant is the extracted protein.

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

[0285] 2.7.2.2、Western blot

[0286] The steps are the same as 1.2.10.2.

[0287] 3. Statistical processing

[0288] The steps are the same as 1.3.

[0289] 4. Results

[0290] 4.1. Screening of safe concentration of Corylin for keratinocytes

[0291] 4.1.1. Screening of safe concentration of Corylin for HaCaT cells

[0292] Fig.14 The results showed that the cell viability of the 100 μM Corylin solution group was 109.42±10.57%, which was significantly improved compared with the Control group (P<0.01); there was no significant change in the cell viability of the 1 and 10 μM Corylin solution groups compared with the Control group (P>0.05), and 1 and 10 μM Corylin concentrations were selected for subsequent experiments.

[0293] 4.1.2 Screening of safe concentration of Corylin for HEK cells

[0294] Fig.15 The results showed that the cell viability of the 100 μM Corylin solution group was 114.55±11.50%, which was significantly improved compared with the Control group (P<0.01); there was no significant change in the cell viability of the 1 and 10 μM Corylin solution groups compared with the Control group (P>0.05), and 1 and 10 μM Corylin concentrations were selected for subsequent experiments.

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

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

[0297] Fig.16The results showed that compared with the Control group, after TNF-α / IFN-γ stimulation of HaCaT cells for 6 hours, the mRNA levels of cytokines such as IL-6, IL-8, MDC, and TSLP did not change significantly, while after TNF-α / IFN-γ stimulation of HaCaT cells for 24 hours, 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 hours was selected as the modeling condition for subsequent experiments on HaCaT cells.

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

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

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

[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 of 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 downregulated (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. -3 The -1μM Cylin solution group significantly increased cell viability (P<0.001, P<0.01, P<0.05) and reduced LDH release (P<0.001, P<0.01, P<0.05). Fig.18 , Fig.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 of 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 downregulated (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. -3 The -1μM Cylin solution group significantly increased cell viability (P<0.001, P<0.01) and reduced LDH release (P<0.05). Fig. 20 , Fig.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 Fig. 22 As shown in the figure, compared with the Control group, the mRNA levels of cytokines such as IL-4, IL-1β, IL-6, IL-8, MDC, and TSLP in the Model group were significantly increased (P<0.001, P<0.01). -3 After administration of -1μM Cytokine, the mRNA levels of cytokines 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 Fig.23 As shown in the figure, compared with the Control group, the mRNA levels of cytokines such as IL-4, IL-1β, IL-6, IL-8, MDC, and TSLP in the Model group were significantly increased (P<0.001, P<0.01, P<0.05). -3 After -1μM Cytokine administration, the cytokine mRNA levels were significantly decreased (P<0.001, P<0.01, P<0.05).

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

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

[0312] In HaCaT cells, compared with the Control group, the mRNA and protein expression levels of skin barrier function-related indicators such as Filaggrin, Involucrin, and Loricrin in the Model group were significantly decreased (P<0.001). -3 -10 -1 Corylin at a concentration of 1 μM can significantly increase the mRNA and protein expression levels of skin barrier function-related indicators such as Filaggrin, Involucrin, and Loricrin (P<0.001, P<0.01). Fig.24 , Fig.25 , Fig.26 , Fig. 27 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 mRNA and protein expression levels of skin barrier function-related indicators such as Filaggrin, Involucrin, and Loricrin in the Model group were significantly decreased (P<0.001, P<0.01). -1 -10 -3 Corylin at a concentration of 1 μM can significantly increase 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). Fig.28 , Fig.29 , Fig.30 , Fig.31 shown.

[0315] 5. Summary

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

[0317] Example 3

[0318] Study on the improvement effect of corylin on AD model through PDE4B molecule

[0319] This example first uses Western blot experiments to explore the changes in PDE4B expression in AD mice and keratinocytes stimulated by TNF-α / IFN-γ by Corylin. In order to clarify the key role of PDE4B in AD, PDE4B was overexpressed using plasmid transfection technology to explore the effects of PDE4B expression changes on biomarkers related to inflammation and barrier function in AD; qRT-PCR and Wetsern blot were used to clarify the regulatory effect of Corylin on PDE4B in HEK cells, and further explore whether the protective effect of Corylin on the HEK inflammation model stimulated by TNF-α / IFN-γ is achieved by regulating PDE4B molecules.

[0320] 1. Materials

[0321] 1.1 Reagents and consumables

[0322] Overexpression plasmid China Genetron Health Co., Ltd.

[0323] GPtransmate transfection reagent China Genetron Health Co., Ltd.

[0324] Shanghai Bioengineering

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

[0330] 2. Methods

[0331] 2.1. Preparation of solution: the steps are the same as those in Example 2.

[0332] 2.2. Culture and treatment of keratinocytes

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

[0334] 2.2.2. Keratinocytes were passaged in the same manner as in Example 2.

[0335] 2.2.3. Administration treatment of keratinocytes, the steps are the same as those in Example 2.

[0336] 2.3 Effect of Corylin on PDE4B Expression in AD Model

[0337] 2.3.1. Western blot was used to detect the expression of PDE4B protein. The operation was the same as in Example 2.

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

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

[0340] HEK cells with good growth status were taken and 4×10 5 The cells were inoculated at a density of 100 / mL in a 60mm culture dish, and the PDE4B overexpression plasmid transfection experiment was performed when the cells grew to about 60-80%. Control group, transfection reagent group (Mock group), and model group (TNF-α / IFN-γ co-stimulation) were set up, and the transfection reagent was prepared. 500μL of serum-free medium was added to tube A and 15μL of GP transmate transfection reagent was added to mix well. 500μL of serum-free medium was added to tube B and 15μL of PDE4B-OE was added to mix well. After standing for 5min, the solution in tube A was directly added to tube B, and gently blown. After incubation at room temperature for 15-20min, 4mL of DMEM complete medium was added and added to the culture dish. The cells were placed in an incubator for transfection for 5-7h. After the transfection was completed, the transfection solution was discarded, and 5mL of DMEM complete medium was added to terminate the transfection. qRT-PCR and Western blot were used to detect the cell transfection.

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

[0342] Table 3 Primer sequences

[0343]

[0344] The steps of protein sample preparation and Western blot detection were the same as those in Example 2.

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

[0346] HEK cells with good growth status were taken and 4×10 5The cells were inoculated at a density of 100 μg / mL in a 60 mm culture dish, and the PDE4B overexpression plasmid transfection experiment was performed when the cells grew to about 60-80%. Control group, transfection reagent group (Mock group), model group (TNF-α / IFN-γ co-stimulation), TNF-α / IFN-γ co-stimulation + Corylin group, TNF-α / IFN-γ co-stimulation + Corylin + PDE4B-OE group were set up. Prepare the transfection reagent, add 500 μL of serum-free medium to tube A and add 15 μL of GPtransmate transfection reagent to mix, add 500 μL of serum-free medium to tube B and add 15 μL of PDE4B-OE to mix, let it stand for 5 minutes, then add the solution in tube A directly to tube B, blow it gently, incubate at room temperature for 15-20 minutes, add 4 mL of DMEM complete medium and add it to the culture dish, and place the cells in the incubator for transfection for 5-7 hours. After transfection, the transfection solution was discarded, and 5 mL of DMEM complete medium was added to terminate the transfection. qRT-PCR and Western blot were used to detect the cell transfection. Subsequent experiments were performed after 48 h of culture. The expression of PDE4B and cytokines was subsequently detected using qRT-PCR.

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

[0348] 2.4.3 Effect of Corylin on the Expression of Skin Barrier-Related Proteins in HEK Cells Stimulated by TNF-α / IFN-γ after Overexpression of PDE4B

[0349] The experimental steps of overexpression PDE4B plasmid transfection were the same as 3.2.5.1. Subsequently, the skin barrier-related proteins were detected by Western blot.

[0350] The steps of protein sample preparation and Western blot detection were the same as those in Example 2.

[0351] 3. Statistical processing

[0352] The steps are the same as 1.3.

[0353] 4. Results

[0354] 4.1 Effect of Corylin on PDE4B Expression in AD Model

[0355] like Fig.32 As shown. 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, the Corylin and Crisaborole administration groups could significantly reduce the expression level of PDE4B protein (P<0.05).

[0356] Depend on Fig.33 , Fig.34 The results showed that in vitro experiments showed that in HaCaT cells and HEK cells, the expression level of PDE4B protein in the Model group was significantly increased compared with the Control group (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 Effect of Corylin on TNF-α / IFN-γ-stimulated HEK cell inflammation model after overexpression of PDE4B

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

[0359] like Fig.35 , Fig.36 As shown in the figure, 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 the PDE4B overexpression plasmid was transferred into HEK cells, the expression levels of PDE4B mRNA and protein in the PDE4B overexpression plasmid transfection group were significantly increased compared with the Control group (P<0.001, P<0.05), which indicates that the PDE4B overexpression plasmid was successfully transferred into HEK cells and can be used for subsequent experiments.

[0360] 4.2.2 Effect of Corylin on Cytokine Expression in HEK Cells after Overexpression of PDE4B

[0361] like Fig.37 As shown in the figure, in HEK cells, compared with the Control group, the expression of cytokines in the Model group was significantly increased (P<0.001, P<0.01), proving that the model was successfully constructed. Compared with the Model group, the administration of Corylin can significantly reduce the expression level of cytokines (P<0.001, P<0.01), indicating that Corylin has an ameliorative effect on the HEK cell inflammation model stimulated by TNF-α / IFN-γ. Compared with the group stimulated by TNF-α / IFN-γ and administered with Corylin, the cytokine release in the group administered with PDE4B overexpression plasmid and Corylin drug was significantly increased (P<0.001, P<0.01), indicating that the ameliorative effect of Corylin on the HEK cell inflammation model stimulated by TNF-α / IFN-γ was eliminated by the PDE4B overexpression plasmid. The above results indicate that Corylin may play an ameliorative role in the HEK cell inflammation model stimulated by TNF-α / IFN-γ through the potential target of PDE4B.

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

[0363] like Fig.38 , Fig.39 , Fig.40 , Fig.41 As shown in the figure, 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 that the model was successfully constructed. Compared with the Model group, the administration of Corylin can significantly upregulate the expression of skin barrier-related indicators (P<0.001, P<0.01), which indicates that Corylin has an improvement effect on the TNF-α / IFN-γ stimulated HEK cell inflammation model. Compared with the group receiving TNF-α / IFN-γ stimulation and given Corylin, the expression of skin barrier-related indicators in the group given PDE4B overexpression plasmid and Corylin drug was significantly reduced ((P<0.001, P<0.01, P<0.05), indicating that the improvement effect of Corylin on the TNF-α / IFN-γ stimulated HEK cell inflammation model was eliminated by the PDE4B overexpression plasmid. The above results indicate that Corylin will play an improvement role in the inflammatory response caused by TNF-α / IFN-γ stimulated cells through the potential target of PDE4B.

[0364] 5. Summary

[0365] Corylin significantly inhibited the protein expression of PDE4B in DNCB-induced AD mice and TNF-α / IFN-γ stimulated keratinocytes. Overexpression of PDE4B restored the trend of Corylin to reduce the expression of cytokines in keratinocytes stimulated by TNF-α / IFN-γ, and restored the trend of Corylin to increase the expression of Filaggrin, Loricrin, and Involucrin. The improvement 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 descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary engineers and technicians in the field should all fall within the protection scope of the present invention.

Claims

1. Use of psoralea corylifolia isoflavones in the preparation of PDE4B inhibitors.

2. Use of psoralea corylifolia isoflavones in the preparation of medicines or skin care products for preventing and treating atopic dermatitis.

3. Use of psoralea corylifolia isoflavones in the preparation of skin barrier repair drugs or skin care products.

4. The use of psoralea corylifolia isoflavones according to claim 3, characterized in that: The skin barrier repair is to improve skin redness, scaling and ulceration.

5. The use of psoralea corylifolia isoflavones according to claim 3, characterized in that: The skin barrier repair is to relieve itching.

6. The use of psoralea corylifolia isoflavones according to claim 3, characterized in that: The skin barrier repair is to reduce the loss of skin water.

7. The use of psoralea corylifolia isoflavones according to claim 3, characterized in that: The skin barrier repair is to reduce the thickness of the skin epidermis.

8. The use of psoralea corylifolia isoflavones according to claim 2 or 3, characterized in that: The medicine is for external use.

9. A medicine / skin care product for preventing and treating atopic dermatitis, characterized in that: The main active ingredients include psoralea corylifolia isoflavones.

10. A skin barrier repair medicine / skin care product, characterized in that: The main active ingredients include psoralea corylifolia isoflavones.

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