Application of tetrahydrobiopterin
By adding tetrahydrobiopterin to shrimp feed, the adverse effects of ammonia nitrogen accumulation on shrimp in aquaculture environment are solved, its survival rate and antioxidant ability under ammonia nitrogen stress are improved, and the damage caused by ammonia nitrogen toxicity is reduced. The potential application value of healthy shrimp farming.
Patent Information
- Application Number
- CN202411173430.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-26
- Publication Date
- 2025-01-14
AI Technical Summary
Ammonia nitrogen accumulation in aquaculture environments adversely affect shrimps, leading to an increased risk of red tides and disease outbreaks. Existing research lacks methods to effectively reduce ammonia nitrogen toxicity.
Tetrahydrobiopterin was used as a metabolite and added to shrimp feed to improve its antioxidant ability and reduce ammonia nitrogen toxicity. By regulating the metabolic pathway of Vannah shrimp, it enhances its survival rate and cell proliferation ability under ammonia nitrogen stress.
It significantly improves the survival rate and antioxidant ability of shrimps under ammonia nitrogen stress, reduces hepatopancreatic damage caused by ammonia nitrogen toxicity, and enhances the adaptability of shrimps.
Smart Images
Figure CN119302959A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to an application of tetrahydrobiopterin. Background Art
[0002] In order to solve the aquaculture environmental pollutants (such as microplastics, nitrite, ammonia nitrogen, nonylphenol, heavy metals, etc.), especially the ammonia nitrogen pollution caused by accumulation pathways (feed, feces, dead individuals, breeding density, ammonia excretion and other nitrogen-containing exogenous substances), which in turn causes eutrophication of shrimp aquaculture water, leading to the occurrence of red tides, increased risk of shrimp disease outbreaks and large-scale shrimp deaths, and other negative effects brought about by the expansion of shrimp aquaculture, one of the key links is to strengthen basic research on shrimp, reveal the mechanism of shrimp's own resistance to ammonia nitrogen, find potential metabolites that reduce ammonia nitrogen toxicity, and breed excellent ammonia nitrogen resistant varieties. However, there are few reports on how shrimp reduce ammonia nitrogen toxicity. To this end, actively carrying out research in this area will help find potential targets for the prevention and control of ammonia nitrogen, which has important theoretical significance and potential application value for achieving healthy shrimp aquaculture. Summary of the invention
[0004] The purpose of the present invention is to solve the problem of adverse effects of ammonia nitrogen accumulation on shrimps in an aquaculture environment.
[0005] The following technical solutions are adopted:
[0006] A tetrahydrobiopterin (C9H 15 N5O3, chemical structure see Figure 1 ) for preparing a preparation for improving the survival rate of crustaceans.
[0007] Studies have shown that tissue damage caused by ammonia nitrogen toxicity is the main factor causing shrimp death. Therefore, by finding the metabolite tetrahydrobiopterin that can reduce shrimp blood ammonia toxicity, adding it to the feed to enhance the shrimp's physique and improve the shrimp's ability to resist ammonia nitrogen, it can better solve the adverse effects of ammonia nitrogen accumulation in the aquaculture environment on shrimp.
[0008] Preferably, it is used to prepare a preparation for improving the survival rate of crustaceans under ammonia nitrogen stress.
[0009] Preferably, it is used to prepare a preparation for improving the antioxidant capacity of crustaceans under ammonia nitrogen stress.
[0010] Preferably, it is used to prepare a preparation for increasing the proliferation rate of crustacean cells under ammonia nitrogen stress.
[0011] Preferably, the invention is used for preparing a preparation for reducing damage to crustaceans caused by ammonia nitrogen toxicity.
[0012] Preferably, it is used to prepare a preparation for reducing crustacean hepatopancreas damage caused by ammonia nitrogen toxicity.
[0013] Preferably, the ratio of the added amount of tetrahydrobiopterin to the body weight of the crustacean is 20-40 μg / 6g; and the crustacean includes shrimp.
[0014] The invention provides a metabolite tetrahydrobiopterin, and exogenous tetrahydrobiopterin regulates the antioxidant capacity of Penaeus vannamei and reduces tissue damage, thereby improving the survival rate of the shrimp under ammonia nitrogen stress. The added dosage is 20-40 μg / 6g.
[0015] A preparation obtained by the above application.
[0016] Preferably, the preparation comprises one or more of a medicine and a feed.
[0017] The above-mentioned preparation products include injections, which can be used for immune conditioning or enhancement of shrimps and crabs, especially prawns.
[0018] Compared with the prior art, the present invention has the following beneficial effects:
[0019] The present invention is based on the injection of hemocyanin degradation fragment HMCs27 combined with ammonia nitrogen stress for 24 hours, and through transcriptomics, it is found that the tetrahydrobiopterin metabolic pathway is significantly enhanced. The rate-limiting enzyme of tetrahydrobiopterin synthesis, guanosine triphosphate cyclohydrolase gene GTPCH I (GTP cyclohydrolase 1-like), is significantly upregulated, and further analysis shows that the content of tetrahydrobiopterin is significantly increased. After knocking down its key synthase gene GTPCH I, the content of tetrahydrobiopterin is significantly downregulated, and the antioxidant capacity and cell proliferation rate of shrimp are significantly reduced, causing hepatopancreatic tissue damage, and reducing the survival rate of shrimp under ammonia nitrogen stress. Through the analysis of injection of tetrahydrobiopterin combined with ammonia nitrogen stress, it is found that compared with the control group, after the injection of 20μg or 40μg of tetrahydrobiopterin, the antioxidant capacity and cell proliferation rate of shrimp under ammonia nitrogen stress can be improved, the hepatopancreatic damage caused by ammonia nitrogen toxicity can be reduced, and the survival rate of shrimp under ammonia nitrogen stress can be improved, which may be the mechanism for tetrahydrobiopterin to exert its biological function. The tetrahydrobiopterin for reducing ammonia nitrogen toxicity of the present invention can be used as a preparation for reducing ammonia nitrogen toxicity and can be added to feed as an additive to improve the adaptability of shrimp under ammonia nitrogen stress. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 It is the chemical structural formula of tetrahydrobiopterin.
[0021] Figure 2This is a diagram showing the screening and verification results of the metabolic pathways against ammonia nitrogen toxicity based on the injection of HMCs27 combined with ammonia nitrogen stress. Among them: The heat map analysis is based on the changes in genes related to the tetrahydrobiopterin metabolic pathway under HMCs27 injection combined with ammonia nitrogen stress ( Figure 2 A); Schematic diagram of tetrahydrobiopterin metabolic pathway ( Figure 2 B); Changes in tetrahydrobiopterin content under HMCs27 injection combined with different ammonia nitrogen concentrations ( Figure 2 C); Based on the HMCs27 injection combined with different ammonia nitrogen concentrations, the key tetrahydrobiopterin synthase gene GTP cyclohydrolase 1-like ( Figure 2 D) Synthetic gene Carbonylreductase [NADPH] ( Figure 2 E), metabolic gene Protein henna-like ( Figure 2 F) Relative expression levels.
[0022] Figure 3 This is a graph showing the changes in the antioxidant capacity of shrimps based on the interference with GTPCH I combined with ammonia nitrogen stress. Figure 3 A); Analysis of the effect of interfering with GTPCH I on tetrahydrobiopterin content ( Figure 3 B).
[0023] Figure 4 This is an analysis chart based on the effect of interfering with the key tetrahydrobiopterin synthase gene GTPCH I combined with ammonia nitrogen stress on various physiological indicators. Among them: T-SOD (superoxide dismutase) ( Figure 4 A), T-AOC (total antioxidant capacity) Figure 4 B), CAT (catalase) Figure 4 C), Malondialdehyde ( Figure 4 D), glutathione S-transferase ( Figure 4 E), alanine aminotransferase ( Figure 4 F).
[0024] Figure 5 This is an analysis chart based on the effects of interfering with the key tetrahydrobiopterin synthase gene GTPCH I combined with ammonia nitrogen stress on the proliferation of hepatopancreatic cells.
[0025] Figure 6 This is an analysis chart based on the effects of interfering with the key tetrahydrobiopterin synthase gene GTPCH I combined with ammonia nitrogen stress on hepatopancreatic damage.
[0026] Figure 7 This is an analysis chart based on the effects of interfering with the key tetrahydrobiopterin synthase gene GTPCH I combined with ammonia nitrogen stress on shrimp survival.
[0027] Figure 8 This is an analysis chart based on the effects of tetrahydrobiopterin injection combined with ammonia nitrogen stress on various physiological indicators.
[0028] Among them: T-SOD (superoxide dismutase) ( Figure 8 A), T-AOC (total antioxidant capacity) Figure 8 B), CAT (catalase) Figure 8 C), Malondialdehyde ( Figure 8 D), glutathione S-transferase ( Figure 8 E), alanine aminotransferase ( Figure 8 F).
[0029] Fig. 9 This is an analysis chart based on the effect of tetrahydrobiopterin injection combined with ammonia nitrogen stress on the proliferation of hepatopancreatic cells.
[0030] Fig.10 This is an analysis chart based on the effects of tetrahydrobiopterin injection combined with ammonia nitrogen stress on hepatopancreatic damage.
[0031] Fig.11 This is an analysis chart based on the effects of tetrahydrobiopterin injection combined with ammonia nitrogen stress on shrimp survival. DETAILED DESCRIPTION
[0032] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.
[0033] Unless otherwise specified, the tetrahydrobiopterin (BH4) used in the following examples has the chemical formula C9H 15 N5O3, structural formula Figure 1 shown.
[0034] Example 1: Screening and verification of metabolic pathways against ammonia nitrogen toxicity based on injection of HMCs27 combined with ammonia nitrogen stress
[0035] (1) Inject HMCs27 combined with ammonia nitrogen stress and take samples 24 hours after stress. The samples were sent to Shanghai Meiji Biotechnology Co., Ltd. for transcriptome sequencing analysis. Heat map analysis was performed to analyze genes related to metabolic pathways with significant changes, such as Figure 2 As shown in A and B, significant changes were found in enzyme genes related to tetrahydrobiopterin metabolism, among which the key tetrahydrobiopterin synthase gene GTPCH I was significantly upregulated, and the metabolic enzyme gene Protein henna-like was significantly downregulated.
[0036] (2) Subsequently, the changes in tetrahydrobiopterin in implementation case (1) were detected, and it was found that the content of tetrahydrobiopterin increased significantly, such as Figure 2As shown in C.
[0037] (3) Figure 2 As shown in DF, the genes related to the tetrahydrobiopterin metabolic pathway in implementation case (1) were verified and found to be consistent with the transcriptome results.
[0038] Example 2: Analysis of changes in the antioxidant capacity of shrimp based on interference with GTPCH I combined with ammonia nitrogen stress
[0039] (1) Interference with the key tetrahydrobiopterin synthase gene. Figure 3 As shown in A. After successful interference, the tetrahydrobiopterin content was measured, as shown in Figure 3 As shown in B, its content was significantly reduced. Then different concentrations of ammonia nitrogen stress experiments were carried out, and after 24 hours, 5 vannamei shrimps were randomly selected to take hepatopancreas tissue samples.
[0040] (2) Then, the hepatopancreas total superoxide dismutase (T-SOD) test (Cat. No. A001-1-1, Nanjing Jiancheng), total antioxidant capacity (T-AOC) test (Cat. No. A015-3-1, Nanjing Jiancheng), catalase (CAT) test kit (Cat. No. A007-1-1, Nanjing Jiancheng), malondialdehyde test kit (Cat. No. A003-1-2, Nanjing Jiancheng), glutathione S-transferase test kit (Cat. No. A004-1-1, Nanjing Jiancheng), alanine aminotransferase test kit (Cat. No. C009-2-1, Nanjing Jiancheng) were performed according to the instructions. The tissue protein content was determined according to the BCA method. The results are as follows. Figure 4 As shown in the figure, after interfering with the key tetrahydrobiopterin synthase GTPCH I, the hepatopancreas T-SOD, T-AOC, CAT and other indicators of shrimp ( Figure 4 AC) were significantly reduced, and malondialdehyde, glutathione S-transferase, alanine aminotransferase and other indicators ( Figure 4 The results showed that the reduction of tetrahydrobiopterin inhibited the antioxidant system of shrimp under ammonia nitrogen stress.
[0041] Example 3: Analysis of the proliferation of shrimp hepatopancreatic cells based on interference with GTPCH I combined with ammonia nitrogen stress
[0042] (1) The same treatment conditions as in Example 2 (1) were used to culture Penaeus vannamei. At the same time, EdU in a cell proliferation detection kit (Cat. No. C0071S, Bio-Tech) was injected into the shrimp to label the proliferating cells, and 5 Penaeus vannamei were randomly selected to obtain hepatopancreas tissue samples 24 hours later.
[0043] (2) Then, the shrimp hepatopancreas was transferred into a 1.5 mL sterile centrifuge tube, 500 μL of pre-cooled sterile phosphate buffered saline (PBS) (0.01 M, pH 7.4) was added, and the hepatopancreas was gently squeezed with a grinding rod to allow its own pancreatic enzymes to separate the hepatopancreatic cells. Then, the hepatopancreas was washed three times with 500 μL of pre-cooled sterile PBS and filtered through a flow tube to obtain the cells in PBS. Then, the shrimp hepatopancreas cells were processed according to the instructions of the cell proliferation detection kit and detected by flow cytometry. The results are shown in Figure 2. Figure 5 As shown in the figure, after interfering with the key tetrahydrobiopterin synthase gene under different ammonia nitrogen concentrations, the proliferation rate of shrimp hepatopancreas cells was significantly reduced. This shows that the reduction of tetrahydrobiopterin content leads to a weakening of cell proliferation ability.
[0044] Example 4: Analysis of the effects of GTPCH I interference combined with ammonia nitrogen stress on shrimp hepatopancreas damage and survival
[0045] (1) The same treatment conditions as in Example 2 (1) were used to culture Penaeus vannamei. After 24 hours, 5 Penaeus vannamei were randomly selected to obtain hepatopancreas tissue samples, which were sent to Wuhan Bios Biotechnology for pathological section analysis. At the same time, the survival rate of the shrimp was calculated.
[0046] The results are as follows Figure 6 As shown in the figure, after interfering with the key tetrahydrobiopterin synthase gene under different ammonia nitrogen concentrations, the degree of damage to the hepatopancreas tissue of shrimp was significantly worse than that of the control group. At the same time, the survival rate of shrimp was also significantly reduced compared with the control group, as shown in the figure. Figure 7 shown.
[0047] Example 5: Analysis of changes in the antioxidant capacity of shrimp based on tetrahydrobiopterin injection combined with ammonia nitrogen stress
[0048] (1) After injection of tetrahydrobiopterin (20 and 40 μg / prawn) under different ammonia nitrogen concentrations (50, 100, and 150 mg / L), 5 shrimp were randomly selected for hepatopancreas tissue sampling 24 hours later.
[0049] (2) The sample was treated by the same method as in Example 2 (2), and the test results were as follows: Figure 8 As shown in Figure 2, when tetrahydrobiopterin (20, 40 μg / shrimp) was injected under different ammonia nitrogen concentrations (50, 100, 150 mg / L), the hepatopancreas T-SOD, T-AOC, CAT and other indicators of shrimp ( Figure 8 AC) were significantly increased, and malondialdehyde, glutathione S-transferase, alanine aminotransferase and other indicators ( Figure 8 The results showed that tetrahydrobiopterin significantly enhanced the antioxidant capacity of shrimp under ammonia nitrogen stress.
[0050] Example 6: Analysis of the proliferation of shrimp hepatopancreatic cells based on tetrahydrobiopterin injection and ammonia nitrogen stress
[0051] (1) The same treatment conditions as in Example 5 (1) were used to culture Penaeus vannamei. At the same time, EdU in a cell proliferation detection kit (Cat. No. C0071S, Bio-Tech) was injected into the shrimp to label the proliferating cells, and 5 Penaeus vannamei were randomly selected to obtain hepatopancreas tissue samples 24 hours later.
[0052] (2) Subsequently, the sample was treated in the same manner as in Example 3 (2), and the test results were as follows: Fig. 9 As shown, the proliferation rate of shrimp hepatopancreatic cells was significantly increased after injection of tetrahydrobiopterin (20, 40 μg / shrimp) under different ammonia nitrogen concentrations (50, 100, 150 mg / L).
[0053] Example 7: Analysis of the effect of tetrahydrobiopterin injection on hepatopancreatic damage and survival of shrimp
[0054] (1) The same treatment conditions as in Example 5 (1) were used to culture Penaeus vannamei. After 24 hours, 5 Penaeus vannamei were randomly selected to obtain hepatopancreas tissue samples, which were sent to Wuhan Bioas Biotechnology for pathological section analysis. At the same time, the survival rate of the shrimp was calculated.
[0055] The results are as follows Fig.10 As shown in the figure, after injection of tetrahydrobiopterin (20, 40 μg / shrimp) under different ammonia nitrogen concentrations (50, 100, 150 mg / L), the hepatopancreas tissue damage of shrimp under ammonia nitrogen stress was significantly reduced in the tetrahydrobiopterin injection group compared with the control group. At the same time, the survival rate of shrimp was also significantly higher than that of the control group, as shown in the figure. Fig.11 shown.
[0056] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.
Claims
1. An application of tetrahydrobiopterin, characterized in that: Used to prepare preparations for improving the survival rate of crustaceans.
2. The use of tetrahydrobiopterin according to claim 1, characterized in that: Used for preparing preparations for improving the survival rate of crustaceans under ammonia nitrogen stress.
3. The use of tetrahydrobiopterin according to claim 2, characterized in that: Used for preparing preparations for improving the antioxidant capacity of crustaceans under ammonia nitrogen stress.
4. The use of tetrahydrobiopterin according to claim 2, characterized in that: Used for preparing preparations for increasing the proliferation rate of crustacean cells under ammonia nitrogen stress.
5. The use of tetrahydrobiopterin according to claim 2, characterized in that: Used for preparing preparations for reducing damage to crustaceans caused by ammonia nitrogen toxicity.
6. The use of tetrahydrobiopterin according to claim 5, characterized in that: Used for preparing preparations for reducing crustacean hepatopancreatic damage caused by ammonia nitrogen toxicity.
7. The use of tetrahydrobiopterin according to claim 1, characterized in that: The ratio of the added amount of tetrahydrobiopterin to the body weight of the crustacean is 20-40 μg / 6g; the crustacean includes shrimp.
8. A preparation obtained by using tetrahydrobiopterin according to claim 1.
9. The preparation according to claim 8, characterized in that The preparation comprises one or more of medicine and feed.
10. The preparation according to claim 8, characterized in that The medicament includes an injection.