A method for alleviating hydrogen sulfide poisoning in zebrafish larvae using exogenous nitric oxide

Abstract

A method for alleviating hydrogen sulfide poisoning in zebrafish juveniles using exogenous nitric oxide includes the following steps: Step 1: Determining the median lethal concentration of hydrogen sulfide for zebrafish juveniles; Step 2: Determining the safe concentration range of exogenous nitric oxide and the applicable concentration for effectively alleviating hydrogen sulfide toxicity. This invention provides a method for alleviating hydrogen sulfide poisoning in zebrafish in aquatic ecological pollution. 2 This method for treating H poisoning is rapid, effective, inexpensive, safe, and harmless, making it suitable for widespread application. It can be used as research material in the field of environmental monitoring and also for clinical treatment of H poisoning. 2 Drug screening and evaluation model for S poisoning.

Description

A method for alleviating hydrogen sulfide poisoning in zebrafish juveniles using exogenous nitric oxide. Technical Field

[0001] This invention relates to a method for alleviating hydrogen sulfide (H2S) poisoning in juvenile zebrafish using exogenous nitric oxide (NO), and belongs to the field of biotechnology. Background Technology

[0002] As human industrialization continues, large amounts of nutrients such as nitrogen and phosphorus enter slow-moving water bodies like lakes and rivers, causing rapid proliferation of algae and other plankton, leading to a rapid decrease in dissolved oxygen and the death of some oxygen-intensive aquatic animals. Under the fermentation of anaerobic microorganisms, the large amounts of hydrogen sulfide (H2S) produced by the decomposition of these animal carcasses cause extremely serious harm to other aquatic animals, further deteriorating water quality. Meanwhile, H2S is a common pollutant in urban drainage pipes. As a foul-smelling, toxic, and corrosive gas, its generation and release in sewage pipes can cause foul odor pollution, lead to large-scale corrosion and damage to pipes, and even seriously threaten the safety and health of people in the surrounding area. Zebrafish, initially used as an experimental animal model in embryonic developmental biology and genetics, have been used in a range of environmental toxicology studies on many chemicals in the environment, including developmental toxicity and teratogenicity, carcinogenicity, embryotoxicity, cardiovascular toxicity, neurotoxicity, and behavioral toxicity. They have wide applications in the identification of environmental pollutant hazards and risk assessment. Currently, the solution to H2S pollution is usually to add chemical agents such as strong alkalis and biological inhibitors, but these can all pose a significant threat to the aquatic ecosystem of the water body. Summary of the Invention

[0003] The purpose of this invention is to provide a method for alleviating hydrogen sulfide poisoning in juvenile zebrafish using exogenous nitric oxide.

[0004] To achieve the above and other related objectives, the technical solution provided by this invention is: a method for alleviating hydrogen sulfide poisoning in zebrafish juveniles using exogenous nitric oxide, comprising the following steps:

[0005] Step 1: Determine the median lethal concentration of hydrogen sulfide for juvenile zebrafish;

[0006] Step 2: Determine the safe concentration range of exogenous nitric oxide and the applicable concentration to effectively mitigate hydrogen sulfide toxicity.

[0007] The preferred technical solution is as follows: In step 1, a concentration gradient experiment is conducted using a multi-well plate to detect the median lethal concentration of zebrafish juveniles.

[0008] The preferred technical solution is as follows: ① Collected zebrafish fertilized eggs are placed into the wells of a multi-well plate containing zebrafish embryo culture medium, with 300 zebrafish fertilized eggs per well and 3 wells forming a parallel replicate; ② The first group serves as the control group and is cultured normally, while the other groups are treated with NaHS solution. During subsequent culture, the zebrafish embryo culture medium is changed and NaHS solution is added daily; ③ The hatching rate of fertilized eggs and the survival rate of zebrafish larvae are recorded. The survival rate and malformation rate of zebrafish larvae in different groups are recorded daily. The results show that the median lethal concentration (LD50) of zebrafish larvae exposed to H2S solution is 30 μM / L. Furthermore, as the NaHS treatment concentration increases, the probability of malformations such as notochord curvature and severe edema in larvae increases, leading to death. Therefore, 30 μM / L NaHS solution is selected as the treatment concentration for zebrafish larvae.

[0009] The preferred technical solution is as follows: Determining the safe concentration range of exogenous NO includes: ① Placing the collected zebrafish fertilized eggs into the wells of a multi-well plate containing zebrafish embryo culture medium, with 30 zebrafish fertilized eggs per well, and 3 wells forming a parallel replicate; ② The first group serves as the control group and is cultured normally, while the other groups are cultured with nitric oxide solution. During subsequent culture, the zebrafish embryo culture medium is changed and nitric oxide solution is added daily; ③ The hatching rate of fertilized eggs and the survival rate of zebrafish larvae are counted. Subsequently, the survival rate and malformation rate of zebrafish larvae in different groups are counted daily. The statistical results show that: in nitric oxide solution culture, a concentration of 20 μM / L or below is the safe concentration for zebrafish larvae.

[0010] The preferred technical solution is as follows: Determining the applicable concentration for effectively alleviating hydrogen sulfide toxicity includes: using a multi-well plate to conduct a concentration gradient experiment, and exploring the optimal therapeutic concentration of nitric oxide to alleviate H2S poisoning in zebrafish juveniles within the determined safe concentration range of exogenous nitric oxide. The specific method is as follows: ① Collected zebrafish fertilized eggs are placed into the wells of a multi-well plate containing zebrafish embryo culture medium, with 30 zebrafish fertilized eggs per well, and 3 wells forming a parallel replicate; ② The first group serves as the control group and is cultured normally, while the other groups are cultured in 30 μM / L NaHS solution, and in solutions containing 0.1 μM / L, 0.5 μM / L, 1 μM / L, 1.5 μM / L, 3 μM / L, and 5 μM / L NaHS solution, respectively. ③ The fertilized egg hatching rate and zebrafish juvenile survival rate were statistically analyzed. Subsequently, the survival rate and malformation rate of zebrafish juveniles in different groups were statistically analyzed daily. The results showed that, in terms of survival rate, hatching rate and malformation rate, the 3 μM / L NO solution had the best effect in mitigating H2S toxicity.

[0011] Due to the application of the above technical solution, the advantages of this invention compared with the prior art are:

[0012] This invention provides a method for alleviating H2S poisoning in zebrafish in aquatic ecological pollution. This method is rapid, effective, low-cost, safe and harmless, and suitable for widespread application. It can be used as research material in the field of environmental monitoring, and can also be used as a drug screening and evaluation model for the clinical treatment of H2S poisoning. Attached Figure Description

[0013] Figure 1 shows the survival rate, hatching rate, and deformity rate of zebrafish under different H2S concentrations.

[0014] Figure 2 shows the survival rate of zebrafish juveniles under different NO solution concentrations.

[0015] Figure 3 shows the survival rate of zebrafish exposed to H2S under different NO concentrations.

[0016] Figure 4 shows the hatching rate of zebrafish exposed to H2S under different NO concentrations.

[0017] Figure 5 shows the overall malformation rate of zebrafish exposed to H2S under different NO concentrations. Detailed Implementation

[0018] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in these embodiments.

[0019] Please refer to Figures 1-5. It should be noted that the structures, proportions, sizes, etc., illustrated in the accompanying drawings are merely for illustrative purposes and to aid those skilled in the art in understanding and reading the invention. They are not intended to limit the scope of the invention and therefore have no substantial technical significance. Any modifications to the structure, changes in proportions, or adjustments to size are not permitted. The following embodiments are provided to better understand the invention, but are not intended to limit it. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

[0020] Unless otherwise specified, all reagents or materials described in the following examples are commercially available.

[0021] The technical solution of the present invention will be further described in detail below with reference to examples.

[0022] Example 1: A method for alleviating hydrogen sulfide poisoning in zebrafish juveniles using exogenous nitric oxide.

[0023] Determining the median lethal concentration (LD50) of H2S to simulate aquatic ecological pollution: H2S solutions of different concentrations were prepared using embryo culture medium, and a concentration gradient experiment was conducted using a six-well plate to construct a model of zebrafish embryos. The LD50 of H2S was determined by survival rate, hatching rate, and malformation rate.

[0024] Determine the safe concentration range for exogenous NO, and determine the applicable concentration for effectively mitigating H2S toxicity:

[0025] Different concentrations of NO solutions were prepared using embryo culture medium, and a concentration gradient experiment was conducted using a six-well plate to construct a model of zebrafish embryos. The safe concentration range of NO was determined by measuring survival rate, hatching rate, and malformation rate.

[0026] Within the safe concentration range of NO, explore suitable concentrations that can mitigate H2S toxicity.

[0027] 1. Determine the median lethal concentration (LD50) of H2S.

[0028] (1) Use at least 5 pairs of male and female wild-type zebrafish (type AB) to mate and spawn, and collect the fertilized eggs;

[0029] (2) A concentration gradient experiment was conducted using a six-well plate to determine the median lethal concentration (LC50) of zebrafish larvae within 9 days. The specific method was as follows: ① The collected zebrafish fertilized eggs were placed into the wells of a six-well plate containing 10 ml of zebrafish embryo culture medium, with 30 eggs per well and 3 wells forming a parallel replicate; ② The first group served as the control group and was cultured normally, while the other groups were treated with NaHS solution (0.1 mM / ml). - With H in water + NaHS solutions (containing toxic gas H2S) were prepared at concentrations of 2.5 μM / L, 5 μM / L, 10 μM / L, 20 μM / L, 30 μM / L, 40 μM / L, 50 μM / L, and 60 μM / L (equal amounts of NaHS solution were added to all three wells in each group). During the subsequent culture process, the zebrafish embryo culture medium was changed daily and NaHS solution was added again. The hatching rate of fertilized eggs and the survival rate of zebrafish larvae were statistically analyzed. The survival rate and malformation rate of zebrafish larvae in different groups were then recorded daily. The results showed that after 9 days of exposure to H2S solution, the median lethal concentration (LC50) for zebrafish larvae was 30 μM / L. Furthermore, with increasing NaHS concentration, the probability of malformations such as notochordine curvature and severe edema increased, leading to death. Therefore, 30 μM / L NaHS solution was selected as the treatment concentration for zebrafish larvae (see Figure 1).

[0030] 2. Determine the safe concentration range for exogenous NO.

[0031] (1) Use at least 5 pairs of male and female wild-type zebrafish (type AB) to mate and spawn, and collect the fertilized eggs;

[0032] (2) A concentration gradient experiment was conducted using six-well plates to determine the safe concentration range of NO. The specific method was the same as that for determining the median lethal concentration of H2S. The exogenous NO solution was prepared by SNP (NO donor) at concentrations of 0.1 μM / L, 1 μM / L, 10 μM / L, 20 μM / L, 30 μM / L, and 40 μM / L. Statistical results showed that after 9 days of culture in NO solution, a concentration of 20 μM / L and below was the safe concentration for zebrafish juveniles (Figure 2).

[0033] 3. Explore suitable NO concentrations that can mitigate H2S toxicity.

[0034] (1) Use at least 5 pairs of male and female wild-type zebrafish (type AB) to mate and spawn, and collect the fertilized eggs;

[0035] (2) A concentration gradient experiment was conducted using a six-well plate to explore the optimal therapeutic concentration of NO to alleviate H2S poisoning in zebrafish juveniles within a safe concentration range. The specific method was as follows: ① Collected zebrafish fertilized eggs were placed into the wells of a six-well plate containing 10 ml of zebrafish embryo culture medium, with 30 eggs per well and 3 wells as a parallel replicate; ② The first group served as the control group and was cultured normally, while the other groups were cultured in 30 μM / L NaHS solution and solutions containing 0.1 μM / L, 0.5 μM / L, 1 μM / L, 1.5 μM / L, 3 μM / L, and 5 μM / L NaHS solution, respectively. ③ The fertilized egg hatching rate and zebrafish juvenile survival rate were statistically analyzed. Subsequently, the survival rate and malformation rate of zebrafish juveniles in different groups were statistically analyzed daily. The statistical results showed that, in terms of survival rate, hatching rate and malformation rate, the 3 μM / L NO solution had the best effect in mitigating H2S toxicity (Figure 3-5).

[0036] The above description is merely a preferred embodiment for explaining the present invention and is not intended to limit the present invention in any way. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included within the scope of protection intended by the present invention.

Claims

1. The application of a nitric oxide solution in the preparation of a therapeutic agent to alleviate hydrogen sulfide poisoning in juvenile zebrafish, characterized in that: During treatment, zebrafish fertilized eggs are immersed in a nitric oxide solution.

2. The application of the nitric oxide solution according to claim 1 in the preparation of a therapeutic agent to alleviate hydrogen sulfide poisoning in zebrafish juveniles, characterized in that: The concentration of the nitric oxide solution is 1 μM / L, 1.5 μM / L, 3 μM / L, or 5 μM / L.

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