CN111481674A - Application of TRPA1 inhibitor in preparation of medicine for treating multiple sclerosis - Google Patents
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Abstract
The invention discloses an application of an inhibitor of TRPA1 in preparing a medicine for treating multiple sclerosis, wherein the inhibitor of TRPA1 can be used for preparing a medicine for treating multiple sclerosis caused by lysophosphatidylcholine (L PC). The invention researches expression of TRPA1 on O L N-93 oligodendrocyte and oxidative stress and cell injury induced by TRPA1 in L PC (human peripheral component protein) by establishing an O L N-93 oligodendrocyte oxidative stress and cytotoxicity model, and shows that the inhibitor of TRPA1 can obviously inhibit calcium ion inflow in cytoplasm and mitochondria, obviously reduce the generation of mitochondrial active oxygen, obviously inhibit the reduction of mitochondrial membrane potential, obviously inhibit the generation of NO, obviously inhibit the increase of superoxide and effectively inhibit the increase of oligodendrocyte toxicity, so the inhibitor of TRPA1 can be used for treating demyelinating diseases such as multiple sclerosis caused by TRPA L PC and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an application of an inhibitor of TRPA1 in preparation of a medicine for treating multiple sclerosis, in particular to an application of an inhibitor of TRPA1 in preparation of a medicine for treating multiple sclerosis caused by lysophosphatidylcholine.
Background
L PC is often used for construction of demyelinating animal models in experiments, L PC can cause the generation of astrocyte and microglia inflammation and cause oligodendrocyte damage, in the cerebrospinal fluid of demyelinating diseases such as multiple sclerosis patients, the content of L PC is remarkably increased, the specific action mechanism of L PC as an important demyelinating medium is not clear, two hypotheses exist, one is that L PC can cause cell membrane rupture and cell damage, the other is that L PC can cause downstream signals such as oxidative stress and inflammatory reaction by acting on receptors on cell membranes and finally cause cell damage, and the main receptors are G Protein Coupled Receptors (GPCRs) and TRP ion channel families.
Transient receptor potential Anchor protein ion channel 1(TRPA1) is one of the constituent members of the transient receptor potential superfamily (TRP), a nonselective cation channel protein that mediates influx of calcium and sodium ions. TRPA1 is an important exogenous stimulatory receptor that responds to a variety of stimuli, such as hydrogen peroxide, hydrogen sulfide, carbon dioxide, and the like. There are articles reporting that TRPA1 is involved in oligodendrocyte injury under hypoxic conditions, although the specific mechanism is not clear. Oligodendrocyte is a kind of glial cell existing in central nervous system, and it can form myelin sheath to wrap around neuron axon, and can provide energy substance for neuron axon, thus maintaining normal physiological activity of neuron.
Disclosure of Invention
The invention aims to provide application of an inhibitor of TRPA1 in preparing a medicament for treating multiple sclerosis.
The invention detects the expression of TRPA1 and neural nitric oxide synthase (nNOS) in O L N-93 oligodendrocyte through immunofluorescence, detects the calcium influx of TRPA1 participating in mediating L PC, and further discovers that after being treated by TRPA1 inhibitor, the TRPA1 inhibitor can obviously inhibit the calcium influx of cell cytoplasm and mitochondria, obviously inhibit the generation of mitochondrial active oxygen, obviously inhibit the reduction of mitochondrial membrane potential, obviously inhibit the generation of Nitric Oxide (NO) and the generation of superoxide, effectively inhibit the increase of cytotoxicity and reduce the damage of oligodendrocyte, and speculate that L PC stimulates TRPA1 to cause calcium overload, promote the mitochondrial damage and promote NO generation to trigger oxidative stress to damage oligodendrocyte, thus causing demyelinating diseases such as multiple sclerosis and the like.
Therefore, the first object of the present invention is to provide the use of an inhibitor of TRPA1 in the manufacture of a medicament for the treatment of multiple sclerosis. In particular to an application of an inhibitor of TRPA1 in preparing a medicament for treating multiple sclerosis caused by lysophosphatidylcholine.
It is a second object of the present invention to provide a medicament for treating multiple sclerosis, in particular to provide a medicament for treating multiple sclerosis caused by lysophosphatidylcholine, which comprises an inhibitor of TRPA1 as an active ingredient.
The inhibitor of TRPA1 is HC030031, AP-18 or A967079.
The research result shows that TRPA1 is widely expressed on O L N-93 oligodendrocytes, TRPA1 is involved in O L N-93 oligodendrocyte calcium ion overload, mitochondrial active oxygen generation, mitochondrial membrane potential reduction, NO generation, superoxide increase and cytotoxicity increase caused by L PC, the inhibitor of TRPA1 can obviously inhibit calcium ion inflow in cytoplasm and mitochondria, obviously reduce mitochondrial active oxygen generation, obviously inhibit mitochondrial membrane potential reduction, obviously inhibit NO generation, obviously inhibit superoxide increase and effectively inhibit oligodendrocyte toxicity increase, and therefore the inhibitor of TRPA1 can be used for treating demyelinating diseases such as multiple sclerosis and the like caused by L PC.
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FIG. 1A shows the result of protein expression of TRPA1 on O L N-93 oligodendrocyte, FIG. 1B shows the result of protein expression of nNOS on O L N-93 oligodendrocyte, and Myelin Basic Protein (MBP) is specifically expressed on oligodendrocyte and is the tag protein thereof.
FIG. 2A shows that TRPA1 participates in regulating and controlling the cytoplasmic and mitochondrial calcium ion influx induced by L PC, FIG. 2B shows that L PC can significantly promote the cytoplasmic and mitochondrial calcium ion influx, and inhibition of TRPA1 can effectively inhibit the increase of cytoplasmic and mitochondrial calcium ions;
FIG. 2C shows that L PC has significant effects on the promotion of cytoplasmic and mitochondrial calcium influx and the inhibition of TRPA1 cytoplasmic and mitochondrial calcium influx****p<0.0001)；
FIG. 3A shows that TRPA1 participates in the regulation of L PC to cause the production of mitochondrial reactive oxygen by O L N-93 oligodendrocyte, FIG. 3B shows that L PC significantly promotes the production of mitochondrial reactive oxygen by O L N-93 oligodendrocyte and inhibits TRPA1 to significantly inhibit the production of mitochondrial reactive oxygen (****p<0.0001)；
FIG. 4A shows that TRPA1 participates in L PC to control the reduction of mitochondrial membrane potential of O L N-93 oligodendrocyte, FIG. 4B shows that L PC can obviously promote the reduction of mitochondrial membrane potential of O L N-93 oligodendrocyte, and inhibition of TRPA1 can obviously inhibit the generation of mitochondrial membrane potential reduction (3)***p<0.001，****p<0.0001), FIG. 4C shows that the mitochondrial membrane potential decrease caused by L PC is enhanced with time, and inhibition of TRPA1 can significantly inhibit this process;
FIG. 5A shows TRPA1 involved in regulationL PC induced generation of O L N-93 oligodendrocyte nitric oxide, FIG. 5B is L PC can significantly promote generation of O L N-93 oligodendrocyte nitric oxide, inhibit TRPA1 can significantly inhibit generation of nitric oxide: (****p<0.0001)；
FIG. 6A shows that TRPA1 participates in the regulation of O L N-93 oligodendrocyte superoxide production caused by L PC, FIG. 6B shows that L PC can significantly promote O L N-93 oligodendrocyte superoxide production, and inhibition of TRPA1 can significantly inhibit superoxide production (B)****p<0.0001)；
FIG. 7A shows that TRPA1 is involved in regulating L PC-induced decrease in O L N-93 oligodendrocyte activity (****p<0.0001，##p<0.01; # versis L PS, # versis 30. mu.M L PC, # NC indicates no significan; FIG. 7B shows that TRPA1, mitochondrial reactive oxygen, caspase-1 and nNOS are involved in regulating L PC-induced increases in O L N-93 oligodendrocyte cytotoxicity: (*p<0.05，***p<0.001，****p<0.0001，####p<0.0001；*versus 50μM LPC，#versus LPS)
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1TRPA1 regulates L PC-induced O L N-93 oligodendrocyte oxidative stress and cell injury
1. Material
1.1 Main instruments and reagents
1.2 major cell sources
Rat oligodendrocyte cell line O L N-93, purchased from Saururus (Shanghai) Biotech GmbH, Inc., cat # iCell-r 023.
2. Method of producing a composite material
2.1 culture of O L N-93 oligodendrocytes
Culturing O L N-93 oligodendrocyte in DMEM complete medium containing 10 wt% fetal calf serum at 37 deg.C under 5% CO2。
2.2 TRPA1, nNOS immunofluorescence assay in O L N-93 oligodendrocytes
(1) Culturing O L N-93 oligodendrocyte in 4-grid 3.5cm glass-bottom dish, the cell amount in each grid is 1 × 105And then cultured in a cell culture box overnight.
(2) Rinse 3 times with PBS and fix cells with 4 wt% paraformaldehyde for 15min at room temperature.
(3) Rinsed 3 times with PBS for 3min each time, and permeabilized with 0.1 wt% TritonX-100 for 5min at room temperature.
(4) Rinse 3 times with PBS and block with 10 wt% goat serum (containing 0.1 wt% tween-20) for 1h at room temperature.
(5) anti-Rabbit anti-TRPA1(1:200), Rabbit anti-nNOS (1:200) and Human anti-MBP (1:100) formulated with 10 wt% goat serum were added and incubated overnight at 4 ℃.
(6) Wash 3 times with PBS for 3min each.
(7) Add 10 wt% goat serum prepared secondary Alexa Fluor 488 goat anti-rabbit IgG (1:100) and Alexa Fluor594 goat anti-human IgG (1:100) and incubate for 1h at room temperature in the dark.
(8) Incubate with DAPI at room temperature for 15 min.
(9) Washing with PBS for 3 times, 3min each time, sealing with anti-quenching sealing agent, and observing with fluorescence microscope or confocal microscope.
2.3 calcium ion imaging detection
(1) O L N-93 oligodendrocytes were cultured in 4-well 3.5cm glass plates with the amount of cells in each well being 1 × 105And then cultured in a cell culture box overnight.
(2) Rinse 3 times with calcium free D-Hank's solution.
(3) Add 2.5 u M Fluo 4-AM and 2.5 u M Rhod 2-AM Hank's solution, in the cell culture box incubation for 40 min.
(4) Rinsing with calcium-free D-Hank's solution for 3 times, incubating with calcium-containing Hank's solution in cell culture box for 30min, and observing with confocal microscope.
(5) Preparing 30 μ M L PC containing or not containing calcium in advance or mixing 30 μ M L PC with 10 μ M A967079(CAS No.:1170613-55-4), 10 μ M AP-18(CAS No.:55224-94-7), 10 μ M ruthenium red or 100 μ M GdCl3And (4) mixing.
(6) The fluorescence signal was recorded by confocal microscopy for 30s without drug stimulation, followed by continuous recording for 3min with the addition of drug, and pictures were taken every 1 s. The change in calcium ion fluorescence was obtained by image processing software ImageJ analysis.
2.4 mitochondrial reactive oxygen species detection
(1) O L N-93 oligodendrocytes were cultured in 4-well 3.5cm glass plates with the amount of cells in each well being 1 × 105And then cultured in a cell culture box overnight.
(2) The medium was incubated for 30min with 10. mu. M A967079 or 10. mu.M HC030031(CAS No.:349085-38-7) and for 1h with 30. mu.M L PC.
(3) 2.5. mu.M mitoSOX was added and incubated in a cell incubator for 10min, rinsed 3 times with Hank's solution.
(4) Adding Hoechst 33342 dye solution, incubating at 37 deg.C for 30min, rinsing with Hank's solution for 3 times, and observing with confocal microscope. The mean fluorescence intensity was obtained by image processing software ImageJ analysis.
2.5JC-1 method for detecting mitochondrial membrane potential
(1) O L N-93 oligodendrocytes were cultured in 4-well 3.5cm glass plates with the amount of cells in each well being 1 × 105And then cultured in a cell culture box overnight.
(2) Cells were treated with 100ng/m L lipopolysaccharide (L PS) for 12h and rinsed 3 times with PBS.
(3) Cells were treated with 10 μ M A967079 for 30min and then L PC for 1h 30 min.
(4) Adding JC-1 into the culture medium, treating for 20min in a cell culture box, rinsing with PBS for 2 times, adding serum-free DMEM culture medium, and observing by a confocal microscope. The mean fluorescence intensity was obtained by image processing software ImageJ analysis.
2.6 detection of mitochondrial Membrane potential by TMRM method
(1) O L N-93 oligodendrocytes were cultured in 4-well 3.5cm glass plates with the amount of cells in each well being 1 × 105And then cultured in a cell culture box overnight.
(2) Cells were treated with 100ng/m L lipopolysaccharide (L PS) for 12h and rinsed 3 times with PBS.
(3) 30nM TMRM was added and incubated in cell incubator for 30min, rinsed 2 times with PBS.
(4) 30 μ M L PC was prepared in advance or 30 μ M L PC was mixed with 10 μ M A967079.
(5) The calcium containing Hank's solution was added and the fluorescence change was recorded for 30s under a confocal microscope, followed by addition of 30 μ M L PC or a mixture of 30 μ M L PC and 10 μ M A967079, followed by a confocal measurement of fluorescence change at intervals of 30s for 19min30 s.
2.7 detection of nitric oxide content in cells
(1) O L N-93 oligodendrocytes were cultured in 4-well 3.5cm glass plates with the amount of cells in each well being 1 × 105And then cultured in a cell culture box overnight.
(2) Cells were treated with 10. mu. M A967079, 100. mu.M spermidine for 30min and L PC for 2 h.
(3) Adding 5 μ M DAF-FM DA, incubating in cell culture box for 30min, adding Hoechst 33342 dye solution, incubating at 37 deg.C for 20min, washing with PBS for 1 time, covering cells with Hank's solution, and observing under confocal microscope. The mean fluorescence intensity was obtained by image processing software ImageJ analysis.
2.8 detection of superoxide content
(1) O L N-93 oligodendrocytes were cultured in 4-well 3.5cm glass plates with the amount of cells in each well being 1 × 105And then cultured in a cell culture box overnight.
(2) Cells were treated with 1. mu.g/m L lipopolysaccharide (L PS) for 12h and rinsed 3 times with PBS.
(3) Cells were treated with 10 μ M A967079 for 30min and then L PC for 2 h.
(4) Add 5 μ M DHE to incubate in cell incubator for 30min, add Hoechst 33342 stain to incubate at 37 ℃ for 20min, wash 1 time with PBS, cover cells with Hank's solution, and observe under confocal microscope. The mean fluorescence intensity was obtained by image processing software ImageJ analysis.
2.9CCK-8 method for detecting cell activity
(1) Culturing O L N-93 oligodendrocyte in 96-well plate with cell amount of 8 × 104And then cultured in a cell culture box overnight.
(2) Cells were treated with 1. mu.g/m L lipopolysaccharide (L PS) for 16h and rinsed 3 times with PBS.
(3) Cells were treated with either 5. mu.M, 10. mu.M and 30. mu. M A967079 for 30min, then 30. mu.M L PC for 3h, or 1. mu.M, 10. mu.M, 30. mu.M, 50. mu.M, 100. mu.M L PC alone for 3 h.
(4) Adding CCK-8, incubating for 2h in a cell incubator, and detecting the absorbance value by using an enzyme-linked immunosorbent assay.
2.10L DH method for detecting cytotoxicity
(1) Culturing O L N-93 oligodendrocyte in 96-well plate with cell amount of 5 × 104And then cultured in a cell culture box overnight.
(2) Cells were treated with 1. mu.g/m L lipopolysaccharide (L PS) for 16h and rinsed 3 times with PBS.
(3) 0.5. mu.M, 2. mu.M, 5. mu.M and 30. mu.M AP-18, respectively; 30 μ M Ac-YVAD-cmk; 100 μ M spermidine; 100 μ M GdCl3Cells were treated with either 50. mu.M L PC for 3h after 30min at 10. mu.M mitoTEMPO or 10. mu.M, 50. mu.M, 100. mu.M L PC alone for 3 h.
(4) Adding L DH detection reagent, incubating for 30min at room temperature in dark place, and detecting absorbance value with enzyme labeling instrument.
2.11 statistical treatment
Statistical analysis and data mapping were performed on the data in this study using GraphPad Prism 8 software, with each statistical data being represented by mean plus minus standard error (± SEM). The statistical significance is that Holm-Sidak's are used for multi-factor variance analysis, and the judgment standard of significance difference is that data have significance when the p value is smaller than 0.05.
3. Results
3.1 expression of TRPA1 and nNOS in O L N-93 oligodendrocytes
The expression of TRPA1 and neural nitric oxide synthase (nNOS) on O L N-93 oligodendrocytes was examined by immunofluorescence (FIG. 1). The results indicate that TRPA1 (FIG. 1A) and nNOS (FIG. 1B) are widely expressed on O L N-93 oligodendrocytes.
3.2 participation of TRPA1 in L PC-induced intracellular and mitochondrial calcium ion influx
The dynamic change condition of cytoplasm and mitochondrial calcium ions is detected by calcium ion imaging (figure 2). The result shows that L PC can obviously cause the inner flow of cytoplasm and mitochondrial calcium ions in O L N-93 oligodendrocyte, and TRPA1 specific inhibitor AP-18 or A967079 can obviously inhibit the inner flow of calcium ions caused by L PC3Inhibition of calcium influx induced by L PC (FIG. 2A). L PC can continuously stimulate O L N-93 to produce calcium influx, which is inhibited by TRPA1 inhibitor, TRPV broad-spectrum inhibitor and cation channel inhibitor (FIG. 2B). statistical analysis shows that the inhibition of calcium influx by TRPA1 inhibitor, TRPV broad-spectrum inhibitor and cation channel inhibitor is significant compared with that induced by L PC alone (the formula****p<0.0001) (fig. 2C).
3.3TRPA1 is involved in regulating O L N-93 oligodendrocyte mitochondrial reactive oxygen species production
The mitobox probe for detecting the production of mitochondrial active oxygen of O L N-93 oligodendrocytes under the induction of L PC (figure 3) shows that L PC can obviously cause the production of mitochondrial active oxygen, and TRPA1 inhibitor A967079 or HC030031 can obviously inhibit the production of mitochondrial active oxygen caused by L PC (figure 3A) statistical analysis shows that the inhibition effect of the TRPA1 inhibitor on the production of mitochondrial active oxygen is significant compared with the stimulation of only L PC (figure 3A)****p<0.0001) (fig. 3B).
3.4TRPA1 is involved in regulating the decrease in mitochondrial membrane potential of O L N-93 oligodendrocytes
Detection Using mitochondrial Membrane potential probes JC-1 and TMRMThe results of measuring the change of the mitochondrial membrane potential of O L N-93 oligodendrocyte (oligodendrocyte) under the induction of L PC (figure 4) show that L PC can obviously cause the reduction of the mitochondrial membrane potential, while the TRPA1 inhibitor A967079 can obviously inhibit the reduction of the mitochondrial membrane potential caused by L PC (figure 4A) statistical analysis shows that compared with the stimulation of L PC only, the TRPA1 inhibitor has obvious inhibition effect on the reduction of the mitochondrial membrane potential (figure 4A) (the TRPA1 inhibitor has the obvious inhibition effect on the reduction of the mitochondrial membrane potential)***p<0.001) (fig. 4B), L PC is time-dependent in the process of O L N-93 mitochondrial membrane potential reduction, while TRPA1 inhibitors could suppress this trend (fig. 4C).
3.5 Co-participation of TRPA1 and nNOS in the modulation of O L N-93 oligodendrocyte nitric oxide production
The NO generation condition of O L N-93 oligodendrocytes under the induction of L PC (shown in figure 5) is detected by using a Nitric Oxide (NO) specific fluorescent probe DAF-FM DA (digital addressable domain-FM-DA)), the result shows that L PC can obviously cause NO generation, and the TRPA1 inhibitor A967079 and the nNOS specific inhibitor speramine can obviously inhibit the NO generation caused by L PC (shown in figure 5A), statistical analysis shows that the inhibition effect of the TRPA1 and the nNOS inhibitor on the NO generation is obvious compared with that only stimulated by L PC (shown in figure 5A) ((the inhibition effect of the TRPA1 and the nNOS inhibitor on the NO generation is obvious)****p<0.0001) (fig. 5B).
3.6TRPA1 is involved in regulating O L N-93 oligodendrocyte superoxide formation
The generation situation of O L N-93 oligodendrocyte superoxide is detected by using a superoxide fluorescent probe DHE under the induction of L PC (figure 6). The result shows that L PC can obviously cause the generation of superoxide, while the TRPA1 inhibitor A967079 can obviously inhibit the generation of superoxide caused by L PC (figure 6A). statistical analysis shows that compared with the stimulation of L PC only, the TRPA1 inhibitor has obvious inhibition effect on the generation of superoxide (as shown in figure 6A) (the)****p<0.0001) (fig. 6B).
3.7TRPA1 is involved in regulating the production of O L N-93 oligodendrocyte cytotoxicity
The change in cellular activity of O L N-93 oligodendrocytes induced by L PC was examined using CCK-8 (FIG. 7A). The results show that lipopolysaccharide (L PS) does not cause significant damage to the activity of O L N-93 while causing inflammation at a concentration of 100ng/m L. the results show that the activity of O L N-93 oligodendrocytes gradually decreases as the concentration of L PC increases, whereas TR is usedThe PA1 inhibitor A967079 can obviously inhibit activity reduction caused by L PC, statistical analysis shows that compared with L PC stimulation, the TRPA1 inhibitor has significant inhibition effect on cell activity reduction caused by L PC (the method is shown in the specification)****p<0.0001，##p<0.01; # vers L PS, # vers 30. mu.M L PC; NC denotes no signalicicane) (FIG. 7A.) the change in cytotoxicity of O L N-93 oligodendrocytes under L PC induction was examined using L DH (FIG. 7B). The results indicated that the toxicity of O L N-93 oligodendrocytes gradually increased with increasing L PC concentration, whereas the TRPA1 inhibitor AP-18, the caspase-1 inhibitor Ac-YVAD-cmk, the nNOS inhibitor speramidine, the mitochondrial active oxygen inhibitor mitoTEMPO or the cationic channel protein inhibitor GdCl3The activity reduction caused by L PC can be obviously inhibited, statistical analysis shows that compared with the stimulation of L PC, the inhibition effect of TRPA1 inhibitor, caspase-1 inhibitor, nNOS inhibitor, mitochondrial active oxygen inhibitor or cation channel protein inhibitor on the cytotoxicity increase caused by L PC is significant (the inhibition effect of the inhibitor on the cytotoxicity increase caused by the L PC is obvious)*p<0.05，***p<0.001，****p<0.0001，####p<0.0001; # versus 50. mu.M L PC, # versus L PS) (FIG. 7B).
The research result shows that TRPA1 has wide expression in O L N-93 cells, TRPA1 is involved in O L N-93 oligodendrocyte calcium ion influx caused by L PC, mitochondrial active oxygen generation, mitochondrial membrane potential reduction, nitric oxide generation, superoxide increase, cell activity reduction and cytotoxicity increase, and the results show that TRPA1 may have an important role in demyelinating diseases such as multiple sclerosis caused by L PC and the like and can be used as a potential target for treating multiple sclerosis caused by L PC.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Claims (6)
- Use of an inhibitor of TRPA1 in the manufacture of a medicament for the treatment of multiple sclerosis.
- 2. The use according to claim 1, for the manufacture of a medicament for the treatment of multiple sclerosis caused by lysophosphatidylcholine.
- 3. The use according to claim 1 or 2, wherein said inhibitor of TRPA1 is HC030031, AP-18, or a 967079.
- 4. A medicament for treating multiple sclerosis, comprising an inhibitor of TRPA1 as an active ingredient.
- 5. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition is a lysophosphatidylcholine-induced multiple sclerosis drug.
- 6. The medicament for treating multiple sclerosis according to claim 4, wherein said inhibitor of TRPA1 is HC030031, AP-18 or A967079.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114903995A (en) * | 2022-06-02 | 2022-08-16 | 中国人民解放军军事科学院军事医学研究院 | Application of TRPA1 ion channel as drug target in amide herbicide poisoning |
| CN114903995B (en) * | 2022-06-02 | 2023-07-28 | 中国人民解放军军事科学院军事医学研究院 | Application of TRPA1 ion channel as drug target in amide herbicide poisoning |
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