Application of disulfiram and / or irisin in preparation of medicine for treating aconitine poisoning

Abstract

The invention discloses an application of disulfiram and / or irisin in preparation of a medicine for treating aconitine poisoning. It is found through a large number of attempts that disulfiram can inhibit abnormal expression of alpha-syn in central nervous system caused by aconitine, irisin can inhibit aggregation of alpha-syn, both disulfiram and irisin can rescue PI3K / Akt / mTOR pathway abnormity caused by aconitine, disulfiram or irisin is applied to dyskinesia caused by aconitine, the remarkable improvement effect can be achieved, and the application range of disulfiram or irisin is widened. And an effective strategy is provided for toxicity reduction treatment of clinical aconitine-containing drug poisoning.

Description

Technical Field

[0001] The present invention specifically relates to the use of disulfiram and / or irisin in the preparation of a medicament for treating aconitine poisoning. Background Art

[0002] Chinese medicinal herbs of the genus Aconitum in the Ranunculaceae family, such as Radix Aconiti, Radix Aconiti Kusnezoffii, Lateralis Radix Aconiti, Herba Aculeti, and Radix Aconiti Szechenyiani, can be clinically used for analgesia, anti - inflammation, anesthesia, blood pressure reduction, etc. However, improper processing, insufficient decocting time, accidental ingestion, or excessive drinking are likely to cause aconitine poisoning. Aconitine is a highly toxic component, and its effect on the nervous system is mainly manifested as excitation first and then inhibition, which can lead to convulsions, coma, and even death in severe cases. When using drugs containing aconitine, caution must be exercised and medical advice and processing methods must be strictly followed to avoid poisoning.

[0003] Aconitine poisoning is a serious clinical emergency, mainly manifested in three major symptom complexes of the nervous system, cardiovascular system, and digestive system. In patients with mild nervous system poisoning, paresthesia and numbness around the mouth and face are manifested, and some patients may experience dizziness, tinnitus, and sweating; in severe patients, general numbness, limb stiffness, restlessness, blurred vision, headache, convulsions, and even coma may occur. Palpitation and chest tightness in the cardiovascular system are extremely common. When blood pressure drops and shock occur, it can be manifested as pale complexion, cold extremities, profuse sweating, and various arrhythmias, resulting in cardiogenic shock and cardiac arrest. The digestive system shows symptoms such as nausea, vomiting, abdominal pain, and diarrhea. Therefore, rapid recognition and timely treatment are required. For patients with nervous system symptoms such as convulsions and seizures, currently, sedative drugs such as diazepam are generally used for symptomatic treatment; for patients with respiratory failure, respiratory support treatments such as endotracheal intubation and mechanical ventilation are required; for patients with severe poisoning symptoms and high blood drug concentrations, blood purification methods such as hemoperfusion and hemodialysis can be used to remove the absorbed aconitine in the body and reduce the blood drug concentration.

[0004] Disulfiram, also known as tetraethylthiuram disulfide or antabuse, is an organic compound (chemical formula C 10 H 20 N2S4). It was initially used as a vulcanization accelerator and fungicide in the rubber industry. Modern pharmacological studies show that it interferes with the alcohol metabolism process, causing discomfort reactions in drinkers after drinking, thus achieving the purpose of alcohol abstinence. Due to the neurotoxicity and psychiatric side effects of disulfiram itself, there is currently no report on its application in the attenuation treatment of drug - induced neurotoxicity.

[0005] Irisin is a hormone secreted by muscles and has various physiological functions, showing extensive application potential in aspects such as energy metabolism, nervous system protection, bone health, cardiovascular protection, and anti-tumor. Modern pharmacological studies have shown that irisin can promote the formation of brown adipose tissue, increase energy consumption, and help reduce body weight; it can regulate blood glucose metabolism, improve insulin sensitivity, and has potential value for the management of type 2 diabetes; it has shown the effects of protecting neurons and improving mitochondrial function in models such as Parkinson's disease and cerebral ischemia-reperfusion injury. However, there is currently no pharmacological data supporting the use of irisin in the treatment of drug-induced neurotoxicity. Summary of the Invention

[0006] To solve the above problems, the present invention provides the use of disulfiram and / or irisin in the preparation of a drug for treating aconitine poisoning.

[0007] Furthermore, the drug is a drug for reducing the central neurotoxicity caused by aconitine.

[0008] Furthermore, the drug is a drug for relieving neuron damage caused by aconitine.

[0009] Furthermore, the drug is a drug for relieving neuronal pyroptosis caused by aconitine.

[0010] Even further, the drug is a drug for relieving neuronal pyroptosis caused by aconitine by inhibiting the expression or aggregation of α-syn.

[0011] Even further, the drug is a drug for relieving neuronal pyroptosis caused by aconitine by rescuing the PI3K / Akt / mTOR pathway.

[0012] Furthermore, the drug is a drug for relieving the motor ability disorder caused by aconitine.

[0013] Even further, the motor ability disorder includes difficulties in motor control, coordination, strength, endurance, and / or flexibility

[0014] Furthermore, the drug is a preparation prepared from disulfiram and / or irisin as the active ingredient plus pharmaceutically acceptable excipients.

[0015] Even further, the preparation is an oral preparation or an injection.

[0016] The use of disulfiram and / or irisin in the preparation of a medicament for treating aconitine poisoning is based on the total alkaloids before and after processing Aconitum pendulum Busch with tsampa, as well as the main alkaloid monomers aconitine and benzoylaconine as the research objects. It is found that the manifestation of aconitine-induced neuronal damage is a large number of pyroptosis, and neuronal pyroptosis is also related to the abnormal accumulation of α-syn and the up-regulation of the PI3K / AKT / mTOR pathway. Through a large number of attempts, it is found that disulfiram can inhibit the abnormal expression of α-syn in the central nervous system induced by aconitine, and irisin can inhibit the aggregation of α-syn. Both can rescue the abnormal PI3K / Akt / mTOR pathway caused by aconitine. Applying disulfiram or irisin to the motor ability disorder caused by aconitine can significantly improve the damaged motor function, providing an effective strategy for the detoxification treatment of clinical aconitine-containing drug poisoning.

[0017] Obviously, based on the above content of the present invention, according to the common general knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0018] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Brief Description of the Drawings

[0019] Figure 1 Expression and distribution of neuronal p-α-syn(Ser129) under different drug interventions; (A) Schematic diagram of the distribution of p-α-syn(Ser129)+NeuN+ double-positive signals (red signal points) in different coronal sections of mice; (B) p-α-syn(Ser129) + (green) NeuN + (red) Detailed expression map in different specific brain regions; (C) p-α-syn(Ser129)+NeuN + Statistical expression of co-labeling in specific brain regions;

[0020] Figure 2 Effect of α-syn knockdown on pyroptosis of primary neurons; (A and B) Verification and statistics of siRNA knockdown efficiency; (C and D) Flow cytometry detection of the effect of different drug interventions and Snca knockdown on the proportion of Casp1 + PI + Cells and its statistics;

[0021] Figure 3Investigation on the interaction relationship between nucleolin and α-Syn after aconitine administration; (A) WB expression verification of bait protein; (B) Venn diagram of the statistical quantity of differential proteins in IP / MS; (C) Protein silver staining map; (D) PPI interaction map; (E) WB map of Co-IP experimental results; (F and G) Detection and statistical chart of α-Syn phosphorylation and Ncl expression levels under different drug interventions;

[0022] Figure 4 Protein expression and its statistics of α-syn and Ncl in the cerebral cortex of mice at different time points after administration under different drug interventions (A: AC, B: AC + Irisin, C: AC + AS1411);

[0023] Figure 5 Effects of different drugs on the PI3K / Akt / mTOR pathway and pyroptosis in the cerebral cortex of mice (A) and its statistics (B);

[0024] Figure 6 Evaluation of the motor ability of mice under different drug interventions; (A and B) Classic schematic diagram of the tail suspension test mice at 8 h after administration and score statistics under different drug interventions and at different time points; (C) Rotarod test of mice under different drug interventions; (D) Grip strength test of mice under different drug interventions. Detailed implementation method

[0025] Example 1 Research on reducing the central nervous toxicity of aconitine

[0026] Aconitum pendulum Busch is a plant of the genus Aconitum in the family Ranunculaceae. It contains diester alkaloid components such as aconitine and has certain toxicity. However, after stir-frying with zanba, the diester alkaloids undergo ester hydrolysis to form monoester alkaloids such as benzoylaconine, which can reduce the toxicity of Aconitum pendulum Busch. Based on this, the total alkaloids before and after zanba processing of Aconitum pendulum Busch, as well as the main alkaloid monomers aconitine and benzoylaconine, were respectively applied to a mouse model. Through the observation and analysis of the behavior, brain tissue morphology, protein and neurotransmitter-related metabolite expression, and neuronal ultrastructure of mice, it was found that the manifestation of aconitine-induced neuronal damage was a large number of cell pyroptosis. Further analysis of the cerebral cortex tissue of mice after aconitine administration based on transcriptome combined with proteomics found that aconitine-induced neuronal pyroptosis may be related to the abnormal accumulation of α-syn and the upregulation of the PI3K / AKT / mTOR pathway. Therefore, drugs related to the abnormal expression of α-syn or the PI3K / AKT / mTOR pathway were tried to intervene in the aconitine poisoning mouse model to verify the mechanism conjecture of aconitine-induced central nerve damage and simultaneously explore the treatment targets for aconitine poisoning, providing an effective strategy for the detoxification treatment of clinical aconitine-containing drug poisoning.

[0027] I. Effects of drugs intervening in aconitine poisoning on the phosphorylation expression and distribution of α-syn

[0028] 1 Experimental materials

[0029] 1.1 Experimental drugs

[0030] Aconitine (batch number: DSTDW000602) and benzoylaconine (batch number: DSTDB005502) were both purchased from Chengdu Dest Biotechnology Co., Ltd., and their purities were both greater than 98%. Disulfiram (DSF, HY - B0240) with a purity of 99.78% and Irisin (HY - P72534, 95%) were both purchased from MedChemExpress.

[0031] 1.2 Experimental animals

[0032] Male C57BL / 6 mice, 8 - 10 weeks old, with body weights controlled between 20 - 25 g, were from the Experimental Animal Center of Xi'an Jiaotong University. They were fasted for 12 h before the experiment with free access to water. The filing number for the ethical review application for the welfare of experimental animals in this study was No: 202301126.

[0033] 2 Experimental methods

[0034] 2.1 Experimental grouping and drug intervention

[0035] The mice were randomly divided into a control group (Ctrl), an aconitine group (AC), a benzoylaconine group (BAC), an aconitine + disulfiram group (AC + DSF), and an aconitine + irisin group (AC + Irisin). Aconitine and benzoylaconine were both administered by gavage at a dose of 0.8 mg / kg, disulfiram was administered by gavage at a dose of 50 mg / kg, irisin was injected intraperitoneally at a dose of 0.1 mg / kg, and the control group was gavaged with the same volume of 1×PBS.

[0036] 2.2 Sampling

[0037] At 8 h after drug administration, the mice were quickly perfused with pre - cooled 1×PBS and 4% PFA through the heart, and then the whole brain tissues of the mice were quickly taken out on ice and immersed in 4% PFA for post - fixation for 24 h. Subsequently, they were embedded with OCT. After each brain tissue was embedded, coronal sections were made from the rostral end to the caudal end, and 6 coronal sections were selected for mounting and observation of typical brain regions, including the olfactory bulb (OB), medial prefrontal cortex (mPFC), striatum (Str), piriform cortex (Pir), lateral amygdala (LA), and substantia nigra (SN).

[0038] 2.3 Immunohistochemical staining

[0039] (1) After taking out the slices from the refrigerator and restoring them to room temperature, bake the slices in an oven at 50 °C for 30 min, then take them out and restore them to room temperature;

[0040] (2) Wash with 1×PBS three times, 10 min each time. Then perform heat antigen retrieval (Tris-EDTA, pH = 9), microwave at low power for 8 min, and take it out and restore it to room temperature;

[0041] (3) Block and punch holes. Draw a circle around the tissue and then prepare the hole-blocking solution with the following formula: 0.2% Triton X-100 + 10% donkey serum + 1×PBS. Place the slices in a wet box containing a small amount of deionized water, add the hole-blocking solution, and let it stand at room temperature for 1.5 h;

[0042] (4) Incubate the primary antibody at low temperature. Prepare the primary antibody with the following formula: 0.1% Triton X-100 + 10% donkey serum + 1×PBS + primary antibody (appropriate dilution ratio). After discarding the hole-blocking solution, add the primary antibody and place it in the dark in a 4 °C refrigerator overnight;

[0043] (5) Incubate the secondary antibody at room temperature. After discarding the primary antibody, wash with 1×PBS three times, 10 min each time. Prepare the secondary antibody with the following formula: 0.01% Triton X-100 + 5% donkey serum + 1×PBS + secondary antibody (appropriate dilution ratio), and incubate at room temperature in the dark for 2 h;

[0044] (6) Stain the nucleus. After discarding the secondary antibody, wash with 1×PBS three times, 10 min each time. Incubate with 1 μg / mL DAPI for 10 min;

[0045] (7) Mount the slides. After discarding the DAPI, wash with 1×PBS three times, 10 min each time, then add an anti-fluorescence quencher to mount the slides, air-dry in the dark at room temperature, and store at 4 °C.

[0046] 2.4 Statistical methods

[0047] Statistical analysis was performed using GraphPad Prism 9.5, and the data were all expressed as mean ± standard error of the mean (means ± SEMs). One-way RMANOVA was used for inter-group analysis. *p < 0.05 was considered to have a significant difference, **p < 0.01 and ***p < 0.001 were considered to have a highly significant difference in statistical significance, and ns indicated no significant difference.

[0048] 3 Experimental results

[0049] 3.1 Expression and distribution of p-α-syn(Ser129) in the central nervous system (CNS) after aconitine administration

[0050] p-α-syn(Ser129) in 6 different levels of mouse brain tissue + (green) and NeuN + (red) co-labeling results (shown as red signal dots in the schematic diagram). ( Figure 1 A), after aconitine administration, compared with the control group, the p-α-syn(Ser129) signal in neurons was mostly distributed in the cerebral cortex region of mice, and was extremely significantly increased in the OB, mPFC, Pir, and LA regions. In the benzoylaconine group, the ester hydrolysis product, compared with the control group, there was only a significant difference in the OB region, and no significant differences in the mPFC, Pir, and LA regions. Subsequently, under the intervention of DSF and irisin, we found that compared with the aconitine group, the co-labeling results of p-α-syn(Ser129) + and NeuN + and NeuN + significantly decreased, indicating that after the alleviation of pyroptosis, the phosphorylation level of α-syn at Ser129 could also be alleviated. It should be noted that we further saw in the detailed images of each brain region ( Figure 1 B–C), although the double-positive signals of p-α-syn(Ser129) + and NeuN + were widely distributed in the cerebral cortex, in all drug treatment groups, p-α-syn(Ser129) + was not detected in the Str and SN regions.

[0051] 4 Discussion

[0052] p-α-syn(Ser129) has a high co-labeling rate with NeuN and is concentrated in the cerebral cortex region. The abnormal accumulation of α-syn in neurons ultimately leads to neuronal death. As an abnormal phosphorylation form of α-syn, p-α-syn(Ser129) may induce toxic aggregation in neurons, thereby leading to the occurrence of pyroptosis.

[0053] In addition, it is worth mentioning that different brain regions have different sensitivities to aconitine. p-α-syn(Ser129) is only expressed in the OB, mPFC, Pir, and LA, but not in the Str and SN. The OB, the starting region of the olfactory system, may be more vulnerable to damage due to direct exposure to environmental toxins or a relatively weak blood-brain barrier. The mPFC (medial prefrontal cortex), which is involved in cognition, decision-making, and emotion regulation, has neurons that are highly sensitive to oxidative stress and mitochondrial dysfunction. The Pir (piriform cortex) is a key region for olfactory processing and may form a functional network with the OB to respond to toxic stimuli synergistically. The LA (lateral amygdala) is the core region of emotional memory, and its neurons are rich in glucocorticoid receptors and are vulnerable to stress-related toxicity. The striatum mainly contains dopaminergic and GABAergic neurons. Aconitine may indirectly affect its function by inhibiting dopamine transporters or interfering with mitochondrial function, but it may not directly induce α-syn phosphorylation. The substantia nigra does not express this, which may be different from Parkinson's disease (PD), and the reason remains to be further explored.

[0054] 5 Summary

[0055] The experimental verification results show that aconitine administration can indeed cause abnormal expression of α-syn phosphorylation, mainly concentrated in the cerebral cortex region of mice, and the main brain regions are the OB, mPFC, Pir, and LA, and not expressed in the Str and SN. There is no significant difference between its ester hydrolysis product benzoylaconine and the control group, indicating that the processing after ester hydrolysis prevents the over-phosphorylation of α-syn. When DSF intervention inhibits pyroptosis, p-α-syn(Ser129) is also alleviated, indicating that pyroptosis is one of the reasons for the abnormal increase of p-α-syn(Ser129). As an inhibitor of GSDMD, DSF may have a good detoxification effect. Surprisingly, irisin, as a hormone that regulates muscle energy metabolism, can also inhibit α-syn aggregation and is also expected to have a positive effect on reducing the neurotoxicity of aconitine.

[0056] II. Effects of drugs intervening in aconitine poisoning on neuronal pyroptosis

[0057] 1 Experimental materials

[0058] 1.1 Experimental drugs

[0059] Aconitine (AC) (batch number: DSTDW000602) and benzoylaconine (batch number: DSTDB005502) were both purchased from Chengdu Deste Biotechnology Co., Ltd., and their purities were both greater than 98%. Disulfiram (DSF, HY-B0240, 99.78%) was purchased from MedChemExpress (USA).

[0060] 1.2 Experimental animals

[0061] C57BL / 6 male and female mice, 10 - 12 weeks old, with body weight controlled between 20 - 25 g, were sourced from the Experimental Animal Center of Xi'an Jiaotong University. Pregnant mice were specifically bred within the group. The filing number for the ethical review application of experimental animal welfare in this study is No: 202301126.

[0062] 2 Experimental methods

[0063] 2.1 Primary neuron culture

[0064] (1) Preparation: Coating of cell culture dishes or sterilized cell slides: Add an appropriate amount of 0.1 mg / ml polylysine to cover the bottom or the slide, incubate overnight at 37°C in an incubator, and wash 3 times or more with sterile water or 1×PBS the next day, then seal and set aside. Polylysine can be recycled 1 - 3 times for repeated use. Surgical instruments (including a set of precision ophthalmic instruments) are cleaned, sterilized, and dried for later use;

[0065] (2) Use the consumables and instruments after ultraviolet irradiation for 30 min before sampling;

[0066] (3) Isolation of fetal mice: Pregnant wild - type C57BL / 6J mice containing embryos at 14 - 15 days are deeply anesthetized, and under sterile conditions, the abdominal skin and subcutaneous fascia are cut open to expose and separate the uterus. The uterus is taken out and placed in a culture dish containing D - Hanks solution for washing. Use scissors and forceps to separate the fetal mice;

[0067] (4) Isolation of the cerebral cortex: Place the fetal mouse in a 10 - cm dish, fix the mouse's head with a fine curved forceps (insert near the eyes, not too tightly), make an incision in the center of the mouse's brain with a fine straight forceps, peel off the skin on it until the entire brain is exposed, take out the brain with forceps, remove the cerebellum, striatum and other remaining parts, and further dissect out the complete fetal mouse cerebral cortex;

[0068] (5) Tissue digestion: Cut the cortex tissue into small pieces with fine scissors and transfer it to pre - cooled digestive solution. Use a 10 - mL pipette to gently pipette the cells several times on ice and then let it stand on ice for 5 min; after fully digesting into single cells, filter through a 0.45 - μm cell sieve, collect them, and centrifuge at 1000 rpm for 5 min to collect the cell pellet;

[0069] (6) Wash the cells: Gently pipette with D - Hanks solution to resuspend the cell pellet for washing. Then centrifuge to collect the pellet;

[0070] (7) Resuspend with culture medium and inoculate in the pre - coated culture dish at the required density. Culture the neurons in a 5% CO2 incubator at 37°C for 3 - 7 days, and change the culture medium every three and a half days.

[0071] 2.2 Construction and verification of siRNA plasmids

[0072] To study the effect of α-syn (Snca) knockdown on pyroptosis, three kinds of siRNA Snca were constructed by Hanheng Biotechnology Co., Ltd. and 1.5 μg was transfected into primary neurons by electroporation. Cell proteins were collected, and then their efficiency was verified by WB. The most effective siRNA was selected for further experiments. The sequences are shown in Table 1.

[0073] Table 1 Sequences of siRNA Snca

[0074]

[0075] 2.3 Electroporation and drug intervention of primary neurons

[0076] Primary neurons have relatively fragile cell structures and highly differentiated characteristics, and belong to cell types that are difficult to transfect. Therefore, the siRNA was transfected using the electroporation technique of Buchser et al. Electroporation was performed using 800 μL of electroporation buffer and Petri pulse electrodes, with a pulse of 340 V for 900 μs. After electroporation, the cells were switched back to the normal culture medium for primary neurons and continued to be cultured for use.

[0077] Experimental groups: control group, aconitine group, benzoylaconine group, aconitine + DSF group, aconitine + siRNA Snca group, aconitine + siRNA NC group. According to the experimental analysis, drug intervention was carried out. The administration concentrations of aconitine and benzoylaconine were 400 μM, the administration concentration of DSF was 10 nM, and 400 μM of aconitine was administered immediately after electroporation of siRNA Snca or NC. The drugs were treated for 48 h, and flow cytometry was prepared for detection.

[0078] 2.4 Detection of Casp1 by flow cytometry + PI + Number of cells

[0079] According to Operate according to the instruction manual of the Caspase-1(WEHD) detection kit: (1) Neurons after drug treatment and electroporation for 48 h were used as the samples to be treated; (2) Dilute the 10× wash buffer 1:10 with cell-grade deionized water, dissolve FLICA with 50 μL of dimethyl sulfoxide (DMSO), and add 200 μL of PBS to dilute FLICA 1.5 times; (3) Add the diluted FLICA to each sample at a ratio of 1:30; (4) Incubate for about 1 h; (5) Dilute propidium iodide (PI) to 1× for staining; (6) Aspirate the culture medium, wash the cells 3 times with 1× wash buffer, then centrifuge to collect the cells, and filter through a nylon filter membrane for loading onto the machine; (7) Analyze with a flow cytometer. The excitation wavelength of FAM-FLICA is 492 nm, and the emission wavelength is 520 nm.

[0080] 2.5 Statistical methods

[0081] The experimental data in this study were statistically analyzed using GraphPad Prism 9.5, and the data were expressed as mean ± standard error of the mean (means ± SEMs). When there were two independent variables in the experiment, two-way repeated measures (RM) ANOVA was used. When comparing data among multiple groups (>2) of the same variable, one-way repeated measures (RM) ANOVA was used. When comparing two groups of experimental data, an independent t-test was used for statistics. *p < 0.05 was considered to have a significant difference, **p < 0.01 and ***p < 0.001 were considered to have a highly significant difference in statistical significance, and ns was considered to have no significant difference.

[0082] 3 Experimental results

[0083] 3.1 siRNA1 had the best knockdown efficiency

[0084] From the WB results, we could clearly see that all 3 siRNAs had good knockdown effects. According to the statistical results of 3 replicates, we finally selected siRNA1 to continue the subsequent experiments ( Figure 2 A–B).

[0085] 3.2 Knockdown of siRNASnca alleviated aconitine-induced neuronal pyroptosis

[0086] As Figure 2 C–D shown, Casp1 + PI +The cells were mainly concentrated in region Q2. From the perspective of the number of cells in region Q2, compared with the control group, the aconitine group was more than three times higher, showing a highly significant difference. The benzoylaconine group at 10.2% was almost the same as 10.5% in the control group, showing no significant difference. After treatment with aconitine and simultaneous intervention with the pyroptosis inhibitor DSF, it was found that the proportion of Q2 decreased significantly compared with the simple aconitine group, showing an obvious effect of inhibiting pyroptosis. It is worth noting that after knocking down α-syn in the aconitine treatment group, it was found that the degree of cell pyroptosis decreased significantly, with Q2 decreasing from 36.8% in the aconitine group to 25.3%, while the siRNA control group was 33.0%.

[0087] 4 Discussion

[0088] Knocking down α-syn on primary neurons alleviated the aconitine-induced cell pyroptosis phenomenon, which fully demonstrated that the abnormal expression of α-syn would exacerbate the effect of aconitine on neuronal pyroptosis.

[0089] By comparing region Q2 between the AC group and the control group, AC was significantly increased by nearly three times, confirming that aconitine could strongly activate the neuronal pyroptosis pathway, which was consistent with previous research results. For example, aconitine might disrupt cell homeostasis and activate Casp1 to trigger cell membrane rupture and the release of inflammatory factors. Moreover, the significant decrease in Q2 after DSF intervention also proved that DSF had the effect of inhibiting aconitine-induced neuronal pyroptosis.

[0090] After knocking down α-syn, Q2 decreased significantly, indicating that α-syn was a key mediator of aconitine-induced cell pyroptosis. It was speculated that it might play a role through the following pathways. For example, abnormal aggregation of α-syn could bind to inflammasomes, promote their self-assembly process, accelerate Casp1 cleavage, and thus induce cell pyroptosis.

[0091] For the benzoylaconine group, there was no significant difference compared with the control group. This indicated that aconitine-induced neuronal pyroptosis had structural specificity, and its toxicity might be related to the unique diester structure of aconitine. It also showed that the ester hydrolysis process promoted by the processing of zanba could indeed reduce neuronal toxicity.

[0092] 5. Conclusion

[0093] The experimental results verified that the administration of aconitine would induce the occurrence of neuronal cell pyroptosis; α-syn was positively correlated with cell pyroptosis, and inhibiting the expression of α-syn would alleviate neuronal pyroptosis after aconitine administration. Targeting α-syn and GSDMD-NT could be used as a treatment strategy for aconitine poisoning clinically. The pyroptosis pore-forming protein GSDMD inhibitor DSF and the α-syn inhibitor irisin were expected to be applied to the clinical treatment of aconitine poisoning.

[0094] III. Molecular Mechanisms of the Interaction Targets between Drugs for Intervention of Aconitine Poisoning and α-syn

[0095] 1 Experimental Materials

[0096] 1.1 Experimental Drugs

[0097] Aconitine (AC, batch number: DSTDW000602) and Benzoylaconine (BAC, batch number: DSTDB005502) were both purchased from Chengdu Dest Biotechnology Co., Ltd., and their purities were both greater than 98%. Irisin (HY-P72534, 95%) and AS1411 (HY-147081, 93.11%) were both purchased from MedChemExpress.

[0098] 1.2 Experimental Animals

[0099] Male C57BL / 6 mice, 8 - 10 weeks old, with body weights controlled between 20 - 25 g, were sourced from the Experimental Animal Center of Xi'an Jiaotong University. They were fasted for 12 h before the experiment with free access to water. The filing number for the ethical review application for the welfare of experimental animals in this study was No: 202301126.

[0100] 2 Experimental Methods

[0101] 2.1 IP in the Cerebral Cortex after Administration of Aconitine and Corresponding Intervention Drugs

[0102] 2.1.1 Preparation of Protein Samples

[0103] (1) The cerebral cortex of mice at 8 h after administration of aconitine and corresponding intervention drugs, 30 - 50 mg (about the size of mung bean - soybean grains); (2) Add 250 μL of weak lysis buffer, containing PMSF and phosphatase inhibitors; (3) Gently grind and homogenize, let stand on ice for 30 min to fully lyse the tissue; (4) Centrifuge at 4°C, 13000 rpm for 15 min. Collect the supernatant and measure BCA; (5) Dilute the protein to 2 mg / mL for use according to the quantification result. At this concentration, about 8 μL of antibody (4 μg) was added to 500 μL of supernatant protein (1000 μg) (volume dilution ratio is about 1:50). (Note: 1 - 2 μg of antibody is used for 100 - 500 μg of protein, then 4 μg = 800 μg of protein).

[0104] 2.1.2 Pretreatment of Magnetic Beads

[0105] (1) Equilibrate the magnetic beads. Add the required protein A / G magnetic beads to a 1.5 mL EP tube, wash with 500 μL of lysis buffer on a magnetic rack by inverting (10 - 20 times), discard the liquid, and repeat 3 times; (Note: Cut off the tip of a 200 μL pipette tip by 0.5 - 1 cm to protect the integrity of the magnetic beads.

[0106] (2) Pre-clearing. Add 5 μL of IgG antibody of the same species and 10 μL of equilibrated magnetic beads to each group of samples (including total proteins of IP and IgG), and mix them in a rotary incubator at 4°C for 1 h;

[0107] (3) Take input after pre-clearing. Transfer the magnetic bead-separated lysate to an EP tube. Take 50 μL of input sample, add 5× loading buffer, denature it in a metal bath at 100°C for 10 min, and store it at -80°C; (Steps 2 - 3 can be omitted if there are no specific bands in the non-IgG group).

[0108] (4) Continue with the following steps for the remaining supernatant.

[0109] 2.1.3 IP reaction

[0110] (1) Add specific antibody p-a-syn(Ser) to the IP sample. For 500 μL of protein supernatant, add 8 μL of antibody (protein amount: antibody amount = 200:1 = 800 μg:4 μg = 500 μL:8 μL). For the negative control IgG sample, add IgG sample of the same species as the IP sample, that is, add 4 μL of antibody to 500 μL of protein supernatant, and mix them in a rotary incubator at 4°C for 3 - 4 h;

[0111] (2) Add 100 μL of equilibrated magnetic beads to the well-mixed IP sample and IgG sample respectively, and mix them in a rotary incubator at 4°C overnight;

[0112] (3) The next day, place the samples on a magnetic stand, gently invert them up and down 10 - 20 times, discard the liquid part, and wash the magnetic beads 3 times repeatedly with IP lysis buffer; (At this time, the magnetic beads adsorbed on the magnetic stand bind the specific molecules pulled down by IP and several proteins bound to them). Add 7.5 μL of 5× loading buffer to 30 μL of the magnetic beads, shake well, and dissociate all the proteins bound to the magnetic beads into the loading buffer in a metal bath at 100°C for 10 min, and then verify its effectiveness by WB experiment, etc.

[0113] 2.2 HPLC-MS / MS qualitative detection of proteins bound by IP

[0114] The identification of IP-bound proteins was entrusted to Beijing Qinglian BioTech Co., Ltd. to complete.

[0115] 2.2.1 Protein extraction quality control

[0116] To detect the content of protein samples obtained after IP and identify the purity, etc., the protein samples are separated by polyacrylamide gel electrophoresis (PAGE). Different protein bands are formed in the gel according to the characteristics of proteins such as size and charge, enabling different proteins to be separated from each other, which facilitates the subsequent detection and analysis of specific proteins. Then, steps such as fixation, sensitization, rinsing, silver staining, color development, and termination of color development are carried out for protein visualization imaging of the gel.

[0117] 2.2.2 Enzymatic desalting

[0118] Protein reduction alkylation: 5 mM DTT (1 h at 37 °C), 10 mM iodoacetamide (45 min at room temperature in the dark). Trypsin digestion: Dilute 4-fold with 25 mM ammonium bicarbonate, protein:trypsin = 50:1 (overnight at 37 °C), terminated with formic acid (pH < 3). C18 desalting: Activate with acetonitrile, equilibrate with 0.1% formic acid, load the sample, wash with 0.1% formic acid, elute with 70% acetonitrile, and lyophilize.

[0119] 2.2.3 Instrument operation

[0120] (1) Mass spectrometry chromatographic conditions: Liquid chromatography - mass spectrometry detection method: Mobile phase A (0.1% formic acid in water) / B (80% acetonitrile + 0.1% formic acid). Dissolve the lyophilized sample in 10 μL of mobile phase A, centrifuge at 14000 g for 20 min at 4 °C, and inject 1 μg of the supernatant. Orbitrap Exploris 480 mass spectrometer (FAIMS Pro CV - 45 / -65 V switching): NSI source (2.0 kV, 320 °C), data - dependent acquisition (m / z 350 - 1500). Primary parameters: Resolution 120000 (at 200 m / z), AGC 300%, IT 50 ms; Secondary Top Speed mode: Resolution 15000 (at 200 m / z), AGC 75%, IT 22 ms, CE 33%, generating.raw data. Liquid chromatography elution conditions are as follows: 0 min, 8% mobile phase B; 5 min, 12% mobile phase B; 35 min, 30% mobile phase B; 44 min, 40% mobile phase B; 45 min, 95% mobile phase B; 60 min, 95% mobile phase B.

[0121] (2) Database search parameters

[0122] The selection of the database is based on the required species, the completeness of database annotation, and the reliability of sequences. Database used this time: Mus_musculus_uniprot database. Search the database using Proteome Discoverer 2.4 software, and set the search parameters as follows:

[0123] Table 2 Database search parameters

[0124]

[0125] 3 Experimental Results

[0126] 3.1 Expression Verification of Bait Protein

[0127] First, the protein samples obtained after the IP reaction were subjected to WB experiments to detect whether the bait protein could be successfully expressed, whether it had the correct structure and activity, and to verify whether it could effectively capture the proteins that interacted with it in subsequent IP experiments. As Figure 3 shown in A, the bait protein p-α-syn(Ser) was expressed in both the Input and IP groups in the control group and the aconitine group, but not in the IgG group. This indicates that the bait protein was working properly during the IP reaction.

[0128] 3.2 PPI Suggests That Nucleolin May Interact with p-α-Syn(Ser129)

[0129] As Figure 3 shown in B, compared with the control group, there were specific proteins that bound to p-α-syn(Ser) in the aconitine group, and approximately 26 were detected. And through the silver staining map, we found differences in the expression of proteins with different molecular weights. For example, there was an up-regulated expression in the aconitine IP group at a protein molecular weight of around 100 ± 10 kD( Figure 3 C). Subsequently, through PPI network interaction analysis and combined with the results of literature research, it was speculated that there might be an interaction between nucleolin (NUCL) and α-syn (SYUA)( Figure 3 D).

[0130] 3.3 Verification of the Interaction between Nucleolin and p-α-syn(Ser129)

[0131] Through the Co-IP results, it was found that when p-α-syn(Ser) was used as the bait protein, a high expression of nucleolin could be detected in the precipitated proteins, indicating an interaction between the two( Figure 3 E). Subsequently, by detecting BAC, as well as the changes in the phosphorylation level of α-syn and the expression level of Ncl after the intervention of AS1411 (nucleolin inhibitor) and irisin (α-syn inhibitor). The results are as Figure 3As shown in F - G, compared with the control group, the phosphorylation level of α - syn and the expression level of Ncl both showed a significant upward trend after aconitine administration, while no difference was observed after BAC administration. Interestingly, after irisin intervention, after the phosphorylation level of α - syn decreased, nucleolin also showed a downward trend. After AS1411 intervention, the expression level of nucleolin showed a significant downward trend compared with the aconitine group. However, at the same time, the phosphorylation level of α - syn showed an opposite increasing trend. This indicates that nucleolin is very likely to play a role in clearing the abnormal accumulation of α - syn.

[0132] 4 Discussion

[0133] After verifying that aconitine administration could cause an abnormal increase in the phosphorylation level of α - syn, IP / MS technology was used to identify another target Ncl that interacts with α - syn. Intervention was carried out with irisin and a nucleic acid aptamer AS1411. AS1411 can specifically bind to Ncl, thereby inhibiting the expression of Ncl and its protein function. As expected, when α - syn was inhibited, the expression of Ncl was not abnormally expressed; when Ncl was inhibited, the phosphorylation level of α - syn was significantly increased compared with the aconitine group. This shows that Ncl can act as a "scavenger" for the abnormal accumulation of α - syn under pathological conditions.

[0134] 5 Summary

[0135] Nucleolin played a mitigating role in the abnormal increase in the phosphorylation level of α - syn caused by aconitine, promoting the clearance of the abnormal accumulation of α - syn. Irisin intervention decreased both the phosphorylation level of α - syn and the expression level of nucleolin, indicating that irisin can clear the abnormal accumulation of α - syn, and at the same time, nucleolin can function normally, thus playing a detoxifying role.

[0136] III. Verification of the drug effects of intervening aconitine poisoning

[0137] 1 Experimental materials

[0138] 1.1 Experimental drugs

[0139] Aconitine (AC, batch number: DSTDW000602), benzoylaconine (BAC, batch number: DSTDB005502) were both purchased from Chengdu Dest Biotechnology Co., Ltd., and the purity was greater than 98%. Disulfiram (DSF, HY - B0240, 99.78%), Irisin (HY - P72534, 95%), AS1411 (HY - 147081, 93.11%) were all purchased from MedChemExpress.

[0140] 1.2 Experimental animals

[0141] C57BL / 6 male mice, 8 - 10 weeks old, with body weight controlled between 20 - 25 g, were sourced from the Experimental Animal Center of Xi'an Jiaotong University. They were fasted for 12 h before the experiment without water restriction. The filing number for the ethical review application of experimental animal welfare in this study was No: 202301126.

[0142] 2 Experimental methods

[0143] 2.1 Protein extraction and quantification

[0144] Pre-cool the pre-prepared PIRA containing 1 mM PMSF and 1% phosphatase inhibitor in advance; after administration of aconitine and its intervention drugs to mice, directly and rapidly collect the cerebral cortex of the mouse brain tissue according to the experimental design without cardiac perfusion, weigh it, add 200 μL of pre-cooled pre-prepared PIRA to 20 mg of brain tissue, then perform tissue homogenization, and place it on ice for lysis for 30 min; centrifuge at 12000 rpm and 4 °C for 15 min; discard the precipitate, aspirate the supernatant, and then perform BCA protein quantification according to the instructions of the BCA kit.

[0145] 2.2 Western blotting

[0146] (1) Gel preparation: Wash the glass plates, and select the appropriate concentration of separating gel and stacking gel for preparation according to the molecular weight of the antibody to be detected. Pour 10% separating gel into the fixed glass plates, and flatten the surface of the gel with about 200 μL of isopropanol, and let it stand for 50 min until it solidifies; then discard the isopropanol, invert the gel preparation rack, add the 5% stacking gel mixture to the upper layer of the separating gel, quickly insert the comb, and let it stand at room temperature for 30 min to completely solidify the stacking gel;

[0147] (2) Protein loading: Add 1×running buffer to the vertical electrophoresis tank, place the gel plate, vertically pull out the comb teeth upwards, and load the protein at a quantity of 30 - 50 ng / well;

[0148] (3) Electrophoresis: After loading, first perform electrophoresis at a low voltage of less than 90 V for 30 min, aiming to align the samples in the stacking gel so that they enter the separating gel at the same starting line. After reaching the separating gel, adjust the voltage to about 120 V and continue electrophoresis until the target protein band runs to the appropriate position. If the target band is small, when it runs to the appropriate position, adjust to a low voltage and electrophoresis for about 10 min to make its band pattern beautiful;

[0149] (4) Transfer membrane: The 1× transfer buffer needs to be pre-cooled in advance, then poured into the transfer tank. After taking out the electrophoresed gel plate, peel off the complete gel strip and remove the stacking gel part, and soak it in the transfer buffer for equilibration. Immediately afterwards, take a PVDF membrane of appropriate size, put it into methanol to make the membrane transparent and quickly activate it, and then soak it in the transfer buffer for equilibration. Then, use the "sandwich" installation method to assemble the black plate - sponge pad - filter paper - gel strip - PVDF membrane - filter paper - sponge pad - white plate in sequence. Then, place it into the transfer tank according to the positive and negative electrode indications, put an ice pack to cool down, and cool down in an ice bath throughout the process to prevent protein degradation caused by excessive heat during the transfer membrane process. Set a constant current of 250 mA for transfer membrane, and set the transfer membrane time at a rate of approximately 1 KD / min according to the protein size;

[0150] (5) Blocking: After the transfer membrane is completed, according to the characteristics of the target protein, select an appropriate blocking solution and place it on a shaker to slowly shake for room temperature blocking for about 1 - 2 h. Generally, 5% skim milk powder is used as the blocking solution, but for phosphorylated proteins, etc., casein in the milk powder may cause dephosphorylation, so 5% BSA is used for blocking;

[0151] (6) Primary antibody binding: After blocking, transfer the membrane to the primary antibody and incubate it on a shaker at 4℃ overnight with slow shaking;

[0152] (7) Washing the membrane: Discard the primary antibody, soak it in 1×TBST, shake it quickly on a shaker at room temperature at about 100 rpm, wash it 3 times, 10 min each time;

[0153] (8) Secondary antibody binding: Place the membrane in the secondary antibody diluted with 1×TBST with corresponding properties and incubate it on a shaker at room temperature with slow shaking for 2 h;

[0154] (9) Washing the membrane: Discard the secondary antibody, soak it in 1×TBST, shake it quickly on a shaker at room temperature at about 100 rpm, wash it 3 times, 10 min each time;

[0155] (10) Development: Evenly drip the ECL luminescent solution onto the PVDF membrane and perform imaging analysis using a chemiluminescence imager;

[0156] (11) Data analysis: Use the software Image J to analyze the gray value of the protein bands and perform statistics.

[0157] 2.3 Motor behavior

[0158] 2.3.1 Rotarod test

[0159] Both during training and formal detection, mice were placed in the behavior detection room 2 hours in advance for environmental adaptation, and the time was fixed from 19:00 to 24:00 at night. Before the experiment, each mouse was trained for 3 days, twice a day with an interval of 1 hour. During training, the rotarod started at 4 revolutions per minute (r / min) and was uniformly accelerated to 40 r / min within 5 minutes. During training, if a mouse fell off the rotarod continuously three times, it was considered fatigued and the training was terminated. After training, intragastric administration was performed, and experiments were carried out at 0, 4, 8, 12, and 24 hours after administration. The recording of the time on the rod was carried out with an initial rotation speed of 4 r / min and uniformly accelerating to 40 r / min within 5 minutes as the experimental conditions.

[0160] 2.3.2 Grip strength experiment

[0161] Mouse forelimb grip strength detection experiments were carried out at 0, 4, 8, 12, and 24 hours after intragastric administration. The four limbs of the mouse were placed on the grip strength net, and its forelimbs were placed at the front end. Subsequently, the experimenter clicked to record and then moved backward horizontally along the grip strength net at a uniform speed until the mouse was completely separated. Each mouse was detected 3 times, and the average value was the final detection result.

[0162] 2.3.3 Tail suspension experiment

[0163] Performed according to the method described by Wang et al., the mouse was suspended by grasping the root of its tail with the hand and observed for 10 - 15 seconds. Each mouse was independently tested three times a day and scored according to the following criteria: 0 points for normal hindlimb opening, 1 point for brief grasping of one hindlimb, 2 points for brief grasping of both hindlimbs, 3 points for severe and continuous grasping of one hindlimb, 4 points for severe and continuous grasping of both hindlimbs. The higher the score, the more severe the phenotype of hindlimb grasping in the mouse.

[0164] 3 Experimental results

[0165] 3.1 Expressions of α-syn and Ncl proteins in the cerebral cortex of mice at different drug intervention time points

[0166] The phosphorylation level of α-syn in the cerebral cortex of mice administered with aconitine at a dose of 0.8 mg / kg at 2, 4, 8, 12, and 24 hours showed an expression trend of increasing first and then decreasing. Compared with the control group, the phosphorylation level of α-syn was the highest and showed a significant difference at 8 hours after administration. The protein expression trend of Ncl was consistent with that of nascent mRNA, also showing an expression trend of increasing first and then leveling off, and the inflection point was at 8 hours after administration. Compared with the control group, there were significant differences at 8, 12, and 24 hours after administration ( Figure 4 A).

[0167] On the basis of aconitine administration, after intervention with Irisin, the phosphorylation level of α-syn showed a trend of transient decrease at 2 h, followed by a slight increase and then a decrease, but there was no significant difference overall, confirming the inhibitory effect of Irisin. At this time, Ncl was consistent with the change trend of the phosphorylation level of α-syn, also showing a trend of transient decrease at 2 h, followed by a slight increase and then a decrease, and there was no significant difference overall ( Figure 4 B).

[0168] On the basis of aconitine administration, after intervention with AS1411, the phosphorylation level of α-syn also showed a trend of first increasing and then decreasing compared with the aconitine group. The difference was that the peak time of expression advanced from the original 8 h to 4 h, and there were significant differences at 4 h, 8 h and 12 h. Due to the inhibitory effect of AS1411, there was no significant change in Ncl overall( Figure 4 C).

[0169] 3.2 Expression of PI3K / Akt / mTOR pathway and pyroptosis-related proteins in the cerebral cortex of mice under different drug interventions

[0170] Subsequently, at the time point of 8 h after drug administration, the changes in the phosphorylation level of α-syn, the protein level of Ncl, and the expression of PI3K / Akt / mTOR pathway and pyroptosis-related proteins were investigated under the intervention of the ester hydrolysis products BAC, DSF and Irisin. As Figure 5 A and Figure 5 shown in B, compared with the control group, the phosphorylation level of α-syn and Ncl in the aconitine group were significantly increased, while there was no obvious increase in the BAC group, and after the intervention of DSF and Irisin. At the same time, the PI3K / Akt / mTOR pathway was also activated by aconitine, and the phosphorylation levels of each target were significantly increased. Moreover, the pyroptosis-related indexes (GSDMD-NT / FL, p20 / pro-Casp1 and ASC) were significantly up-regulated as in the previous study. There was no significant difference between BAC administration and the control group. Interestingly, when DSF and Irisin were used for intervention at the same time as aconitine administration, aconitine did not cause a series of protein level increases caused by itself, and the two inhibitors showed obvious rescue effects.

[0171] 3.3 Effects of different drug interventions on the motor behavior of mice

[0172] The above research results have identified target drugs that can be applied to the clinical detoxification of aconitine. Subsequently, from the perspective of motor behavior, the motor behavior of different drug interventions was evaluated. As Figure 6 A and Figure 6The results of the mouse tail suspension test are shown in Figure B. From the grasping situation of the hind limbs of the mice, it can be clearly seen that compared with the control group, in the aconitine group, the mice curled up their bodies and their hind limbs were in a continuous grasping state. In the BAC group, the degree of extension of the mice was similar to that of the Ctrl group. Under the intervention of DSF and Irisin, although the mice had slight salivation like the aconitine group, their body postures were relatively natural, and the extension of the hind limbs of the mice was between the aconitine group and the control group, suggesting that after the combined treatment of DSF / Irisin and aconitine, the stress response of the mice caused by aconitine alone was alleviated to a certain extent. It should be noted that compared with the control group, the grasping of the hind limbs of the mice in the AS1411 group was more obvious, and the body posture was similar to that of the aconitine group, indicating that when AS1411 was combined with aconitine, the effect on the mice was more similar to that of aconitine alone, and there may be no obvious alleviation of the stress response caused by aconitine. From the scoring situation of the mouse tail suspension test within 24 hours, the total score of the control group was the least, the BAC group was quite close to the control group, the DSF and Irisin intervention groups were between the control group and the aconitine group, and after the AS1411 intervention, it was close to the aconitine group and even higher than the aconitine group.

[0173] Furthermore, the same phenomenon was also observed in the rotarod and grip strength tests. The motor ability disorders caused by aconitine in the mice were alleviated under the intervention of the BAC group and DSF and Irisin. However, after the AS1411 intervention, it not only did not alleviate but aggravated the motor defects caused by aconitine ( Figure 6 C and Figure 6 D).

[0174] 4 Discussion

[0175] At the protein level, the phosphorylation level of α-syn and the change trend of Ncl within 24 hours under the administration dose of 0.8 mg / kg of aconitine were consistent with the trend of nascent mRNA, and the peak expression time point was 8 hours. This indicates that the regulatory processes of these two proteins from the mRNA level to the protein level (such as transcription, post-transcriptional processing, translation, and post-translational modification, etc.) are relatively stable. Moreover, from the results of exploring the expression trends of pyroptosis-related proteins in the early stage, the highest expression time point of neuronal pyroptosis was also 8 hours. This implies that the time point when aconitine causes the most serious damage to the body's neurons is speculated to be around 8 hours under the administration dose of 0.8 mg / kg.

[0176] From the perspective of the protein expression in the cerebral cortex of mice 8 hours after aconitine administration (α-syn phosphorylation level and proteins related to the Ncl, PI3K / Akt / mTOR pathway and pyroptosis), the ester hydrolysis product BAC of aconitine has a good detoxifying effect, indicating that the "processing to reduce toxicity" of tsampa can still be concluded to relieve neurotoxicity from the perspective of molecular biology. Surprisingly, not only was the scientific theoretical basis for the processing of tsampa to reduce toxicity found, but also two target detoxifying drugs (DSF and Irisin) for pyroptosis and abnormal α-syn accumulation were discovered. After these two drugs were combined with aconitine, the neuronal damage was significantly alleviated compared with the aconitine alone group. At the same time, the dynamic relationship between Ncl and α-syn phosphorylation was clarified, and it was found that Ncl has the role of a "scavenger". When the α-syn phosphorylation level is abnormally elevated, Ncl also increases to prevent abnormal accumulation of α-syn, and when the α-syn phosphorylation level is normal, the expression of Ncl is relatively stable.

[0177] The conclusion obtained from the perspective of molecular biology was verified again by behavioral experiments. From the results of motor behavior, good motor function was shown after BAC, DSF, and Irisin intervention, while the motor function of the AS1411 intervention group showed worse motor disorders compared with the aconitine group.

[0178] 5 Summary

[0179] The peak time of the protein expression levels of Ncl and α-syn phosphorylation after aconitine administration was 8 hours, and Ncl and α-syn had the same expression trend as the nascent transcripts. Moreover, aconitine could activate the PI3K / Akt / mTOR pathway, and the benzoylaconine group and the DSF and irisin intervention groups could rescue the abnormality of this pathway. Motor behavior experiments showed that BAC, DSF, Irisin, and the activator of Ncl could be used as effective clinical treatment means.

[0180] In summary, based on the previous research on the pathological mechanism of aconitine-induced central nerve poisoning, it was found through animal experiments that disulfiram can inhibit the abnormal expression of α-syn in the central nerve caused by aconitine, and irisin can inhibit the aggregation of α-syn. Both can rescue the abnormality of the PI3K / Akt / mTOR pathway caused by aconitine. Applying disulfiram or irisin to the motor ability disorder caused by aconitine can significantly improve the damaged motor function, providing an effective strategy for the detoxification treatment of clinical aconitine-containing drug poisoning.

Claims

1. Use of disulfiram and / or irisin in the preparation of a medicament for treating aconitine poisoning.

2. The use according to claim 1, characterized in that: The medicament is a medicament for reducing the central neurotoxicity caused by aconitine.

3. The use according to claim 2, wherein: The medicament is a medicament for relieving the neuronal damage caused by aconitine.

4. The use according to claim 3, characterized in that: The medicament is a medicament for relieving the neuronal pyroptosis caused by aconitine.

5. The use according to claim 4, characterized in that: The medicament is a medicament for relieving the neuronal pyroptosis caused by aconitine by inhibiting the expression or aggregation of α-syn.

6. The use according to claim 4, wherein: The medicament is a medicament for relieving the neuronal pyroptosis caused by aconitine by rescuing the PI3K / Akt / mTOR pathway.

7. The use according to any one of claims 2 to 6, characterized in that: The medicament is a medicament for relieving the motor impairment caused by aconitine.

8. The use according to claim 7, wherein: The motor impairment includes difficulties in motor control, coordination, strength, endurance and / or flexibility.

9. The use according to claim 1, characterized in that: The medicament is a preparation prepared from disulfiram and / or irisin as active ingredients plus pharmaceutically acceptable excipients.

10. The use according to claim 1, wherein: The preparation is an oral preparation or an injection.

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