Application of hydrated oxidized purpurin in the preparation of MrgprA1 itch receptor agonist

Abstract

The present invention provides the use of hydrated oxidized desperadoxin in the preparation of MrgprA1 itch receptor agonists. Hydrated oxidized desperadoxin can be used as a tool molecule to construct an itch animal model and applied to the preclinical pharmacodynamic evaluation of anti-itch drugs. The present invention shows that hydrated oxidized desperadoxin has potent MrgprA1 itch receptor agonist activity and selectivity, and can be used as a tool molecule of MrgprA1 itch receptor agonist in the screening of modulators targeting MrgprA1 itch receptors on primary sensory neurons of mice; in vitro experiments have shown that hydrated oxidized desperadoxin can specifically activate MrgprA1 itch receptors on mouse DRG neuronal cells, causing itch, while having no agonist activity on other members of the Mrgpr family, thus realizing the use of hydrated oxidized desperadoxin in the preclinical pharmacodynamic evaluation of anti-itch drugs targeting MrgprA1 itch receptors.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to the application of hydrated oxidized despermatin in the preparation of MrgprA1 itch receptor agonist. Background Art

[0002] Itch is an unpleasant feeling that triggers the desire to scratch. Like pain, itch is an unbearable feeling, but there are obvious differences in behavioral manifestations. Pain manifests as an avoidance reflex, while itch causes a scratching reflex. Many chronic inflammatory skin diseases (such as atopic dermatitis) can cause patients to itch and scratch repeatedly. Some systemic diseases such as cholestasis, allergies, diabetes, chronic kidney disease, AIDS and tumors are also accompanied by symptoms of itching, which seriously affects the patient's quality of life and physical and mental health. However, the mechanisms of pruritus caused by different diseases are different, and the causes of these itches are very complex, and most of the formation mechanisms have not yet been elucidated. Therefore, the discovery of exogenous and endogenous itch-causing substances will help to find itch-related targets, thereby providing new ideas for the development of antipruritic drugs.

[0003] Mrgpr family receptors are a new type of G protein-coupled receptors discovered in recent years, also known as sensory neuron-specific receptors. The expression of this receptor has been found in mice, rats, humans, gerbils, red-tailed monkeys and macaques. Except for a few members found in non-neuronal cells such as mast cells, the vast majority of Mrgprs receptor family members are only distributed in noxious small-diameter neurons of the trigeminal nerve and dorsal root ganglion, which are closely related to pain, and participate in the formation of pain and itch sensations. MrgprA3 is the earliest discovered itch-related Mrgpr family receptor, which can be activated by the drug (chloroquine) for the treatment of malaria and produce itch. In recent years, MrgprA1, MrgprC11, MrgprD and MrgprB2 have been found to be new receptors that sense itch stimulation. Finding specific agonists and antagonists of these itch receptors will help to analyze the mechanism of itch formation, thereby providing new targets for the development of antipruritic drugs.

[0004] Oxypeucedanin hydrate is a furanocoumarin compound with a molecular formula of C16H16O6, CAS number: 2643-85-8, English name: Oxypeucedanin hydrate, and the structural formula is as follows (Formula 1):

[0005]

[0006]

[0007] Oxypeucedanin hydrate (OXY) can be isolated and extracted from natural herbs such as Peucedanum chinense, Angelica dahurica root, and Angelica pubescens. Studies in recent years have shown that OXY has antibacterial, antioxidant, and anti-tumor activities, but its mechanism of action is not clear. The role and application of this compound in the field of neurobiology have not been reported at home and abroad. Summary of the invention

[0008] The object of the present invention is to provide the use of hydrated oxidized purpurin as the only active ingredient to activate dorsal root neurons and cause itch, and the effect is achieved through the receptor MrgprA1 on the neurons.

[0009] The invention discloses an application of hydrated oxidized desperadoxin in the preparation of MrgprA1 itch receptor agonist. The hydrated oxidized desperadoxin is used as a tool molecule of MrgprA1 itch receptor agonist for the screening of tool drugs targeting MrgprA1 itch receptor.

[0010] The hydrated oxidized despermatin is used in the preparation of MrgprA1 itch receptor agonist, and the MrgprA1 itch receptor agonist is a medicament for constructing an animal itch model.

[0011] The hydrated oxidized despermatin is used in the preparation of MrgprA1 itch receptor agonist, and the model is used for preclinical pharmacodynamic evaluation of anti-itch drugs.

[0012] The hydrated oxidized despermatin is used in the preparation of MrgprA1 itch receptor agonist. The hydrated oxidized despermatin is prepared into one of cream, spray and injection by conventional method.

[0013] The present invention shows that hydrated oxidized desperadoxin has potent MrgprA1 itch receptor agonist activity and selectivity, and can be used as a MrgprA1 itch receptor agonist tool molecule in the screening of modulators targeting the MrgprA1 itch receptor on mouse primary sensory neurons; in vitro experiments have shown that hydrated oxidized desperadoxin can specifically activate the MrgprA1 itch receptor on mouse DRG neuronal cells to cause pruritus, while having no agonist activity on other members of the Mrgpr family, thereby realizing the application of hydrated oxidized desperadoxin in the preclinical pharmacodynamic evaluation of anti-itch drugs targeting the MrgprA1 itch receptor. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a calcium imaging diagram of hydrated oxidized praeruptoside in the present invention activating dorsal root neurons cultured in vitro.

[0015] Figure 2This is a calcium imaging diagram of Hek293 cells transfected with pcDNA3.1(+)-MrgprA1 plasmid activated by hydrated oxidized purpurin as an agonist in the present invention.

[0016] Figure 3 The number of scratching reactions of mice induced by different concentrations (500um, 1mM, 2mM) of hydrated oxidized despermatin as an itch-inducing agent in the present invention. DETAILED DESCRIPTION

[0017] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, but these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0018] Example 1: Hydrated oxidized purpurin can activate dorsal root ganglion neurons cultured in vitro.

[0019] (1) Experimental Materials

[0020] Hydrated oxypephedrine (CAS No.: 2643-85-8, MW304.299, Shanghai Yuanye Biotechnology Co., Ltd., purity ≥98%), Pirt-Gcamp3 transgenic mice were donated by Professor Xinzhong Dong (Johns Hopkins University School of Medicine, Baltimore, Maryland, USA) and are currently kept in the SPF Animal Experiment Center of Nanjing University of Chinese Medicine.

[0021] (2) Experimental methods

[0022] DRG primary cell culture: Pirt-GCaMP3 mice express the genetically encoded calcium ion indicator GCaMP3 in almost all sensory neurons. It is a fusion of a green fluorescent protein and the calcium-binding protein calmodulin. When a neuron is excited and discharged, the protein will emit light when the intracellular calcium ion concentration increases. After the mice of this strain were anesthetized with isoflurane and killed, the DRG cell bodies were acutely isolated on ice and placed in DH10 culture medium (90% DMEM / F-12, 10% fetal bovine serum, 1% antibiotics). The mixed enzyme solution (5mg / ml trypsin, 1mg / ml collagenase dissolved in HBSS solution without Ca 2+ and Mg 2+) was digested in a 37℃ water bath for 30 minutes, and the cells were turned upside down every 5 minutes until the cell bodies were loose. After digestion, the supernatant was discarded, an appropriate amount of DH10 culture medium was added, and the cells were gently blown with a gun to disperse and suspend the cells, and filtered with a cell sieve with a diameter of 100μm. Centrifuge at 1000rpm for 5 minutes, discard the supernatant, add an appropriate amount of DH10 solution (containing 20ng / ml NGF) and gently blow to make it a uniform cell suspension solution, then plant the cell suspension on a glass slide coated with Poly-D-Lysine (0.5mg / ml) and Laminin (10g / ml). Put it in a 37℃ (95% O 2, 5% CO 2) CO 2 incubator. After 1 hour, the cells adhere to the wall and add an appropriate amount of 37℃ preheated DH10 cell culture medium.

[0023] Calcium ion imaging: After culturing the above DRG neurons for 12 hours, they were rinsed twice with calcium imaging buffer solution and then moved to the perfusion tank of the imaging system. The calcium imaging system was controlled by Hamamatsu calcium imaging software and measured the changes in intracellular free calcium at an excitation wavelength of 470nm. The composition of the calcium imaging buffer solution (mM): 137NaCl, 5.4KCl, 1.2MgCl2, 1.2NaH2PO 4, 1CaCl2, 10Glucose, 20HEPES, PH 7.38, osmotic pressure 300mOsm. Hydrated oxidized praeruptosin was directly added to the calcium imaging buffer solution perfused by the cells, and the changes in the gray value of the cells were continuously collected at intervals of 1 second, and the data analysis was completed in the system.

[0024] (3) Experimental results: Figure 1 As shown, Figure 1 A is the background, Figure 1 B shows the increase in intracellular calcium ion concentration after perfusion with 200 μM hydrated oxidized praeruptoside. Figure 1 C is a graph showing the real-time changes in intracellular calcium ion concentration. Figure 1 A and Figure 1 The cells indicated by arrows in B are cells activated by hydrated oxidized praeruptoside.

[0025] (4) Experimental conclusion: OXY can cause an increase in calcium ion concentration in DRG neurons, indicating that OXY can excite DRG neurons.

[0026] Example 2: Experiment on the effect of OXY on intracellular calcium ion concentration

[0027] (1) Experimental Materials

[0028] Hydrated oxypurpurin (CAS No. 2643-85-8, MW304.299, Shanghai Yuanye Biotechnology Co., Ltd., purity ≥98%), pcDNA3.1(+)-MrgprA1, pcDNA3.1(+)-MrgprA3 and pcDNA3.1(+)-Gcamp6 plasmids were donated by Professor Xinzhong Dong (Johns Hopkins University School of Medicine, Baltimore, MD, USA), and Hek293 cells were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0029] (2) Experimental methods

[0030] Hek293 cells in the logarithmic growth phase were inoculated into a 6-well plate with an 8mm round glass slide. When the cells grew to 85%-95% confluence, a cell transfection experiment was performed. The experiment was divided into 3 groups: blank control group: only transfected with pcDNA3.1(+)-Gcamp6(1ug) plasmid, experimental group: co-transfected with pcDNA3.1(+)-Gcamp6(1ug) and pcDNA3.1(+)-MrgprA1(1ug) plasmid, negative control group: co-transfected with pcDNA3.1(+)-Gcamp6(1ug) and pcDNA3.1(+)-MrgprA3(1ug) plasmid. Calcium imaging experiments were performed 48 hours after transfection (refer to the experimental method in

[0017] ). The above cells were cultured in a 37°C (95% O2, 5% CO2) CO2 incubator.

[0031] (3) Experimental results

[0032] like Figure 2 Calcium imaging results showed that hydrated oxidized purpurin can act as an agonist to activate Hek293 cells transfected with MrgprA1 plasmid. When pcDNA3.1(+)-Gcamp6 plasmid was transfected alone, water and oxidized purpurin did not cause changes in calcium ion concentration in Hek293 cells ( Figure 2 A, Figure 2 B, Figure 2 C). However, 24 hours after co-transfection of pcDNA3.1(+)-Gcamp6 and pcDNA3.1(+)MrgprA1 plasmids into Hek293 cells ( Figure 2 D), perfusion of 200 μM hydrated oxidized praeruptoside, the calcium ion concentration in the cells increased rapidly ( Figure 2E, Figure 2 F), indicating that OXY can activate MrgprA1 receptors and cause an increase in calcium ion concentration in cells. However, 24 hours after co-transfection of pcDNA3.1(+)-Gcamp6 and pcDNA3.1(+)MrgprA3 plasmids into Hek293 cells ( Figure 2 G), perfusion of OXY did not cause changes in intracellular calcium concentration ( Figure 2 H, Figure 2 I), proving that OXY cannot activate MrgprA3 receptor. In summary, OXY can specifically activate MrgprA1 receptor, causing an increase in calcium ion concentration in transfected cells.

[0033] (4) Experimental conclusion: OXY can activate MrgprA1 receptor, causing an increase in intracellular calcium ion concentration.

[0034] Example 3: Experiment on the effect of hydrated oxidized despermatin on the itch behavior of mice in vivo

[0035] (1) Experimental Materials

[0036] Hydrated oxypurpurin (CAS No.: 2643-85-8, MW304.299, Shanghai Yuanye Biotechnology Co., Ltd., purity ≥98%), SPF grade C57B6 / J mice (18-20g, Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.).

[0037] (2) Experimental methods

[0038] Acute itch test: Different doses of hydrated oxidized purpurin were injected subcutaneously on the neck or back of mice, and the scratching behavior of mice caused by these itch-inducing substances was observed, and the behavioral changes were recorded. The experiment was divided into 4 groups, with 6 mice in each group. The control group was injected with normal saline, and the experimental groups were injected with 500μM, 1mM, and 2mM hydrated oxidized purpurin, respectively, with each mouse injected with 25μl. After recording, the number of scratches of each mouse was counted by reviewing the video.

[0039] (3) Experimental results

[0040] like Figure 3 As shown, the injection of hydrated oxidized purpurin into the back of the mouse neck can cause the number of scratching reactions in the mice to be significantly different from that in the saline group.

[0041] (4) Experimental conclusion: This indicates that hydrated oxidized despermatin can induce itch behavior in mice.

[0042] Example 4: Preparation of cream

[0043] Take 10 grams of hydrated oxidized praeruptoside, add 1 gram of stearic acid, 1 gram of vaseline, and 2 grams of glycerol, and stir to prepare a cream.

[0044] Example 5: Preparation of spray

[0045] Take 10 grams of hydrated oxidized praeruptosin, add 20 grams of ethanol until it is completely dissolved, and prepare it into a spray.

[0046] Example 6: Preparation of injection

[0047] Take 1 gram of hydrated oxidized praeruptosin, add 100 grams of water for injection until it is completely dissolved, and prepare an injection.

[0048] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. The use of hydrated oxidized despermatin in the preparation of a medicament for constructing an animal pruritus model, characterized in that: Hydrated oxidized proserpine is used as a tool molecule of MrgprA1 itch receptor agonist for screening tool drugs targeting MrgprA1 itch receptor.

2. The use of hydrated oxidized despermatin according to claim 1 in the preparation of a medicament for constructing an animal pruritus model, characterized in that: The model is used for preclinical pharmacodynamic evaluation of anti-pruritic drugs.

3. The use of hydrated oxidized despermatin according to claim 1 in the preparation of a medicament for constructing an animal pruritus model, characterized in that , hydrated oxidized praeruptoside is prepared into one of cream, spray and injection using conventional methods.

Images