CN114903995B

CN114903995B - Application of TRPA1 ion channel as drug target in amide herbicide poisoning

Info

Publication number: CN114903995B
Application number: CN202210625128.1A
Authority: CN
Inventors: 王永安; 骆媛; 王小宁; 隋昕; 杨军; 杨扬; 高翔; 肖振宇; 刘凤英; 姚舜
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2022-06-02
Filing date: 2022-06-02
Publication date: 2023-07-28
Anticipated expiration: 2042-06-02
Also published as: CN117180431A; CN114903995A

Abstract

The invention belongs to the field of biological medicine, and relates to application of a TRPA1 ion channel as a drug target in amide herbicide poisoning. The research results of the invention show that TRPA1 and amide herbicide poisoning are closely related, and the inhibition or knocking-out of TRPA1 targets can reduce the increase of TRPA1 expression and calcium ion inflow caused by the amide herbicide, and finally reduce the alveolar epithelial cytotoxicity caused by the amide herbicide. Therefore, the invention provides a new thought of a broad-spectrum treatment means for diagnosing amide herbicide poisoning and researching and developing medicaments based on TRPA1 targets.

Description

Application of TRPA1 ion channel as drug target in amide herbicide poisoning

Technical Field

The invention belongs to the field of biological medicine, and relates to application of a TRPA1 ion channel as a drug target in amide herbicide poisoning.

Background

The amide herbicide is a weeding pesticide containing typical benzene ring structure, mainly comprises propanil, iprovalicarb, metolachlor, alachlor, acetochlor, pretilachlor, butachlor and other electrophilic organic small molecular compounds, has high-efficiency and high-selectivity weeding effect and is widely applied in annual output of China up to more than hundred thousand tons. The amide herbicide is easy to migrate and enrich in the environment, has high environmental residue and long hydrolysis half-life, can be transmitted into human body through a food chain, interferes endocrine function and even causes canceration of lung, liver and the like, threatens human health and ecological safety, and does not have specific detoxification drugs for poisoning the amide herbicide at present. Therefore, the research on the poisoning mechanism of the amide herbicide and the research on the amide herbicide target point therapeutic drug have important significance on the treatment of amide herbicide poisoning.

Disclosure of Invention

In some embodiments, the invention provides an application of a medicament taking TRPA1 as a medicament action target in preparing a medicament for preventing or treating symptoms caused by amide compound poisoning.

In some embodiments, the condition includes, but is not limited to, symptoms of lung injury, liver injury, thyroid injury, immunotoxicity, or embryogenic toxicity.

In some embodiments, the lung injury comprises alveolar epithelial cell injury.

In some embodiments, the alveolar epithelial cell injury includes decreased cell viability, increased cytotoxicity, increased intracellular calcium influx.

In some embodiments, the amide is an amide herbicide.

In some embodiments, the amide includes propanil, iprovalicarb, metolachlor, alachlor, acetochlor, pretilachlor, butachlor, prometryn, metolachlor, biszoxamide, naphalamide, diflufenican, dimethenamid, naphalam, dimethenamid, barnacle, naphalamide, clothianidin, mefenacet, metazachlor, bromobutachlor, flufenacet, dimethenamid, flumetsulam, dimethenamid, penoxsulam, clomazone, high-efficiency methyl or high-efficiency benfuracil, but is not limited thereto.

In some embodiments, the agent that targets TRPA1 for drug action comprises an inhibitor that inhibits the TRPA1 gene, TRPA1mRNA, or TRPA1 protein.

In some embodiments, the inhibitor comprises an inhibitor that inhibits TRPA1 ion channel function.

Transient receptor potential ion channels (transient receptor potential ion channels, TRPs) are nonselective cation channels found in recent years to exist on cell membranes or intracellular organelle membranes, for Ca2 ⁺ The plasma cations have high permeability and are associated with pain or itching induced by the chemical substance. Wherein, the transient receptor potential cation channel subfamily A member 1 (transient receptor potential cation channel subfamily A member 1, TRPA 1) is widely distributed on cell membranes or organelle membranes of organs such as lungs, brains and the like, is considered as a chemical switch, and can be activated by various existing compounds such as cinnamaldehyde, allicin, formaldehyde, acrolein, allyl isothiocyanate and the like, thereby causing calcium ion inflow and producing inflammatory or pain reaction. The invention provides application of TRPA1 as a novel target for diagnosing and controlling amide herbicide poisoning. The research results of the invention show that TRPA1 and amide herbicide poisoning are closely related, and the inhibition or knocking-out of TRPA1 targets can reduce the increase of TRPA1 expression and calcium ion inflow caused by the amide herbicide, and finally reduce the alveolar epithelial cytotoxicity caused by the amide herbicide. Therefore, the invention provides a new thought of a broad-spectrum treatment means for diagnosing amide herbicide poisoning and researching and developing medicaments based on TRPA1 targets.

In some embodiments, the inhibitor is selected from at least one of the group consisting of a substance that inhibits TRPA1 activity, a substance that degrades TRPA1, and a gene tool that reduces TRPA1 levels.

In some embodiments, the inhibitor comprises a compound, a peptide antagonist, a second antibody specific for TRPA1, an siRNA, or an antisense molecule specific for TRPA 1.

In some embodiments, the substance that inhibits or degrades TRPA1 comprises a protein or compound.

In some embodiments, the gene tools that reduce TRPA1 levels comprise RNA interference, microRNA, gene editing, or gene knockout materials.

In some embodiments, the gene editing or gene knockout uses the crispr-cas9 gene knockout technique.

In some embodiments, the inhibitor comprises a TRPA1 ion channel blocker or cardamomin.

In some embodiments, the TRPA1 ion channel blocker comprises at least one of HC-030031, A-967079, TCS5861528, or Chembridge-5861528.

In some embodiments, the medicament further comprises pharmaceutically acceptable excipients and/or carriers.

In some embodiments, the dosage form of the medicament comprises a dry powder injection, an injection, a tablet, a capsule.

In some embodiments, the present invention provides a use of a composition in the preparation of a medicament for preventing or treating a condition caused by amide poisoning, wherein the composition comprises an effective amount of the medicament, and the medicament is a medicament with TRPA1 as a medicament action target.

In some embodiments, the lung injury comprises alveolar epithelial cell injury.

In some embodiments, the amide is an amide herbicide.

In some embodiments, the amide comprises at least one of propanil, iprovalicarb, metolachlor, acetochlor, pretilachlor, butachlor, prometryn, metolachlor, biszoxamide, naphalamide, diflufenican, dimethenamid, naphalam, dimethenamid, barnacle, naphalamide, clothianidin, mefenacet, metazachlor, bromobutachlor, flufenacet, dimethenamid, flumetsulam, fluobutachlor, dimethenamid, penoxsulam, clomazone, high-efficiency methyl or high-efficiency benfuracil.

In some embodiments, the compound, peptide antagonist, second antibody specific for TRPA1, siRNA or antisense molecule specific for TRPA 1.

In some embodiments, the present invention provides an application of a detection reagent of TRPA1 in preparing a diagnostic reagent for detecting amide poisoning.

In some embodiments, the detection reagent is a detection of gene expression level of TRPA 1.

In some embodiments, the detection reagent is a reagent that detects mRNA expression of TRPA 1.

In some embodiments, the detection reagent is a reagent that detects the amount of TRPA1 protein expressed.

In some embodiments, the detection reagent comprises one or more of a fluorescent quantitative PCR dye, a fluorescent quantitative PCR primer, a fluorescent quantitative PCR probe, an antibody functional fragment, and a conjugated antibody.

In some embodiments, when pesticide or herbicide poisoning, particularly amide herbicide poisoning, is suspected in a patient, a TRPA1 detection reagent may be used to detect, e.g., an increased expression level of TRPA1 is detected, suggesting a high likelihood of herbicide poisoning. In some embodiments, TRPA1 inhibitors may be administered for treatment when the patient is suspected or identified as being amide herbicide intoxication. In some embodiments, when herbicide poisoning, particularly amide herbicide poisoning, is suspected in a patient, TRPA1 detection may be performed first, and TRPA1 inhibitors are administered when the results show increased amounts of TRPA1 expression.

Drawings

FIGS. 1A-1G are graphs showing the effect of 7 amide herbicides on lung epithelial A549 cell viability and the effect of inhibiting or knocking out TRPA1 on amide herbicide-induced cytotoxicity in the experiments of the present invention. Fig. 1A: propanil (propanil); fig. 1B: propisochlor; fig. 1C: metolachlor; fig. 1D: alachlor; fig. 1E: acetochlor; fig. 1F: pretilachlor; fig. 1G: butachlor.

FIGS. 2A-2G are graphs showing that amide herbicides induced A549 cell damage in the experiments of the present invention. Fig. 2A: propanil (propanil); fig. 2B: propisochlor; fig. 2C: metolachlor; fig. 2D: alachlor; fig. 2E: acetochlor; fig. 2F: pretilachlor; fig. 2G: butachlor.

FIGS. 3A-3G are graphs showing the effect of an amide herbicide on TRPA1mRNA expression and the effect of inhibiting or knocking out TRPA1 on TRPA1mRNA expression induced by an amide herbicide in an experiment of the present invention. Fig. 3A: propanil (propanil); fig. 3B: propisochlor; fig. 3C: metolachlor; fig. 3D: alachlor; fig. 3E: acetochlor; fig. 3F: pretilachlor; fig. 3G: butachlor.

FIGS. 4A-4G are graphs showing the effect of an amide herbicide on the calcium ion influx of A549 cells in the experiments of the present invention, and the effect of inhibiting or knocking out TRPA1 on the amide herbicide-induced calcium ion influx of cells. Fig. 4A: propanil (propanil); fig. 4B: propisochlor; fig. 4C: metolachlor; fig. 4D: alachlor; fig. 4E: acetochlor; fig. 4F: pretilachlor; fig. 4G: butachlor.

Detailed Description

The technical solution of the present invention is further illustrated by the following specific examples, which do not represent limitations on the scope of the present invention. Some insubstantial modifications and adaptations of the invention based on the inventive concept by others remain within the scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same definition as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention, the preferred methods and materials being described in the detailed description.

As used herein, "a" and "an" refer to the grammatical indefinite articles "a," "an," or "a plurality," "a plurality" (i.e., "at least one," "at least one"). For example, "an element" refers to one or more elements.

As used herein, unless otherwise indicated, the term "preventing" refers to treating or administering an inhibitor provided herein to a patient, particularly at risk of poisoning and/or other conditions described herein, prior to the onset of symptoms. The term "prevention" includes inhibition or alleviation of symptoms of a particular disease. In addition, patients with a history of symptom recurrence are also potential candidates for prophylaxis. In this regard, the term "prevention" may be used interchangeably with the term "prophylactic treatment".

As used herein, unless otherwise indicated, the term "treating" refers to the elimination or alleviation of a disease or disorder, or one or more symptoms associated with a disease or disorder. In certain embodiments, the term refers to minimizing the spread or exacerbation of a disease or disorder by administering one or more prophylactic or therapeutic agents to a subject suffering from the disease or disorder.

In the present invention, the components in the "composition" may be present in a mixed form or may be packaged separately. The separately packaged components may also contain their respective adjuvants. The adjuvant refers to a means for assisting the curative effect of the medicine in pharmacy. In the case of separate packages for the components of the composition, the individual components of the separate packages may be administered simultaneously or in any order, wherein the patient is treated with one drug and then administered with the other. The patient refers to a mammalian subject, particularly a human.

In some embodiments, the components of the compositions of the present invention may be provided separately or mixed together in unit dosage form, e.g., as a dry form, as a lyophilized powder or anhydrous concentrate in a closed container such as an ampoule or sachet, indicative of the amount of active agent. When the composition is administered by infusion, it may be used with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration.

An "effective amount" is an amount sufficient to achieve a beneficial or desired result, such as an enhanced immune response, treatment, prevention, or amelioration of a medical condition (disease, infection, etc.). The effective amount may be administered in one or more administrations, applications or dosages. The appropriate dosage will vary depending upon the weight, age, health, disease or condition to be treated, and the route of administration.

In this disclosure, "comprising," "including," "having" can mean "including," "covering," etc., and "consisting essentially of," "consisting of," etc., have the meanings given in the patent statutes, which are open-ended, allowing more than the recited items to exist, so long as the basic or novel characteristics of the recited items are not altered by the presence of more than the recited items, but rather preclude embodiments of the prior art.

Pharmaceutically acceptable carriers include saline, aqueous buffer solutions, solvents, and/or dispersion media. The use of such vectors is well known in the art. Preferably, the solution is stable under conditions of manufacture and storage and preserved against the contaminating action of microorganisms of bacteria and fungi through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.

Example 1

1. Experimental materials

1.1 experimental cells: human type II alveolar epithelial cell A549 cells are purchased from the cell resource center of basic medical institute of China medical science center; TRPA1crispr-cas9 gene knockout A549 cells (TRPA 1-KO A549 cells) were purchased from Kirschner Biotech Co.

1.2McCoy's 5A medium, fetal bovine serum, 0.25% Trypsin-EDTA, all purchased from Gibco corporation; amide herbicides (propanil, iprovalicarb, metolachlor, alachlor, acetochlor, pretilachlor, and butachlor), all purchased from Shanghai alaa Ding Shenghua technologies limited; TRPA1 channel blocker HC-030031, available from Selleck Chemicals company; the CCK-8 cell viability detection kit and the LDH cell damage detection kit are both purchased from Dojindo corporation; reverse transcription kit and fluorescent real-time quantitative RT-PCR reaction kit are purchased from TaKaRa company;reagent and Fluo-4 Direct ^TM The Calcium kit was purchased from ThermoFisher Scientific company.

2. Experimental method

2.1 cell culture and treatment

A549 cells of Wild Type (WT) and a549 cells of TRPA1 gene knockout (TRPA 1-KO) were cultured in McCoy's 5A complete medium containing 10% fbs, respectively. Inoculating 5×10 respectively ⁴ cell/mL WT A549 cells and TRPA1-KO A549 cell suspensions were placed at 37℃at 5%CO ₂ Culturing in a wet incubator.

Normal control group (WT a549 cell group): cell replacement is carried out on the complete culture medium, and the culture is carried out for 24 hours;

toxicological group treatment (TRPA 1-KO a549 cell group): when WT/TRPA1-KO A549 cells are in logarithmic growth phase, the original medium (McCoy's 5A complete medium containing 10% FBS) was aspirated, and complete medium containing amide herbicides (propanil, iprovalicarb, alachlor, pretilachlor and butachlor) at concentrations of 0, 150, 300, 600, 1200. Mu.M was added, and cultured for 24 hours;

inhibitor addition group treatment (WT a549 cell+hc-030031 50 μm group): when WT A549 cells were in logarithmic growth phase, the original medium (McCoy's 5A complete medium containing 10% FBS) was aspirated, and complete culture with 50. Mu.M HC-030031 was added based on 37℃and 5% CO ₂ Pre-culturing in an incubator for 0.5h; then respectively adding amide herbicide to make HC-030031 final concentration be 50mM, and making amide herbicide concentration be 0, 150, 300, 600 and 1200 mu M respectively, placing them at 37 deg.C and 5% CO ₂ Culturing in an incubator for 24 hours.

2.2 cell viability assay

The CCK-8 kit is adopted for measuring the cell viability. The 96-well plate was removed, the supernatant was aspirated off from the cells, and CCK-8 reagent was mixed with complete medium at a volume ratio of 1:9, and 100. Mu.L/well was added. The 96-well plate was placed at 37℃with 5% CO ₂ Culturing in an incubator for 4 hours. The absorbance at 450nm was measured with a microplate reader and the percent cell viability was calculated.

2.3 cell injury assay

The measurement of cell damage was continued using LDH kit. The 96-well plate was removed, and after 10. Mu.L/well Lysis Buffer was added to the high control well, the 96-well plate was placed at 37℃with 5% CO ₂ Culturing in a wet incubator for 30min. After 100. Mu.L/well Working Solution was added to each well, the wells were incubated at room temperature for 30min in the dark. Immediately after 50. Mu.L/well Stop Solution was added to each well, absorbance at 490nm was measured with a microplate reader, and the cell damage rate was calculated.

2.4 real-time fluorescent quantitative RT-PCR

The 6-well plate was removed, the medium was aspirated, and 1 mL/sample TRIzol reagent was added. Total RNA from WT/TRPA1-KO A549 cells was extracted according to TRIzol instructions for subsequent reverse transcription experiments or frozen at-80 ℃. cDNA reverse transcription was performed with reference to reverse transcription kit instructions. The reaction conditions were 42℃for 2min to 4℃for infinity (removal reaction of genomic DNA) and 37℃for 15min to 85℃for 5s to 4℃for infinity (reverse transcription reaction), respectively. The real-time fluorescent quantitative PCR reaction is carried out according to the instructions of a TaKaRa real-time fluorescent quantitative PCR kit. The primer sequences and the product length sizes are respectively as follows: hTRPA1 (107 bp) Forward primer 5'AGTATATTTGGGTATTGCAAAGAAGC 3' (SEQ ID NO: 1), reverse primer 5'ATGCCCGTCGTGTAGATAATCC 3' (SEQ ID NO: 2); h.beta. -actin (184 bp) Forward primer 5'AGAGCTACGAGCTGCCTGAC 3' (SEQ ID NO: 3), reverse primer 5'AGCACTGTGTTGGCGTACAG 3' (SEQ ID NO: 4). The reaction conditions were 95℃for 30s→ (95℃for 5s→60℃for 30s→72℃for 30s+Plate Read). Times.40 move times→melt Curve 65℃to 95℃increment 0.5℃for 5s+Plate read→END. All operations were performed on ice.

2.5 calcium imaging of cells

Using Fluo-4 Direct ^TM The Calcium kit is used for measuring the content of Calcium ions in cells. Taking out the 96-well plate, mixing one bottle of component A and component C, adding 200 μl of component B working solution (77 mg of component B is completely dissolved in 1mL of component C), and vortex mixing to obtain Fluo-4 Direct ^TM Calcium reagent working solution. Directly adding the working solution into the cells, and blowing the cells under 3-4. The 96-well plate was placed at 37℃with 5% CO ₂ Incubation is carried out for 30min in a moist incubator, and the incubator is protected from light after being taken out. Single channel FITC fluorometry and data processing by confocal high content imaging systems.

3. Experimental results

3.1 A549 cell viability results

As shown in FIGS. 1A-1G, the activity of A549 cells was compared with that of 0. Mu.M amide herbicides, respectively, propanil (150. Mu.M, P < 0.001; 300. Mu.M, P < 0.0001; 600. Mu.M, P < 0.0001; 1200. Mu.M, P < 0.0001), metolachlor (150. Mu.M, P < 0.01; 300. Mu.M, P < 0.0001; 600. Mu.M, P < 0.0001), metolachlor (150. Mu.M, P < 0.0001; 300. Mu.M, P < 0.0001; 600. Mu.M, P < 0.0001; 1200. Mu.M, P < 0.0001), and metolachlor (150. Mu.M, P < 0.001; 300. Mu.M, P < 0.001; 600. Mu.M, P < 0.0001; 1200. Mu.M, P < 0.0001), acetochlor (150. Mu.M, P < 0.0001; 300. Mu.M, P < 0.0001; 600. Mu.M, P < 0.0001; 1200. Mu.M, P < 0.0001), pretilachlor (150. Mu.M, P < 0.001; 300. Mu.M, P < 0.001; 600. Mu.M, P < 0.001; 1200. Mu.M, P < 0.0001) and butachlor (150. Mu.M, P < 0.01; 300. Mu.M, P < 0.0001) significantly decreased A549 cell viability following exposure.

Compared with 150, 300 and 600 mu M amide herbicides in the corresponding groups, HC-030031 (50 mu M) can obviously improve the decrease of A549 cells caused by propanil (150 mu M, P < 0.05;300 mu M, P < 0.05;600 mu M, P < 0.01), metolachlor (150 mu M, P < 0.01;600 mu M, P < 0.01), alachlor (300 mu M, P < 0.0001;600 mu M, P < 0.0001), acetochlor (150 mu M, P < 0.001;300 mu M, P < 0.01;600 mu M, P < 0.001), pretilachlor (600 mu M, P < 0.05) and butachlor (300 mu M, P < 0.01) and can also improve the decrease of A549 cell viability caused by metolachlor.

Compared with the corresponding groups of 150, 300 and 600 mu M amide herbicides, after TRPA1 gene knockout, propanil (300 mu M, P < 0.05;600 mu M, P < 0.01), metolachlor (150 mu M, P < 0.001;600 mu M, P < 0.01), metolachlor (150 mu M, P < 0.01;300 mu M, P < 0.01;600 mu M, P < 0.05), acetochlor (150 mu M, P < 0.001;300 mu M, P < 0.0001;600 mu M, P < 0.05), pretilachlor (150 mu M, P < 0.05) and butachlor (150 mu M, P < 0.01;300 mu M, P < 0.001;600 mu M, P < 0.01) can be significantly improved.

3.2 A549 cell injury results

As shown in FIGS. 2A-2G, the results of cell damage of A549 are shown, respectively, in comparison with 0. Mu.M amide herbicides, propanil (150. Mu.M, P < 0.0001; 300. Mu.M, P < 0.0001; 600. Mu.M, P < 0.0001; 1200. Mu.M, P < 0.0001), metolachlor (150. Mu.M, P < 0.0001; 300. Mu.M, P < 0.0001; 600. Mu.M, P < 0.0001), metolachlor (150. Mu.M, P < 0.001; 300. Mu.M, P < 0.0001; 600. Mu.M, P < 0.0001; 1200. Mu.M, P < 0.0001), metolachlor (150. Mu.M, P < 0.0001; 300. Mu.M, P < 0.0001; 600. Mu.M, P < 0.0001; 1200. Mu.M, P < 0.0001), acetochlor (150. Mu.M, P < 0.0001; 300. Mu.M, P < 0.0001; 600. Mu.M, P < 0.0001; 1200. Mu.M, P < 0.0001), pretilachlor (150. Mu.M, P < 0.0001; 300. Mu.M, P < 0.0001; 600. Mu.M, P < 0.0001; 1200. Mu.M, P < 0.0001) and butachlor (150. Mu.M, P < 0.05; 300. Mu.M, P < 0.0001; 600. Mu.M, P < 0.0001) were significantly elevated in A549 cytotoxicity after exposure.

In combination with the results of fig. 1 and 2, it was found that 7 types of AHs (amide herbicides) each resulted in a significant decrease in a549 cell viability, respectively, and that there was a significant cytotoxic effect.

3.3 Results of TRPA1mRNA expression

The results of TRPA1mRNA expression are shown in FIGS. 3A-3G. The A549 cell TRPA1mRNA expression induced by propanil (300. Mu.M, P < 0.05; 600. Mu.M, P < 0.01), metolachlor (300. Mu.M, P < 0.01; 600. Mu.M, P < 0.0001), alachlor (300. Mu.M, P < 0.01; 600. Mu.M, P < 0.01), acetochlor (600. Mu.M, P < 0.01), pretilachlor (600. Mu.M, P < 0.05) and butachlor (150. Mu.M, P < 0.05; 300. Mu.M, P < 0.05; 600. Mu.M, P < 0.001) was significantly increased compared to the 0. Mu.M amide herbicide, respectively.

The TRPA1 specific inhibitor HC-030031 (50. Mu.M) significantly reduced propanil (150. Mu.M, P < 0.01), metolachlor (150. Mu.M, P < 0.05), alachlor (600. Mu.M, P < 0.05), acetochlor (150. Mu.M, P < 0.01; 600. Mu.M, P < 0.05) and butachlor (600. Mu.M, P < 0.01) induced increases in TRPA1mRNA expression of A549 cells, and possibly also reduced increases in TRPA1mRNA expression induced by metolachlor, pretilachlor, compared to the corresponding group of 150, 300, 600. Mu.M amide herbicides, respectively. While TRPA1-KO a549 cells barely expressed TRPA1mRNA after exposure to seven classes of amide herbicides at each concentration.

3.4 A549 cell calcium ion influx results

The results of calcium ion influx in A549 cells are shown in FIGS. 4A-4G. Propanil (150. Mu.M, P < 0.01; 300. Mu.M, P < 0.001; 600. Mu.M, P < 0.001; 1200. Mu.M, P < 0.0001), propisochlor (150. Mu.M, P < 0.01; 300. Mu.M, P < 0.0001; 600. Mu.M, P < 0.0001; 1200. Mu.M, P < 0.0001), propisochlor (150. Mu.M, P < 0.001; 300. Mu.M, P < 0.0001; 600. Mu.M, P < 0.0001; 1200. Mu.M, P < 0.0001), alachlor (150. Mu.M, P < 0.001; 300. Mu.M, P < 0.0001; 600. Mu.M, P < 0.0001; 1200. Mu.M, P < 0.0001), propisochlor (150. Mu.M, P < 0.0001; 1200. Mu.M, P < 0.0001; 600. Mu.M, P < 0.0001; P < 0.0001) and P < 0.0001) lead to a significant increase in the cells compared with the 0. Mu.M amide herbicides, respectively.

HC-030031 (50. Mu.M) can significantly reduce propanil (300. Mu.M, P < 0.05; 600. Mu.M, P < 0.01), metolachlor (150. Mu.M, P < 0.01; 300. Mu.M, P < 0.0001; 600. Mu.M, P < 0.0001; 1200. Mu.M, P < 0.01), metolachlor (150. Mu.M, P < 0.01; 300. Mu.M, P < 0.05; 600. Mu.M, P < 0.001; 1200. Mu.M) after dry-pre-treatment compared to the corresponding group of 150, 300, 600, 1200. Mu.M amide herbicides, respectively, P < 0.0001), alachlor (150. Mu.M, P < 0.01; 300. Mu.M, P < 0.0001; 600. Mu.M, P < 0.0001), acetochlor (150. Mu.M, P < 0.0001; 300. Mu.M, P < 0.0001; 600. Mu.M, P < 0.0001; 1200. Mu.M, P < 0.0001), pretilachlor (150. Mu.M, P < 0.05; 300. Mu.M, P < 0.001; 600. Mu.M, P < 0.0001), and butachlor (300. Mu.M, P < 0.0001; 600. Mu.M, P < 0.0001; 1200. Mu.M, P < 0.05).

Propanil (150. Mu.M, P < 0.001; 600. Mu.M, P < 0.05; 1200. Mu.M, P < 0.0001), metolachlor (150. Mu.M, P < 0.0001; 300. Mu.M, P < 0.001; 600. Mu.M, P < 0.0001; 1200. Mu.M, P < 0.001), metolachlor (150. Mu.M, P < 0.0001; 300. Mu.M, P < 0.001; 600. Mu.M, P < 0.0001; 1200. Mu.M, P < 0.01), metolachlor (150. Mu.M, P < 0.001; 300. Mu.M, P < 0.0001; 600. Mu.M, P < 0.01; 1200. Mu.M, P < 0.0001), acetochlor (150. Mu.M, P < 0.0001; 300. Mu.M, P < 0.0001; 600. Mu.M, P < 0.0001; 1200. Mu.M, P < 0.0001), pretilachlor (150. Mu.M, P < 0.0001; 300. Mu.M, P < 0.0001; 600. Mu.M, P < 0.001; 1200. Mu.M, P < 0.0001) and butachlor (150. Mu.M, P < 0.0001; 300. Mu.M, P < 0.0001; 600. Mu.M, P < 0.0001) resulted in an increase in calcium ion influx in A549 cells.

The invention discovers that 7 kinds of amide herbicides respectively lead to the obvious reduction of the activity of A549 cells and the obvious increase of cell damage, and the mRNA expression of TRPA1 is obviously increased; while intervention of TRPA1 inhibitor HC-030031 (50 mu M) or knocking out of TRPA1crispr-cas9 gene can improve the activity of A549 cells infected by the amide herbicide and reduce the increase of TRPA1mRNA expression induced by the amide herbicide; it is demonstrated that TRPA1 is closely related to A549 cytotoxicity caused by amide herbicides.

Meanwhile, 7 kinds of amide herbicides can cause the increase of the calcium influx of A549 cells, and intervention with a TRPA1 inhibitor HC-030031 (50 mu M) or TRPA1 gene knockout can obviously inhibit the increase of the calcium influx of cells mediated by TRPA1, and again, TRPA1 is an important target point for the amide herbicides to induce the cytotoxicity of A549 cells.

In conclusion, the experiment proves that the TRPA1 target is closely related to lung epithelial cytotoxicity caused by amide herbicide poisoning, and inhibiting or gene knockout of TRPA1 can reduce the cytotoxicity of the amide herbicide by reducing the increase of TRPA1 expression and the mediated calcium ion inflow thereof. Therefore, the invention provides a new target point for diagnosing amide herbicide poisoning and also provides a new thought for researching and developing amide herbicide poisoning drugs based on the TRPA1 target point.

Claims

Use of a TRPA1 ion channel blocker in the manufacture of a medicament for preventing or treating lung injury caused by amide herbicide poisoning, wherein the TRPA1 ion channel inhibitor is HC-030031, a-967079, TCS5861528 or Chembridge-5861528.
2. The use of claim 1, wherein the lung injury is selected from alveolar epithelial cell injury.
3. The use of claim 2, wherein said alveolar epithelial cell injury is selected from the group consisting of decreased cell viability, increased cytotoxicity, increased intracellular calcium influx.
4. The use according to any one of claims 1 to 3, wherein, the amide herbicide is selected from propanil, iprovalicarb, metolachlor, acetochlor, pretilachlor, butachlor, prometryn, ethofenpyr-ethyl, bispyribac-sodium, naproxen, diflufenican, dimethenamid, naproxen, dichlormid, barnyard grass, naproxen, benthiavalicarb-sodium, pyriftalid, bromobutachlor, flufenacet, dimethenamid, flumetsulam, bezoxamide, dimethenamid, penoxsulam, high-efficiency metolachlor, high-efficiency methyl or high-efficiency propyl.
5. The use according to any one of claims 1 to 3, wherein the medicament further comprises pharmaceutically acceptable excipients and/or carriers.
6. The use according to any one of claims 1 to 3, wherein the pharmaceutical dosage form is selected from the group consisting of a lyophilized powder for injection, an injection, a tablet, a capsule.
7. Use of a composition for the preparation of a medicament for preventing or treating lung injury caused by amide herbicide poisoning, wherein the composition comprises an effective amount of the medicament which is a TRPA1 ion channel blocker, and the TRPA1 ion channel inhibitor is HC-030031, a-967079, TCS5861528 or Chembridge-5861528.
8. The use of claim 7, wherein the lung injury is selected from alveolar epithelial cell injury.
9. The use of claim 8, wherein said alveolar epithelial cell injury is selected from the group consisting of decreased cell viability, increased cytotoxicity, increased intracellular calcium influx.
10. The use according to any one of claims 7 to 9, wherein, the amide herbicide is selected from propanil, iprovalicarb, metolachlor, acetochlor, pretilachlor, butachlor, prometryn, ethofenpyr-ethyl, bispyribac-sodium, naproxen, diflufenican, dimethenamid, naproxen, dichlormid, barnyard grass, naproxen, benthiavalicarb-sodium, pyriftalid, bromobutachlor, flufenacet, dimethenamid, flumetsulam, bezoxamide, dimethenamid, penoxsulam, high-efficiency metolachlor, high-efficiency methyl or high-efficiency propyl.
11. The use according to any one of claims 7 to 9, wherein the medicament further comprises pharmaceutically acceptable excipients and/or carriers.
12. The use according to any one of claims 7 to 9, wherein the pharmaceutical dosage form is selected from the group consisting of dry powder injection, tablet, capsule.
Use of trpa1 as a target in screening diagnostic reagents for the detection of amide herbicide poisoning.