Amphiphotic agent for regulating and controlling animal chemical perception receptor and application thereof in green prevention and treatment

Abstract

The invention provides a repellent for regulating and controlling animal chemical perception receptors and application of the repellent in green prevention and control, and belongs to the technical field of agriculture and biological medicine. The invention provides an application of sodium houttuyfonate in preparation of an animal repellent. An electrophysiology experiment shows that SH can activate TRPA1 receptors of various species including nematodes and herbivores and initiate behavior response of animals to external stimulation. A behavioral experiment shows that the SH treatment can induce agricultural pests and herbivores to generate avoidance behaviors, reduce the harm of the agricultural pests and the herbivores to crops, enable wild mice to initiate an aversion reaction, show a remarkable avoidance effect on nematodes, generate an avoidance effect on herbivores such as cattle and sheep, and the like. In conclusion, the SH initiates the avoidance reaction of animals by activating the TRPA1 ion channel of the spine and invertebrate, can be developed into a potential insect repellant or biopesticide as a resource, and is applied to the field of agricultural pest control.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of agriculture and biomedicine, and particularly relates to a repellent for regulating animal chemosensory receptors and application thereof in green control. BACKGROUND

[0002] Sodium houttuyfonate (SH) is a compound extracted from the plant Houttuynia cordata or chemically synthesized, which has a wide range of biological activities. Existing studies have shown that SH has certain antibacterial and anti-inflammatory effects, but its function in regulating animal chemosensory behavior, especially in affecting the repellent reactions of various species through ion channel mechanisms, has not been systematically reported. SUMMARY

[0003] In view of the defects in the prior art, the present application aims to provide the application of sodium houttuyfonate in the preparation of an animal repellent. The sodium houttuyfonate targets the TRPA1 ion channel and then regulates various animal behavioral reactions, so that the animals produce a repellent reaction.

[0004] The purpose of the present application is achieved by the following technical solutions: The present application provides the application of sodium houttuyfonate in the preparation of an animal repellent; the animals include vertebrates and / or invertebrates.

[0005] Preferably, the invertebrates include nematodes; the vertebrates include any one or two or more of mice, cows and sheep.

[0006] Preferably, the sodium houttuyfonate makes the animals produce a repellent effect by activating the TRPA1 ion channel.

[0007] The present application provides a repellent for regulating animal chemosensory receptors, and the effective component includes sodium houttuyfonate.

[0008] The present application provides a biological pesticide, and the effective component includes sodium houttuyfonate.

[0009] The present application provides a TRPA1 ion channel activator, and the effective component includes sodium houttuyfonate.

[0010] The present application provides the application of the repellent, the biological pesticide or the TRPA1 ion channel activator in the repellent agricultural pests and / or in making herbivorous animals produce a repellent behavior.

[0011] The present application provides the application of sodium houttuyfonate in the preparation of a medicine for treating TRPA1 related ion channel diseases; the medicine includes an antipruritic; the TRPA1 related ion channel diseases include diseases of the sensory nervous system related to abnormal channel functions.

[0012] The application provides application of a hTRPA1 point mutation in preparation of a treatment for diseases caused by activation of a TRPA1 related ion channel; the hTRPA1 point mutation comprises a 621th amino acid mutation and / or a 641th amino acid mutation of a hTRPA1 amino acid sequence.

[0013] The application provides a method for repelling agricultural pests and / or making herbivorous animals produce repelling behavior, comprising: applying sodium houttuyniae extract on an area to be protected and / or crops to be protected.

[0014] The application has the following beneficial effects: The application provides application of sodium houttuyniae extract in preparation of an animal repellent. The application provides a compound for affecting chemical perception of vertebrates and invertebrates, i.e. sodium houttuyniae extract (SH), which can significantly activate TRPA1 ligand-gated ion channels of different species. Electrophysiological experiments show that SH acts on TRPA1 receptors of multiple species including nematodes and herbivorous animals. After TRPA1, a key ion channel in the animal chemical perception system, is activated, the animal will trigger a behavioral response to external stimuli. Through behavioral experiments, it is found that SH treatment can significantly induce agricultural pests and herbivorous animals to produce repelling behavior and reduce their harm to crops. Sodium houttuyniae extract, a derivative of the main stimulant compound houttuyniae extract in wild houttuyniae, triggers an aversive response in wild-type mice and activates a subset of trigeminal ganglion neurons sensitive to allyl isothiocyanate (AITC). Through electrophysiological experiments or animal experiments such as gene knockout, it is found that TRPA1 in the nasal cavity mediates the aversive response caused by stimulants from houttuyniae. In addition, SH significantly activates the TRPA1 channel of nematodes and exhibits a significant repelling effect on agricultural pests. It also has a repelling effect on herbivorous animals such as cattle and sheep. In summary, SH triggers the repelling response of vertebrates and invertebrates by activating the TRPA1 ion channel, and can be developed as a potential insect repellent or biological pesticide in the field of agricultural pest control. At the same time, the specific activation of SH on the TRPA1 channel makes it can be used as a research tool for related ion channel pathogenesis research and has the potential to be developed as a therapeutic drug. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 It is a result graph of the activation of SH on TRPA1; Figure 2 It is a result graph of the activation of 500 μM SH on TRPA1; Figure 3 It is a result graph of the targeting effect of SH on other related receptors; Figure 4Figure 1. Single-channel patch-clamp recording results of different compounds on the membrane of HEK293T cells transfected with TRPA1. Figure 5 The TRPA1 current map is obtained in whole-cell recording mode, excited by 500 μM SH, 5 mM dithiothreitol and 10 μM A96. Figure 6 This is a structural model diagram of TRPA1; Figure 7 Here is a magnified view of the TRPA1 structural model and the SH binding site. Figure 8 A plot showing the normalized ratio of 500 μM SH-induced current to 4-A-induced current in HEK293 cells expressing wild-type hTRPA1 and mutant hTRPA1. Figure 9 This is a dose-response curve of wild-type and mutant TRPA1 channels to SH. Figure 10 The figure shows the effect of SH on the activation of TRPA1 in nematodes and the effect on the repulsion of nematodes. Figure 11 Figure 1 shows the results of single-cell calcium imaging experiments on trigeminal ganglion cells from wild-type mice and TRPA1 knockout mice using SH. Figure 12 The diagram shows the avoidance effect of SH on wild-type mice and TRPA1 knockout mice; Figure 13 The graph shows the effect of SH on TRPA1 activation in cattle and sheep. Figure 14 The graph shows the effect of SH on the avoidance of cattle and sheep. Detailed Implementation

[0016] This invention provides the application of sodium houttuynin in the preparation of animal repellents; the animals include vertebrates and / or invertebrates. In this invention, SH targets the TRPA1 ion channel and significantly activates TRPA1 receptors in various vertebrates and invertebrates, thereby inducing repellent behavior in animals in response to chemical stimuli. In this invention, SH can be obtained through synthesis or extraction from Houttuynia cordata. Electrophysiological and behavioral experiments have verified that SH can activate TRPA1 channels in nematodes, mice, agricultural pests, and herbivores such as cattle and sheep, and significantly induce their repellent responses. Therefore, the compound can be used to regulate animal behavior and can be developed into related products in the agricultural or pharmaceutical fields.

[0017] As an optional embodiment of the present invention, the animals include vertebrates and / or invertebrates; the invertebrates include nematodes; the vertebrates include any one or more of mice, cattle, and sheep.

[0018] As an optional embodiment of the present invention, the sodium houttuynia cordata induces a repulsive response in animals by activating the TRPA1 ion channel. In this invention, the sodium houttuynia cordata specifically activates the TRPA1 receptor, a type of non-selective cation channel widely expressed in sensory neurons and involved in chemosensory and nociceptive responses. This invention demonstrates the activation effect of SH on TRPA1 channels in nematodes, agricultural pests, and herbivores through electrophysiological experiments; and proves through behavioral experiments that SH treatment significantly reduces the damage to crops caused by herbivores and pests, and induces significant repulsive behavior. The necessity of TRPA1 in the SH-induced behavioral response is further verified using a gene knockout animal model.

[0019] This invention provides a repellent that regulates animal chemosensory receptors, the active ingredient of which includes sodium houttuynin. As an optional embodiment of this invention, the repellent further includes excipients. This invention does not specifically limit the dosage form of the repellent; conventional dosage forms in the art can be used, such as sprayable formulations, slow-release granules, or coatings. Preferably, this invention applies the repellent to agricultural crops, stored grains, or specific habitats by spraying, dispersing, or coating to control pests and animals harmful to crops.

[0020] This invention provides a biological pesticide, characterized in that the active ingredient includes sodium houttuynin.

[0021] This invention provides a TRPA1 ion channel activator, the active ingredient of which includes sodium houttuynia cordata. In cell lines, overexpression of the mouse and nematode TRPA1 receptors was performed. Whole-cell patch-clamp techniques were used to record that SH could activate TRPA1 currents in a concentration-dependent manner, and that this activation could be inhibited by TRPA1 antagonists. Molecular docking and point mutation experiments were used to preliminarily explore the potential binding region between SH and the TRPA1 channel protein, providing a structural basis for subsequent mechanism studies and drug design.

[0022] This invention provides the application of the repellent agent, the biopesticide, or the TRPA1 ion channel activator described in the above-mentioned technical solutions in repelling agricultural pests and / or inducing repellent behavior in herbivores. As an optional embodiment of this invention, the agricultural pests include nematodes and / or mice; the herbivores include cattle and / or sheep.

[0023] This invention provides the use of sodium houttuynia cordata in the preparation of medicaments for treating TRPA1-related ion channel disorders. As an optional embodiment of this invention, the medicament includes an antipruritic agent; the TRPA1-related ion channel disorders include sensory nervous system disorders related to channel dysfunction; the sensory nervous system disorders related to channel dysfunction include any two or more of pain, chronic cough, asthma, atopic dermatitis, and chronic pruritus. This invention does not specifically limit the dosage form of the medicament, which may include injections, patches, or inhalers, etc.

[0024] This invention provides the application of hTRPA1 point mutations in the preparation of treatments for diseases caused by activation of TRPA1-related ion channels; the hTRPA1 point mutations include amino acid mutations at positions 621 and / or 641 of the hTRPA1 amino acid sequence. As an optional embodiment of this invention, the amino acid mutation at position 621 includes a mutation from cysteine ​​to alanine at position 621 of hTRPA1. As an optional embodiment of this invention, the amino acid mutation at position 641 includes a mutation from cysteine ​​to alanine at position 641 of hTRPA1. As an optional embodiment of this invention, diseases caused by activation of TRPA1-related ion channels include chronic pain and / or asthma.

[0025] This invention provides a method for repelling agricultural pests and / or inducing repulsive behavior in herbivores, comprising: applying sodium houttuynin to the area to be protected and / or the crop to be protected. This invention does not specifically limit the method of applying the sodium houttuynin; any conventional application method in the art may be used.

[0026] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0027] All HEK293T cell lines were cultured in DMEM (Dulbecco's modified Eagle's medium) with 10% fetal bovine serum and 1% penicillin / streptomycin, at 37°C and 5% CO2.

[0028] In the following technical solutions, the TRPA1 expression sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd.; the mouse TRPA1 gene number (GeneID is 277328); and the nematode TRPA1 gene number (GeneID is 178118).

[0029] Example 1: Electrophysiological experiment on the targeting of SH and TRPA1 and other pruritus-related receptors 1. TRPA1 gene transfection into HEK293T cells, as follows: Preparation of TRPA1-containing plasmid: The empty pcDNA3.1 plasmid and the template containing the target cDNA (hTRPA1 gene) were double-digested using restriction endonucleases XhoI and HindIII to generate compatible sticky ends. After the digestion reaction, the target DNA fragment was separated by agarose gel electrophoresis, and the large vector fragment and the cDNA insert (TRPA1 gene sequence) were purified using a gel extraction kit. Subsequently, the purified vector and insert fragment were directionally ligated at a 3:1 molar ratio using T4 DNA ligase, and the reaction system was incubated overnight at 16°C. The ligation product was transformed into competent DH5α Escherichia coli using the heat shock method. The transformed bacterial culture was plated on LB agar plates containing ampicillin (100 µg / mL) for antibiotic selection. After single colonies were picked and amplified by shaking, plasmid DNA was extracted using a plasmid miniprep kit. Finally, the successfully constructed recombinant plasmids were validated by restriction endonuclease digestion mapping and DNA sequencing to ensure that the cDNA sequence was correct and the reading frame was accurate. All validated recombinant plasmids were prepared in large quantities using an endotoxin-free plasmid bulk extraction kit for subsequent cell transfection experiments.

[0030] Cell transfection was performed using the Lipofectamine 3000 Transfection Kit (Invitrogen). TRPA1 was transfected with HEK293T cells. The specific steps were as follows: HEK293T cells in T25 culture flasks were observed 24 hours before transfection to confirm that the cell confluence was 80%~90%.

[0031] When the cell density in the T25 culture flask reaches 80%–90%, a single-cell suspension is prepared by digestion (trypsin). The cells in the culture flask are then transferred to 3.5 cm dishes and incubated in a cell culture incubator at 37°C and 5% CO2 for 6 hours. When the cells in the dishes adhere to the dish and the density reaches 80%, transfection begins.

[0032] Take two 1.5 mL EP tubes, A and B. Add 200 µL of Opti-MEM serum-depleted medium and 6 µL of Lipofectamine 3000 to tube A, mix well, and let stand at room temperature for 3 min. Add 200 µL of Opti-MEM serum-depleted medium, 6 µL of P3000, 5 µg of plasmid DNA (TRPA1 plasmid DNA, recombinant plasmid), and 0.5 µg of EGFP plasmid to tube B, mix well, and let stand at room temperature for 3 min.

[0033] Transfer the mixture in tube B into tube A, mix thoroughly, and let stand at room temperature for 15 minutes.

[0034] Before transfection, aspirate the old culture medium from the 3.5 cm dish and rinse the cells. Slowly add the mixed solution from tube A to the dish, gently shake the dish to distribute the complex evenly, and incubate at 37°C in a 5% CO2 incubator for 24 h (without changing the culture medium).

[0035] Remove the transfected cells from the small dish and passage them into a 3.5 cm culture dish containing a 10 mm cell spread. Incubate the dish for 2 hours and, once the cells have adhered, begin the single-cell patch-clamp experiment.

[0036] 2. Patch-clamp Experiment: Cells with relatively smooth membranes and homogeneous cytoplasm were selected under an inverted microscope and patch-clamped experiments were performed at room temperature (20–25°C). WPI 0.86 mm thin-walled borosilicate glass capillaries were used as the glass electrode material. The glass electrodes were drawn in five steps using a drawing apparatus (P-97, Shutter). After heat polishing, the electrode tip diameter was 1.5–3.0 μm. Intracellular fluid was then injected into the glass electrode after drawing. The initial resistance of the glass electrode was 1.5–2.5 MΩ.

[0037] After a high-impedance kingieumic (GΩ) seal is established between the electrode and the cell membrane, the fast capacitance of the electrode is compensated. Then, a short but strong negative voltage is applied to rapidly break the cell membrane clamped in the electrode, followed by compensation of the slow capacitance of the cell. After establishing whole-cell recording mode, the cell is clamped at 0 mV. After the cell has stabilized for 10 minutes, current recording begins using an appropriate pulse voltage (80 / -80 mV). Drugs are perfused using a Biolab RS200, with a switching speed of 50 ms between drugs. The series resistance (Rs) remains constant within the range of 5–8 MΩ throughout the experiment, and the system series resistance compensation is generally between 30–60%. Experimental data are analyzed using Patch Master software, and further analysis is performed using Igor software. All results are expressed as average ± standard error (SEM), where n represents the number of data points in the experiment.

[0038] Cells are slowly lifted using a motorized micromanipulation system until they are flush with the lower edge of the drug delivery port on the rapid drug delivery switching system (RSC-200). The drug delivery switching system is directly connected to the amplifier, and all experimental data are recorded via the amplifier in the PatchMaster computer software. Cells are stimulated and drug-treated using PatchMaster and the switching drug delivery system, and changes in channel current are recorded to detect the activity of different drugs on the channels. To ensure the accuracy of the experimental data, stable sealing and series resistances must be maintained throughout the recording process.

[0039] TRPA1 channel internal and external solutions: 130 mM NaCl, 3 mM HEPES, 0.2 mM EDTA, adjusted to pH 7.2 with NaOH. Drugs used in the experiment must be dissolved and prepared using the above external electrode solutions.

[0040] Baseline currents of cells in extracellular fluid were recorded without drug stimulation. Gradient concentration SH stimulation: 100, 300, 350, 400, 450, and 500 µM SH solution were sequentially perfused onto cells using a gravity perfusion system. After each drug addition, the current was allowed to stabilize, and the TRPA1 current induced by different concentrations of SH was recorded.

[0041] A96 inhibition assay: After the current induced by 500 µM SH stabilized, 10 µM A967079 was perfused into the cells, and the inhibitory effect of the antagonist on the TRPA1 current was recorded.

[0042] The effect of SH on TRPA1 activation is as follows: Figure 1 As shown. Figure 1 In the diagram, A represents the current trajectory of the TRPA1 channel, and B represents the dose-effect curve of the TRPA1 channel.

[0043] Furthermore, in whole-cell recording mode, the basal current of cells in the extracellular fluid was first recorded to ensure current stability. 4-A stimulation: 1 mM 4-A solution was applied to the extracellular fluid to stimulate cells, and the 4-A-induced current was recorded. SH stimulation: After 4-A stimulation, 500 μM SH solution was applied to the extracellular fluid, and the regulatory effect of SH on TRPA1 channel current was recorded. A96 stimulation: Finally, 10 μM A96 solution was applied to verify whether the current was mediated by the TRPA1 channel. The detection results are as follows: Figure 2 As shown.

[0044] To confirm the direct activation effect of SH on TRPA1, whole-cell patch-clamp experiments were performed on HEK 293T cells expressing TRPA1. The results are as follows: Figures 1-2 As shown, perfusion of 500 µM SH induced a current greater than that of 4-aminodiphenylamine (4-A), a reversible TRPA1 agonist, which could be blocked by 10 µM A-967079 (A-96), a specific TRPA1 inhibitor. Furthermore, SH activated the TRPA1 current in a dose-dependent manner, with an EC50 value of 343.10 ± 12.54 μM (n = 6). These results suggest that TRPA1 is an endogenous target of SH.

[0045] Example 2: Targeting effects of SH with other related receptors Cell source: The experimental cells were HEK293T cells transfected with the target channel genes TRPV1 (NM_080706), TRPV2 (NM_016113), TRPV3 (NM_145068), TRPV4 (XM_011248235), TRPM8 (NM_001397617), GlyR α1 (NM_001290821), ASIC2a (NM_001094), and OTOP1 (NM_177998). The transfection method was the same as in Example 1. The cells were used for experiments 24-48 hours after transfection.

[0046] The patch-clamp experiment method is the same as in Example 1, as detailed below: Electrophysiological experiments were performed at room temperature (approximately 22°C) using a HEKA EPC10 amplifier and Patch Master software. The resistance of the glass microelectrodes perfused with the internal fluid was recorded as 3–5 MΩ. The bath and internal fluid composition (mM) was: NaCl 130, HEPES 3, EDTA 0.2 (pH 7.4). In whole-cell mode, the membrane potential was clamped at 0 mV, and current was induced alternately with +80 mV, 250 ms steps followed by −80 mV, 250 ms steps, with a 1 s interval. In inside-out and outside-out modes, the membrane potential was also clamped at 0 mV, and current was induced with ±80 mV, 500 ms steps. The solution was rapidly switched using a gravity perfusion system RSC-200 (Bio-Logic, France). The TRPV1, TRPV2, TRPV3, TRPV4, and TRPM8 channels used the same voltage protocol as TRPA1. The bath solution for the GlyRα1 channel contains (mM): NaCl 140, KCl 5, MgCl2 1, CaCl2 2, HEPES 10, glucose 10 (adjusted to pH 7.4 with NaOH); the inner solution contains (mM): CsCl 130, NaCl 5, CaCl2 0.5, MgCl2 1, EGTA 5, HEPES 30 (adjusted to pH 7.2 with CsOH). The bath solution for the ASIC2a channel contains (mM): NaCl 150, KCl 5, MgCl2 1, CaCl2 2, glucose 10, HEPES 10 (pH 7.4); the inner solution contains (mM): KCl 120, NaCl 30, MgCl2 1, CaCl2 0.5, EGTA 5, Mg... 2+ -ATP 2, HEPES 10 (pH 7.25). The bath and internal fluid of the OTOP1 channel are (mM): NMDG-Cl 160, CaCl2 2, HEPES 10 (pH 7.4).

[0047] Results of the targeting effects of SH with other related receptors, such asFigure 3 As shown.

[0048] Depend on Figure 3 Therefore, SH is a selective agonist of the TRPA1 channel.

[0049] Example 31. The source of the TRPA1 gene and the method of transfecting HEK293T cells are the same as in Example 1.

[0050] 2. The patch-clamp detection method is the same as in Example 1, except that after sealing, the electrode is quickly lifted, allowing a small piece of cell membrane to form an inside-out pattern at the electrode tip, with the intracellular side of the membrane exposed in the bath solution. The patch-clamp amplifier voltage is clamped at +80 mV, and the channel basal current is recorded. 500 µM SH is applied to the bath, and the channel current response is recorded, observing current changes. After the current stabilizes, a bath solution containing 1 mM AITC is applied, and the channel current response is recorded. Finally, 10 µM A96 is applied to the bath, and data is recorded multiple times for each cell, for a total of n=12 cells. The results are as follows: Figure 4 As shown in 'a'.

[0051] 3. The patch-clamp detection method is the same as in Example 1, except that after sealing and rupturing the membrane under negative pressure, the electrode is slowly lifted to detach the cell membrane from the cell. At this time, the ruptured cell membrane will spontaneously fuse to form an outer-to-outer closed patch (similar to a "small vesicle" structure). The voltage of the patch-clamp amplifier is clamped at +80 mV, and the baseline current of the channel is recorded. 500 µM SH, 1 mM AITC, and 10 µM A96 are sequentially applied to the bath, and the channel current response is recorded. The changes in channel current after each reagent application are observed and recorded. Data for a total of n=11 cells are recorded. The results are as follows: Figure 4 As shown in b in the figure.

[0052] Depend on Figure 4 As shown in a and b, single-channel patch-clamp recordings performed on the membranes of TRPA1-transfected HEK293T cells revealed that in both outside-out mode (n = 11) and inside-out mode (n = 12), bath application of SH (500 µM), AITC (1 mM), and A96 (10 µM, A967079) evoked channel responses. These results indicate that SH exerts its effect by penetrating the cell membrane and reaching the intracellular region of the TRPA1 channel.

[0053] 4. Observation revealed that the SH-induced TRPA1 current amplitude could not be eluted after perfusion, indicating an irreversible activation mechanism for the TRPA1 channel. Many harmful compounds activate TRPA1 channels through covalent modification of cysteine ​​residues. For example, cinnamaldehyde can bind to the cysteine ​​residue of the TRPA1 channel via Michael addition, methyl sulfonate ethylamine (MTSEA) can form a disulfide bond with the cysteine ​​residue of TRPA1, and the alkylating agent iodoacetamide can also bind to TRPA1 by modifying cysteine ​​residues. Given that the irreversible activation of TRPA1 by SH is similar to that of other electrophiles, we hypothesized that SH might activate TRPA1 through covalent binding to cysteine. We tested whether SH binds to TRPA1 through disulfide bond modification. Dithiothreitol (DTT), a cell-permeable reducing agent, can reverse the disulfide bond modification of cysteine ​​but cannot reverse the Michael addition or cysteine ​​conjugation reaction. Therefore, DTT (5 mM) was added after the application of SH.

[0054] The patch-clamp detection method is the same as in Example 1. Specifically, after forming the GΩ seal, the cell membrane is ruptured by applying a brief strong negative pressure or electroporation to form a whole-cell model. The voltage of the patch-clamp amplifier is clamped at an appropriate potential, and the basal current of the cell is recorded first. Then, 500 µM SH is applied to the bath, and the changes in channel current are recorded. After the current stabilizes, 5 mM DTT is added to the bath, and the changes in current are observed. Finally, 10 µM A96 is added. The results are as follows: Figure 5 As shown.

[0055] Depend on Figure 5 It was found that when DTT (5 mM) was applied to SH-induced whole-cell TRPA1 current, the current did not decrease significantly, indicating that SH covalently modified the cysteine ​​of TRPA1 in other ways.

[0056] Example 4: Point Mutation Experiment of TRPA1 I.hTRPA1 point mutation This study investigated the effect of hTRPA1 (Gene ID: 8989) on the target binding site of SH using point mutagenesis experiments. All mutant hTRPA1 channels used in this invention were constructed using homologous recombination, as detailed in the instruction manual (https: / / bio.vazyme.com / product / 79.html). Site-directed mutagenesis was performed using the Mut-Express-II Fast Mutagenesis Kit-V2 primer-mediated method, and all mutants were verified by DNA sequencing. The specific experimental procedures are as follows: 1. Experimental Materials Plasmid: The preparation method of the plasmid containing hTRPA1 is the same as in Example 1.

[0057] 2. Primer design Reverse complementary primer pairs were designed targeting key cysteine ​​residues in mTRPA1, namely C414, C421, C621, C641, C727, C773, C786, C834, and C856. Primer design principles: Introducing a single-base site-directed mutation into a plasmid only requires designing a pair of primers to perform reverse PCR amplification of the plasmid, mutating the cysteine ​​codon to the alanine codon.

[0058] Primer sequences: C414A-F: 5'-gaagacaacgatgggtgtactcctctacattatgcatgt-3', SEQ ID NO.1; C414A-R: 5'-ataatgtagaggagtacacccatcgttgtcttcatccat-3', SEQ ID NO.2; C421A-F: 5'-cctctacattatgcatgtagacaggggggccctggttct-3', SEQ ID NO.3; C421A-R: 5'-agggcccccctgtctacatgcataatgtagaggagtaca-3', SEQ ID NO.4; C621A-F: 5'-tctccaggcaataaatctccaattacagaaatgatagaa-3', SEQ ID NO.5; C621A-R: 5'-catttctgtaattggagaatttattgcctggagaattatg-3', SEQ ID NO.6; C641A-F: 5'-gtacttttagatttctctatgttgcattccacagaagac-3', SEQ ID NO.7; C641A-R: 5'-tgtggaatgcaacatagagaaatctaaaagtaccttcat-3', SEQ ID NO.8; C727A-F: 5'-aatttaggatcttactgtcttggtctcatacctatgacc-3', SEQ ID NO.9; C727A-R: 5'-aggtatgagaccaagacagtaagatcctaaattcatcat-3', SEQ ID NO.10; C773A-F: 5'-tatctaataaaaacttgtatgattttagtgtttttatca-3', SEQ ID NO. 11; C773A-R: 5'-aattcatatctaataaaaacttgtatgattttagtgttt-3', SEQ ID NO. 12; C786A-F: 5'-agtatatttgggtattgcaaagaagcggggcaaattttc-3', SEQ ID NO. 13; C786A-R: 5'-ttgccccgcttctttgcaatacccaaatatacttgataa-3', SEQ ID NO. 14; C843A-F: 5'-catctacagtggcaatgtggagcaattgctgtttacttc-3', SEQ ID NO. 15; C834A-R: 5'-aacagcaattgctccacattgccactgtagatgagctgg-3', SEQ ID NO. 16; C856A-F: 5'-caaagatttgaaaattgtggaatttttattgttatgttg-3', SEQ ID NO. 17; C856A-R: 5'-aacaataaaaattccacaattttcaaatctttgaagata-3', SEQ ID NO. 18.

[0059] 3. PCR amplification of mutant plasmids Reaction system: 1 μL template DNA sequence (mTRPA1 plasmid), 8.5 μL deionized water, 2 μL 2×Max buffer, 0.5 μL dNTP mixture, 1 μL 5' PCR primer, 1 μL 3' PCR primer and 0.5 μL DNA polymerase were centrifuged in a centrifuge tube for reaction.

[0060] The PCR instrument was used for amplification according to the following program: 95℃ pre-denaturation for 30s; 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 9min, 30 cycles; 72℃ extension for 5min.

[0061] 4. DpnⅠ digestion and recombination DpnI enzyme was added to the PCR product to remove methylated template plasmids. The mixture was incubated at 37°C for 1 h to obtain the DpnI digest.

[0062] The DpnI digestion product was added to recombinase for homologous recombination. The reaction system consisted of 2 μL DpnI digestion product, 2 μL C E II buffer, 1 μL Exnase II and 5 μL deionized water. The reaction was carried out at 37℃ for 30 min, followed by cooling in an ice-water bath for 5 min to obtain the recombinant product.

[0063] 5. Transformation and Sequencing Validation The recombinant product was transformed into 100 µL of Dh5α competent cells, plated and cultured overnight, and single colonies were picked, amplified by shaking culture, and sequenced to verify that only the target cysteine ​​residue was mutated to alanine, with no other random mutations.

[0064] II. HEK293 cell transfection and culture The method for transfecting HEK293T cells with wild-type hTRPA1 plasmid and mutant plasmid is the same as in Example 1.

[0065] III. Whole-cell patch-clamp detection of SH-induced current The detection method is the same as in Example 1, except that the drug stimulation and current recording are as follows: First, administer 1 mM 4-A (TRPA1 activator) stimulation and record the activation current (I0). 4-A After the current stabilizes, elute. Then administer 500 µM SH stimulation and record the SH-induced current (IL). SH ).

[0066] Calculate the standardized ratio: Ratio = |I 4−A ∣ / ∣I SH | is used to quantify the activation efficiency of SH.

[0067] IV. Results as follows Figures 6-8 As shown. Figure 6 The TRPA1 structural model; Figure 7 Here is a magnified view of the TRPA1 structural model and SH binding site. Figure 8 The ratio of the 500 µM SH-induced current to the 4-A-induced current was standardized in HEK293 cells expressing wild-type hTRPA1 or mutant hTRPA1. Statistical analysis was performed using one-way ANOVA (F = 6.889), followed by Sidak multiple comparison tests: wild-type vs. C621A mutant, t = 4.271, p = 0.0002; wild-type vs. C641A mutant, t = 4.250, p = 0.0003. p<0.05, p<0.01, p < 0.001. Data are expressed as mean ± standard error.

[0068] The activating activity of SH on all point-mutated hTRPA1 channels was examined, and the activation efficiency of SH on wild-type and each point-mutated channel was compared. The amino acid mutation at position C621 / C641 significantly reduced the activation efficiency of SH on hTRPA1 channels. Here, C621 represents a cysteine ​​residue at position 621.

[0069] Example 5, based on Example 4, investigated the effects of different concentrations of SH on wild-type (WT), C621A mutant hTRPA1 channels, and C641A mutant hTRPA1 channels. 1. HEK293 cell transfection and culture The method for transfecting HEK293T cells with wild-type hTRPA1 plasmid and mutant plasmid is the same as in Example 1.

[0070] 2. Whole-cell patch-clamp detection of SH-induced current The detection method is the same as in Example 1, except that the drug stimulation and current recording are as follows: For each cell, its basal current in the extracellular fluid was first recorded as the background current. A 1 mM 4-A solution was then applied to the extracellular fluid to stimulate the cells, and the 4-A-induced current (I4A) was recorded. 4−A After the current stabilized, the cells were flushed with extracellular fluid to restore the current to baseline levels. Following a predetermined concentration sequence, different concentrations of SH solution (100, 300, 350, 400, 450, and 500 µM) were sequentially added to the extracellular fluid. After each addition of a different concentration of SH solution, a waiting period was allowed for the current to stabilize, and then the SH-induced current (|IL) was recorded. SH |).

[0071] After recording the current under the action of one concentration of SH solution, the cells were thoroughly rinsed with extracellular fluid to remove residual SH. After the cells returned to their basal state, the next concentration of SH solution was applied for recording.

[0072] For cells expressing wild-type hTRPA1 (WT), C621A, and C641A mutant hTRPA1, the above steps were followed, and the current under different concentrations of SH solution was recorded.

[0073] 3. Results For each cell, calculate the current ratio I under different SH concentrations.SH / I 4−A This refers to the standardized ratio of SH-induced current to 4-A-induced current. For each genotype (WT, C621A, C641A), Ig was calculated at different SH concentrations. SH / I 4−A The mean and standard deviation. Plot Log[SH]M on the x-axis, I... SH / I 4−A Using the vertical axis as the ordinate, a curve was plotted to show the response of different genotypes of hTRPA1 at different SH concentrations, and statistical analysis was performed to compare the differences between wild-type and mutant types.

[0074] The dose-response curves of wild-type and mutant TRPA1 channels to SH are shown in the figure. Figure 9 As shown.

[0075] Depend on Figure 9 Therefore, for wild-type (WT): with increasing SH concentration, I... SH / I 4−A The concentration-effect relationship gradually increased, showing a clear concentration-dependent relationship, indicating that the activation of wild-type TRPA1 channels is sensitive to SH concentration, and SH can effectively activate wild-type TRPA1 channels. C621A mutant and C641A mutant: Compared with wild type, at the same SH concentration, I... SH / I 4-A The levels were significantly lower than those of the wild type. This indicates that mutations at the C621 and C641 sites greatly weakened the activation effect of SH on the TRPA1 channel, suggesting that C621 and C641 are key sites for SH to activate the TRPA1 channel, and that SH exerts its activation function on the TRPA1 channel by binding to these two sites.

[0076] Example 61. Effect of SH on TRPA1 activation in nematodes (1) Preparation of experimental materials: Nematode TRPA1 receptor sequence information: GeneID, 178118; Gene number, NP_001294083.1. The plasmid containing nematode TRPA1 was prepared using the same method as in Example 1. The nematode TRPA1 channel was transiently expressed in HEK293T cells via transfection, using the same method as in Example 1. Whole-cell electrophysiological methods were used to detect the activation activity of SH.

[0077] (2) Reagent preparation SH solutions: Prepare SH solutions of 100, 300, 350, 400, and 500 µM respectively.

[0078] 4-AP solution: Prepare a 100 μM solution as a positive control reagent.

[0079] A96 solution: Prepare a 10 μM working solution of A967079 (TRPA1 antagonist).

[0080] (3) Investigate the effect of SH on the activation of TRPA1 channel in nematodes using patch-clamp experiments. The detection was performed in whole-cell mode using a patch-clamp method, specifically as follows: Basic current recording: The voltage of the patch-clamp amplifier is clamped to an appropriate potential to record the basic current of nematode neurons.

[0081] Drug stimulation and recording: First, add 100 μM 4-AP and observe and record the changes in current.

[0082] Different concentrations of SH solution were added sequentially, namely 100, 300, 350, 400, and 500 μM. After each addition, the current was allowed to stabilize, and the current changes induced by different concentrations of SH solution were recorded.

[0083] After the current induced by 500 μM SH stabilized, 10 μM A96 was added, and the inhibitory effect of the antagonist on the current was recorded.

[0084] The results are as follows Figure 10 As shown in A in the diagram.

[0085] Depend on Figure 10 As shown in section A, firstly, 100 µM of 4-AP (4-aminopyridine, positive control) induced a current response, indicating that the TRPA1 channel can be activated. Subsequently, different concentrations of SH (100, 300, 350, 400, 500 µM) induced currents in a concentration-dependent manner, and the current amplitude increased with increasing SH concentration. Finally, the current was significantly inhibited upon the addition of 10 µM of A96. These results indicate that SH can activate the nematode TRPA1 channel in a concentration-dependent manner, and this activation can be blocked by the TRPA1 antagonist A96, demonstrating the specificity of SH in activating the TRPA1 channel.

[0086] 2. SH repulsion experiment on nematodes Two-sided preference assay for SH-induced repulsion in nematodes: A longitudinal midline was drawn in the center of a 9 cm plate. The experimental group (containing SH stimulant, 500 μM SH solution) and the control group (solvent) were symmetrically placed on both sides of the midline at a distance of 2 cm. Then, 50 synchronously metamorphosed adult N2 nematodes were released at the midline. After standing at 20°C in the dark for 1 h, the number of nematodes on both sides was counted. The repulsion index CI = (N control - N experiment) / N total × 100% was used to quantify the SH-induced repulsion effect.

[0087] The aversion index is as follows Figure 10 As shown in B in the diagram. Figure 10 "in "The differences are extremely significant."

[0088] Depend on Figure 10 As shown in B, the avoidance rate of nematodes to 500 μM SH solution increased significantly, indicating that 500 μM SH solution can significantly increase the avoidance behavior of nematodes.

[0089] Example 71. Effect of SH on TRPA1 activation in mice Single-cell calcium imaging experiments were performed on trigeminal ganglion cells from TRPA1-KO mice (TRPA1 gene knockout mice, prepared by knocking out CRISPR-Cas9 technology).

[0090] Newborn wild-type (WT) and TRPA1-KO mice were euthanized, and their trigeminal ganglion (TG) tissues were harvested and dissociated and washed with Hank's buffer (Gibco). The tissues were then minced into small pieces and placed in DMEM solution containing type I collagenase and trypsin (Sigma), and digested at 37°C for 30 minutes. A trypsin inhibitor (Sigma) was added, and the suspension was centrifuged at 3000 rpm for 10 minutes. The resulting precipitate was washed twice with pre-chilled phosphate-buffered saline (PBS) and once with 1 mL of cell culture medium. The cells were resuspended in Neurobasal A medium (Invitrogen) containing 2% B27 and seeded onto round coverslips in 35 mm culture dishes. The culture dishes were kept at 37°C for subsequent experiments.

[0091] Calcium imaging was used to detect the dynamic changes of intracellular calcium ions in trigeminal ganglion neurons. Trigeminal neurons from TRPA1-KO mice and wild-type mice were subjected to 2 mM Ca2+ imaging. 2+ After incubation with Fluo-4 AM dye prepared with Ringer's solution, Fluo-4 AM enters the cell and is hydrolyzed by intracellular esterases into Fluo-4, which has fluorescent activity, thereby enabling the detection of changes in intracellular calcium ion concentration.

[0092] After incubation, place the cells in a container containing 2 mM Ca 2+ In Ringer's bath solution. Ringer's solution formula: 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 10 mM glucose, 2 mM CaCl2 and 10 mM HEPES (pH 7.4).

[0093] Cells were observed under a microscope, and experiments were conducted on cells derived from wild-type (WT) mice and TRPA1 knockout (TRPA1-KO) mice. Neuronal cells were sequentially stimulied with 500 μM SH, 1 mM AITC, and 70 mM KCl, and fluorescence images of the cells under different stimuli were captured simultaneously. Changes in calcium fluorescence in trigeminal ganglion cells were acquired using a Hamamatsu R2 charge-coupled camera controlled by MetaFluor Software. An LED light source (X-Cite 120LED) filtered through a 500 / 20 nm excitation filter was used to excite Fluo-4 in trigeminal ganglion cells. The emission fluorescence of Fluo-4 was filtered through a 535 / 30 nm emission filter and then acquired and recorded by the camera. Fluo-4 fluorescence images were processed and analyzed using MetaMorph software.

[0094] Finally, fluorescence time-series curves were constructed using MetaMorph software, and quantitative analysis was performed using Igor Pro software to calculate the changes in fluorescence intensity under different stimuli and to compare between groups. SH was used as the agonist to be tested in trigeminal nerve cells; AITC was used as a TRPA1 channel agonist; and KCl was used as a positive control for calcium signaling.

[0095] Figure 11 In the diagram, A represents the fluorescence images of trigeminal nerve cells from TRPA1-KO mice under different stimuli; B represents the time-series curves showing the fluorescence intensity changes of cells from wild-type mice and TRPA1-KO mice over time; and C represents the statistical bar charts of fluorescence intensity of cells from wild-type mice and TRPA1-KO mice under different stimuli.

[0096] Depend on Figure 11 It was found that in wild-type (WT) mouse cells, the F / F0 value gradually increased after the addition of 500 μM SH, further increased after the addition of 1 mM AITC, and increased sharply after the addition of KCl. In contrast, in TRPA1-KO mouse cells, the increase in F / F0 value after the addition of 500 μM SH was smaller, the increase after the addition of 1 mM AITC was also not significant, and the increase in F / F0 value after the addition of KCl was similar to that in WT mice. This indicates that SH and AITC promote the increase of intracellular calcium ion concentration in wild-type mouse cells, and this effect is weakened in TRPA1-KO mouse cells, suggesting that SH induces the increase of intracellular calcium ion concentration through the TRPA1 channel.

[0097] 2. Free-feeding test in mice (1) Grouping of experimental animals Healthy wild-type (WT) mice and TRPA1 gene knockout (TRPA1-KO, prepared by gene knockout CRISPR-Cas9 technology) mice were selected to ensure that the mice were similar in age, sex, and weight, and that they had been adapted to the breeding environment for more than one week before the experiment.

[0098] Mice were housed individually in standard cages with a constant temperature (22-25°C) and humidity (50-60%) environment, a 12-hour light / 12-hour dark cycle, and free access to food, but water intake was restricted 12 hours before the experiment.

[0099] (2) Preparation of reagents and equipment Preparation of test solutions: Prepare 500 μM SH solution and 1000 μM SH solution with sterile distilled water respectively. Use sterile distilled water as a blank control group. All solutions should be prepared and used immediately to ensure accurate concentration.

[0100] Prepare a dual-bottle selection device: Place two identical water bottles symmetrically in each cage, with the same spout height, to avoid differences in drinking difficulty.

[0101] (3) Experimental operation procedure Two-bottle selection test The tests were conducted on WT mice and TRPA1-KO mice separately. Each mouse was assigned to one set of two-bottle combinations, and WT mice were assigned to three sets of two-bottle combinations (n=8 per set). Combination 1: Water (blank control) + 500 μM SH solution; Combination 2: Water (blank control) + 1000 μM SH solution; Combination 3: Water (blank control) + Water.

[0102] The TRPA1-KO mice were configured with the same three double-bottle combinations as the WT mice, with the same sample size in each group as the WT mice.

[0103] Two water bottles were placed in the mouse cage at the same time, and the initial liquid volume in the bottles was recorded (accurate to 0.1 mL). The mice were then allowed to drink freely in an undisturbed environment for 3 hours.

[0104] (4) Data recording and analysis After the experiment, the remaining liquid volume in each water bottle was recorded again, and the "consumption of target liquid (SH solution or water)" in each pair of bottles was calculated: Consumption = Initial volume - Remaining volume. Mouse preference percentage: [Test solution consumption / (Test solution consumption + Water consumption)] × 100%. The water preference percentages of TRPA1-KO mice and WT mice are shown below. Figure 12 As shown. Figure 12 In the table, A represents the percentage of wild-type mice that prefer drinking water; B represents the percentage of WT mice that prefer drinking water.

[0105] The results are as follows Figure 12 The results showed that 500 μM and 1000 μM SH produced a significant aversion effect in WT mice during the two-bottle selection test. The 500 μM SH and 1000 μM SH groups consumed significantly less water than the blank control group. The 1000 μM SH group also consumed significantly less water than the 500 μM SH group and the water group. Compared with WT mice, TRPA1-KO mice showed no significant preference among the water, 500 μM SH, and 1000 μM SH groups. These results indicate that SH selectively acts on the highly expressed TRPA1 channel in animal chemosensory neurons, thereby exerting an aversion effect.

[0106] Example 81. Effect of SH on the activation of bovine TRPA1 Bovine receptor sequence information: GeneID, 505317; Gene number, XP_015329969.1. The preparation method of the plasmid containing bovine TRPA1 was the same as in Example 1. The bovine TRPA1 channel was transiently expressed in HEK293T cells via transfection, as in Example 1. The activation activity of SH was detected using whole-cell electrophysiological methods. Results are as follows... Figure 13 As shown in A in the diagram.

[0107] 2. Effects of SH on the activation of TRPA1 in sheep Sheep receptor sequence information: GeneID, 102170065; Gene number, XP_017913911.1. The plasmid containing sheep TRPA1 was prepared using the same method as in Example 1. The sheep TRPA1 channel was transiently expressed in HEK293T cells via transfection, using the same method as in Example 1. Whole-cell electrophysiological methods were used to detect the activation activity of SH. Results are as follows... Figure 13 As shown in B in the diagram.

[0108] 3. The avoidance effect of SH on cattle and sheep Houttuynia cordata was evaluated through a free-feeding selection test. H. cordata The aroma of Thunb. leaves affects cattle ( Bos taurus L.) and goats ( Capra hircus The avoidance effect of L.

[0109] During the experiment, the experimental animals were allowed free contact with items including houttuynia cordata leaves and fresh corn in an undisturbed environment. Zea mays L.) leaves and sweet potato ( Ipomoea batatasA variety of plant materials were used, including (L.) Lam. leaves. To reduce initial alertness and potential bias, highly palatable corn and sweet potato leaves were introduced first in the experiment, and each test animal underwent two independent repeated trials. The repellency effect of houttuynia cordata odor was analyzed by quantifying its feeding selection behavior.

[0110] Plant materials: Houttuynia cordata leaves, sweet potato leaves and sweet potato leaves sprayed with SH, referred to as sweet potato leaves + SH (the concentration of SH solution is 1mM, and the relative amount of spraying is to cover one side of the leaf).

[0111] Cattle and sheep were divided into different groups, and each group underwent multiple plant presentation experiments.

[0112] For each animal, three plant materials were presented sequentially: houttuynia cordata leaves, sweet potato leaves, and sweet potato leaves + SH. The animal's "accepted" and "refused" responses to each plant material were recorded. "Accepted" was defined as the animal actively consuming the plant material, while "refused" was defined as the animal not consuming the plant material or exhibiting avoidance behavior.

[0113] Repeat the experiment to obtain a sufficient sample size and improve the reliability of the test results.

[0114] The number of times each plant material was "accepted" and "rejected" in the cattle and sheep trials was counted, and the proportion of each plant material presented was calculated.

[0115] The results are as follows Figure 14 As shown.

[0116] Depend on Figure 14 It was found that both sheep and cattle showed a high acceptance of sweet potato leaves, with almost no rejection; however, they exhibited a higher rejection rate towards houttuynia cordata leaves. When SH was sprayed onto the sweet potato leaves, the rejection rate in both sheep and cattle increased significantly, while the acceptance rate decreased sharply, and the rejection rate became close to that of houttuynia cordata leaves. The results indicate that SH has a repulsive effect on both cattle and sheep, significantly reducing their acceptance of sweet potato leaves, which they normally prefer, and causing them to exhibit a rejection behavior similar to that towards the naturally repellent plant houttuynia cordata when SH was added to sweet potato leaves.

[0117] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of sodium houttuyniae in the preparation of an animal repellent; the animal includes any one or more of a vertebrate and / or an invertebrate.

2. Use according to claim 1, characterized in that, The invertebrate includes a nematode; the vertebrate includes any one or more of a mouse, a cow and a sheep.

3. Use according to claim 1, characterized in that, The sodium houttuyniae causes the animal to have a repellent effect by activating a TRPA1 ion channel.

4. A repellent for modulating a chemosensory receptor of an animal, characterized in that, The active ingredient includes sodium houttuyniae.

5. A biopesticide, characterized by, The active ingredient includes sodium houttuyniae.

6. A TRPA1 ion channel activator, characterized by, The active ingredient includes sodium houttuyniae.

7. Use of the repellent of claim 4, the biological pesticide of claim 5 or the TRPA1 ion channel activator of claim 6 in repelling an agricultural pest and / or causing a herbivore to have a repellent behavior.

8. Use of sodium houttuyniae in the preparation of a medicament for treating a TRPA1 -related ion channel disease; the medicament includes an antipruritic; the TRPA1 -related ion channel disease includes a sensory nervous system disease associated with abnormal channel function.

9. Use of a hTRPA1 point mutation in the preparation of a medicament for treating a disease caused by activating a TRPA1 -related ion channel; the hTRPA1 point mutation includes a mutation of an amino acid at position 621 and / or a mutation of an amino acid at position 641 of a hTRPA1 amino acid sequence.

10. A method of repelling agricultural pests and / or inducing a repellent behavior in herbivores, characterized in that, including: applying sodium houttuyniae on an area to be protected and / or a crop to be protected.

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