Application of alpha-boswellic acid as target itch receptor MRGPRX1 antagonist

Abstract

The invention belongs to the field of traditional Chinese medicines, relates to application of alpha-masticinic acid as a targeted itch receptor MRGPRX1 antagonist, and relates to application of alpha-masticinic acid in targeted therapy of specific dermatitis. The invention gets rid of the constraint of the traditional concept, and a BRET experiment proves that the alpha-boswellic acid can specifically inhibit the activation of the MRGPRX1 receptor, has an obvious effect, and is expected to be developed into a medicine for specifically inhibiting the activation of the MRGPRX1 receptor.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and particularly relates to the application of α-boswellic acid as a targeted antagonist of the pruritus receptor MRGPRX1. Background Art

[0002] MRGPRs belong to the G protein-coupled receptor (GPCR) family, with approximately 50 members in rodents and humans. They are divided into eight subfamilies in humans. MRGPRX1 is one of the members of the Mas-related pruritus receptor family, mainly expressed in the dorsal root ganglion (DRG). It can be activated by γ2-melanocyte-stimulating hormone (γ2-MSH), bovine adrenal medullary peptide (BAM8-22), and the antimalarial drug chloroquine (CQ) with specific dermatitis side effects, etc. It can trigger downstream Gi and Gq signaling pathways and participate in physiological processes such as pruritus. Targeting MRGPRX1 to understand the structural basis of pruritus generation and the sensory process of the peripheral nervous system will be of great significance for the development of drugs for treating pruritus-related diseases and central nervous system pain.

[0003] The molecular formula of α-boswellic acid is C 15 H 24 O3, which is a pentacyclic triterpenoid compound extracted from frankincense resin. The pure product is a white crystalline powder, soluble in organic solvents such as methanol and ethanol, and insoluble in water. The melting point is about 263 - 267°C. It has significant anti-inflammatory, antioxidant, antitumor, immunomodulatory and other pharmacological activities, and can inhibit the release of inflammatory mediators and regulate cell signaling pathways. Clinically, α-boswellic acid shows potential value in the treatment of chronic inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease; in the cosmetic field, it is also used to soothe sensitive skin due to its anti-inflammatory properties. In addition, its derivatives are expected to further improve bioavailability and therapeutic effects through structural modification.

[0004] Atopic dermatitis (AD) is a common skin disease in clinical practice, with a wide range of symptoms and signs, resulting in severe functional impairment, affecting daily life, and causing psychological distress. Currently, the incidence of atopic dermatitis is increasing year by year. Existing drugs for treating atopic dermatitis, such as topical corticosteroids, topical calcineurin inhibitors, systemic immunosuppressants, phototherapy, and moisturizing creams, but their therapeutic effects are not satisfactory. Studies have shown that commonly used antihistamines are ineffective in relieving scratching caused by AD. Long-term use may affect the sleep quality of children and produce drug resistance. This means that new, effective, and safe drugs are needed to treat chronic itching caused by AD. The clinical application of traditional Chinese medicine has the advantages of high efficacy and low toxic side effects, and is safe and effective in treating AD and has broad research prospects. Summary of the Invention

[0005] The object of the present invention is to provide the application of α-boswellic acid as an antagonist of the itch receptor MRGPRX1. The present invention discloses that α-boswellic acid has a significant inhibitory effect on the overactivation of the MRGPRX1 receptor. The present invention breaks away from the shackles of traditional concepts and proves through BRET experiments that α-boswellic acid can specifically inhibit the activation of the MRGPRX1 receptor, and the effect is obvious, and it is expected to be developed into a drug for specifically inhibiting the activation of the MRGPRX1 receptor.

[0006] Furthermore, the present invention provides the application of α-boswellic acid in the preparation of drugs for targeted treatment of atopic dermatitis. There has been no previous report that α-boswellic acid has definite antipruritic activity, nor has it been reported through which signaling pathway antipruritic effects are achieved. It is expected to be developed into a drug for specifically treating atopic dermatitis by targeting a single receptor in specific populations.

[0007] Furthermore, the present invention provides the application of α-boswellic acid in relieving atopic dermatitis-related symptoms in MC903-induced mice. The atopic dermatitis-related symptoms described are selected from ear swelling, ear desquamation, ear thickening, and an increase in the plasma total IgE content.

[0008] The present invention provides the application of α-boswellic acid as an antagonist of the itch receptor MRGPRX1. The present invention has the following advantages: 1. Targeted treatment: Through a large number of experiments, it has been proved that plasma samples of patients with atopic dermatitis can specifically activate the itch receptor MRGPRX1. α-Boswellic acid can accurately target the source of atopic dermatitis for inhibition, and the curative effect is significant.

[0009] 2. Safe and reliable: α-Boswellic acid is a natural plant extract and has relatively high safety.

[0010] 3. Clear mechanism: The structure of the MRGPRX1 receptor has been analyzed by our laboratory, and its mechanism of action has been published in the journal Nature Communications.

[0011] 4. High economic benefits: Compared with existing treatment methods, the drug of the present invention is simple to prepare, has low costs, and broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings forming a part of this invention are used to provide a further understanding of the invention. The schematic embodiments and descriptions thereof of the invention are used to explain the invention and do not constitute an improper limitation of the invention.

[0013] Figure 1 : Effect of α-boswellic acid on MRGPRX1 activity; Figure 2 : Inhibitory effect of α-boswellic acid on the inflammatory phenotype of atopic dermatitis model mice Figure 3 : Effect of α-boswellic acid on the inflammatory score of atopic dermatitis model mice Figure 4 : Effect of α-boswellic acid on ear thickening of atopic dermatitis model mice Figure 5 : Effect of α-boswellic acid on the total plasma IgE content of atopic dermatitis model mice DETAILED DESCRIPTION OF THE INVENTION

[0014] The embodiments of the present invention will be described in detail below. The embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation manners and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0015] Example 1: Effect of α-boswellic acid on MRGPRX1 activity Experimental method: 1. Cultivate 293T cells (human embryonic kidney cells) (1) Resuscitation Preheat the culture medium in a 37 °C water bath, and add the culture medium to a centrifuge tube for standby. Take out the cryopreserved cells from liquid nitrogen, quickly thaw them by shaking in the water bath, and transfer the cells to the centrifuge tube in a laminar flow hood after thawing. Centrifuge at 800 rpm for 5 min. After discarding the supernatant, resuspend with the culture medium, transfer to a culture dish, mix by drawing an "8" shape, label the name and date, and place in an incubator. Finally, clean and tidy the laminar flow hood.

[0016] (2) Cryopreservation Preheat the culture medium, trypsin, and PBS buffer in a 37°C water bath; let the cryo box thaw at room temperature. Take out the cells from the incubator and observe the density using a microscope. If the cells cover the culture dish, the excess cells can be cryopreserved. Discard the culture medium in the laminar flow hood, add 2 mL of PBS buffer to wash and aspirate, then add 1 mL of 0.25% trypsin to digest the cells until large pieces of cells detach from the culture dish wall, and then add 2 mL of DMEM medium (containing 10% FBS) to terminate the digestion and resuspend. Then transfer the resuspended cells to a 15 mL centrifuge tube and centrifuge at 800 rpm for 3 min. After discarding the supernatant, add cell cryopreservation solution (10 mL = 5 mL DMEM medium + 4 mL FBS + 1 mL DMSO) to resuspend the cells, transfer to cryotubes at 1 mL / tube, seal with parafilm, place in the cryo box, then put in the -80°C refrigerator, and transfer to liquid nitrogen for storage after 48 hours. Finally, clean and tidy the laminar flow hood.

[0017] (3) Subculture Preheat the culture medium, trypsin, and PBS buffer in a 37°C water bath. Take out the cells from the incubator and observe the cell density using a microscope to determine whether subculture is needed and how many generations are required. Discard the culture medium in the laminar flow hood, add 2 mL of PBS buffer to wash and aspirate, then add 1 mL of 0.25% trypsin to digest the cells until large pieces of cells detach from the culture dish wall, and then add 2 mL of medium to terminate the digestion and resuspend. Transfer to a 15 mL centrifuge tube and centrifuge at 800 rpm for 3 min. After discarding the supernatant, add fresh DMEM. Divide the cells evenly into culture dishes and make up to 8 mL of medium, and mix by drawing an "8" shape. Label the name and date and place in the incubator. Finally, clean and tidy the laminar flow hood.

[0018] 2. Transfection (1) The 293T cells subcultured the previous day are basically in the logarithmic growth phase after 24 hours and can be transfected within five generations. We select cells at P2 or P3 generations for transfection. Observe the cells under the microscope, and the cell density should be between 60% - 80% as much as possible, and then put the cells back into the 37°C incubator.

[0019] (2) Turn on the water bath in advance and set it to 37°C to preheat the SFM medium. Take out the PEI and dissolve it at 56°C, and use it after it reaches room temperature. (The pH value of PEI is 7.0, and it cannot be repeatedly frozen and thawed. The unused part can be stored at 4°C for a short time.) (3) Turn off the ultraviolet lamp, turn on the laminar flow hood, wipe the workbench from the inside out with a dust-free paper dipped in 75% alcohol, and then wipe the PBS, SFM medium, pipette, and vacuum pump port.

[0020] (4) Take a sterile 1.5 mL EP tube and divide it into two groups. Add 100 μL of preheated SFM basal medium to the tube. Calculate the volume of the plasmid to be added, add the plasmid to one group of EP tubes, and then add PEI to another group of EP tubes at a ratio of plasmid (1.5 μg): PEI (4.5 μL) = 1:3. After adjusting the volume of the pipette to 1 / 2 of the total volume, gently pump out the liquid above the liquid surface, add drop by drop, shake and add, mix repeatedly 6 to 8 times, and leave at room temperature for 5 minutes. Among them, 1.5 μg of the plasmid is receptor: Gα-RLuc: Gβ3: Gγ9-GFP = 2:1:1:1.

[0021] (5) Add the diluted PEI to the diluted plasmid, adjust the pipette volume to 1 / 2 of the total volume, then aspirate the mixture from the bottom and add it dropwise into the EP tube above the liquid surface. Repeat the mixing for 6 to 8 times. Gently shake the EP tube during the addition process. After mixing, let it stand at room temperature for 15 min.

[0022] (6) Take out the cells after subculturing, use a vacuum pump to discard the original culture medium, and slowly add 0.5 mL of HBSS along the wall to wash the cells. Be gentle when adding to avoid blowing up the cells. After discarding the PBS with a vacuum pump, add 2 mL of serum-free SFM culture medium to starve the cells, which will increase the transfection efficiency.

[0023] (7) After standing for 15 min, add the transfection solution drop by drop evenly into the culture dish, draw an “∞” or “+” to mix the cells, and place the cells in a 37°C carbon dioxide incubator.

[0024] (8) 4-6 h after transfection, replace the cell medium, discard the transfection medium with a vacuum pump, add 2 mL of complete culture medium, and place the cells in a 37°C carbon dioxide incubator for 48 h for BRET assay.

[0025] (9) Place the used SFM culture medium, PBS, and PEI in a 4-degree refrigerator, clean the clean bench, turn off the alcohol lamp, wipe the table from the inside out with a dust-free paper dipped in 75% alcohol, close the clean bench, and turn on the UV lamp.

[0026] 3. Diluting the ligand (1) In twelve rows (the first to twelfth wells are named A1 to A12, respectively), the α-boswellic acid ligand is diluted with HBSS starting from 1 mM. Each 10-fold decrease is a concentration gradient. The ligand concentration decreases from A1 to A7. A8 and A9 are pure HBSS and do not require ligand dilution.

[0027] (2)In a 12-well row (the first well to the twelfth well are named A1 - A12 in sequence), dilute the agonist BAM8 - 22 ligand with HBSS starting from 10 mM, with each 10-fold decrease as a concentration gradient. The ligand concentration decreases successively from A1 - A7, and A8 and A9 are pure HBSS without the need for ligand dilution.

[0028] (3)In a 12-well row (the first well to the twelfth well are named A1 - A12 in sequence), dilute the agonist BAM8 - 22 ligand with HBSS to the EC80 concentration (based on the dose curve measured for BAM8 - 22 this time, and A9 is pure HBSS without the need for ligand dilution).

[0029] (4)After taking out the substrate coelenterazine 400a powder from -20°C, centrifuge at 12000 rpm, 4°C for 3 min. Pipette 638 μL of absolute ethanol into the tube and pipette to dissolve it into a stock solution with a final concentration of 2 mM. Pay attention to avoiding light throughout the process and store it in the dark at 4°C.

[0030] 4. Digest the cells (1)48 hours after transfection, aspirate the original medium and add 0.5 mL of HBSS buffer for washing. Pay attention to gentle addition to avoid blowing up the cells.

[0031] (2)Add 250 μL of pre-warmed trypsin to digest the cells. When large chunks of cells are observed to fall off the bottom of the culture dish or the cells are floating, add 1 mL of complete medium to terminate the digestion.

[0032] (3)Gently pipette the adherent cells at the bottom of the culture dish to make all the cells completely detached and transfer them to a 15 mL centrifuge tube, then centrifuge quickly for 5 min.

[0033] (4)Aspirate the supernatant, add 1 mL of HBSS, gently pipette and mix well, then centrifuge quickly for 5 min.

[0034] 5. Detection on the machine (1)Take a sterile white 96-well enzyme-linked immunosorbent assay (ELISA) plate and add 70 μL of the digested cell suspension to each well, with about 10,000 - 50,000 cells per well.

[0035] (2)Take a brown EP tube, add 975 μL of BRET buffer, and then add 25 μL of coelenterazine 400a and pipette to mix well. The working concentration of coelenterazine 400a is 5 μM. (1:40 dilution).

[0036] (3)Add BAM8 - 22 from low to high concentration simultaneously from A1 - A8, with 10 μL of gradient ligand per well, and immediately add 10 μL of coelenterazine 400a. Note that coelenterazine needs to be kept away from light throughout the process.

[0037] (4) Immediately place the white 96-well enzyme-linked immunosorbent assay (ELISA) plate on the Mithras LB940 microplate reader for detection.

[0038] (5) Export the emission light intensities detected at λ = 410 nm and λ = 515 nm, calculate the BRET Ratio value, and fit the curve.

[0039] (6) Fit the curve to calculate the EC80 of the BAM8-22 activation curve, multiply by ten, and dispense the corresponding constant concentration of BAM8-22 into a twelve-well strip (the concentration in the twelve-well strip should be 10 times the EC80 because it will be diluted tenfold when added to the 96-well plate).

[0040] (7) Take a sterile white 96-well ELISA plate, add 70 μL of the digested cell suspension to each well, with approximately 10,000 - 50,000 cells per well. Add 9 wells in a row (A1 - A9). Use a multichannel pipette to take 10 μL of the BAM8-22 with a constant concentration of EC80 and 10 μL of the gradient concentration of α-boswellic acid. Add the mixed ligands simultaneously from A1 - A9, 20 μL of ligands per well, and immediately add 10 μL of coelenterazine 400a. Note that coelenterazine needs to be protected from light throughout the process.

[0041] (8) Fit the α-boswellic acid antagonistic curve following the same steps as in (4) and (5).

[0042] Experimental results: Through further viability testing of α-boswellic acid, as Figure 1 shown, it was confirmed that α-boswellic acid exhibited a significant Gq antagonistic effect on MRGPRX1. The above results suggest that we can develop drugs for the treatment of AD targeting MRGPRX1.

[0043] Example 2: Inhibitory effect of α-boswellic acid on the inflammatory phenotype of atopic dermatitis model mice Experimental method: Select C57BL / 6 mice at 6 - 8 weeks of age, in good health and with similar body weights. Before the experiment, the mice were housed in a standard environment for 1 week to adapt. The environmental conditions were temperature (22 ± 2) °C, relative humidity (50 ± 5)%, and a 12 h light / 12 h dark cycle. The mice had free access to food and water. Normal control group, model group, α-boswellic acid group (8 mg / kg, intraperitoneal injection), upadacitinib group (1.8 mg / kg, gavage) (n = 5 in each group). Induce the AD model by applying MC903 (100 μM, 20 μL) to both ears continuously for 12 days. Start administering drugs on the 13th day, once a day for 7 days. After the last drug administration, anesthetize the mice with isoflurane, place the mice on a fixed photographing table, adjust the light and angle to ensure that the inflamed area is clearly visible. The entire ear should be included in the photograph, and a clear scale in the photograph is used to measure the lesion area.

[0044] Experimental results: As Figure 2 shown, after modeling with MC903, obvious inflammatory symptoms such as ear swelling and desquamation were observed in mice. After using upadacitinib and α-boswellic acid, the symptoms were significantly alleviated.

[0045] Example 3: Effect of α-boswellic acid on the inflammatory score of atopic dermatitis model mice Experimental method: The modeling method used in Example 2 was used for modeling. After the last administration, photos were taken. Trained experimental personnel independently scored the ear inflammation photos of the mice, avoiding interference with each other during the scoring process, and the statisticians were not informed of the animal grouping situation. The scores of different personnel at the same modeling stage were statistically analyzed respectively to evaluate the severity of the AD model and the therapeutic effect of drugs or intervention measures.

[0046] Experimental results: As Figure 3 shown, after modeling with MC903, the inflammatory score of the mice's ears increased significantly. After using upadacitinib and α-boswellic acid, the score decreased significantly.

[0047] Example 4: Effect of α-boswellic acid on ear thickening of atopic dermatitis model mice Experimental method: The modeling method used in Example 2 was used for modeling. On the 0th, 4th, 8th, 12th, 16th, and 20th days of modeling, the mice were anesthetized with isoflurane. An electronic vernier caliper with a precision of 0.01 mm was calibrated before use to ensure the accuracy of the measurement data. The most obvious part of the ear inflammation of the mice (usually the middle part of the auricle) was gently clamped with the electronic vernier caliper, taking care to avoid additional squeezing or damage to the ear. The skin thickness value was read and recorded. Each ear was measured 3 - 5 times, and the average value was taken as the skin thickness of that ear. At the same time, the skin thickness of the same part of the ears of the normal control group mice was measured as a control. By comparing the differences in the ear skin thickness between the AD model mice and the normal control group mice at different time points, the development and remission of skin inflammation were evaluated.

[0048] Experimental results: As Figure 4 shown, after modeling with MC903, the ears of the mice thickened significantly. After using upadacitinib and α-boswellic acid, the degree of thickening decreased significantly.

[0049] Example 5: Effect of α-boswellic acid on the total plasma IgE content of atopic dermatitis model mice Experimental method: The animal model was established using the same method as in Example 2. After the last administration, the required indicators were measured, and blood samples were collected and placed in centrifuge tubes containing anticoagulants. The blood samples were centrifuged at a speed of 3000 - 4000 revolutions per minute for 10 - 15 minutes at 4°C to separate the plasma. The plasma was transferred to a new sterile centrifuge tube and stored in an -80°C refrigerator for further testing. The content of IgE in the plasma was detected by enzyme-linked immunosorbent assay (ELISA). According to the requirements of the ELISA kit (purchased from Jianglai Biotech), the kit was taken out of the 4°C refrigerator 10 minutes in advance, and materials such as gradient concentration standards, samples, enzyme-linked immunosorbent assay plates, washing solutions, and substrates were prepared. After diluting the plasma samples 2-fold with a universal diluent, 100 μL / well was added to the wells of the enzyme-linked immunosorbent assay plates. At the same time, standard wells and blank control wells were set. The operations were carried out according to the incubation time, temperature, and washing steps specified in the kit. Finally, the substrate was added for color development, and the absorbance values of each well were measured using an enzyme-linked immunosorbent assay reader at a wavelength of 450 nm. A standard curve was plotted based on the concentration and absorbance values of the standards, and the content of IgE in the samples was calculated through the standard curve, with the unit of ng / mL. By comparing the differences in the plasma IgE content between the AD model mice and the normal control group mice, the changes in the IgE content in the AD model were evaluated.

[0050] Experimental results: As Figure 5 shown, after establishing the model using MC903, the total IgE content in the plasma of mice increased significantly. After using upadacitinib and α-boswellic acid, the degree of increase decreased significantly.

Claims

1. Application of α-boswellic acid as an antagonist targeting the itch receptor MRGPRX1.

2. Application of α-boswellic acid in the preparation of targeted drugs for the treatment of atopic dermatitis.

3. Application of α-boswellic acid in alleviating symptoms related to atopic dermatitis in MC903 model mice.

4. The use according to claim 3, characterized in that: The atopic dermatitis-related symptoms are selected from ear redness and swelling, ear desquamation, ear thickening, and increased plasma total IgE content.

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