Application of N-acetylcysteine amide as preparation for relieving or relieving pesticide poisoning of bees

Abstract

The invention belongs to the cross technical field of biological medicine and agricultural pest control, and particularly relates to application of N-acetylcysteine amide as a preparation for relieving or relieving pesticide poisoning of bees. The invention finds that N-acetylcysteine amide has a remarkable detoxification function on the bee toxic effect induced by dinotefuran. Experimental data confirm that by adjusting redox balance and repairing metabolic steady state, N-acetylcysteine amide can effectively improve death, dyskinesia, apoptosis, oxidative stress injury, metabolic disorder and other multi-dimensional toxicity characterization of bees caused by exposure of dinotefuran. The N-acetylcysteine amide can obviously reverse the destructive effect of dinotefuran on the physiological system of bees, provides a novel solution for preventing and treating the colony poisoning syndrome caused by dinotefuran, and has important application value in the field of ecotoxicology.

Description

Technical Field

[0001] The present invention belongs to the cross-technical field of biomedicine and agricultural pest control, and more specifically, relates to the application of N-acetylcysteine ​​amide as a preparation for reducing or relieving pesticide poisoning of bees. Background Art

[0002] As an important pollinating insect, bees play an irreplaceable role in maintaining biodiversity and ecological balance. They pollinate many plants, promote plant reproduction and seed formation, and are crucial to global food security. According to statistics, about one-third of crops rely on pollinating insects such as bees for pollination.

[0003] With the development of modern agriculture, the widespread use of pesticides has effectively prevented and controlled pests, but has also caused serious harm to beneficial insects such as bees. The frequent occurrence of bee pesticide poisoning not only affects the normal physiological functions and survival and reproduction of bees, but also has a negative impact on the pollination process of plants in the ecosystem, and may also endanger human health through the food chain of bee products. Due to its high toxicity, long persistence, high systemicity and high mobility, neonicotinoid insecticides can cause bee poisoning through floating dust, pollen and dew, causing a series of chronic toxic symptoms such as behavioral disorders, disorientation and slow activity, which in turn leads to a decrease in the number of bees and the occurrence of colony collapsed is order (CCD).

[0004] As a third-generation neonicotinoid pesticide, dinotefuran and its two enantiomers, R-(–)-dinotefuran and S-(+)-dinotefuran, can competitively inhibit nicotinic acetylcholine receptors, causing neurotoxicity in bees, leading to motor dysfunction, oxidative stress damage, metabolic disorders and other multi-dimensional toxic effects in bees. In severe cases, it can even cause the death of bees, affecting the survival and reproduction of the bee colony. Compared with imidacloprid, dinotefuran has a more significant sublethal effect on bees due to its higher water solubility and residuality.

[0005] At present, the detoxification methods and preparations for bee pesticide poisoning are relatively limited and have certain limitations. For example, the Ma Minglin team at Cornell University (Caserto SJ, Wright L, Reese C, et al. Ingestible hydrogel microparticles improve bee health after pesticide exposure [J]. Nature Sustainability, 2024, (prepublish): 1-11.) developed ingestible hydrogel microparticles (IHM) to reduce toxic exposure by physically adsorbing imidacloprid in the digestive tract of bees. Experiments have shown that IHM can absorb 78% of imidacloprid in vitro, increase the survival rate of bees by 30% at a lethal dose, and improve their feeding behavior and motor ability. However, those skilled in the art cannot confirm the detoxification effect of the above-mentioned hydrogel microparticles on the more toxic imidacloprid. For example, the Chinese Academy of Agricultural Sciences (Chen Z, Yao X, Dong F, et al. Ecological toxicity reduction of dinotefuran to honeybee: New perspective from an enantiomeric level [J]. Environment International, 2019, 130104854.) separated the chiral enantiomers of dinotefuran and found that R-(–)-dinotefuran was less toxic to honeybees than S-(+)-dinotefuran. It is recommended to use a single low-toxic enantiomer (R-(–)-dinotefuran) to replace traditional racemic pesticides to reduce harm to bees. Another example is the team led by Li Xuesheng of Guangxi University (Zhang Y, Li H, Chen L, et al. Non-contact exposure to dinotefuran disrupts honey bee homing by altering MagR and Cry2 gene expression [J]. Journal of Hazardous Materials, 2025, 484136710-136710.) who used RNA interference technology to silence bee magnetic induction-related genes (MagR and Cry2) to alleviate the interference of dinotefuran on homing ability. Studies have found that non-contact exposure to dinotefuran can impair the navigation ability of bees by affecting the expression of nicotinic acetylcholine receptors (nAChR) and flightin genes. However, genetic intervention may destroy the natural physiological functions of bees, pose ethical and ecological risks, and cannot directly detoxify, but can only reduce the negative impact at the behavioral level.

[0006] It can be seen that the current mainstream methods focus on preventing or reducing the effects of exposure, and lack rapid detoxification methods for poisoned bees. Therefore, it is urgent to develop an efficient, safe, and environmentally friendly detoxification preparation to provide a new solution for bee protection and sustainable agricultural development. Summary of the invention

[0007] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the prior art that there is a lack of rapid detoxification means for poisoned bees, and to provide an application of N-acetylcysteine ​​amide or a pharmaceutically acceptable salt thereof as a preparation for alleviating or relieving pesticide poisoning of bees.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] The invention protects the use of N-acetylcysteine ​​amide as a preparation for alleviating or relieving pesticide poisoning of honey bees, wherein the pesticide is dinotefuran.

[0010] As a new type of thiol antioxidant and neuroprotectant, N-Acetylcysteineamide (NACA) has received widespread attention in the fields of medicine and biology in recent years. NACA has a unique chemical structure and biological activity, can cross the blood-brain barrier, has high lipophilicity, antioxidant properties and cell membrane permeability, and thus plays an important role in the central nervous system. Although NACA has shown good application prospects in many fields, its application in the field of detoxification of bee pesticide poisoning has not been studied. Regarding the detoxification method of bee pesticide poisoning, the inventor team creatively discovered that NACA has a significant detoxification function. On the one hand, NACA can alleviate cell apoptosis in the bee brain; on the other hand, it can also effectively improve the multi-dimensional toxicity of bees caused by exposure to dinotefuran, such as motor dysfunction, oxidative stress damage and metabolic disorders.

[0011] Furthermore, the dinotefuran is R-(–)-dinotefuran, S-(+)-dinotefuran or a mixture thereof.

[0012] Furthermore, the method of alleviating or relieving bee pesticide poisoning is to increase the survival rate of poisoned bees.

[0013] Furthermore, the method of alleviating or relieving bee pesticide poisoning is to improve the motor performance of the poisoned bees.

[0014] Furthermore, the method of alleviating or relieving bee pesticide poisoning is to inhibit cell apoptosis of poisoned bees.

[0015] Furthermore, the reducing or relieving of pesticide poisoning of bees is to reduce the oxidative stress damage of the poisoned bees.

[0016] Furthermore, the said reducing or relieving of bee pesticide poisoning is to regulate the metabolic disorder of the poisoned bees.

[0017] Furthermore, the concentration of N-acetylcysteine ​​amide is 5 to 100 μg / mL. Within this range, NACA can more effectively exert its detoxification effect.

[0018] Preferably, the concentration of N-acetylcysteine ​​amide is 50-100 μg / mL, more preferably 60-80 μg / mL.

[0019] Furthermore, the preparation also includes a pesticide-acceptable carrier and / or excipient.

[0020] Furthermore, the formulation of the preparation is a solution, emulsion, suspension, powder, foam, paste, granule, sustained-release agent or aerosol. The preparation of the present invention can be combined into a variety of formulations to adapt to different application scenarios. For example, it can be compounded with pesticides to form a nano sustained-release agent to achieve the synergistic effect of pesticides and antidotes and prolong the efficacy; it can be made into a microemulsion for foliar spraying, which is convenient for uniform spraying on the surface of plants, so that bees can contact the antidote when collecting nectar; it can also be made into a sugar water suspension for feeding, and the antidote can be directly given by feeding bees; in addition, it can also be used in combination with other antidotes, such as in combination with a vitamin C complex system, to exert a synergistic effect; it can also be based on the synergistic detoxification mechanism regulated by the intestinal flora of bees to further enhance the detoxification effect.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The present invention finds that N-acetylcysteine ​​amide has a significant detoxification function for the toxic effects of dinotefuran induced by bees. Experimental data confirm that by regulating redox balance and repairing metabolic homeostasis, N-acetylcysteine ​​amide can effectively improve the multi-dimensional toxicity characteristics of bees such as death, motor dysfunction, apoptosis, oxidative stress damage, and metabolic disorders caused by dinotefuran exposure. That is, N-acetylcysteine ​​amide can significantly reverse the destructive effect of dinotefuran on the physiological system of bees, providing a new solution for the prevention and control of bee colony poisoning syndrome caused by dinotefuran, and has important application value in the field of ecotoxicology. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The figures are statistical diagrams of the effect of NACA on the survival rate of honey bees induced by the racemic dinotefuran and its two enantiomers; A is a statistical diagram of the survival rate of honey bees in each group at different feeding days; B is a statistical diagram of the survival rate of honey bees in the blank control group after 3 days of feeding; C is a statistical diagram of the survival rate of honey bees in the rac-(±)-dinotefuran group after 3 days of feeding; D is a statistical diagram of the survival rate of honey bees in the R-(–)-dinotefuran group after 3 days of feeding; E is a statistical diagram of the survival rate of honey bees in the S-(+)-dinotefuran group after 3 days of feeding.

[0023] Figure 2 The figures are statistical diagrams of the effects of NACA on the motor performance of honey bees induced by the racemic dinotefuran and its two enantiomers; wherein, A is a statistical diagram of the walking routes of honey bees in each group; B is a statistical diagram of the average acceleration of honey bees in each group; C is a statistical diagram of the average speed of honey bees in each group; and D is a statistical diagram of the total distance traveled by honey bees in each group.

[0024] Figure 3 This is a DAPI staining image showing the changes in apoptosis of honeybee brain tissue cells induced by NACA and the two enantiomers of dinotefuran.

[0025] Figure 4 This is a DHE staining image showing the effect of NACA on the ROS content level in honey bee brain tissue induced by the two enantiomers of dinotefuran.

[0026] Figure 5 The mass spectrometry imaging data statistics of the effect of NACA on the metabolite PE (36:2) induced by the two enantiomers of dinotefuran in the brain tissue of honey bees; the left figure represents the mass spectrometry imaging of the metabolite in the honey bee brain, and the right figure represents the data statistics of the signal intensity of the mass spectrometry imaging of the metabolite.

[0027] Figure 6 The mass spectrometry imaging data statistics of the effect of NACA on the metabolite PE (38:6e) induced by the two enantiomers of dinotefuran in the brain tissue of honey bees; the left figure represents the mass spectrometry imaging of the metabolite in the honey bee brain, and the right figure represents the data statistics of the signal intensity of the mass spectrometry imaging of the metabolite. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0029] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0030] Rac-(±)-Dinotefuran is a racemic mixture of S-(+)-Dinotefuran and R-(–)-Dinotefuran.

[0031] Figure 1 A means Figure 1 Figure A in Figure 2 A means Figure 2 This is Figure A in the figure. The meanings of the other figures are similar.

[0032] rac-(±)-dinotefuran, S-(+)-dinotefuran, and R-(–)-dinotefuran were purchased from Shanghai Qinlu Biotechnology Co., Ltd.;

[0033] N-acetylcysteine ​​amide was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0034] Example 1 Detoxification of NACA on the reduction of honey bee survival rate induced by the racemic dinotefuran and its two enantiomers

[0035] 1. Detection method

[0036] The enantiomers of dinotefuran were dissolved in acetone to prepare a 10000 μg / mL stock solution, and then the enantiomers were diluted to 6 μg / mL with acetone. The tested bees were anesthetized with CO2 for 20 s, and then 1 μL of the solution was dripped on the back of each bee, and then transferred to the experimental beehive and fed with 75 μg / mL NACA sucrose solution (50%, w / v) for 3 consecutive days. The group fed with R-(–)-dinotefuran was referred to as the R-NACA group (or RN group), the group fed with S-(+)-dinotefuran was referred to as the S-NACA group (or SN group), and the group fed with rac-(±)-dinotefuran was referred to as the rac-NACA group (or rac-N group). In addition, a blank control group (not exposed to pesticides and not fed with NACA, referred to as the CK group) and a NACA-only treatment group (not exposed to pesticides but fed with NACA, referred to as the NACA group) were set up. The number of bees in each group was the same, and each group had 3 replicates. During the experiment, the number of deaths of bees in each group was observed and recorded every day, and the survival rate was calculated. After the experiment, the results were statistically analyzed and a survival rate comparison chart was drawn.

[0037] 2. Experimental results

[0038] from Figure 1 It can be seen that the survival rate of bees after NACA feeding was significantly improved compared with the group treated with pesticides only. Specifically, the original mortality rate of bees was about 15% in the S-(+)-dinotefuran group, about 50% in the rac-(±)-dinotefuran group, about 70% in the R-(–)-dinotefuran group, and about 85% in the control group ( Figure 1 A). After taking NACA, rac-(±)-dinotefuran ( Figure 1 C) and R-(–)-dinotefuran group ( Figure 1 D) the survival rate was increased to the control group (CK) ( Figure 1 B) No difference, S-(+)-dinotefuran group ( Figure 1The survival rate of E) was increased to 60%. This result shows that NACA has a certain mitigating effect on the death of honey bees caused by dinotefuran and its two enantiomers.

[0039] Example 2 Detoxification of NACA on honey bee motor performance disorders induced by dinotefuran racemate and its two enantiomers

[0040] 1. Detection method

[0041] The bees were grouped according to the method of Example 1, and the bees were treated according to the settings of each group. The bees that survived after being fed for 3 days in each group were subjected to the following tests.

[0042] The test bees were gently placed in a glass culture dish with a diameter of 9 cm, with one bee placed in each dish, and 15 parallel samples were set up to ensure the reliability of the data. First, the glass culture dish containing the bees was placed in a quiet, undisturbed environment, and the bees were allowed to rest for 30 minutes to eliminate the stress response that may have occurred during the move and restore their normal behavior. After the resting time, the recording device was immediately started to continuously record the free activities of the bees on the glass culture dish for 15 minutes. During this process, the stability of the experimental environment was ensured to avoid interference from external factors on the bees' behavior. The recorded video was analyzed to obtain key motion parameters such as the bees' motion acceleration, motion speed, and motion distance.

[0043] 2. Experimental results

[0044] The results are as follows Figure 2 The results showed that bees in the NACA treatment group had a higher movement acceleration ( Figure 2 B) Movement speed ( Figure 2 C) Total distance traveled ( Figure 2 Dinotefuran and its enantiomers weaken the navigation ability of bees, making their walking routes chaotic and even unable to complete a complete circular path ( Figure 2 A). The degree of influence was in the order of S-(+)-dinotefuran>rac-(±)-dinotefuran>R-(–)-dinotefuran>control group. However, after taking NACA, this decline in navigation ability was significantly alleviated, indicating that NACA can improve the impairment of dinotefuran and its enantiomers on the motor performance of honey bees.

[0045] Given that the racemic compound rac-(±)-dinotefuran is a mixture of R-(–)-dinotefuran and S-(+)-dinotefuran, and that the two enantiomers have significantly different toxicities to non-target insects, bees, the following examples will focus on the possible detoxification mechanism of NACA against the two enantiomers of dinotefuran.

[0046] Example 3 Detoxification effect of NACA on apoptosis of honey bee brain tissue cells induced by two enantiomers of dinotefuran

[0047] 1. Detection method

[0048] The bees were grouped according to the method of Example 1, and the bees were treated according to the settings of each group. The bees that survived after being fed for 3 days in each group were subjected to the following tests.

[0049] DAPI (4',6-diamidino-2-phenylindole) can specifically bind to DNA in the cell nucleus and emit blue fluorescence. The degree of cell apoptosis can be quantitatively evaluated by judging the intensity and distribution changes of the DAPI fluorescence signal. Take the bee brain tissue, carefully cut off the bee's head with a blade, remove the antennae, fill the tissue with OCT (optimal cuttingtemperature compound) embedding medium, and use a freezing microtome to cut into 10μm thick slices at -20℃ and transfer to poly-lysine anti-shedding slides for histological staining. Follow the instructions of the DAPI staining kit, observe the changes in the morphology of the cell nucleus under a fluorescence microscope, judge the cell apoptosis, and take pictures of the staining ( Figure 3 ).

[0050] 2. Experimental results

[0051] By DAPI staining ( Figure 3 ) observed that the DAPI fluorescence intensity of the honey bee brain tissue sections was weakened and cell apoptosis was obvious only in the pesticide-treated group, while the cell apoptosis in the NACA-treated group was alleviated, indicating that NACA has the effect of inhibiting cell apoptosis.

[0052] Example 4 Detoxification effect of NACA on the elevated ROS content in honey bee brain tissue induced by two enantiomers of dinotefuran

[0053] 1. Detection method

[0054] The bees were grouped according to the method of Example 1, and the bees were treated according to the settings of each group. The bees that survived after being fed for 3 days in each group were subjected to the following tests.

[0055] DHE (Dihydroethidium) staining is a commonly used method to detect the level of intracellular reactive oxygen species (ROS). The intensity of red fluorescence detected by fluorescence microscopy can indirectly reflect the level of intracellular ROS. Similarly, the bee brain tissue was taken, the bee's head was carefully cut off with a blade, the antennae were removed, the tissue was filled with OCT embedding medium, and sliced ​​into 10μm thick slices at -20°C using a freezing microtome and transferred to poly-lysine anti-shedding slides for histological staining. Use the DHE staining kit and stain according to the instructions. Use a fluorescence microscope to observe the production of ROS and obtain the staining image ( Figure 4 ).

[0056] 2. Experimental results

[0057] DHE staining image ( Figure 4 ) The results showed that only the DHE fluorescence intensity of the bee brain tissue slices in the pesticide treatment group increased, indicating that the intracellular ROS level increased and oxidative stress damage was obvious. The DHE fluorescence intensity of the NACA treatment group decreased significantly, indicating that NACA can effectively reduce the generation of intracellular ROS and alleviate oxidative stress damage.

[0058] Example 5 Detoxification of NACA on metabolic disorders of honey bee brain tissue induced by two enantiomers of dinotefuran

[0059] 1. Detection method

[0060] The bees were grouped according to the method of Example 1, and the bees were treated according to the settings of each group. The bees that survived after being fed for 3 days in each group were subjected to the following tests.

[0061] Mass spectrometry imaging (MSI) can obtain the distribution and location of key differential metabolites in biological tissues stimulated by drugs, which helps to gain a deeper understanding of the changes in various metabolic processes related to toxicity. The bee brain tissue was obtained, the bee's head was carefully cut off with a blade, the antennae were removed, the tissue was filled with 10% (w / v) gelatin, and sliced ​​into 15μm thick slices at -20°C using a freezing microtome. The slices were transferred to indium tin oxide (ITO) slides and sprayed with AuNPs suspension or NEDC matrix using an ultrasonic sprayer for subsequent MSI analysis, which was performed on a MALDI time-of-flight mass spectrometer using a 355nmNd:YAG laser. In order to avoid pixel overlap, the laser spot size was set to "Min" (about 10μm) during MSI analysis. Considering the imaging resolution and imaging time, a 15μm step range was selected to obtain a complete whole-body imaging image. The mass spectrometry imaging image ( Figure 5 and Figure 6 ).

[0062] 2. Experimental results:

[0063] PE (phosphatidylethanolamine) is an important component of the cell membrane and plays a key role in maintaining the fluidity and stability of the cell membrane. Mass spectrometry imaging ( Figure 5 and Figure 6 ) revealed that NACA could regulate the content of PE after NACA treatment, which suggests that NACA may exert its neuroprotective effect in the honey bee brain by regulating the level of PE to maintain the normal function and health of nerve cells.

[0064] In summary, this example, through a series of experiments, confirms that N-acetylcysteine ​​amide has a significant effect in alleviating the poisoning of dinotefuran racemate and its two enantiomers to bees, can improve the survival rate of bees, improve motor performance, inhibit cell apoptosis and oxidative stress damage, and regulate metabolic disorders, which provides a strong experimental basis for the development of preparations for preventing and treating bee pesticide poisoning.

[0065] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. The use of N-acetylcysteine ​​amide as a preparation for alleviating or relieving pesticide poisoning in bees, characterized in that: The pesticide is dinotefuran.

2. The application according to claim 1, characterized in that: The dinotefuran is R-(–)-dinotefuran, S-(+)-dinotefuran or a mixture thereof.

3. The use according to claim 1 or 2, characterized in that: The method of alleviating or relieving bee pesticide poisoning is to increase the survival rate of poisoned bees.

4. The use according to claim 1 or 2, characterized in that: The method of alleviating or relieving the bee pesticide poisoning is to improve the motor performance of the poisoned bees.

5. The use according to claim 1 or 2, characterized in that: The method of reducing or relieving the pesticide poisoning of bees is to inhibit the apoptosis of the bees after poisoning.

6. The use according to claim 1 or 2, characterized in that: The method of reducing or relieving the pesticide poisoning of bees is to reduce the oxidative stress damage of the poisoned bees.

7. The use according to claim 1 or 2, characterized in that: The method of alleviating or relieving the bee pesticide poisoning is to regulate the metabolic disorder of the poisoned bees.

8. The use according to any one of claims 1 to 7, characterized in that: The concentration of the N-acetylcysteine ​​amide is 5 to 100 μg / mL.

9. The use according to claim 1, characterized in that: The formulation also includes a pesticidally acceptable carrier and / or excipient.

10. The use according to claim 1, characterized in that: The preparation is in the form of solution, emulsion, suspension, powder, foam, paste, granule or aerosol.

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