阅读说明：本技术 citrusinine-II在制备影响人和动物对痒和痛感知的试剂中的应用 (Use of citrusinine-II for the preparation of agents affecting the perception of itch and pain in humans and animals ) 是由 赖仞 罗雷 韩亚蓝 罗安纳 于 2020-11-20 设计创作，主要内容包括：本发明提供了citrusinine-II在制备影响人和动物对痒和痛感知的试剂中的应用,涉及生物医学技术领域。所述citrusinine-II能够显著抑制人类或动物TRPV3配体门控型离子通道的2-APB激活,从而在一定时间内会钝化对瘙痒和疼痛的感应。citrusinine-II可以增强人类或动物对瘙痒和疼痛的抵抗力,可以作为资源开发成一种潜在的止痒剂、止痛剂或护肤品,应用于人类和其他动物缓解瘙痒和疼痛。同时,citrusinine-II靶向作用于受体TRPV3,可以应用于相关离子通道病的研究及作为潜在治疗药物。(The invention provides application of citrusinine-II in preparation of a reagent influencing human and animal perception of itch and pain, and relates to the technical field of biomedicine. The citrusinine-II can remarkably inhibit 2-APB activation of TRPV3 ligand gated ion channels of human or animal, so that the response to pruritus and pain can be inactivated within a certain time. The citrusinine-II can enhance the resistance of human or animals to pruritus and pain, can be used as a resource to be developed into a potential antipruritic, analgesic or skin care product, and is applied to human and other animals to relieve pruritus and pain. Meanwhile, the citrusinine-II is targeted on a receptor TRPV3, and can be applied to the research of related ion channel diseases and used as a potential therapeutic drug.)

Use of citrusinine-II in the preparation of a reagent for affecting the perception of itch and pain in humans and animals, said citrusinine-II having the structural formula shown in formula I:

2. an agent for affecting the perception of itch and pain in humans and animals, wherein the active ingredient of said agent comprises citrusinine-II, the structural formula of said citrusinine-II being represented by formula I:

3. an antipruritic agent, which is characterized in that the active ingredient of the antipruritic agent comprises citrinin-II, wherein the structural formula of the citrinin-II is shown as a formula I:

4. an analgesic, characterized in that the active ingredient of the antipruritic comprises citrusinine-II, the structural formula of citrusinine-II is shown in formula I:

5. an antipruritic skin care preparation comprising the agent according to claim 2 or the antipruritic according to claim 3 as an active ingredient.

The application of citrusinine-II in preparing a reagent for inhibiting a TRPV3 ion channel, wherein the structural formula of the citrusinine-II is shown as the formula I:

7. an inhibitor of a TRPV3 ion channel, wherein the effective component of the inhibitor comprises citrusinine-II, and the structural formula of the citrusinine-II is shown as a formula I:

8. the use of the inhibitor of claim 6 for the manufacture of a medicament for the treatment of a TRPV3 ion channel related disorder.

9. The use according to claim 7, wherein said TRPV3 ion channel-associated disease comprises skin inflammation or chronic pruritus.

10. A medicament for treating a TRPV3 ion channel related disease, wherein the active ingredient of the medicament comprises the inhibitor according to claim 6.

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to application of citrinine-II in preparation of a reagent for influencing human and animal perception of itch and pain.

Background

Citrustinine-II, molecular formula C₁₅H₁₃NO₅Molecular weight 287.27. The citrusinine-II is an acridone alkaloid derived from Atalantia monophyllla of Citruideae. Experimental research shows that the citrusinine-II has an anti-tumor promoting effect, can obviously inhibit the activity of EBV-EA, and has an inhibition rate of 100% at a molecular ratio of 1000 to TPA. However, there is currently no study on citrusinine-ii and itch perception.

Disclosure of Invention

In view of the above, the invention aims to provide an application of citrusinine-II in preparation of an agent for influencing itching and pain perception of human and animals, wherein the citrusinine-II can remarkably inhibit 2-APB activation of TRPV3 ligand gated ion channels of different species, passivate induction on external itching stimulation within a certain time, can be used as a resource to develop a potential itching relieving agent, an analgesic or a skin care product, and is applied to relieve itching and pain of human and other animals.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an application of citrusinine-II in preparation of a reagent for influencing itching and pain perception of human and animals, wherein the structural formula of the citrusinine-II is shown as a formula I:

the invention provides a reagent for influencing the perception of itch and pain of human and animals, the effective component of the reagent comprises citrusinine-II, the structural formula of the citrusinine-II is shown as formula I:

the invention provides an antipruritic, which comprises the effective components of citrinin-II, wherein the structural formula of the citrinin-II is shown as a formula I:

the invention provides an antipruritic skin care product, and the active ingredients of the skin care product comprise the above reagent or the above antipruritic.

The invention provides an analgesic, wherein the active ingredient of the analgesic comprises citrinin-II, the structural formula of the citrinin-II is shown as a formula I:

the invention provides an application of citrinine-II in preparing a reagent for inhibiting a TRPV3 ion channel, wherein the structural formula of the citrinine-II is shown as a formula I:

the invention provides an inhibitor of a TRPV3 ion channel, wherein the effective component of the inhibitor comprises citrusinine-II, and the structural formula of the citrusinine-II is shown as a formula I:

the invention provides application of the inhibitor in preparing a medicament for treating TRPV3 ion channel related diseases.

Preferably, the TRPV3 ion channel related diseases include skin inflammation or chronic pruritus.

The invention provides a medicament for treating TRPV3 ion channel related diseases, and the effective component of the medicament comprises the inhibitor.

The invention provides an application of citrusinine-II in preparation of a reagent for influencing itching and pain perception of human and animals, wherein the citrusinine-II can obviously inhibit 2-aminoethoxy dibenzoborate (2-APB) activation of ligand-gated ion channels of TRPV3(Transient receptor potential vanilloid 3) of different species. Electrophysiological experiments have shown that citrusinine-II acts on TRPV3 receptors in other species, including humans. TRPV3 is inhibited as a receptor on the surface of animal skin closely related to chronic pruritus and pain sense, and can inactivate the response to pruritus and pain in a certain period of time. Experiments on model animal mice show that the tolerance of mice to histamine-induced acute pruritus (VEH) can be enhanced by injecting and applying the citrusinine-II, the number of scratching behaviors of mice to pruritus is remarkably reduced, and the spontaneous scratching behaviors of mice of a mouse skin pruritus model (AEW) can be inhibited by injecting and applying the citrusinine-II dose-dependently, so that the resistance of the mice to chronic pruritus is remarkably enhanced; the injection of citrusinine-II can inhibit formalin and acetic acid induced acute pain and heat pain dose-dependently, and the number of pain behaviors of mice is remarkably reduced. In conclusion, the citrusinine-II enhances the resistance of the species such as mice to itch and pain, can be developed into a potential antipruritic, analgesic or skin care product as a resource, and is applied to human beings and other animals to relieve itch and pain. Secondly, the citrusinine-II is targeted on a receptor TRPV3, and can be applied to the research of related ion channel diseases and used as a potential therapeutic drug.

Drawings

FIG. 1 is a graph of the effect of citrusinine-II on TRPV3, where A represents the current at which mTRPV3 is inhibited by 50 μ M citrusinine-II; b represents the Hill equation fitting of a dose-effect curve of citrusinine-II to mTRPV3 (n ═ 6);

FIG. 2 is a graph of the selectivity of citrusinine-II on several representative ion channels, wherein A to F represent the effect of 100. mu.M citrusinine-II on TRPV1, TRPA1, TRPV4, TRPM8, Nav1.7, Nav1.8 channels;

FIG. 3 is a diagram of the mimic structure of mTRPV3 and its site of action of citrusinine-II, where A represents a side view of multiple citrusinine-II docking to mTRPV3 channel; b represents a representative plan view of the interaction of the amino acids involved in citrusinine-II and TRPV 3; c and D represent side and magnified views of 1 citrusinine-II binding to one mTRPV3 subunit, respectively, with mTRPV3 marked grey, Y564 residue side chain marked blue, citrusinine-II marked orange; e represents the inhibition rate of 50 μ M citrusinine-II on TRPV3 mutant (n ═ 5, ×) p < 0.01);

FIG. 4 is a graph of the inhibition of the activation of TRPV3 by citrusinine-II, which inhibits the whole-cell current of 2-APB activation of TRPV3, a representative species of a different group, where A is the inhibition of the activation current of human TRPV3 by citrusinine-II and B is the inhibition of the activation current of rat TRPV3 by citrusinine-II;

FIG. 5 is a pathological observation of skin tissues of AEW model, wherein A and B are neck skin of mice treated for 5 days in VEH group (water) and AEW group (acetone-ethyl ether-water), respectively; c and D are HE staining of neck skin of mice 5 days after VEH (water) and AEW (acetone-ether-water) treatment, respectively;

FIG. 6 is a graph of the effect of citrusinine-II on the relief of itch in mice, where A and B are the number of scratching and total distance of movement of mice within 1 hour after subcutaneous injection of experimental drugs in VEH group (water) and AEW group (acetone-ether-water), respectively; c and D represent the aging curve of the mice scratching behavior after subcutaneous administration and statistics of the number of scratching times for each experimental group, vehicle is a control group (0.05% DMSO), CII is citrusine-II, (n ═ 5-6, n.s. represent no significant difference,. p <0.05,. p < 0.01);

figure 7 is a graph of the effect of citrusinine-II on pain relief in mice. A represents the statistics of the number of licks of formalin-induced pain in mice in stage I (0-5min) and stage II (15-30) after 30min of intraperitoneal predosing of the mice (n-5, p < 0.05); b represents statistics of the number of acetic acid-induced painful writhing behaviors in mice after 30min of intraperitoneal predose (n-5,. sp < 0.05); c represents the statistics of the reaction time of the mouse on the foot lifting due to heat pain after the mouse is pre-dosed in the abdominal cavity for 30min (n is 5, p is less than 0.05); d represents the statistics of the hot tail flick response time of mice 30min after the mice were pre-dosed in the abdominal cavity (n-5, p < 0.05).

Detailed Description

The invention provides an application of citrusinine-II in preparation of a reagent for influencing itching feeling of human and animals, wherein the structural formula of the citrusinine-II is shown as a formula I:

the citrusinine-II is preferably an acridone alkaloid derived from Atalantia monophyllla of the family Citruidae. The source of the citrusinine-II is not particularly limited in the invention, and the preferred extraction method is the method according to Yannai and the like: leaves of a. monophyllla were identified and collected from the profusion Phuwieng region in thailand, air-dried and pulverized into powder, and then extracted with hexane, EtOAc and MeOH in this order. The reagent leach solution was evaporated in vacuo to obtain dry extracts of hexane, EtOAc and MeOH. After functional screening, the EtOAc fractions were subjected to flash column chromatography on silica gel (FCC) and eluted with a gradient of hexanes, EtOAc and MeOH with increasing polarity. The eluate was collected every 50ml and checked by Thin Layer Chromatography (TLC) to give 10 fractions designated F1-F10. F7 was purified by silica FCC to give F7.1-F7.4. F7.2 was isolated by Sephadex LH20 gel filtration eluting with MeOH to give citrusinine-II (Yannai, Shmuel. (2004) Dictionary of food compounds with CD-ROM: Additives, flavors, and ingredients. boca Raton: Chapman & Hall/CRC.). In the application of the invention, the purity of the citrusinine-II is preferably more than or equal to 95%.

The invention provides a reagent for influencing itch perception of human and animals, wherein the effective component of the reagent comprises citrinin-II, and the structural formula of the citrinin-II is shown as a formula I:

the citrusinine-II can obviously inhibit the activation of 2-aminoethoxy diphenyl borate (2-APB) of ligand gated ion channels of different species of TRPV3(Transient receptor potential vanilloid 3), and TRPV3 is inhibited as a receptor on the skin surface of a human or an animal, which is closely related to chronic pruritus diseases, and can inactivate the induction of pruritus within a certain time, thereby influencing the perception of itch of the human or the animal. Specifically, the embodiment of the invention provides a mouse model experiment, and the resistance of the citrusinine-II to pruritus can be improved after the citrusinine-II is injected or smeared, so that the citrusinine-II can be prepared into an agent for influencing or improving the itching feeling of people and animals. The source and preparation method of the citrusinine-II of the present invention are preferably the same as those described above, and are not described herein again.

The citrusinine-II disclosed by the invention can specifically target a receptor TRPV3, wherein the TRPV3 is a temperature-sensitive nonselective cation channel, is expressed in a large amount in skin keratinocytes, particularly in the outer root sheath and the epidermal basal layer of hair follicle epithelial cells, and the TRPV3 is closely related to various physiological and pathological functions of the skin due to the special distribution of TRPV 3. The invention verifies the effect of citrusinine-II on relieving itching of a mouse through experiments of a mouse chronic itching model and an acute itching model. The method comprises the steps of synthesizing TRPV3 plasmids of mouse and human species and the like, verifying that the citrinin-II inhibits 2-APB activation of TRPV3 of mammals by using a patch clamp technology, and confirming the universality of the citrinin-II in application of different species.

The invention provides an antipruritic, which comprises the effective components of citrinin-II, wherein the structural formula of the citrinin-II is shown as a formula I:

the content of the active ingredients in the antipruritic is not particularly limited, and the antipruritic preferably also comprises auxiliary materials, and the types of the auxiliary materials are not particularly limited. The source and preparation method of the citrusinine-II of the present invention are preferably the same as those described above, and are not described herein again.

The invention provides an analgesic, wherein the active ingredient of the analgesic comprises citrinin-II, the structural formula of the citrinin-II is shown as a formula I:

the content of the active ingredients in the analgesic is not particularly limited, and the analgesic preferably further comprises auxiliary materials, and the types of the auxiliary materials are not particularly limited. The source and preparation method of the citrusinine-II of the present invention are preferably the same as those described above, and are not described herein again.

The invention provides an antipruritic skin care product, and the active ingredients of the skin care product comprise the above reagent or the above antipruritic. The preparation form of the itching relieving skin care product is not particularly limited, and the conventional preparation form in the field can be utilized. The itching-relieving skin care product disclosed by the invention is preferably applied to human beings and animals in modes of external application or injection and the like to relieve pruritus, overcome acute pruritus and chronic pruritus in a short time and the like.

The invention provides an application of citrinine-II in preparing a reagent for inhibiting a TRPV3 ion channel, wherein the structural formula of the citrinine-II is shown as a formula I:

in the invention, the citrusinine-II can specifically target a receptor TRPV3, wherein TRPV3 is a temperature-sensitive nonselective cation channel and is expressed in a large amount in skin keratinocytes, particularly in the outer root sheath and epidermal basal layer of hair follicle epithelial cells, and TRPV3 is found to be closely related to various physiological and pathological functions of the skin due to the specific distribution of TRPV3, so that the citrusinine-II can be used for preparing an agent for inhibiting TRPV3 ion channels.

The invention provides an inhibitor of a TRPV3 ion channel, wherein the effective component of the inhibitor comprises citrusinine-II, and the structural formula of the citrusinine-II is shown as a formula I:

according to the invention, a mouse TRPV3 receptor (mTRPV3) is over-expressed on a HEK293T cell line, and the action target of the citrusinine-II is found to be the TRPV3 receptor through a patch clamp technology, so that the 2-APB activation of the TRPV3 receptor can be inhibited; the molecular structure of mTRPV3 was simulated using Rosetta molecular modeling software, and chimeras and point mutations of mTRPV3 sequence were found to affect citrusinine-II binding at mutation site Y564A, and thus the compounds inhibited TRPV3 channel. The dosage form of the inhibitor is not particularly limited in the invention, and the source, preparation method and the like of the citristinine-II are preferably the same as those described above, and are not described again.

The invention provides application of the inhibitor in preparing a medicament for treating TRPV3 ion channel related diseases.

The dosage form of the drug is not particularly limited in the present invention, and when the drug is used, the drug can be injected and administered orally. The TRPV3 ion channel related diseases preferably comprise skin inflammation or chronic pruritus.

The invention provides a medicament for treating TRPV3 ion channel related diseases, and the effective component of the medicament comprises the inhibitor.

The use of the citrusinine-II provided by the present invention for the preparation of agents affecting the perception of itch and pain in humans and animals is described in detail below with reference to the examples, but they should not be construed as limiting the scope of the invention.

Example 1

Electrophysiological experiment of targeting effect of cistrustine-II and mTRPV3 and other itch-related receptors

Transient overexpression of plasmids such as mTRPV3, hTRPV3, ratTRPV3, TRPV1, TRPA1, TRPV4, TRPM8, Nav1.7 and Nav1.8 in HEK29On the 3T cell line, the above plasmids were synthesized by Shanghai Bioengineering Co., Ltd. All HEK293T cell lines were cultured in DMEM (Dulbecco's modified Eagle's medium) with the addition of 10% fetal bovine serum, 1% penicillin/streptomycin, 37 ℃, 5% CO₂And (5) incubation and culture.

And selecting cells with smooth cell membranes and uniform cytoplasm under an inverted microscope, and performing a patch clamp experiment at the room temperature of 20-25 ℃. The method comprises the steps of selecting a WPI 0.86mm thin-wall borosilicate glass capillary tube as a glass electrode material, drawing the glass electrode on a drawing instrument (P-97, Shutter) for 5 steps, wherein the caliber of the tip of the electrode after the glass electrode is subjected to thermal polishing is 1.5-3.0 mu m, and filling intracellular fluid into the glass electrode after drawing is finished. The initial resistance of the glass electrode is 1.5-2.5M omega. And after the electrode and the cell membrane form high-impedance Jingou (G omega) sealing, the electrode fast capacitor is supplemented. Then a short and powerful negative pressure is given to rapidly break the cell membrane clamped in the electrode and compensate the slow capacitance of the cell. After the whole cell recording mode was established, the cells were clamped to 0mV and the cells stabilized for 10min and the recording of current was started using the appropriate pulse voltage (80/-80 mV). The drugs were perfused using Biolab RS200 with a switching speed between drugs of 50 ms. The series resistance (Rs) is kept constant within the range of 5-8M omega all the time in the experimental process, and the series resistance compensation of the system is generally between 30-60%. The experimental data were analyzed using Patch Master software, the data were further analyzed using Igor software, and n represents the number of experimental data.

The cells are slowly lifted to the lower edge of a dosing port of a rapid dosing switching system (RSC-200) to be flush through an electric micromanipulation system. The dosing switching system is directly connected with an amplifier, and all experimental data are recorded in a computer software Patch Master through the amplifier. Cell stimulation and drug treatment are given through a Patch Master and a switching drug adding system, channel current change is recorded, and the activity of different drugs on channels is detected. In order to ensure the accuracy of experimental data, the whole recording process needs to maintain stable sealing resistance and series resistance.

TRPV3 channel internal and external fluids: 130mM NaCl, 3mM HEPES, 0.2mM EDTA, adjusted to pH 7.2 with NaOH. The medicine used in the experimental process needs to be dissolved and prepared by the electrode external liquid.

Sodium ion channel internal liquid: 20mM NaCl, 5mM EGTA, 1mM MgCl₂135mM CsF, 10mM glucose and 10mM HEPES, pH adjusted to 7.4 with CsOH solution.

Sodium ion channel external liquid: 130mM NaCl, 0.1mM NiCl₂、20mM TEA-Cl、1.8mM CaCl₂5mM 4-AP, 0.01mM propylamine valerate hydrochloride, 5mM CsCl, 1mM CdCl₂And 10mM HEPES, adjusted to pH 7.4 with NaOH solution.

As shown in FIG. 1, citrusinine-II inhibited 2-APB activation of mTRPV3 channel and exhibited a concentration-dependent relationship; targeting of citrusinine-II to other itch-associated receptors (mTRPV3 receptor in vitro) failed to activate or inhibit these receptors (figure 2).

Example 2

Structure simulation and point mutation experiment of mTRPV3

Structural simulation of mTRPV3

modeling of mTRPV3 was constructed using Rosetta molecular modeling software version 2020.37. The cryomicroscopic structure of mouse TRPV3 in the open state (PDB:6DVW) was used as a template. And (3) extending the structure of the strain by using Rosetta, and selecting a model with the lowest energy as a final structure model to carry out molecular docking with the citrusinine-II. Performing molecular Docking operation of the compound and a TRPV3 channel by using a Ligand-Docking calculation method in Rosetta to obtain 10000 compound and channel composite structures, grading the energy of candidate structure models by using an energy function based on cell membrane environment specificity, and selecting a compound structure model with the lowest binding energy as a final structure model of interaction between citrinusine-II and TRPV3 (figure 3).

mTRPV3 chimeras

Chimera experiments were used to explore the effect of mTRPV3 on the targeting region of citrusinine-II. A partial amino acid residue fragment of TRPV3 is substituted to the homologous position of TRPV1 by using TRPV1 as an acceptor and TRPV3 as a donor. The specific operation method comprises the following steps:

primer design, TRPV1 receptor channel plasmid is linearized by PCR reaction, 15bp base sequence homologous to donor TRPV1 is added at 5' end of upstream primer and downstream primer:

TRPV1 primer (SEQ ID NO. 1: 5'-GAATACCTCGCCTGCCGAGGATTCCAGCAGATGGGC-3' and SEQ ID NO. 2: 5'-GGTGAGGATGACATAATAGGCCAGTAACAGGATGAT-3');

TRPV3 primer (SEQ ID NO. 3: 5'-CTGTTACTGGCCTATTATGTCATCCTCACCTTCGTC-3' and SEQ ID NO. 4: 5'-CTGCTGGAATCCTCGGCAGGCGAGGTATTCTTTGTA-3');

dpn1 restriction enzyme was added to the PCR product and the remaining template in the PCR product was removed.

Linearized TRPV1 receptor channel plasmids were obtained by DNA purification kits.

Primer design 15bp base sequences homologous to the receptor TRPV1 embedding part are added at the 5' ends of the upstream primer and the downstream primer, and amplification is carried out through PCR reaction:

50 μ l system: mu.l template DNA, 19. mu.l deionized water, 25. mu.l 2 XPrimerStar Max, 2. mu.l 5 'PCR primers and 2. mu.l 3' PCR primers.

PCR procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 9min, and 30 cycles; extension at 72 ℃ for 5 min.

The amplification product was separated by 2% agarose gel electrophoresis, and a purified TRPV3 donor insert DNA fragment (SEQ ID No.5) was obtained using an agarose gel purification kit:

TATGTCATCCTCACCTTCGTCCTCCTCCTCAACATGCTCATTGCCCTGATGGGGGAGACGGTGGAGAACGTCTCCAAAGAAAGTGAGCGGATCTGGCGCTTGCAGAGAGCCAGGACCATCTTGGAGTTTGAGAAAATGTTACCAGAATGGCTGAGAAGCAGATTCCGCATGGGCGAGCTGTGCAAAGTAGCAGATGAGGACTTCCGGCTGTGTCTGCGGATCAACGAGGTGAAGTGGACGGAATGGAAAACACACGTGTCCTTCCTTAATGAAGACCCGGGACCCATAAGACGGACAGCAGATTTAAACAAGATTCAAGATTCTTCCAGGAGCAATAGCAAAACCACCCTCTATGCGTTTGATGAATTAGATGAATTCCCAGAAACGTCGGTGTAG。

the process of homologous recombination cyclization was carried out by inserting the linearized TRPV1 receptor channel plasmid and TRPV3 donor into the DNA fragment using a homologous recombinase. The connection conditions were as follows: 50ng of linearized TRPV1 receptor channel DNA fragment, 150ng of linearized TRPV3 donor DNA fragment, 2. mu.L of Exnase II, addition of ddH2O to 10. mu.L, and ligation at 37 ℃ for 30 min. The ligation products were transformed into 100. mu.L Dh 5. alpha. competent cells, plated overnight, picked single colonies, shake-cultured for amplification, and then verified by sequencing.

mTRPV3 point mutation

A point mutation experiment is utilized to explore the target binding site of mTRPV3 affecting citrisine-II.

All mutant mTRPV3 channels were constructed using homologous recombination and obtained using the Fasta site-directed mutagenesis kit from seebeck corporation. The specific experimental operation steps are as follows:

introducing single base site-directed mutation into a plasmid, and designing a pair of primers:

mutant 5' primer (SEQ ID NO. 6):

GGGCTGGGCGGCTATGCTCTACTACACGAGAGGCTTCC；

mutant 3' primer (SEQ ID NO. 7):

AGTAGAGCATAGCCGCCCAGCCCAGGGCCATGGCCAGC；

the plasmid was subjected to reverse PCR amplification, and 1. mu.l of the template DNA sequence, 8.5. mu.l of deionized water, 2. mu.l of 2 XMax buffer, 0.5. mu.l of dNTP mix, 1. mu.l of 5 'PCR primer, 1. mu.l of 3' PCR primer and 0.5. mu.l of DNA polymerase centrifuge tube were reacted.

Amplification was performed in a PCR instrument according to the following procedure: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 9min, 30 cycles; extension at 72 ℃ for 5 min.

And (3) recombination reaction: mu.l of Dpnl digest, 2. mu.l of CE II buffer, 1. mu.l of Exnase II and 5. mu.l of deionized water were aspirated, reacted at 37 ℃ for 30min, and then cooled in an ice water bath for 5 min. The ligation products were transformed into 100. mu. LDH 5. alpha. competent cells, plated overnight, picked single colonies, shake-cultured for amplification, and then verified by sequencing.

And (3) detecting the inhibitory activity of the citrusinine-II on the activation of all point mutation mTRPV3 channel 2-APB, and comparing the inhibitory rates of the citrusinine-II on wild type and each point mutation channel, wherein the amino acid mutation at the Y564 position obviously weakens the inhibition of the citrusinine-II on the activation of mTRPV3 channel 2-APB.

Example 3

Universality of citrusinine-II on mammals

According to NCBI cash-outThere is a reference genome, synthesizing TRPV3 receptor sequences of human, rat and mouse, and the receptor sequence information is shown in table 1. All the synthesized cdnas were cloned into the pEGFPN1 plasmid vector. Plasmids such as mTRPV3 were transiently overexpressed on the HEK293T cell line. All HEK293T cell lines were cultured in DMEM (Dulbecco's modified Eagle's medium) with the addition of 10% fetal bovine serum, 1% penicillin/streptomycin, 37 ℃, 5% CO₂And (5) incubation and culture. Whole cell patch clamp recordings indicate that citrusinine-II inhibits 2-APB activation of TRPV3 receptors from different species. The citrusinine-II can be used as an inhibitor of different species, and can be used as a potential TRPV3 channel disease drug, an antipruritic and an analgesic.

TABLE 1 TRPV3 receptor sequence information for human, rat and mouse

In FIG. 4 it is shown that citrusinine-II inhibits the 2-APB activation of TRPV3 channel of different species and presents a concentration dependent relationship.

Example 4

Antipruritic activity of citrusinine-II

Citrustinine-II for assisting mice in relieving chronic pruritus

AEW skin itch model: three days prior to the experiment, the hair of the right posterior neck of the C57BL/6 mouse was shaved using an electric shaver. The cotton balls soaked in the acetone-ethyl ether (1:1) mixed solution are applied to the back neck of the right side of the mouse for 15s, the cotton balls soaked in the double distilled water are immediately applied to the same position for 30s, the cotton balls are respectively treated once in the morning and at the evening every day for 5 days continuously, the double distilled water is used for smearing the back neck of the right side of the mouse in a control group, and the treatment time and frequency are the same as those of an experimental group. Mice were placed in clear observation cages on day 6 and randomly divided into five groups. 1 hour before the experiment, 50. mu.L DMSO (0.05%) was injected subcutaneously into the shaved part of the mice in the water-treated group, 50. mu.L DMSO (0.05%), 50. mu.L lcitrosine-II (5. mu.M), 50. mu.L citrisine-II (10. mu.M) were injected subcutaneously into the shaved part of the mice in the AEW-treated group, respectively, the number of itching times of the mice in 1 hour was observed and counted, and the total moving distance of the mice in one hour was recorded by an infrared camera. The front or rear paw of the mouse leaves the ground to grab the right back neck part treated with the drug until the paw is returned to the ground, and one scratching behavior is calculated.

The AEW model group mice showed marked dry, flaky and severe itching behavior in the skin (a-B in fig. 5). HE staining of the treated part and skin pathological observation show that the skin of mice in AEW type group has skin pathological symptoms of hyperkeratosis, epidermal hyperplasia and obvious increase of inflammation, and the skin tissue of mice in water treatment group has no obvious change (C-D in figure 5)

The number of pruritus times in 1h of mice in the AEW model group is 63.6(n is 5) times which is obviously higher than that in the water treatment group which is 5.0 +/-0.6 (n is 5). The number of mice with scratchback behavior after 5 and 10 μ M citrusinine-II treatment in the AEW model group was significantly reduced to 42.0(n 5) and 17.6(n 5) times, and the citrusinine-II compound could dose-dependently reduce spontaneous scratchback behavior in mice. The results of the above animal experiments show that citrusinine-II reduces chronic pruritus caused by skin dryness in mice by directly inhibiting TRPV3 channel (fig. 6).

Citrustinine-II assists mice in relieving acute pruritus

Acute itch model induced by histamine (VEH, a in fig. 5): three days prior to the experiment, the hair of the right posterior neck of the C57BL/6 mouse was shaved using an electric shaver. 1 hour before the experiment, the mice were randomly divided into six groups, and the control group mice were injected with 50. mu.L DMSO (0.05%) subcutaneously at the shaving sites, and the experimental group mice were injected with 50. mu.L of citrusinine-II (5. mu.M), 50. mu.L of citrusinine-II (10. mu.M) subcutaneously at the shaving sites, respectively, and then the control group was injected with physiological saline and 50. mu.L of histamine (100. mu.M) at the same sites, respectively, and the experimental groups were injected with 50. mu.L of histamine (100. mu.M). And observing and counting the itching times of the mouse within 30min, and scratching the right-side back neck part of the mouse by taking the front paw or the back paw of the mouse away from the ground until the paw is put back to the ground for once scratching behavior.

The number of spontaneous ticklers increased to 64.8(n 6) within 30 minutes after intradermal injection of histamine, whereas the blank group was 13.7(n 6). In contrast, various concentrations of citrusinine-II inhibited histamine-induced pruritic behavior dose-dependently. The number of scratching behaviors was reduced to 31.0 (n-6) and 12.0 (n-6) in the 5 μ M and 10 μ M citrusinine-II treated groups of mice. The above experimental results further demonstrate that citrusinine-II reduces histamine-induced acute pruritic behavior in mice by inhibiting TRPV3 channel (fig. 6).

Example 4

Analgesic Activity of citrusinine-II

citrusinine-II-assisted mice relieve formalin-induced pain: before modeling, the mice were injected with drugs such as morphine, citrusinine-II (5. mu. mol/kg, 10. mu. mol/kg, 50. mu. mol/kg) and physiological saline, and 30 minutes later, 20. mu.l of 0.92% (v/v) formalin solution was injected into the right rear sole of the mice. The licking behavior of the mice within 30min was observed and counted.

Plantar injection of formalin can result in the development of a phase 2 pain response in mice, with phase I pain (0-5min), which is referred to as neuropathic pain, and phase II pain (15-30min), which is referred to as chronic pain. The phase I pain licking time of the saline control group mice was 76.8s, and the licking behavior time of the mice in the treated groups, 5 μmol/kg, 10 μmol/kg citrusinine-II and 50 μmol/kg, was reduced by 4.43%, 12.76% and 51.82%, respectively (fig. 7, a, n-5). Furthermore, in stage II pain, the paw licking behavior time was reduced by 4.24%, 22.90% and 58.34% in 5, 10, and 50 μmol/kg citrusinine-II and 50 μmol/kg treated mice, respectively (fig. 7, a, n is 5).

citrusinine-II help mice to relieve acetic acid-induced pain: before molding, the mice were given intraperitoneal injections of drugs such as morphine, citrusinine-II (5. mu. mol/kg, 10. mu. mol/kg, 50. mu. mol/kg) and physiological saline, and 30 minutes later, the mice were intraperitoneally injected with 200. mu.l of 0.8% (v/v) acetic acid solution. And observing and counting the writhing frequency of the mice within 30 min.

Intraperitoneal injection of 5, 10 and 50 μmol/kg citrusinine-II reduced writhing pain behavior in 22.62, 42.86 and 45.24% of mice, respectively (B, n is 5 in fig. 7).

Before modeling, respectively injecting medicaments such as morphine, citrusinine-II (5 mu mol/kg, 10 mu mol/kg and 50 mu mol/kg) and normal saline into the abdominal cavity of the mouse, after 30 minutes, detecting the hot pain foot lifting reaction time of the mouse by using an intelligent hot plate instrument, and simultaneously detecting the hot pain tail-flick reaction threshold of the mouse by using an infrared illumination pain detector.

In a hot plate experiment, the time of the hot pain foot lifting reaction of mice in a normal saline control group is 5s, and the intraperitoneal injection of 5 mu mol/kg, 10 mu mol/kg of citrusinine-II and 50 mu mol/kg can increase the time of the hot pain foot lifting reaction of the mice from 5s to 7.8, 10.8 and 13.8s (C, n is 5 in figure 7) respectively, and can increase the time of the hot stimulation induction tail flick reaction from 3.8s to 5.2, 9.2 and 14s (D, n is 5 in figure 7) respectively.

The mouse-related behavioral experiments show that the animal can be helped to relieve pruritus and pain by smearing or injecting the animal with the citrusinine-II with physiological concentration. The resistance of the species such as mice to pruritus and pain is enhanced by the citrusinine-II, and the citrusinine-II can be used as a resource to be developed into a potential antipruritic, analgesic or skin care product which is applied to human and other animals to relieve pruritus and pain. Secondly, the citrusinine-II is targeted on a receptor TRPV3, and can be applied to the research of related ion channel diseases and used as a potential therapeutic drug.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Kunming animal research institute of Chinese academy of sciences

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