SerpinB3 inhibitor and application thereof

Abstract

The invention discloses a SerpinB3 inhibitor and an application of the SerpinB3 inhibitor. The phloretin can be used as the SerpinB3 inhibitor for the first time, can effectively relieve the symptoms of the rosacea and the psoriasis at the same time, and is used for preparing related drugs for treating inflammatory skin diseases such as the rosacea and the psoriasis. The invention provides a certain theoretical basis for research and development of related treatment products for inflammatory skin diseases such as rosacea or psoriasis.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a SerpinB3 inhibitor and its application. Background Technology

[0002] Rosacea and psoriasis are common chronic inflammatory skin diseases with a high incidence rate and a tendency to relapse, causing great suffering to patients. Therefore, it is urgent to develop new treatment strategies for rosacea and psoriasis.

[0003] Phloretin is a flavonoid compound found in fruits and vegetables such as apples. It has antioxidant, anti-aging and antibacterial biological activities and has great potential in treating various inflammatory or immune-related diseases. However, there are currently no reports on the use of phloretin to treat inflammatory skin diseases such as rosacea and psoriasis. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes the application of phloretin.

[0005] This invention also proposes a SerpinB3 inhibitor.

[0006] The present invention also proposes a product for the prevention and / or treatment of inflammatory skin diseases such as rosacea and psoriasis.

[0007] According to one aspect of the invention, the use of phloretin in any of the following is proposed:

[0008] (1) As a SerpinB3 inhibitor;

[0009] (2) Prepare products for the treatment and / or prevention of inflammatory skin diseases.

[0010] In some embodiments of the present invention, the inflammatory skin disease includes at least one of psoriasis, rosacea, dermatitis (atopic, seborrheic, contact), eczema, parapsoriasis, lichen planus, lichen planus pilaris, acute pustular lichenoid pityriasis, chronic lichenoid pityriasis, pityriasis rubra pilaris, graft-versus-host disease, histiocytosis, drug-induced rash, autoimmune connective tissue disease (e.g., lupus), rosacea, folliculitis, acne, warts, ichthyosis, vitiligo, cicatricial alopecia, and CTCL.

[0011] In some embodiments of the present invention, the product can be used for topical skin application.

[0012] In some embodiments of the present invention, the product has at least one of the following functions:

[0013] 1) Anti-inflammatory;

[0014] 2) Inhibit SerpinB3 expression;

[0015] 3) Reduce symptoms of inflammatory skin diseases.

[0016] In some embodiments of the present invention, the anti-inflammatory effect includes inhibiting the expression of inflammatory factors and reducing the number of inflammatory cells.

[0017] In some embodiments of the present invention, the inflammatory factors include TNF-α, IL-6, CCL2, and CCL20.

[0018] In some embodiments of the present invention, the relief of inflammatory skin disease symptoms includes: reducing erythema and / or edema caused by rosacea, reducing immune infiltration of rosacea and / or reducing epidermal thickening and / or desquamation caused by psoriasis.

[0019] In some embodiments of the present invention, the product is at least one of skin care products, pharmaceuticals, health products, and functional foods.

[0020] In some embodiments of the present invention, the product is a pharmaceutical product, and the pharmaceutical product further includes pharmaceutically acceptable excipients.

[0021] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of diluents, excipients, fillers, binders, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, sweeteners, and flavorings.

[0022] In some embodiments of the invention, the excipient comprises water.

[0023] In some embodiments of the present invention, the filler includes at least one of starch and sucrose.

[0024] In some embodiments of the present invention, the adhesive includes at least one of cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone.

[0025] In some embodiments of the present invention, the wetting agent includes glycerin.

[0026] In some embodiments of the present invention, the disintegrant includes at least one of agar, calcium carbonate, and sodium bicarbonate.

[0027] In some embodiments of the present invention, the absorption enhancer includes a quaternary ammonium compound.

[0028] In some embodiments of the present invention, the surfactant includes hexadecyl alcohol.

[0029] In some embodiments of the present invention, the adsorbent carrier includes at least one of kaolin and soap clay.

[0030] In some embodiments of the present invention, the lubricant includes at least one of talc, calcium stearate, magnesium stearate, and polyethylene glycol.

[0031] In some embodiments of the present invention, the dosage form of the drug is a solid, semi-solid, or liquid, and may be an aqueous solution, a non-aqueous solution, or a suspension.

[0032] In some embodiments of the present invention, the dosage form of the medicine is tablet, capsule, granule, pill, oral liquid, dry suspension, drop pill, dry extract, injection, or transdermal preparation.

[0033] In some embodiments of the present invention, the administration method of the drug can be a conventional administration method in the art, including but not limited to topical application, injection, or oral administration.

[0034] In some embodiments of the present invention, the injection administration can be via intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection.

[0035] In some embodiments of the present invention, the phloretin in the product has a mass fraction of 0.01% to 100%.

[0036] In some embodiments of the present invention, the phloretin in the product has a mass fraction of 0.05% to 95%.

[0037] In some embodiments of the present invention, the phloretin in the product has a mass fraction of 0.05% to 50%.

[0038] According to a second aspect of the invention, a SerpinB3 inhibitor is provided, said SerpinB3 inhibitor containing phloretin.

[0039] In some embodiments of the present invention, the SerpinB3 inhibitor can inhibit the expression of SerpinB3.

[0040] In some embodiments of the present invention, the SerpinB3 inhibitor can be prepared into products for the treatment and / or prevention of inflammatory skin diseases.

[0041] In some embodiments of the present invention, the inflammatory skin diseases include psoriasis, rosacea, dermatitis (atopic, seborrheic, contact), eczema, parapsoriasis, lichen planus, lichen planus pilaris, acute pustular lichenoid pityriasis, chronic lichenoid pityriasis, pityriasis rubra pilaris, graft-versus-host disease, histiocytosis, drug-induced rashes, autoimmune connective tissue diseases (e.g., lupus), rosacea, folliculitis, acne, warts, ichthyosis, vitiligo, cicatricial alopecia, and CTCL.

[0042] In some embodiments of the present invention, the product is at least one of skin care products, pharmaceuticals, health products, and functional foods.

[0043] The present invention also proposes a product for the prevention and / or treatment of inflammatory skin diseases, said product containing phloretin and pharmaceutically acceptable excipients.

[0044] In some embodiments of the present invention, the inflammatory skin disease includes at least one of psoriasis, rosacea, dermatitis, eczema, parapsoriasis, lichen planus, lichen planus pilaris, acute pustular lichenoid pityriasis, chronic lichenoid pityriasis, pityriasis rubra pilaris, graft-versus-host disease, histiocytosis, drug-induced rash, autoimmune connective tissue disease, rosacea, folliculitis, acne, warts, ichthyosis, vitiligo, cicatricial alopecia, and CTCL.

[0045] In some embodiments of the present invention, the phloretin in the product has a mass fraction of 0.01% to 100%.

[0046] In some embodiments of the present invention, the phloretin in the product has a mass fraction of 0.05% to 95%.

[0047] In some embodiments of the present invention, the phloretin in the product has a mass fraction of 0.05% to 50%.

[0048] In some embodiments of the present invention, the product is at least one of skin care products, pharmaceuticals, health products, and functional foods.

[0049] According to some embodiments of the present invention, at least the following beneficial effects are achieved: The present invention proposes for the first time that phloretin can act as a Serpin B3 inhibitor, and can effectively alleviate the symptoms of rosacea or psoriasis, for use in the preparation of related products for treating inflammatory skin diseases such as rosacea or psoriasis. The present invention provides a certain theoretical basis for the research and development of related therapeutic products for inflammatory skin diseases such as rosacea and psoriasis. Attached Figure Description

[0050] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0051] Figure 1 This is a schematic diagram showing the anti-inflammatory effect of phlorizin at the cellular level in an embodiment of the present invention, where *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0052] Figure 2This is a schematic diagram illustrating the results of phloretin inhibiting SerpinB3 expression in vitro in an embodiment of the present invention.

[0053] Figure 3 This is a schematic diagram illustrating the therapeutic effect of phloretin on rosacea model mice in an embodiment of the present invention, wherein ***p<0.001, ****p<0.0001;

[0054] Figure 4 The figure shows the detection results of the effect of phloretin on SerpinB3 expression in rosacea model mice in this embodiment of the invention.

[0055] Figure 5 This is a schematic diagram illustrating the effect of phloretin on the inhibition of skin inflammation in a mouse model of rosacea in an embodiment of the present invention, wherein ****p<0.0001;

[0056] Figure 6 The figure shows the detection results of the effect of phloretin on the expression of inflammatory factors in rosacea model mice in the embodiments of the present invention, wherein ***p<0.001, ****p<0.0001;

[0057] Figure 7 This is a schematic diagram illustrating the effect of phloretin on the inhibition of immune infiltration in a mouse model of rosacea in an embodiment of the present invention, wherein ****p<0.0001;

[0058] Figure 8 This is a graph showing the detection results of the therapeutic effect of phloretin on psoriasis in an embodiment of the present invention;

[0059] Figure 9 The figure shows the detection results of the effect of topical phloretin on the severity (thickening and desquamation) of psoriasis in mice in the embodiments of the present invention, where **p<0.01, ***p<0.001, and “ns” indicates no significant difference;

[0060] Figure 10 The figure shows the detection results of the effect of phloretin on SerpinB3 expression in a mouse model of psoriasis in this embodiment of the invention.

[0061] Figure 11 The figure shows the detection results of the effect of phloretin on the expression of inflammatory factors in psoriasis model mice in the embodiments of the present invention, wherein **p<0.01, ***p<0.001, and ****p<0.0001. Detailed Implementation

[0062] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all obtainable through conventional commercial channels.

[0063] Experimental material: Phloretin, purchased from Selleck.

[0064] Example 1: Application of phlorizin as a Serpin B3 inhibitor

[0065] This embodiment provides the application of phloretin as a Serpin B3 inhibitor, and the specific verification steps are as follows:

[0066] (1) Cell culture: HaCat cells were routinely cultured in a 37℃ constant temperature, 5% CO2 incubator, and the culture medium was changed every 2 days.

[0067] (2) Cell grouping:

[0068] Experimental group (PHL+TNF-α): HaCaT cells were injected at a dose of 1.6 × 10⁻⁶. 6 The number of cells (the same as the control group) were seeded in 6-well plates and pretreated with 75 μm phlorizin (solvent DMSO) for 12 hours, followed by treatment with 100 ng / mL TNF-α for 12 hours.

[0069] Model control group (TNF-α): HaCaT cells were seeded in 6-well plates and treated with TNF-α at a final concentration of 100 ng / mL for 12 hours;

[0070] Control group: HaCaT cells were seeded in 6-well plates and cultured for 24 hours.

[0071] Negative control group (PHL): HaCaT cells were seeded in 6-well plates and pretreated with phlorizin at a final concentration of 75 μm for 12 hours.

[0072] RNA was extracted from cells after treatment in each group, and real-time quantitative PCR was used to detect changes in intracellular inflammatory factors (TNF-α, IL-6, CCL2, CCL20, etc.). At the same time, protein was extracted from cells and Western blot was used to detect changes in SerpinB3 expression levels.

[0073] A. Inflammatory factor detection

[0074] 1) Extracting RNA from cells or tissues:

[0075] a. Remove cell culture medium, wash twice with PBS, add 1 mL Trizol and mix well by pipetting, add 200 μL Trizol and repeatedly grind the tissue on ice with a tissue homogenizer. After the tissue is thoroughly ground, add 800 μL Trizol.

[0076] b. Let stand on ice for 10 minutes to allow for complete lysis, then transfer to a 1.5 mL EP tube.

[0077] c. Add 200 μL of chloroform, shake vigorously for 20 seconds, and let stand on ice for 10-15 minutes until the liquid in the tube separates into layers.

[0078] d. Centrifuge at 4℃ for 12000 rpm for 15 min.

[0079] e. Take the uppermost aqueous phase into a new EP tube, add an equal volume of isopropanol, mix gently, and let stand on ice for 10-15 minutes.

[0080] f. Centrifuge at 4℃, 10000 rpm, for 10 min.

[0081] g. Carefully discard the supernatant. Add 1 mL of 75% ethanol (prepared by mixing 250 μL of DEPC water with 750 μL of anhydrous ethanol) and invert the container.

[0082] Centrifuge at 4℃ for 7500 rpm for 5 minutes.

[0083] i. Carefully aspirate the supernatant, leave the EP tube open for 5-10 minutes to further dry any remaining supernatant. Add 10-30 μL of DEPC water according to the amount of precipitate, and measure the concentration of the obtained RNA using a nucleic acid analyzer.

[0084] 2) Reverse transcription

[0085] After mixing the system according to Table 1 thoroughly, react it in a PCR instrument at 42℃ for 2 min.

[0086] Table 1 Genomic DNA De-Genomic System

[0087] reagents Usage 5×gDNA Eraser Buffer 2μL gDNAEraser 1μL Total RNA 1μg RNase Free ddH2O Make up to 10μL

[0088] After mixing the system according to Table 2, incubate it in a PCR instrument at 37℃ for 15 min, and then at 85℃ for 5 seconds.

[0089] Table 2 Reverse Transcription Reaction System

[0090] reagents Usage Table 1. Reaction solutions obtained from the genomic DNA removal system. 10μL PrimeScriptRT Enzyme Mix I 1μL RT Primer Mix 1μL 5×PrimeScript Buffer 2 4μL <![CDATA[RNase Free ddH2O]]> 4μL total 20μL

[0091] The synthesized cDNA was stored at -20°C.

[0092] 3) Real-time qPCR

[0093] Table 3. RT-qPCR primer sequences

[0094]

[0095]

[0096] Dilute the cDNA 10-fold with DEPC water, prepare the sample according to the system in Table 4 using the RT-qPCR primer sequences in Table 3, and perform RT-qPCR according to the reaction program set in Table 5.

[0097] Table 4. RT-qPCR reaction system

[0098] reagents Usage 2×SYBR GREEN MI 5μL upstream primer 0.5μL Downstream primer 0.5μL cDNA 2μL <![CDATA[dH2O]]> Make up to 10μL

[0099] Table 5. RT-qPCR reaction procedure

[0100]

[0101]

[0102] The results of the inflammatory factor test are as follows Figure 1 As shown in the figure, the phlorizin pretreatment group can significantly reduce the expression levels of intracellular inflammatory factors TNF-α, IL-6, CCL2, and CCL20, indicating that the phlorizin pretreatment group can significantly alleviate TNF-α-induced inflammation in HaCat cells.

[0103] B-WB assay to detect SerpinB3 expression levels

[0104] The expression level of SerpinB3 was detected by Western blotting, and the specific steps are as follows:

[0105] 1) Sample protein extraction

[0106] Wash cells twice with PBS. Add an appropriate amount of protein lysis buffer and protease inhibitor (100:1) according to the cell volume. Lyse on ice for 5-10 min. Then scrape the cells and transfer them to a 1.5 mL EP tube, or place the tissue in a 1.5 mL EP tube, add 200 μL of protein lysis buffer and protease inhibitor (100:1), and repeatedly grind the tissue on ice using a tissue homogenizer. Centrifuge at 13000 rpm for 15 min at 4 °C. Carefully aspirate the supernatant into a new 1.5 mL EP tube and store at -80 °C.

[0107] 2) Prepare protein samples

[0108] After diluting the protein sample obtained through the above steps at an appropriate ratio, take 10 μL of the diluent and add it to a 96-well plate.

[0109] b. Take 10 μL of the protein standard sample used to construct the standard curve and add it sequentially to the same 96-well plate according to the concentration gradient.

[0110] c. Based on the number of samples to be measured, prepare the required BCA working solution at a ratio of 50:1. Add 190 μL of BCA working solution to each well and incubate in a 37°C incubator for 30 min in the dark.

[0111] d. The protein concentration was measured using an enzyme-linked immunosorbent assay (ELISA) reader and calculated based on the standard curve.

[0112] Add an appropriate amount of 6×SDS Loading Buffer according to the protein concentration, and incubate in a metal bath at 95℃ for 10 minutes.

[0113] Store in a refrigerator at -20°C.

[0114] 3) Glue making

[0115] Based on the size of the target protein molecule, a 10% separating gel was selected for subsequent experiments. The gel preparation solution was prepared as shown in Table 6 below.

[0116] Table 6. Proportions of Adhesive Forming Solution

[0117]

[0118] 4) Electrophoresis and development

[0119] a. Sample loading: Select constant voltage 80V mode. After the sample reaches the separating gel, continue electrophoresis at constant voltage 120V for about 60 minutes.

[0120] 10 minutes before the end of electrophoresis, take a PVDF membrane, cut it to a suitable size, and immerse it in a methanol solution.

[0121] c. Transfer: After electrophoresis, carefully remove the gel and fix it in the transfer clamp in the form of sponge-filter paper-gel-PVDF membrane-filter paper-sponge. Place it in the transfer device, select constant current mode, and transfer at 300mA for about 90 minutes.

[0122] d. Sealing: Place the transferred membrane into a pre-prepared 5% milk solution and seal on a shaker for 1 hour.

[0123] e. Pour out the milk, and TBST washes several times to remove excess milk.

[0124] f. Primary antibody incubation: Add an appropriate amount of antibody to TBST and mix thoroughly. Remove the washed membrane, apply the primary antibody, and incubate overnight at 4°C on a shaker.

[0125] The primary antibody was recovered by g and washed three times with TBST for 10 minutes each time.

[0126] h Secondary antibody incubation: Place the membrane in the prepared secondary antibody and incubate at room temperature for 1 hour.

[0127] Discard the secondary antibody, wash three times with TBST, 10 min each time.

[0128] g. Development: Place the washed membrane on a colorimetric plate, add the luminescent solution (prepared according to the reagent instructions), and place it in a luminescence analyzer for development.

[0129] The results are as follows Figure 2 As shown in the figure, the phlorizin pretreatment group can significantly inhibit the TNF-α-induced increase in SerpinB3 expression in HaCat cells.

[0130] Example 2: Application of phloretin in the preparation of a drug for treating rosacea

[0131] This embodiment provides the application of phloretin in the preparation of a drug for treating rosacea. The animal experiments used for validation were all permitted by the Animal Ethics Committee of Xiangya Hospital, Central South University. The specific validation steps are as follows:

[0132] A rosacea model was established using Balb / c mice (purchased from Shanghai Slark Laboratory Animal Co., Ltd. (Shanghai, China)) for in vivo efficacy verification. The specific experimental methods are as follows:

[0133] Experimental animals: Eight 7-week-old female balb / c mice (18-22g) were used, and all mice were kept under specific pathogen-free conditions.

[0134] Preparation of phlorizin solution: Dissolve 25 mg of phlorizin in 1 mL of propylene glycol to prepare a phlorizin solution with a concentration of 25 mg / mL.

[0135] Grouping: 4 female blb / c mice were randomly divided into four groups: a blank control group (CON, no treatment), a negative control group (LL37, 10 μL of phloretin solution was applied to the ears of mice for 7 consecutive days), a positive control group (PHL, no phloretin solution treatment, 4 mice were established by directly injecting LL37 to establish a rosacea model), and an experimental group (LL37+PHL, 10 μL of phloretin solution was applied to the ears of mice for 7 consecutive days, and LL37 was injected at the same time as the application of phloretin solution on the last two days).

[0136] Experimental methods:

[0137] For seven consecutive days, 10 μL of phloretin solution was applied to the ears of mice. Next, to establish a rosacea model, mice received intradermal injections of 20 μL LL37 (640 μM) twice daily in the right ear during the last two days of phloretin application, and intradermal injections of 20 μL PBS twice daily in the left ear. Control group: For 7 consecutive days, 10 μL of propylene glycol was applied to the left ear of mice, and for the last 2 days, 20 μL of PBS was injected intradermally into the left ear of mice twice daily. Negative control group: For 7 consecutive days, 10 μL of phloretin solution was applied to the ears of mice, and for the last 2 days, 20 μL of PBS was injected intradermally into the left ear of mice twice daily. Positive control group: For 5 consecutive days, 10 μL of propylene glycol was applied to the right ear of mice, and 20 μL of LL37 (640 μM) was injected intradermally into the right ear of mice twice daily to establish a rosacea model. Experimental group: For 5 consecutive days, 10 μL of phloretin was applied to the right ear of mice, and 20 μL of LL37 (640 μM) was injected intradermally into the right ear of mice twice daily. Following the final LL37 injection, photographs were taken using a camera and stereoscope to assess skin inflammation and detect the severity of rosacea in the mouse ears (erythema and edema). Skin lesions were collected for Western blotting to detect changes in Serpin B3 expression levels. Skin lesions were also collected for HE staining and real-time quantitative PCR to detect changes in intracellular inflammatory factors (TNF-α, IL-6, CCL2, CCL20, etc.). Immunofluorescence was also performed to detect immune infiltration in the skin lesions of the rosacea model mice.

[0138] (1) Detection of the severity (erythema and edema) of rosacea in mouse ears

[0139] Test results as follows Figure 3 As shown in the figure, topical phloretin can reduce the severity of rosacea (erythema and edema) in mice.

[0140] (2) WB detection

[0141] The WB detection method is as follows:

[0142] 1) Sample protein extraction

[0143] Add an appropriate amount of protein lysis buffer and protease inhibitor (100:1) to the skin lesions and grind on ice for 5-10 min, then centrifuge at 13000 rpm for 15 min at 4℃. Carefully aspirate the supernatant into a new 1.5 mL EP tube and store at -80℃.

[0144] 2) Prepare protein samples

[0145] ① After diluting the protein sample obtained through the above steps at an appropriate ratio, add 10 μL of the diluent to a 96-well plate.

[0146] ② Take 10 μL of the protein standard sample used to construct the standard curve and add it sequentially to the same 96-well plate according to the concentration gradient.

[0147] ③ Based on the number of samples to be measured, prepare the required BCA working solution at a ratio of 50:1. Add 190 μL of BCA working solution to each well and incubate in a 37°C incubator for 30 min in the dark.

[0148] ④ Use an ELISA reader to perform the detection, and calculate the protein concentration based on the standard curve.

[0149] ⑤ Add an appropriate amount of 6×SDS Loading Buffer according to the protein concentration, and incubate in a metal bath at 95℃ for 10 minutes.

[0150] ⑥ Store in a refrigerator at -20℃.

[0151] 3) Glue making

[0152] Based on the size of the target protein molecule, a 10% separating gel was selected for subsequent experiments. The gel preparation solution is shown in Table 6.

[0153] 4) Electrophoresis and development

[0154] ① Sample loading: Select constant voltage 80V mode. After the sample reaches the separating gel, continue electrophoresis at constant voltage 120V for about 60 minutes.

[0155] ② 10 minutes before the end of electrophoresis, take a PVDF membrane, cut it to an appropriate size, and immerse it in a methanol solution.

[0156] ③ Transfer: After electrophoresis, carefully remove the gel and fix it in the form of sponge-filter paper-gel-PVDF membrane-filter paper-sponge. Place it in the transfer device, select constant current mode, and transfer at 300mA for about 90 minutes.

[0157] ④ Sealing: Place the transferred membrane into a pre-prepared 5% milk solution and seal it on a shaker for 1 hour.

[0158] ⑤ Discard the milk and rinse the TBST several times to remove excess milk.

[0159] ⑥ Primary antibody incubation: Add an appropriate amount of antibody to TBST and mix well. Take out the washed membrane, apply the primary antibody, and incubate overnight in a shaker at 4°C.

[0160] ⑦ Recover the primary antibody and wash three times with TBST for 10 minutes each time.

[0161] ⑧ Secondary antibody incubation: Place the membrane in the prepared secondary antibody and incubate at room temperature for 1 hour.

[0162] ⑨ Discard the secondary antibody, wash with TBST 3 times, 10 min each time.

[0163] ⑩ Development: Place the washed membrane on a colorimetric plate, add the luminescent solution (prepared according to the reagent instructions), and place it in a luminescence analyzer for development.

[0164] Test results as follows Figure 4 As shown in the figure, the phlorizin pretreatment group can significantly inhibit the increase in SerpinB3 expression in LL37-induced rosacea-like skin lesions in mice.

[0165] (3) HE staining and inflammatory factor detection

[0166] The steps for HE staining are as follows:

[0167] Mouse skin tissue was immediately fixed in formalin or 4% paraformaldehyde after ex vivo extraction, and sections were prepared to a thickness of 6 μm. Sections were stained with hematoxylin and eosin (H&E) to determine the number of inflammatory cells in the dermis. The number of inflammatory cells was counted in at least four random microscopic fields and then analyzed.

[0168] The method for detecting inflammatory factors is the same as in Example 1.

[0169] The results are as follows Figure 5-6 As shown in the figure, topical phloretin can reduce inflammation of rosacea in mice.

[0170] (4) Immunofluorescence

[0171] After collecting the skin tissue from the model mice, it was fixed with frozen section embedding medium and stored at -80°C. Alternatively, the tissue could be cut into 8μm thick sections on a cryostat and then stored at -80°C.

[0172] (1) Tissue fixation: Remove the sections from the -80℃ freezer and place them in a humidified chamber. Quickly drop 4% paraformaldehyde onto the sections.

[0173] Fix at room temperature for 15 min. Then wash three times in 1×PBS for 5 min each time.

[0174] (2) Blocking: Prepare blocking solution with donkey serum, Triton and 1×PBS (the concentration of donkey serum is 1% and the concentration of Triton is 3%). Carefully drop the prepared blocking solution onto the tissue, ensuring that the tissue is completely covered by the blocking solution, and then block at room temperature for 1 hour. During this period, be careful not to let the slide dry.

[0175] (3) Primary antibody incubation: Remove the blocking solution, carefully circle a region slightly larger than the tissue with a histochemical pen, and drop freshly prepared primary antibody (prepared with blocking solution) into the circle. Incubate at 4°C for 12 to 16 hours. The next day, remove the slides from the cold storage and let them rest at room temperature for 3-5 minutes to allow them to warm up. Then wash the slides three times with 1×PBS, 15 minutes each time.

[0176] (4) Secondary antibody incubation: The slides were then incubated with the secondary antibody (prepared with 1×PBS, diluted 1:500) at room temperature in the dark for 1 hour. The slides were washed three times with 1×PBS, each time for 15 min.

[0177] (5) Nucleus staining: Incubate the cell nuclei with DAPI (prepared with 1×PBS, diluted 1:1000) in the dark for 3 to 5 minutes, and continue to wash the sections with 1×PBS 3 times, each time for 5 minutes.

[0178] (6) Mounting and filming: Use anti-fluorescence quenching mounting medium to mount the film, taking care to avoid air bubbles.

[0179] The results are as follows Figure 7 As shown in the figure, topical phloretin can reduce immune infiltration of skin lesions in mice with rosacea.

[0180] Example 3: Application of phloretin in the preparation of drugs for treating psoriasis

[0181] This embodiment provides the application of phloretin in the preparation of a drug for treating psoriasis. All animal experiments used for validation were permitted by the Animal Ethics Committee of Xiangya Hospital, Central South University.

[0182] The specific verification process is as follows:

[0183] A psoriasis model was established using Balb / c mice (purchased from Shanghai Slark Laboratory Animal Co., Ltd., Shanghai, China) for in vivo efficacy verification. The specific experimental methods are as follows:

[0184] Experimental animals: Eight 7-week-old female balb / c mice (18-22g) were used, and all mice were kept under specific pathogen-free conditions.

[0185] Preparation of phlorizin solution: Dissolve 25 mg of phlorizin in 1 mL of propylene glycol to prepare a phlorizin solution with a concentration of 25 mg / mL.

[0186] Grouping: Balb / c female mice were randomly divided into four groups: blank group (no treatment, n=4), negative control group (10 μL phlorizin solution was applied to the ears of mice for 7 consecutive days, n=4), positive control group (no phlorizin solution treatment, psoriasis model was established directly by applying imiquimod IMQ, n=4), and experimental group (10 μL phlorizin solution was applied to the ears of mice for 12 consecutive days, and imiquimod IMQ was applied at the same time as phlorizin solution on the last 6 days, n=4).

[0187] Experimental methods:

[0188] For 14 consecutive days, 10 μL of phlorizin solution was applied to the ears of mice. Next, to establish a psoriasis model, for the last 7 days, 5% IMQ cream (Sichuan MED-SHINE Pharmaceutical Co., Ltd.) was applied topically to the right ear of mice while the phlorizin solution was applied. An equal amount of petroleum jelly (Vaseline; Unilever, London, UK) was applied topically to the left ear of disease-free control mice. Control group: 10 μL of propylene glycol solution was applied to the left ear of mice for 14 consecutive days, and petroleum jelly was applied topically for the last 6 days; Negative control group: 10 μL of phlorizin solution was applied to the left ear of mice for 14 consecutive days, and petroleum jelly was applied topically for the last 6 days; Positive control group: 10 μL of propylene glycol solution was applied to the right ear of mice for 14 consecutive days, and 5% IMQ cream was applied topically for the last 6 days; Experimental group: 10 μL of phlorizin solution was applied to the right ear of mice for 14 consecutive days, and 5% IMQ cream was applied topically for the last 6 days. After the first application of IMQ, skin inflammation was assessed daily by measuring skin thickness and scaling scores; changes in Serpin B3 expression levels were detected by Western blotting of skin lesions (the detection method was the same as in Example 2); changes in intracellular inflammatory factors (TNF-α, IL-6, CCL2, CCL20, etc.) were detected by real-time quantitative PCR of skin lesions (the detection method was the same as in Example 1).

[0189] Test results as follows Figure 8-11 As shown, from Figure 8-9 As can be seen, topical phloretin can reduce the severity (thickening and desquamation) of psoriasis in mice;

[0190] from Figure 10 As can be seen, topical phloretin can significantly inhibit the expression of Serpin B3 in the skin lesions of the psoriasis model.

[0191] from Figure 11 The results show that topical application of phlorizin can reduce psoriasis-like inflammation in mice.

[0192] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. Application of phloretin in any of the following: (1) As a SerpinB3 inhibitor; (2) Prepare products for the treatment and / or prevention of inflammatory skin diseases.

2. The application according to claim 1, characterized in that, The inflammatory skin diseases include at least one of the following: psoriasis, rosacea, dermatitis, eczema, parapsoriasis, lichen planus, lichen planus pilaris, acute pustular lichenoid pityriasis, chronic lichenoid pityriasis, pityriasis rubra pilaris, graft-versus-host disease, histiocytosis, drug-induced rashes, autoimmune connective tissue diseases, rosacea, folliculitis, acne, warts, ichthyosis, vitiligo, cicatricial alopecia, and CTCL.

3. The application according to claim 1 or 2, characterized in that, The product has at least one of the following functions: 1) Anti-inflammatory; 2) Inhibit SerpinB3 expression; 3) Reduce symptoms of inflammatory skin diseases.

4. The application according to claim 3, characterized in that, The anti-inflammatory effects include inhibiting the expression of inflammatory factors and reducing the number of inflammatory cells; Preferably, the inflammatory factors include TNF-α, IL-6, CCL2, and CCL20; And / or, the relief of symptoms of inflammatory skin diseases includes: relief of erythema and / or edema caused by rosacea, relief of immune infiltration of rosacea and / or relief of epidermal thickening and / or desquamation caused by psoriasis.

5. The application according to claim 1, characterized in that, The product is at least one of the following: skin care products, pharmaceuticals, health products, and functional foods.

6. The application according to claim 5, characterized in that, The medicine also includes pharmaceutically acceptable excipients; Preferably, the pharmaceutically acceptable excipients include at least one of diluents, excipients, fillers, binders, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, sweeteners, and flavorings; And / or, the dosage form of the medicine is tablets, capsules, granules, pills, oral liquids, dry suspensions, drop pills, dry extracts, injections, or transdermal preparations; And / or, the administration methods of the medicine include, but are not limited to, topical application, injection, or oral administration.

7. The application according to claim 1, characterized in that, The phlorizin in the product has a mass fraction of 0.01% to 100%.

8. A Serpin B3 inhibitor, characterized in that, The SerpinB3 inhibitor contains phlorizin.

9. A product for the prevention and / or treatment of inflammatory skin diseases, characterized in that, The product contains phloretin and pharmaceutically acceptable excipients. Preferably, the inflammatory skin disease includes at least one of psoriasis, rosacea, dermatitis, eczema, parapsoriasis, lichen planus, lichen planus pilaris, acute pustular lichenoid pityriasis, chronic lichenoid pityriasis, pityriasis rubra pilaris, graft-versus-host disease, histiocytosis, drug-induced rash, autoimmune connective tissue disease, rosacea, folliculitis, acne, warts, ichthyosis, vitiligo, scarring alopecia, and CTCL.

10. The product according to claim 9, characterized in that, The phlorizin in the product has a mass fraction of 0.01% to 100%.

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