Method for improving hypoxia resistance of pelteobagrus vachelli based on hypoxia induction of liver extracellular vesicles

Abstract

The invention belongs to the field of biotechnology and aquaculture, and particularly relates to a method for improving hypoxia resistance of pelteobagrus vachelli based on hypoxia induction of liver extracellular vesicles of the pelteobagrus vachelli. The method comprises the following steps: extracting high-purity extracellular vesicles EVs in liver tissues of pelteobagrus vachelli subjected to low-oxygen treatment by adopting a method of combining differential centrifugation and high-precision iodixanol density gradient centrifugation; diluting the extracted EVs to 2mu g / mu L by using PBS (Phosphate Buffer Solution) to prepare an EVs suspension; an intraperitoneal injection mode is adopted, the EVs suspension is injected into the body of the pelteobagrus vachelli, and after injection is conducted for 24 hours, the hypoxia resistance of the pelteobagrus vachelli is evaluated through a suffocation point detection test. The invention opens up a brand new path for improving the hypoxia resistance of the pelteobagrus vachelli, provides a new idea and a new method for efficiently enhancing the hypoxia resistance of economic fishes in the field of aquaculture, is expected to play an important role in actual aquaculture production, and assists the sustainable development of the aquaculture industry.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and aquaculture, and particularly relates to a method for improving the hypoxia tolerance of yellow catfish by inducing extracellular vesicles in liver cells under hypoxia. Background Technology

[0002] Yellow catfish (Pelteobagrus vachelli) Pelteobagrus vachelli As an important freshwater economic fish, the yellow catfish (Pelteobagrus vachelli) has long been a focus of attention in aquaculture due to its rapid growth, delicious flesh, and strong adaptability. However, its tolerance to low-oxygen environments is relatively weak, easily leading to hypoxic stress and even death during high-density farming and transportation, seriously affecting farming efficiency and the sustainable development of the industry. Therefore, developing a method to effectively improve the hypoxic tolerance of the yellow catfish is of great significance for increasing farming density, reducing farming risks, and optimizing farming efficiency.

[0003] Currently, although studies have been conducted to improve the hypoxia tolerance of fish through genetic selection and feed formulation optimization, these methods have certain limitations, such as long genetic selection cycles and high costs of feed additives. In recent years, extracellular vesicles (EVs), as an important medium for intercellular communication, have been shown to play a crucial role in various biological processes. EVs are nanoscale, membrane-bound vesicles secreted by all cells. They can act as carriers of various biomolecules, such as miRNAs, proteins, and phospholipids, playing a role in intercellular signal transduction and communication, and participating in regulatory processes such as gene expression, metabolic regulation, and immune responses under normal or pathological conditions. However, there are no reports on the application of EVs to improve the hypoxia tolerance of fish. Therefore, researching the application of EVs derived from the liver tissue of the yellow catfish (Pelteobagrus vachelli) under hypoxic conditions to improve the hypoxia tolerance of fish has significant innovative and practical value. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a method for improving the hypoxia tolerance of extracellular vesicles in liver cells of the yellow catfish by inducing hypoxia.

[0005] This invention is achieved by a method for enhancing the hypoxia tolerance of extracellular vesicles in liver cells of the yellow catfish (Pelteobagrus vachelli) through hypoxia-induced induction. The method includes: S1: Place the yellow catfish in a low-oxygen environment with a dissolved oxygen concentration of 1.0±0.1 mg / L for 4 hours; S2: High-purity extracellular vesicles (EVs) were extracted from the liver tissue of *Pelteobagrus vachelli* after hypoxia treatment using a combination of differential centrifugation and high-precision iodixanol density gradient centrifugation. The extracted EVs were diluted to 2 μg / μL with PBS to prepare an EV suspension. S3: The EV suspension was injected into the body of the yellow catfish using intraperitoneal injection. The hypoxia tolerance of the yellow catfish was evaluated by asphyxiation point detection test 24 hours after injection.

[0006] This invention provides an EVs suspension for improving the hypoxia tolerance of yellow catfish, wherein the EVs suspension is a high-purity EVs suspension extracted from the liver tissue of yellow catfish treated in a hypoxic environment of 1.0±0.1 mg / L for 4 hours.

[0007] Furthermore, the injection volume of the EVs is 6.67 μg / g.

[0008] Furthermore, intraperitoneal injection of liver tissue-derived EVs from yellow catfish under hypoxia can improve the hypoxia tolerance of yellow catfish.

[0009] Furthermore, S2 specifically includes: (1) Weigh the liver tissue after hypoxia treatment and cut it into small pieces with sterile scissors under ice bath; (2) Prepare a solution of collagenase II and IV with a concentration of 2 mg / mL and mix them at a volume ratio of 1:1; (3) Add 5 mM CaCl2 solution to the collagenase mixture; (4) Add collagenase mixture to the minced tissue and incubate at 25 °C with shaking for 100 min to digest into a suspension; (5) Add 1000× protease inhibitor and 100× phosphatase inhibitor; (6) After mixing the sample, centrifuge at 800 g for 5 min and take the supernatant; centrifuge at 5000 g for 20 min and take the supernatant. (7) The collected supernatant was passed through an 800 nm syringe filter and then through a 220 nm syringe filter. The filtrate was collected to obtain the sample. (8) Centrifuge the sample using a combination of ultracentrifugation and high-precision iodixanol density gradient centrifugation.

[0010] Furthermore, step (8) specifically includes: (1) Add the sample to the supernatant tube, add phosphate buffer to 3-4 mm from the tube opening, balance the weight to 3 decimal places, centrifuge at 100,000 g RCF at 4℃ for 2 hours using an SW41-Ti rotor, discard the supernatant, and obtain the precipitate at the bottom. (2) Add 200 μL of PBS to resuspend the precipitate, and then add 175 μL of PBS to resuspend it again to obtain 375 μL of crude EVs suspension; (3) Add 375 μL of EVs crude suspension obtained by ultracentrifugation to a new ultracentrifugation tube, and then add 1.125 mL of 60 w / v% iodixanol solution to the crude suspension. At this time, the density of this density layer is 45 w / v; slowly add 1.5 mL of 40 w / v%, 35 w / v%, 30 w / v%, 25 w / v%, 20 w / v%, 15 w / v%, and 10 w / v% gradient iodixanol solution to the ultracentrifugation tube, and centrifuge at 100,000 g (RCF) and 4 °C for 4 hours using an SW41-Ti rotor; (4) After centrifugation, the first three layers were taken out from top to bottom in 1.5 mL layers, with densities of 20 w / v%, 15 w / v%, and 10 w / v%, and placed in new ultracentrifuge tubes. 11.5 mL of PBS was added, the mixture was balanced, and the tubes were centrifuged for 2 hours at 100,000 g RCF and 4°C using an SW41 Ti ultracentrifuge rotor. Finally, the precipitate in each ultracentrifuge tube was resuspended in PBS to obtain an EVs suspension. The three layers were then combined to obtain a high-purity EVs extract.

[0011] Furthermore, S3 specifically includes: Injection preparation: Healthy yellow catfish of similar weight were selected as test subjects. The EVs extract was diluted with PBS to prepare a suspension of 2 μg / μL. Injection method: The EVs suspension was injected into the body of the yellow catfish by intraperitoneal injection at a dose of 6.67 μg / g. The fish was then temporarily kept in the tank for 24 hours after injection. Asphyxiation point test: 24 hours after injection of EVs, yellow catfish were placed in a closed environment, and the fish's movement and dissolved oxygen content in the water were recorded in real time. The critical asphyxiation point was calculated according to the method in GB / T 18654.8-2008, Germplasm testing of cultured fish - Part 8: Determination of oxygen consumption rate and critical asphyxiation point, in order to evaluate its impact on hypoxia tolerance.

[0012] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows: This invention extracts high-purity endothelial cells (EVs) from the liver tissue of *Pelteobagrus vachelli* by strictly controlling hypoxia-induced conditions (1.0 ± 0.1 mg / L dissolved oxygen, treatment for 4 hours). The EVs were then injected intraperitoneally at a dose of 6.67 μg / g. After 24 hours, asphyxiation point testing confirmed that the critical asphyxiation point of the EVs group was significantly lower than that of the PBS group. This effectively reduces the minimum dissolved oxygen requirement of *Pelteobagrus vachelli*, prolonging its survival time in hypoxic environments and solving the problem of mortality and losses due to hypoxia in high-density aquaculture and transportation. This method boasts high biosafety, low cost, and a short cycle, and can be directly applied to high-density aquaculture and long-distance transportation of *Pelteobagrus vachelli*, helping to increase aquaculture density, expand market coverage, and optimize aquaculture efficiency. This innovative method opens up a new path for improving the hypoxia tolerance of *Pelteobagrus vachelli*, providing new ideas and methods for efficiently enhancing the hypoxia tolerance of economically important fish species in aquaculture. It is expected to play an important role in actual aquaculture production and contribute to the sustainable development of the aquaculture industry.

[0013] (1) The expected benefits and commercial value of the technical solution of this invention after transformation are as follows: This technology can significantly improve the survival rate of high-density cultured yellow catfish, reduce fish mortality due to low oxygen levels, and help increase the yield of yellow catfish in limited water spaces. It also eliminates the need for continuous input of traditional feed additives, directly increasing the economic benefits of the aquaculture process. This technology can reduce the energy consumption for oxygenation and mortality risk during the transportation of yellow catfish, extend the transportation radius, and help aquaculture enterprises overcome geographical limitations, delivering products to a wider market, expanding sales coverage, and indirectly increasing overall revenue. From an industrial transformation perspective, the technology is highly efficient, easy to prepare, and can be applied on a large scale. Aquaculture technology service companies can develop it into a commercial product called "EVs formulation," forming a new profit growth point. At the same time, its technological framework can be applied analogously to improving the stress resistance of other economically important fish species, possessing long-term and broad commercial expansion potential.

[0014] (2) The technical solution of this invention fills a technical gap in the industry both domestically and internationally: Previous domestic and international studies have confirmed the value of EVs in areas such as physiological regulation and disease treatment in mammals, and have also conducted some preliminary explorations into the immune-related aspects of EVs in fish. However, no research has ever applied EVs to enhance the hypoxia tolerance of fish. This invention is the first to combine EV technology with the hypoxia tolerance requirements of the yellow catfish (Pelteobagrus vachelli), filling the application gap of EV technology in this field. Existing fish stress-enhancing technologies lack the exploration and application of the fish's own intrinsic regulatory mechanisms. This invention, for the first time, draws on the regulatory thinking of "netting training" to form a closed-loop path from EV function induction to effect implementation, filling the technical gap in the intrinsic regulation of hypoxia tolerance by fish through their own tissue-derived EVs. It provides a reusable new paradigm for the development of EV technology for other fish species to enhance their resistance to high temperatures, diseases, and other stresses. Attached Figure Description

[0015] Figure 1 This is a flowchart of a method for improving the hypoxia tolerance of yellow catfish by inducing hypoxia in extracellular vesicles of liver cells. Figure 2 This is a transmission electron microscope image of EVs from liver tissue of yellow catfish under hypoxia provided in the embodiments of the present invention; Figure 3 This is a diagram showing the suffocation point test results provided in the embodiments of the present invention.

[0016] Figure 4 This is a diagram showing the asphyxiation point test results for different EV injection volumes provided in the comparative examples of this invention. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0018] like Figure 1 As shown, this embodiment of the invention provides a method for improving the hypoxia tolerance of extracellular vesicles in liver cells of the yellow catfish (Pelteobagrus vachelli) by inducing hypoxia. The method includes: S1: Place the yellow catfish in a low-oxygen environment with a dissolved oxygen concentration of 1.0±0.1 mg / L for 4 hours; S2: High-purity extracellular vesicles (EVs) were extracted from the liver tissue of *Pelteobagrus vachelli* after hypoxia treatment using a combination of differential centrifugation and high-precision iodixanol density gradient centrifugation. The extracted EVs were diluted to 2 μg / μL using phosphate-buffered saline (PBS) to prepare an EVs suspension. S3: The EV suspension was injected into the body of the yellow catfish using intraperitoneal injection. The hypoxia tolerance of the yellow catfish was evaluated by asphyxiation point detection test 24 hours after injection.

[0019] Example: Hypoxia stress test of yellow catfish (Pelteobagrus vachelli) Randomly selected yellow catfish (purchased from Sichuan Rongsen Fishery Co., Ltd., weighing 15±0.94g) were used in the experiment. They were healthy, growing well, and free from disease, injury, and deformity.

[0020] The experimental fish were pre-cultured in a water circulation system for two weeks. They were fasted for 24 hours the day before the experiment. The aeration and water circulation systems were turned off to start low oxygen stress. The dissolved oxygen level was reduced from 6.8 mg / L to 1.0 mg / L by introducing pure nitrogen for 30 minutes. An appropriate amount of nitrogen was continuously introduced to maintain the dissolved oxygen level at 1.0±0.1 mg / L for 4 hours. The liver tissue of 10 fish was taken, treated with liquid nitrogen, and stored at -80 ℃ for later use.

[0021] Extraction and characterization analysis of EVs from liver tissue of yellow catfish (Pelteobagrus vachelli) EVs derived from liver tissue of *Pelteobagrus vachelli* were extracted using a combination of differential centrifugation and high-precision density gradient centrifugation, and their morphology was observed using transmission electron microscopy. The specific steps are as follows: Weigh 2 g of liver tissue after hypoxia treatment and cut it into small pieces with sterile scissors under ice bath. Collagenase II and IV (collagenase II: Servicebio, catalog number GC305014; collagenase IV: Servicebio, catalog number GC305015) were prepared into a 2 mg / mL solution and mixed at a volume ratio of 1:1. Add 5 mM CaCl2 (Comio, analytical grade) solution to the collagenase mixture; Add collagenase mixture to the minced tissue and incubate at 25 °C with shaking for 100 min to digest into a suspension; Add 16 μL of 1000× protease inhibitor (KGI Biotech, catalog number: KGB5101-100) and 160 μL of 100× phosphatase inhibitor (KGI Biotech, catalog number: KGB5101-2); After mixing the samples, centrifuge at 800 g for 5 min and collect the supernatant; centrifuge at 5000 g for 20 min and collect the supernatant. The collected supernatant was passed through an 800 nm syringe filter and then through a 220 nm syringe filter. The filtrate was collected to obtain the sample. The sample was centrifuged using a combination of ultracentrifugation and high-precision iodixanol density gradient centrifugation, including the following four steps: Add the sample to the supernatant tube, add PBS to a depth of 3-4 mm from the tube opening, balance the volume, and weigh to 3 decimal places. Centrifuge at 100,000 g (RCF) for 2 hours using an SW41-Ti rotor at 4°C. Discard the supernatant and obtain the precipitate at the bottom.

[0022] The precipitate was resuspended in 200 μL of PBS, and then resuspended again in 175 μL of PBS to obtain 375 μL of crude EV suspension.

[0023] 375 μL of the EVs coarse suspension obtained by ultracentrifugation was added to a new ultracentrifugation tube, followed by 1.125 mL of 60 w / v% iodixanol solution (Serumwerk Bernburg AG, catalog number: 1893), bringing the density of this density layer to 45 w / v%. A gradient of iodixanol solutions of 40 w / v%, 35 w / v%, 30 w / v%, 25 w / v%, 20 w / v%, 15 w / v%, and 10 w / v% was slowly added sequentially to the ultracentrifugation tube. The mixture was centrifuged at 100,000 g (RCF) for 4 hours using an SW41-Ti rotor at 4°C.

[0024] After centrifugation, the first three layers (20 w / v%, 15 w / v%, and 10 w / v%) were removed from top to bottom in 1.5 mL increments and placed in new ultracentrifuge tubes. 11.5 mL of PBS was added, the mixture was balanced, and the tubes were centrifuged at 100,000 g (RCF) and 4 °C for 2 hours using an SW41 Ti ultracentrifuge rotor. Finally, the precipitate in each ultracentrifuge tube was resuspended in 200 μL of PBS to obtain the EV suspension.

[0025] The first three EV suspensions were combined to obtain a high-purity EV extract, which was then characterized using transmission electron microscopy (TEM). TEM observations are as follows: Figure 2 As shown, EVs derived from liver tissue of the yellow catfish exhibit a typical "cup-shaped" structure.

[0026] Preparation of EV suspension The EVs extract obtained in S2 was diluted with PBS (based on the results of the BCA protein concentration kit) to prepare a suspension with a concentration of 2 μg / μL.

[0027] Grouping information Healthy yellow catfish (15±0.94g) were randomly divided into two groups: the PBS group and the EVs group, with three replicates in each group (i.e., the experiment was repeated three times), and 10 fish in each replicate. Specific information is as follows: PBS group: Each fish was injected intraperitoneally with PBS (3.33 μL / g). EVs group: Each fish was intraperitoneally injected with EVs derived from the liver tissue of the yellow catfish under hypoxia (injection dose of 6.67 μg / g).

[0028] Asphyxiation point determination test The critical asphyxiation point is defined as the measured oxygen content per unit volume of water when 50% of the test fish's gill covers cease movement. Twenty-four hours after injecting EVs, the aeration and water circulation systems of the test tank were shut off, and the tank was covered with a membrane to isolate it from air. The asphyxiation point determination experiment began. Using the cessation of gill cover movement as the criterion, the number of dead fish in each group and the dissolved oxygen content in the water were recorded in real time to calculate the critical asphyxiation point.

[0029] like Figure 4 As shown in the comparative example: the effect of different EV injection doses on the asphyxiation point of yellow catfish. The grouping information is the same as in the previous example.

[0030] Healthy yellow catfish (15±0.94g) were randomly divided into 4 groups: PBS group, EVs-1.67 group, EVs-3.33 group, and EVs-6.67 group, with 3 replicates in each group and 10 fish in each replicate. Specific information is as follows: PBS group: Each fish was injected intraperitoneally with PBS (3.33 μL / g). EVs-1.67 group, EVs-3.33 group and EVs-6.67 group: each fish in each group was intraperitoneally injected with 1.67 μg / g, 3.33 μg / g and 6.67 μg / g of liver tissue-derived EVs from yellow catfish under hypoxia (based on fish body weight).

[0031] Asphyxiation point determination test: Same as the example.

[0032] This invention belongs to the fields of biotechnology and aquaculture, and specifically relates to a method for improving the hypoxia tolerance of yellow catfish by inducing extracellular vesicles in liver cells under hypoxia. Specifically, it is applied to the farming and transportation of yellow catfish, including enhancing the hypoxia tolerance of fish in high-density farming by injecting hypoxia-induced extracellular vesicles into the liver, reducing oxygen deficiency mortality caused by high summer temperatures and eutrophication, and facilitating increased farming density; and reducing the risk of hypoxia-induced mortality during transportation, extending the transportation radius to broaden market coverage.

[0033] Based on the results of the asphyxiation point determination experiment, the experimental data were statistically analyzed using GraphPad Prism 10.4.2. For example... Figure 3 As shown, the critical asphyxiation point in the EVs group was significantly lower than that in the PBS group. Hypoxia treatment of *Pelteobagrus vachelli* with liver tissue-derived EVs for 4 hours significantly reduced the critical asphyxiation point and improved its hypoxia tolerance. Figure 4As shown, different EV injection doses have different effects on the critical asphyxiation point of yellow catfish, which is dose-dependent. With the increase of injection dose, the asphyxiation point gradually decreases and the tolerance to hypoxia increases accordingly. The EVs-6.67 group is statistically significant.

[0034] Enteroblasts (EVs), as natural carriers of intercellular communication, can carry functional molecules such as miRNAs and proteins from source cells and deliver them to target cells via membrane fusion or receptor-mediated pathways, thereby regulating their physiological functions. After hypoxia stimulation, liver EVs are enriched with miRNAs regulating HIF-1α, as well as antioxidant enzymes and glycolytic enzymes. In this invention, after 4 h of hypoxia treatment with 1.0 ± 0.1 mg / L, *Pelteobagrus vachelli* liver EVs also showed the aforementioned differentially expressed molecules in miRNA and proteomics analysis (internal omics experimental data, unpublished). When these EVs are injected intraperitoneally into recipient fish, they can be distributed to organs such as gills, heart, and muscles via body fluid circulation. On the one hand, the miRNAs carried by EVs can activate the body's hypoxia tolerance pathways by inhibiting target gene expression, such as promoting erythropoiesis and enhancing the oxygen-carrying capacity of hemoglobin. On the other hand, the antioxidant proteins they carry can reduce the accumulation of reactive oxygen species (ROS) under hypoxic conditions, reducing cellular oxidative damage. At the same time, energy metabolism-related enzymes can regulate the body's metabolic patterns, efficiently transitioning from aerobic to anaerobic respiration and reducing oxygen consumption. These molecules collectively provide a "pre-adaptation" for the body to cope with hypoxia damage, ultimately manifested in the fact that the critical asphyxiation point of the EV group was significantly lower than that of the PBS group in the experiment, confirming the regulatory role of EVs on the hypoxia tolerance of fish at the functional mechanism level.

[0035] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for improving the hypoxia tolerance of extracellular vesicles in liver cells of the yellow catfish (Pelteobagrus vachelli) by inducing hypoxia, characterized in that... Includes the following steps: S1. Place the yellow catfish in a low-oxygen environment with a dissolved oxygen concentration of 1.0 ± 0.1 mg / L for 4 hours. S2. High-purity extracellular vesicles were extracted from the liver tissue of the hypoxic-treated yellow catfish using a combination of differential centrifugation and iodixanol density gradient centrifugation, and diluted to a concentration of 2 micrograms per microliter with phosphate buffer to obtain an extracellular vesicle suspension. S3. The extracellular vesicle suspension was injected into the body of the yellow catfish via intraperitoneal injection. After injection, the fish was temporarily kept for 24 hours, and its tolerance to low oxygen was evaluated by the asphyxiation point detection method.

2. The method according to claim 1, characterized in that, The amount of extracellular vesicle suspension injected intraperitoneally was 6.67 micrograms per gram of body weight.

3. The method according to claim 1, characterized in that, The evaluation steps include: placing the injected yellow catfish in a closed water environment, monitoring its movement and dissolved oxygen changes in the water in real time, and calculating the critical asphyxiation point according to the national standard GB / T18654.8-2008.

4. The method according to claim 1, characterized in that, The extracellular vesicle suspension, when injected intraperitoneally, can significantly reduce the critical asphyxiation point of yellow catfish and improve its tolerance to low-oxygen environments.

5. An extracellular vesicle suspension for improving the hypoxia tolerance of yellow catfish (Pelteobagrus vachelli), characterized in that, The suspension was derived from the liver tissue of yellow catfish treated for 4 hours in a hypoxic environment with a dissolved oxygen concentration of 1.0 ± 0.1 mg / L. High-purity extracellular vesicles were obtained by differential centrifugation and density gradient centrifugation with iodixanol, and then diluted with phosphate buffer to form a suspension with a concentration of 2 μg / μL.

6. The application of an extracellular vesicle in improving the hypoxia tolerance of *Pelteobagrus vachelli*, characterized in that... The extracellular vesicles were derived from the liver tissue of yellow catfish that had undergone hypoxia-induced treatment.

7. The application according to claim 6, characterized in that, The extracellular vesicles enhance the cell's metabolic regulation capacity in response to hypoxia by modulating the body's oxidative stress-related signaling pathways.

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