Application of FeSO4 in preparation of medicine or feed for promoting phagocytic activity of coelomic cells of stichopus japonicus
By adding ferrous sulfate to the ginseng feed, the expression of hsp70 and c3 genes is promoted and the TLR3 gene pathway is regulated, which solves the problem of germ-corrosive syndrome in ginseng breeding and improves the nonspecific immunity and survival rate of ginseng.
Patent Information
- Application Number
- CN202510633469.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-16
- Publication Date
- 2025-07-25
AI Technical Summary
During the breeding of ginseng ginseng, the economic losses caused by ginseng skin rot syndrome are serious, and the existing technology cannot effectively improve the non-specific immunity of ginseng to prevent and treat this disease.
Add 0.5% to 2% ferrous sulfate to the ginseng feed to promote the expression of hsp70 and c3 genes, regulate the immune signaling pathway mediated by the tlr3 gene, and enhance the identification and removal ability of ginseng to ginseng's exogenous pathogens.
Significantly enhance the non-specific immunity of ginseng, improve the survival rate of Vitiligo infectious disease, improve the structure of the water body bacteria, reduce the number of harmful bacteria, and prevent corrosive skin syndrome.
Smart Images
Figure CN120361043A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of aquaculture, and specifically relates to the application of FeSO4 in the preparation of a drug or feed for promoting the phagocytic activity of coelomocytes of Apostichopus japonicus. Background Art
[0002] Apostichopus japonicus, also known as the sea cucumber, is the main aquaculture variety in China's sea cucumber aquaculture industry. In recent years, with the continuous expansion of the aquaculture scale and the improvement of intensification, disease problems have become increasingly serious, posing challenges to the development of the industry. Apostichopus japonicus skin ulcer syndrome is the most common and serious disease in the process of Apostichopus japonicus aquaculture, causing economic losses of billions of yuan every year and has become a key factor restricting the stable development of the industry. This disease was first discovered in Shandong Province in February 2003 and began to break out on a large scale along the northern coast of China in 2004. In recent years, affected by factors such as global climate change, germplasm degradation, and pathogen evolution, the occurrence of this disease has gradually become normal, posing a severe challenge to the Apostichopus japonicus aquaculture industry.
[0003] As a common inorganic iron salt, ferrous sulfate can promote the synthesis of hemoglobin, has a therapeutic effect on anemia, and can also enhance the immunity of animals. Some studies have shown that ferrous sulfate can effectively induce the cell death of Vibrio harveyi through the influx of Fe 2+ . Moreover, different from traditional antibiotics, the antibacterial mechanism of ferrous sulfate does not rely on the induction of the burst of reactive oxygen species (ROS) and lipid peroxides, but inhibits the growth of Vibrio through a unique iron-dependent cell death mode. This mechanism not only effectively inhibits Vibrio but also avoids the generation of antibiotic resistance. However, Apostichopus japonicus belongs to the phylum Echinodermata, and its coelomic fluid contains some special immune cells and respiratory pigments, such as hemocyanin (hemacyanin), etc., rather than hemoglobin. Hemocyanin is rich in copper ions and plays a role in processes such as oxygen transport, making the blood of Apostichopus japonicus show different colors at different oxygen contents. Therefore, the application effect of ferrous sulfate in Apostichopus japonicus is unpredictable. Summary of the Invention
[0004] The present invention provides the application of ferrous sulfate in the preparation of a drug or feed for promoting the phagocytic activity of coelomocytes of Apostichopus japonicus for the above technical problems. The ferrous sulfate can significantly enhance the non-specific immunity of Apostichopus japonicus.
[0005] The present invention is realized through the following technical solutions:
[0006] Application of FeSO4 in preparing drugs or feeds for promoting phagocytic activity of coelomocytes of Apostichopus japonicus. The application method is to add ferrous sulfate to drugs or feeds to increase the expression of hsp70 and c3 genes in Apostichopus japonicus, thereby improving the pathogen clearance ability of Apostichopus japonicus. At the same time, by regulating the immune signaling pathway mediated by the tlr3 gene, the ability of Apostichopus japonicus to recognize exogenous pathogens is promoted.
[0007] Furthermore, the application is to add ferrous sulfate with a mass ratio of 0.5% - 2% to the feed for Apostichopus japonicus.
[0008] The present invention also provides the application of the FeSO4 in preparing feeds for preventing the skin ulcer syndrome of Apostichopus japonicus. The application is to add ferrous sulfate with a mass ratio of 0.5% - 2% to the basic feed for Apostichopus japonicus to achieve the effect of preventing the skin ulcer syndrome of Apostichopus japonicus.
[0009] Beneficial effects of the present invention compared with the prior art: By adding ferrous sulfate to the feed or drugs for Apostichopus japonicus in the present invention, the iron ions in the ferrous sulfate can enhance the stress tolerance ability of the cells of Apostichopus japonicus by enhancing the expression of hsp70, thereby improving its disease resistance; ferrous sulfate can also promote the expression of the c3 gene by activating the complement system, thereby enhancing the pathogen clearance ability of Apostichopus japonicus; at the same time, ferrous sulfate can also promote the ability of Apostichopus japonicus to recognize exogenous pathogens by regulating the immune signaling pathway mediated by tlr3, thereby enhancing its immune defense response. All these indicate that the present invention can significantly enhance the non-specific immunity of Apostichopus japonicus.
[0010] By adding 0.5% - 2% of ferrous sulfate to the feed for Apostichopus japonicus in the present invention, the non-specific immunity of Apostichopus japonicus can be effectively improved, the survival rate of Apostichopus japonicus when facing Vibrio splendidus infection can be increased, and the water body flora structure of the Apostichopus japonicus breeding environment can also be improved, reducing the proportion of the number of harmful bacteria. Description of the Drawings
[0011] Figure 1 Survival rate diagram of the cultured Apostichopus japonicus of the present invention;
[0012] Figure 2 Relative abundance diagram of the water body flora of the cultured Apostichopus japonicus of the present invention before and after virus challenge; a is before virus challenge, b is after virus challenge;
[0013] Figure 3 Non-specific immunity comparison diagram of the cultured Apostichopus japonicus of the present invention. a is the expression of the hsp70 gene in each experimental group, b is the expression of the c3 gene in each experimental group, c is the expression of the tlr3 gene in each experimental group, d is the phagocytic activity of the coelomocytes of the Apostichopus japonicus in each experimental group, and e is the respiratory burst activity of the coelomocytes of the Apostichopus japonicus in each experimental group. Detailed Embodiments
[0014] The technical solutions in the embodiments of the present invention will be clearly and completely described below. The following described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. The exemplified embodiments do not limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.
[0015] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods.
[0016] Unless otherwise specified, the materials, reagents, etc. used in the following embodiments can all be obtained from commercial channels.
[0017] Example 1: Application of FeSO4 in the preparation of feed for preventing Apostichopus japonicus skin ulcer syndrome
[0018] I. Feed preparation
[0019] (1) Mixing: First, mix 55% of Sargassum powder, 15% of fish meal, 2% of guar gum, 2% of compound vitamins, 2% of compound mineral elements, and 23.5% of sea mud according to the specified weight fractions of each component to make a basic feed; the basic feed is not limited to the above ratio, and any feed that can meet the healthy growth needs of Apostichopus japonicus can be used as the basic feed; in this embodiment, the compound vitamins include vitamin A, D3, E, K3, B1, B2, B6, B12, C, H, nicotinic acid, folic acid, nicotinamide, etc., and the compound vitamins are purchased from Henan Huachu Biotechnology Co., Ltd.
[0020] The compound minerals include iron, copper, zinc, manganese, magnesium, iodine, selenium, cobalt, etc., and are purchased from Henan Huachu Biotechnology Co., Ltd.
[0021] (2) Pelleting: Add 0.5% of ferrous sulfate by weight to the basic feed, and then add 20% of pure water based on the weight of the basic feed raw materials. Mix the raw materials into a dough-like shape, and use a feed pelletizer to press the uniformly mixed raw materials into pellet feed;
[0022] (3) The pellets are sealed and stored after drying.
[0023] II. Effect verification
[0024] Select healthy Apostichopus japonicus with a body weight of about 20 g. Set 3 parallels for each experimental group. The initial feeding amount is 3% of the body weight of Apostichopus japonicus. Feed Apostichopus japonicus once at 7:00 every morning. After feeding, perform bottom suction and water change after 12 hours. Adjust the feeding amount according to the growth of Apostichopus japonicus every 10 days. At the same time, set the feeding of the basic feed product as the control group and feed in the same way for 60 days.
[0025] Example 2: Application of FeSO4 in the preparation of feed for preventing Apostichopus japonicus skin ulcer syndrome, and the application comprises the following steps:
[0026] I. Feed preparation
[0027] (1) Mixing materials: First, mix 55% of sargassum powder, 15% of fish meal, 2% of guar gum, 2% of compound vitamins, 2% of compound mineral elements, and 23% of sea mud evenly according to the specified weight fractions of each component to prepare a basic feed. The basic feed is not limited to the above ratio, and any feed that can meet the healthy growth needs of Apostichopus japonicus can be used as the basic feed. In this example, the compound vitamins include vitamin A, D3, E, K3, B1, B2, B6, B12, C, H, nicotinic acid, folic acid, nicotinamide, etc., and are purchased from Henan Huachu Biotechnology Co., Ltd.
[0028] The compound minerals include iron, copper, zinc, manganese, magnesium, iodine, selenium, cobalt, etc., and are purchased from Henan Huachu Biotechnology Co., Ltd.
[0029] (2) Pelleting: Add 1% of ferrous sulfate based on the weight of the basic feed, and then add 20% of pure water based on the weight of the basic raw materials. Mix the raw materials evenly into a dough-like shape, and use a feed pelletizer to press the evenly mixed raw materials into pellet feed.
[0030] (3) After drying the pellets, store them sealed.
[0031] II. Effect verification
[0032] Select healthy Apostichopus japonicus with a body weight of about 20 g. Set 3 parallels for each experimental group. The initial feeding amount is 3% of the body weight of Apostichopus japonicus. Feed Apostichopus japonicus once at 7:00 every morning. After feeding, perform bottom suction and water change after 12 hours. Adjust the feeding amount every 10 days according to the growth situation of Apostichopus japonicus. At the same time, set the feeding of the basic feed product as the control group and feed it in the same way for 60 days.
[0033] Example 3: Application of FeSO4 in the preparation of feed for preventing Apostichopus japonicus skin ulcer syndrome, and the application method comprises the following steps:
[0034] I. Feed preparation
[0035] (1) Mixing materials: First, mix 55% of sargassum powder, 15% of fish meal, 2% of guar gum, 2% of compound vitamins, 2% of compound mineral elements, and 22.5% of sea mud evenly according to the specified weight fractions of each component to prepare a basic feed. The basic feed is not limited to the above ratio, and any feed that can meet the healthy growth needs of Apostichopus japonicus can be used as the basic feed. In this example, the compound vitamins include vitamin A, D3, E, K3, B1, B2, B6, B12, C, H, nicotinic acid, folic acid, nicotinamide, etc., and are purchased from Henan Huachu Biotechnology Co., Ltd.
[0036] The composite mineral contains iron, copper, zinc, manganese, magnesium, iodine, selenium, cobalt, etc., and is purchased from Henan Huachu Biotechnology Co., Ltd.
[0037] (2) Pelleting: Add ferrous sulfate accounting for 1.5% of the weight of the basic feed, and then add pure water accounting for 20% of the weight of the basic raw materials. Mix the raw materials into a dough-like state, and use a feed pelletizer to press the uniformly mixed raw materials into pellet feed.
[0038] (3) After drying the pellets, store them sealed.
[0039] II. Effect verification
[0040] Select healthy sea cucumbers weighing about 20 g. Set up 3 parallels for each experimental group. The initial feeding amount is 3% of the weight of the sea cucumbers. Feed the sea cucumbers once at 7:00 every morning. After feeding, conduct bottom suction and water change after 12 hours. Adjust the feeding amount according to the growth of the sea cucumbers every 10 days. At the same time, set the group feeding the basic feed product as the control group, and feed in the same way for 60 days.
[0041] Example 4: Application of FeSO4 in the preparation of feed for preventing the skin ulcer syndrome of sea cucumbers. The application method includes the following steps:
[0042] I. Feed preparation
[0043] (1) Mixing: First, stir 55% of sargassum powder, 15% of fish meal, 2% of guar gum, 2% of compound vitamins, 2% of compound mineral elements, and 22% of sea mud evenly according to the specified weight fractions of each component to make a basic feed; the basic feed is not limited to the above ratio, and any feed that can meet the healthy growth needs of sea cucumbers can be used as the basic feed; in this example, the compound vitamins include vitamin A, D3, E, K3, B1, B2, B6, B12, C, H, nicotinic acid, folic acid, nicotinamide, etc., and are purchased from Henan Huachu Biotechnology Co., Ltd.
[0044] The composite mineral contains iron, copper, zinc, manganese, magnesium, iodine, selenium, cobalt, etc., and is purchased from Henan Huachu Biotechnology Co., Ltd.
[0045] (2) Pelleting: Add ferrous sulfate accounting for 2% of the weight of the basic feed, and then add pure water accounting for 20% of the weight of the basic raw materials. Mix the raw materials into a dough-like state, and use a feed pelletizer to press the uniformly mixed raw materials into pellet feed.
[0046] (3) After drying the pellets, store them sealed.
[0047] II. Effect verification
[0048] Select healthy sea cucumbers weighing about 20 g. Set up 3 parallels for each experimental group. The initial feeding amount is 3% of the sea cucumber's weight. Feed the sea cucumbers once at 7:00 every morning. After feeding, perform bottom suction and water change 12 hours later. Adjust the feeding amount every 10 days according to the growth of the sea cucumbers. At the same time, set the feeding of the basic feed product as the control group and feed it in the same way for 60 days.
[0049] The feeding effects of Examples 1-4 are as follows:
[0050] As Figure 1 is the survival rate chart of farmed sea cucumbers in Examples 1-4. Compared with the control group, the survival rate of the experimental group increased significantly. Especially in the group adding Example 4, the survival rate reached 100% (P < 0.05), indicating that the feed of the present invention has a significant positive effect on the disease resistance of sea cucumbers.
[0051] Take some farmed sea cucumbers from Examples 1-4 for a challenge experiment. The challenge method is to add a suspension of Vibrio splendidus with a concentration of 1×10 7 CFU / mL into each water tank so that the concentration of Vibrio splendidus in the seawater is 1×10 5 CFU / ml. Soak continuously for 14 days, change half of the water every day, and re-add the suspension of Vibrio splendidus after water change to maintain the vibrio concentration. Observe the situation of sea cucumbers every day during this period. As Figure 2 shown is the relative abundance chart of water body flora before and after the challenge of farmed sea cucumbers. Before the challenge, there were obvious differences in the flora structures of each experimental group. The relative abundance of Actinobacteria in the control group was 16.7, while in the group of Example 4 it was only 7.15. This may indicate that at the time of not being invaded by pathogens, a higher concentration of ferrous sulfate has an impact on the relative abundance of some bacterial communities (such as Actinobacteria). The relative abundance of Bacteroidetes reached the highest level (26.14%) in the group of Example 4. Ferrous sulfate may have promoted the proliferation of this part of the flora. This phylum has been proven to play an important role in many aquatic ecosystems, including promoting nutrient metabolism, regulating the stability of the microbial community and inhibiting the growth of pathogenic bacteria, which established a defense line for the subsequent invasion of Vibrio splendidus. And the relative abundance of Proteobacteria showed a decreasing trend from 63.55% in the control group to each experimental group step by step, and it was 53.97% in the group of Example 4, indicating that it has a regulatory role in helping to reduce the proportion of potential pathogenic bacteria at the initial stage. Proteobacteria include many opportunistic pathogens, and an excessive proportion of it may lead to flora imbalance and disease occurrence. After the challenge, flora reconstruction occurred in each experimental group, but the change in the group of Example 4 was particularly significant. The relative abundance of Actinobacteria in the group of Example 4 increased from a lower initial abundance to 44.87%. Actinobacteria may inhibit pathogenic bacteria by secreting antibacterial substances and provide protection for sea cucumbers. It shows that the present invention effectively inhibits the proliferation of potential pathogenic bacteria.
[0052] Take a part of the cultured sea cucumbers in Examples 1-4 for real-time fluorescence quantitative qPCR detection. Take a certain amount of the inner body wall of each sea cucumber, add 1 ml of TRIzol reagent, and grind evenly. Let it stand at room temperature for 5 minutes to ensure cell lysis and release of RNA. Then add 0.2 ml of chloroform, mix well by shaking, let it stand at room temperature for 5 minutes, and then centrifuge at 12,000 r / min for 10 minutes, which is divided into three layers. Carefully transfer the supernatant to a new tube, add an equal volume of isopropanol and mix well, let it stand at room temperature for 10 minutes to promote RNA precipitation. Centrifuge again at 12,000 r / min for 10 minutes to remove the supernatant. Add 75% ethanol to wash the RNA precipitate, and then centrifuge at 12,000 r / min for 5 minutes. After removing the ethanol, air-dry the RNA precipitate naturally and dissolve it in RNase-free water. Detect its concentration and quality by spectrophotometer and gel electrophoresis. For cDNA synthesis, use the FastKing One-Step Genomic DNA Removal and cDNA First Strand Synthesis Premix Kit from Tiangen Biochemical Technology (Beijing) Co., Ltd. to reverse transcribe RNA into cDNA. The qPCR reaction uses the FastReal Fast Fluorescent Quantitative PCR Premix Kit (SYBR Green) from Tiangen Biochemical Technology (Beijing) Co., Ltd. for real-time fluorescence quantitative PCR. To ensure the reliability of the data, 3 technical replicates are performed for each experimental group. That is, qPCR reactions are performed with 3 technical replicates for each sample to ensure the accuracy and consistency of the results. The gene expression level is analyzed and compared using the 2 -ΔΔCT -method, which can effectively eliminate operation errors and technical biases, thereby improving the reliability of experimental data. Such as Figure 3The figure shows the comparison chart of the non-specific immunity of cultured sea cucumbers in Examples 1-4. The change in the phagocytic activity of sea cucumber coelomocytes indicates the immune-enhancing effect of ferrous sulfate. The phagocytic activity of sea cucumber coelomocytes increased significantly. The phagocytic activity of sea cucumber coelomocytes is the core link of sea cucumber immune defense. Phagocytes directly participate in the disease resistance process by recognizing, phagocytosing, and degrading pathogenic bacteria, indicating that adding 0.5-2% ferrous sulfate to the sea cucumber feed can effectively enhance the non-specific immune ability of sea cucumbers and enhance their resistance to pathogenic bacteria infection. The heat shock protein 70 gene (hsp70) was significantly up-regulated in all ferrous sulfate experimental groups, especially in the group of Example 4, significantly enhancing the expression of hsp70. As an important stress protein, hsp70 can help cells cope with oxidative stress and pathogen invasion. Iron ions may enhance the stress tolerance of sea cucumber cells by increasing the expression of hsp70, thereby improving their disease resistance. The expression of complement c3 gene also showed a concentration-dependent change in the ferrous sulfate experimental group. The expression of c3 gene increased significantly in the group of Example 4. Long-term feeding of ferrous sulfate may promote the expression of c3 gene by activating the complement system, thereby enhancing the pathogen clearance ability of sea cucumbers. The complement system is a core component of immune defense, and the enhancement of its activity is crucial for improving the disease resistance of sea cucumbers. The expression of tlr3 gene increased significantly in the group of Example 4, indicating that ferrous sulfate may promote the recognition ability of sea cucumbers to exogenous pathogens by regulating the immune signaling pathway mediated by tlr3, thereby enhancing their immune defense response. As an important pattern recognition receptor, the up-regulation of tlr3 indicates that the innate immune response of sea cucumbers is significantly activated. All these indicate that the present invention can significantly enhance the non-specific immunity of sea cucumbers.
Claims
1. The application of FeSO4 in the preparation of drugs or feeds for promoting the phagocytic activity of coelomocytes of Apostichopus japonicus, characterized in that, The application method is to add ferrous sulfate to drugs or feeds to increase the expression of hsp70 and c3 genes in sea cucumbers, thereby enhancing the pathogen clearance ability of sea cucumbers. At the same time, by regulating the immune signaling pathway mediated by the tlr3 gene, the ability of sea cucumbers to recognize exogenous pathogens is promoted.
2. The application according to claim 1, wherein Finally, ferrous sulfate is added to the feed for sea cucumbers at a mass ratio of 0.5% - 2%.
3. Application of FeSO4 in preparing feed for preventing Apostichopus japonicus skin ulcer syndrome, characterized in that, The application is to add ferrous sulfate at a mass ratio of 0.5% - 2% to the basic feed for sea cucumbers to achieve the effect of preventing the skin ulcer syndrome of sea cucumbers. The application method is to add ferrous sulfate to the feed to increase the expression of hsp70 and c3 genes in sea cucumbers, thereby enhancing the pathogen clearance ability of sea cucumbers. At the same time, by regulating the immune signaling pathway mediated by the tlr3 gene, the ability of sea cucumbers to recognize exogenous pathogens is promoted. At the same time, the killing function of FeSO4 on Vibrio splendidus in water is utilized to prevent the skin ulcer syndrome of sea cucumbers.