Application of micromolecular compound ophiopogonin D in psoriasis treatment

Abstract

The invention relates to application of a small molecular compound ophiopogonin D in treatment of psoriasis. The invention belongs to the technical field of biological medicines, and provides application of a reagent for down-regulating KLF3 expression in preparation of a medicine for treating psoriasis, the reagent for down-regulating KLF3 expression comprises a small molecule compound ophiopogonin D, and the structural formula is C44H70O16. By inhibiting the expression of KLF3 on mouse skin, skin lesion of psoriasis can be relieved, wrinkles and scales can be reduced, and the condition of skin thickening at the skin lesion can be relieved, a new treatment mode is provided for psoriasis damage repair, and the application has important application value in psoriasis research.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to the application of ophiopogonin D, and specifically to the application of ophiopogonin D in the treatment of psoriasis. Background Art

[0002] Psoriasis is a common chronic inflammatory skin disease mediated by immunity, and its onset is related to multiple factors such as genetics and environment. In addition to skin involvement, psoriasis can also affect joints and other parts, causing great harm to the health of patients. Excessive proliferation of epidermal cells and infiltration of inflammatory cells and inflammatory mediators are the main pathological manifestations of psoriasis. KLF3 is a member of the transcription factor KLF family, which is involved in various physiological and pathological processes such as adipogenesis and erythropoiesis, B cell development, and regulation of muscle and nervous system functions, and is associated with the occurrence and development of various tumors. Previous studies have shown that KLF3 promotes gastric cancer cell proliferation, migration and invasion by activating the WNT / β-Catenin signaling pathway; in the kidney, KLF3 promotes cell proliferation and aggravates the inflammatory response of diabetic nephropathy by activating the STAT3 pathway; in the skin, KLF3 promotes the expression of keratinocyte differentiation genes, suggesting that KLF3 may aggravate psoriasis by promoting keratinocyte proliferation and inflammation, but there is no report on the role of KLF3 in psoriasis. Ophiopogonin D is isolated from the tuber of Ophiopogon japonicus. It is a rare naturally occurring C29 steroidal glycoside with anti-inflammatory, antioxidant and anti-cancer activities. Studies have shown that Ophiopogonin D can bind to KLF3 and inhibit the function of KLF3, promote angiogenesis and new bone formation, and has potential therapeutic value for osteoporosis model mice. However, its role and mechanism in psoriasis have not been explored. Summary of the invention

[0003] In view of the above problems, the object of the present invention is to provide a new small molecule drug for treating psoriasis by targeting KLF3.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0005] The present invention discloses the use of a small molecule compound ophiopogonin D in the preparation of a drug for treating psoriasis. The molecular formula of the small molecule compound ophiopogonin D is C 44 H 70 O 16 , the chemical structure is as follows: .

[0006] Furthermore, the medicine comprises an effective dose of the small molecule compound ophiopogon saponin D and a pharmaceutically acceptable carrier or excipient.

[0007] Furthermore, the dosage form of the drug includes tablets, capsules, granules, pills, powders, granules, pastes, suspensions, oral solutions or injections.

[0008] Furthermore, the drug plays a role in the treatment of psoriasis by inhibiting the expression of KLF3 in keratinocytes.

[0009] The present invention also discloses a small molecule compound, characterized in that the small molecule compound is used in the preparation of a reagent for inhibiting KLF3 protein; the small molecule compound is ophiopogon saponin D, and the molecular formula is C 44 H 70 O 16 , the chemical structure is as follows: .

[0010] The third aspect of the present invention discloses a use of the above-mentioned small molecule compound in the preparation of a drug for treating psoriasis.

[0011] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0012] Furthermore, the dosage form of the drug is any one of injection, emulsion, tablet, powder, granule, gel, ointment, capsule, and oral solution.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0014] The present invention discovered for the first time that the small molecule compound Ophiopogon japonicus saponin D can alleviate the symptoms of psoriasis mice, and this therapeutic effect is exerted by inhibiting the expression of Krüppel-like factor 3 (KLF3), providing a new method for the treatment of psoriasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The expression of KLF3 protein in keratinocytes after stimulation with IL-17A and Ophiopogon japonicus saponin D.

[0016] Figure 2 This is the immunohistochemistry image of KLF3 in the skin lesions on the back of psoriasis mice modeled with imiquimod, 7 days after continuous application of Ophiopogon japonicus saponin D on the back of mice.

[0017] Figure 3 These are photos of skin lesions on the back of psoriasis mice modeled with imiquimod, on the 7th day after continuous application of Ophiopogon japonicus saponin D on the back of mice.

[0018] Figure 4 This is a picture of HE staining of skin lesions on the back of psoriasis mice modeled with imiquimod, 7 days after continuous application of Ophiopogon japonicus saponin D on the back of mice. DETAILED DESCRIPTION

[0019] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described below in conjunction with specific embodiments. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and therefore, the present invention is not limited to the limitations of the specific embodiments of the following disclosure.

[0020] 1. Experimental methods

[0021] 1. Cells: HaCat keratinocytes (purchased from the Chinese Academy of Sciences Cell Bank) were cultured in DMEM high-glucose medium supplemented with 10% FBS and 1% penicillin / streptomycin antibiotics at 37°C and 5% CO 2 In a constant temperature incubator. When the cells grow to a density of 70-90%, use trypsin to digest at 37°C, and subculture at a ratio of 1:3 or plate according to the corresponding required number for subsequent experiments. Ophiopogon japonicus saponin D (purchased from MCE) was dissolved in DMSO and added to the cells at a concentration of 125nM and 250nM, and then cultured for 5 hours before subsequent experiments.

[0022] 2. Cell protein extraction and protein immunoblotting: Add 60μL of freshly prepared RIPA lysis buffer to each well of the six-well plate, including 58.8μL RIPA (strong) lysis buffer, 0.6μL 100X protease inhibitor and 0.6μL PMSF. Lyse on ice for 10 minutes, then scrape the sample with pre-cooled cells into a 1.5mL EP tube. Centrifuge at 4℃, 15000rpm for 15 minutes, carefully aspirate the supernatant, and transfer it to a new pre-cooled 1.5mL EP tube to obtain the protein sample. The protein sample is quantified using the BCA standard curve method, and the concentration is adjusted and then aliquoted and frozen at -80℃. Use Bio-Rad SDS-PAGE gel preparation kit to prepare gel for western blot detection, specifically (for each gel): mix separation gel A, B solution and concentrated gel A, B solution in a 1:1 ratio (separation gel total 6mL, concentrated gel total 2mL), add 6μL and 4μL of TEMED to the separation gel and concentrated gel respectively, mix well and place in a cool and dark place to exhaust for 10 minutes. During this period, assemble the relevant equipment for vertical gel production for standby. After exhausting, add 30μL and 10μL of 30% ammonium persulfate to the separation gel and concentrated gel respectively, gently blow and mix, and immediately pour the gel into the gel glass tank in turn (separation gel at the bottom, concentrated gel at the top), then slowly insert a 1.0mm sample comb, stand at room temperature for 30 minutes, remove the glass plate after the gel is completely solidified, rinse the residual gel on the surface with tap water, and soak it in deionized water, and store it at 4℃ for standby.

[0023] Install the vertical electrophoresis equipment and SDS-PAGE gel, pour 1L of freshly prepared electrophoresis buffer (14.4g glycine, 3.03g Tris, 1g SDS, deionized water to 1000mL) into the electrophoresis tank, carefully remove the sample comb, and gently blow the sample well with a pipette to expel bubbles and residual gel. Mix the protein solution extracted from the above experiment with 5×SDS loading buffer (4:1) and heat at 99℃ for 10 minutes to denature the protein. Then, spot 20μg of protein per sample well, add 6μL of pre-stained protein molecular weight marker protein, and perform electrophoresis at a constant voltage of 140V for 60 minutes. After the protein sample is fully separated, remove the vertical electrophoresis tank, cut the SDS-PAGE gel, and move it into the protein transfer tank. The installation order of the transfer tank is (from bottom to top): sponge pad-thick filter paper-gel-PVDF membrane (0.45μm, pre-activated with methanol)-thick filter paper-sponge pad. Pour 1 L of freshly prepared electrotransfer buffer (14.4 g glycine, 3.03 g Tris, 200 mL methanol, dilute to 1000 mL with deionized water) into the transfer tank, bury the transfer tank in ice, and transfer at a constant current of 300 mA for 48 minutes.

[0024] After the transfer is completed, remove the transfer device, cut the PVDF membrane according to the molecular weight of the protein to be tested, rinse with TBST, and block with 5% skim milk at room temperature for 2 hours. After the blocking is completed, rinse the PVDF membrane with TBST for 10 minutes, add the primary antibody prepared with the primary antibody diluent to the corresponding PVDF membrane, and place it on a 4°C horizontal shaker to slowly shake and incubate overnight. On the second day, recover the primary antibody and rinse the PVDF membrane with TBST 3 times for 10 minutes each. Then add the secondary antibody configured with TBST, shake and incubate at room temperature for 2 hours, and rinse the PVDF membrane with TBST 3 times for 10 minutes each. After washing, soak the PVDF membrane in a new TBST solution for use.

[0025] Mix ECL luminescent solution A and solution B in equal proportions to prepare ECL developer and keep it away from light for later use. Take out the PVDF membrane, dry it with absorbent paper, and place it in a gel imager. Add a small amount of ECL developer on the surface of the PVDF membrane so that the membrane is just soaked by the developer. Change the imager to chemiluminescence (CL) mode, expose and observe the results, and determine the expression level of the corresponding protein.

[0026] 3. Mouse model: 6-8 week old C57BL / 6 mice were randomly divided into control group and experimental group, with 6 mice in control group and 7 mice in experimental group. The back skin of each mouse was shaved, with an area of ​​2 cm*3 cm. The control group applied 62.5 mg imiquimod cream to the back skin of the mouse for 7 consecutive days. The experimental group applied 62.5 mg imiquimod cream and 20 μL of 10 μM ophiopogon saponin D to the back skin of the mouse for 7 consecutive days.

[0027] 4. HE staining: After sampling, the skin tissue of the mouse back was immersed in tissue fixative and embedded in paraffin the next day. The embedded wax block was fixed on the slicer, and continuous sections were made, and the sections were attached to the slide. After the paraffin sections were placed in a 65°C oven for 2 hours, the sections were placed in xylene (2 times, 15 minutes each time), anhydrous ethanol (2 times, 10 minutes each time), 95% alcohol for 5 minutes, 85% alcohol for 5 minutes, 75% alcohol for 5 minutes, and washed with double distilled water for 3 minutes. Then the sections were stained with hematoxylin for 3 minutes and rinsed with tap water for 10 minutes. The sections were stained in eosin stain for 3 minutes and washed with tap water for 10 minutes. The sections were placed in 75% alcohol for 1 minute, 85% alcohol for 1 minute, 95% alcohol for 1 minute, anhydrous ethanol (2 times, 5 minutes each time), and xylene (twice, 10 minutes each time) to dehydrate and make them transparent. The sections were taken out of xylene and dried slightly, and sealed with neutral gum. Then the images were collected and analyzed under a microscope.

[0028] 5. Immunohistochemistry: The skin tissue from the back of the mouse was collected and immersed in tissue fixative, and then embedded in paraffin the next day. The embedded wax block was fixed on the slicer, and continuous sections were made, and the sections were attached to the slides. After the paraffin sections were placed in a 65°C oven for 2 hours, the sections were placed in xylene (2 times, 15 minutes each time), anhydrous ethanol (2 times, 10 minutes each time), 95% alcohol for 5 minutes, 85% alcohol for 5 minutes, 75% alcohol for 5 minutes, and double distilled water for 3 minutes. The tissue sections were then immersed in a repair box with citric acid antigen repair buffer and placed in a pressure cooker for a high-pressure water bath. After boiling, continue heating for 2 minutes. During this process, the buffer should be prevented from evaporating excessively, and the sections should not be dried. After cooling, the slides were placed in PBS (pH 7.4) and washed 3 times with PBS shaking on a decolorizing shaker for 3 minutes each.

[0029] Use an immunohistochemistry pen to circle the tissue to prevent the antibody from flowing away, add endogenous peroxidase blocker to the circle, and incubate at room temperature for 15 minutes. Wash 3 times with PBS for 3 minutes each time. Add nonspecific staining blocker, incubate at room temperature for 15 minutes, carefully absorb the blocker on the slice with clean absorbent paper, and add a certain proportion of primary antibody to the slice, place the slice flat in a humid box to prevent the antibody from evaporating, and incubate overnight at 4°C. The next day, after placing the wet box at room temperature for 30 minutes, put the slide in PBS and wash it 3 times, 3 minutes each time. Then add biotin-labeled goat anti-mouse / rabbit IgG polymer to the slice and incubate at 37°C for 15 minutes. Wash 3 times with PBS for 3 minutes each time. Add streptavidin-peroxidase, incubate at 37°C for 15 minutes, and then wash 3 times with PBS for 3 minutes each time. Mix the DAB color reagent in proportion to make a working solution, add it to the circle and observe it under a microscope, paying attention to controlling the color development time. Brown-yellow is positive staining. When the color deepens, rinse the slices with tap water to terminate the reaction. Counterstain with hematoxylin for 20 seconds and rinse in tap water for 10 minutes. Finally, place the slices in 75%, 85%, and 95% alcohol for 1 minute each, anhydrous ethanol (2 times, 5 minutes each), and xylene (2 times, 10 minutes each) until dehydrated and transparent. Remove the slices and seal them with neutral gum. Then perform image acquisition and analysis under a microscope.

[0030] 2. Experimental results.

[0031] Figure 1 It can be seen that in the presence of IL-17A, the expression of KLF3 protein in keratinocytes treated with Ophiopogon japonicus saponin D was reduced.

[0032] Figure 2 It can be seen that compared with the control group, the expression of KLF3 in the psoriasis lesions on the back of mice treated with external application of Ophiopogon japonicus saponin D was reduced.

[0033] Figure 3 It can be seen that compared with the control group, the psoriasis lesions on the back of mice treated with external application of Ophiopogon japonicus saponin D were alleviated, and the wrinkles and scales were reduced.

[0034] Figure 4 It can be seen that compared with the control group, the epidermal thickening of the psoriasis lesions on the back of mice treated with external application of Ophiopogon saponin D was significantly reduced.

[0035] In summary, the present invention screened out a small molecule compound that inhibits the expression of KLF3, verified that the compound can inhibit the expression of KLF3 on keratinocytes, and the compound can relieve psoriasis lesions when applied to mouse skin.

[0036] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of a small molecule compound ophiopogonin D in the preparation of a drug for treating psoriasis, wherein the molecular formula of the small molecule compound ophiopogonin D is C 44 H 70 O 16 , the chemical structure is as follows: 。 2. The use according to claim 1, characterized in that The medicine comprises an effective dose of a small molecule compound ophiopogon saponin D and a pharmaceutically acceptable carrier or excipient.

3. The use according to claim 2, characterized in that: The dosage form of the drug includes tablets, capsules, granules, pills, powders, granules, pastes, suspensions, oral solutions or injections.

4. The use according to claim 1, characterized in that The drug plays a role in the treatment of psoriasis by inhibiting the expression of KLF3 in keratinocytes.

5. A small molecule compound, characterized in that: The small molecule compound is used in the preparation of a reagent for inhibiting KLF3 protein; the small molecule compound is ophiopogon saponin D, with a molecular formula of C 44 H 70 O 16 , the chemical structure is as follows: 。 6. Use of the small molecule compound according to claim 5 in the preparation of a drug for treating psoriasis.

7. The use according to claim 6, characterized in that: The drug also includes pharmaceutically acceptable excipients.

8. The use according to claim 6, characterized in that: The dosage form of the drug is any one of injection, emulsion, tablet, powder, granule, gel, ointment, capsule and oral solution.

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