Use of eriodictyol in the prevention of koi herpesvirus disease

Abstract

The application belongs to the technical field of biology, and discloses a use of eriodictyol in preparation of a product for preventing and / or treating fish infected with mandarinfish frog iridovirus, wherein the eriodictyol can activate an Nrf2-Keap1 / SOD pathway in vivo and in vitro, improve antioxidant capacity, remove ROS accumulation caused by MRV infection, inhibit MRV proliferation, reduce tissue damage caused by MRV infection, improve survival rate of large-mouth bass, and play an anti-MRV role. The application provides a basis for application of ER in anti-MRV infection, and provides a new idea for further screening of anti-MRV drugs. Meanwhile, the application also provides a medicine containing eriodictyol, fish feed and fish feed additive.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically the use of sennaol in the treatment of mandarin frog iridovirus. Background Technology

[0002] Mandarinfish ranavirus (MRV), belonging to the family Iridoviridae and the genus Ranavirus, is a linear double-stranded DNA virus with icosahedral viral particles. MRV shares a highly similar genome with largemouthbass ranavirus (LMBV), and they are considered to be isolates of the same virus in different hosts. MRV / LMBV infection in mandarin fish (Siniperca chuatsi) and largemouth bass (Micropterus salmoides) can lead to inflammatory damage in multiple tissues, including the pyloric caeca, liver, spleen, intestines, gills, and kidneys. In the inflammatory response, reactive oxygen species (ROS) act as both inflammatory signaling molecules and inflammatory mediators. ROS mediate the expression of inflammatory genes by activating the NF-κB signaling pathway. MRV / LMBV infection induces a significant increase in ROS levels.

[0003] ROS (Reactive Oxidant Species) is regulated by various enzymes in the intracellular antioxidant system, maintaining a dynamic balance and cellular homeostasis. When invaded by pathogens or stimulated by external factors, excessive ROS is produced intracellularly, causing oxidative stress and potentially inducing inflammatory damage. The antioxidant system, with superoxide dismutase (SOD) and other antioxidant enzymes at its core, plays a crucial role in scavenging ROS and maintaining homeostasis. In the screening of anti-LMBV drugs, anthocyanins and other substances have been found to activate the activity of antioxidant enzymes such as SOD, glutathione peroxidase (GPX), and catalase (CAT), exerting antiviral effects. Furthermore, many substances have been shown to have anti-MRV / LMBV effects, such as baicalin, naringenin, and isoliquiritigenin, all of which have shown some efficacy in inhibiting MRV / LMBV.

[0004] Erodityol (ER) is a flavonoid compound with significant antioxidant and anti-inflammatory activities. ER can activate the nuclear factor E2-related factor 2 (Nrf2) pathway, activating the expression of antioxidant enzyme genes such as SOD and GPX, and reducing tissue damage caused by pathogen infection. Although ER, baicalin, naringenin, and isoliquiritigenin all belong to the flavonoid class, these four substances differ significantly in their sources, chemical structures, and biological activities. The antioxidant activity of ER makes it a potential anti-MRV drug; however, whether ER has an inhibitory effect on MRV has not yet been reported.

[0005] Therefore, the problem that this invention needs to solve is: how to provide a new substance that resists the iridovirus of the mandarin frog. Summary of the Invention

[0006] The purpose of this invention is to provide the use of sennaol in the preparation of products for the prevention and / or treatment of fish infected with mandarin frog iridovirus.

[0007] In addition, the present invention also provides a drug containing sennaol, fish feed and fish feed additive.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] Use of sennaol in the preparation of products for the prevention and / or treatment of fish infected with mandarin frog iridovirus.

[0010] Preferably, the product is a drug with sennaol as its active ingredient.

[0011] More preferably, the dosage form of the drug is an oral dosage form or an injection.

[0012] Preferably, the product is a fish feed or fish feed additive containing sennaol.

[0013] In addition, this invention discloses a drug for treating mandarin frog iridovirus, wherein the drug contains sennaol.

[0014] In addition, this invention discloses a fish feed that resists mandarin frog iridovirus, wherein the fish feed contains sennaol.

[0015] Finally, the present invention provides a fish feed additive for resisting mandarin frog iridovirus, wherein the fish feed additive contains sennaol.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] This invention confirms that SOD activation plays a crucial role in MRV resistance. ER can activate the Nrf2-Keap1 / SOD pathway in vitro and in vivo, enhancing antioxidant capacity, clearing ROS accumulation caused by MRV infection, inhibiting MRV proliferation, reducing tissue damage caused by MRV infection, and improving the survival rate of largemouth bass, thus exerting an anti-MRV effect. This invention provides a basis for the application of ER in MRV infection control and also offers new insights for further screening of anti-MRV drugs. Attached Figure Description

[0018] Figure 1 Results of the effect of ER on the activity of SCB3 cells, CC 50 EC 50The results of the assay and the inhibitory effects of three different administration methods on MRV are shown in the figure; in the figure, a represents the SCB3 cell viability value after administration of different concentrations of ER, and b represents the CC value. 50 c represents the MRV virus copy number, and d represents the EC... 50 e represents the MRV virus copy results for three different administration methods, and f represents a cell state photograph under an optical microscope;

[0019] Figure 2 Figure 1 shows the results of the analysis of ER-regulated expression of antioxidant-related genes and MRV proliferation in SCB3 cells; in the figure, ac represents the relative expression levels of Nrf2, SOD, and GPX4a genes, d represents the MRV copy number, e represents the MRV-MCP fluorescence microscopy image, and f represents the relative fluorescence intensity of MRV-MCP under a fluorescence microscope.

[0020] Figure 3 Figure 1 shows the relative expression levels of antioxidant-related genes Keap1, Nrf2, and SOD in the liver tissue of largemouth bass in different treatment groups; a represents the Keap1 gene, b represents the Nrf2 gene, and c represents the SOD gene.

[0021] Figure 4 The graph shows the ROS level detection results of liver tissue of largemouth bass in different treatment groups. The flow cytometry analysis graph on the left shows the results of one of the three parallel experiments.

[0022] Figure 5 Figure 1 shows the enzyme activity results of T-AOC, SOD and MDA in the liver tissue of largemouth bass in different treatment groups.

[0023] Figure 6 H&E staining results of liver tissue sections from different treatment groups of largemouth bass;

[0024] Figure 7 Figure 1 shows the IFA analysis results of liver tissue sections of largemouth bass from different treatment groups. In the figure, a is an image of MRV-MCP under a laser confocal microscope, and b is the relative fluorescence intensity of MRV-MCP.

[0025] Figure 8 IHC staining results of liver tissue sections from different treatment groups of largemouth bass;

[0026] Figure 9 The figure shows the results of the analysis of MRV virus proliferation and MRV-MCP protein expression. In the figure, a is the MRV virus copy number, b is the MRV-MCP protein expression level in liver tissue, and c is the relative expression level of MRV-MCP protein.

[0027] Figure 10 The survival rate of largemouth bass in different treatment groups is shown in the figure. Detailed Implementation

[0028] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] Materials and product information:

[0030] Largemouth bass were purchased from Bairong Aquaculture Farm in Foshan City, Guangdong Province. A total of 200 healthy largemouth bass with a body weight of 20.08±2.62g and a body length of 13.64±1.68cm were selected. The largemouth bass were temporarily held in a recirculating aquaculture system at 25-28℃ for 7 days before the experiment began.

[0031] SCB3 cells were cultured at 25 °C in Leibovitz L-15 medium (L-15, Gibco) containing 10% fetal bovine serum (FBS, Gibco).

[0032] MRV strains and rabbit anti-MCP (MRV) polyclonal antibodies were isolated and prepared in our laboratory.

[0033] Sacred herbol (ER, CAS: 552-58-9) was purchased from Manster (Chengdu) Biotechnology Co., Ltd.

[0034] Primers and siRNA were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and their sequences are shown in Table 1.

[0035] Table 1 RT-qPCR primer sequences

[0036]

[0037] Part 1: Cell Experiments

[0038] 1.1 Sacred Herbol CC 50 Measurement

[0039] SCB3 cells were seeded into 96-well plates and cultured at 25°C until 90% confluence. ER concentrations of 200 μmol / L, 100 μmol / L, 50 μmol / L, 25 μmol / L, 12.5 μmol / L, 6.25 μmol / L, and 3.15 μmol / L were added to L-15 medium containing 5% FBS. The control group received L-15 medium without ER and 5% FBS. After 96 h of incubation, the medium was discarded, and the cells were washed twice with PBS. L-15 medium containing 10% CCK-8 was added, with cell-free 10% CCK-8 serving as a blank control. The cells were incubated at 28°C, and the absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated using Formula I, and the ER-to-SCB3 cell concentration (CC) was plotted using GraphPadPrism 9.0. 50 Fitted curve.

[0040] Formula I: ;

[0041] 1.2 sennae EC 50 Measurement

[0042] SCB3 cells in good growth condition were seeded into 48-well plates and cultured at 25°C. When the cell confluence reached 90%, the old culture medium was discarded, and ER dilution buffer (diluted in L-15 medium containing 5% FBS at serial dilutions of 50 μmol / L, 25 μmol / L, 12.5 μmol / L, 6.25 μmol / L, and 3.125 μmol / L) was added. After incubation for 4 h, the buffer was washed away, and 1 MOI of MRV dilution buffer was added. After incubation for 4 h, the buffer was washed away, and after washing with PBS, L-15 medium containing 10% FBS was added. The cells were cultured at 25°C for 96 h, and then placed in a -80°C freezer. After three freeze-thaw cycles, the cells were thoroughly mixed, and MRV nucleic acid was extracted using a nucleic acid extractor. The MRV viral copy number was measured by RT-qPCR. The MRV inhibition rate was calculated using Formula II below. The antiviral activity of ER was determined, and the EC50 of ER against MRV was plotted using GraphpadPrism 9.0. 50 Fitted curve.

[0043] Formula II: ;

[0044] 1.3 Evaluation of different administration routes of senna

[0045] Different administration methods have different effects on cell inoculation. In order to find the administration method with the best effect, three different administration methods were tested on cells.

[0046] SCB3 cells in good growth condition were seeded into 48-well cell plates and cultured at 25°C. When the cell confluence reached 90%, the old culture medium was discarded, and the cells were washed with PBS with a light buffer. After washing off the supernatant, the cells were treated according to the groups in Table 2. At the same time, the MRV infection group without drug was set as the positive control group, and the cell group without drug and without challenge was set as the negative control group.

[0047] Table 2 Grouping by different administration routes

[0048] Administration method Specific operations Therapeutic administration Add 1 MOI of MRV dilution to the cell wells, incubate for 4 h, wash off, rinse thoroughly with PBS, add 1 mL of L-15 medium (containing 10% FBS) with 10 μmol / L ER, and incubate at 25°C for 96 h. Prophylactic administration Add 1 mL of L-15 medium (containing 10 μmol / L ER and 10% FBS) to each cell well and incubate at 25°C for 24 h. After washing with PBS, inoculate with 1 MOI of MRV dilution and incubate for 4 h. Wash off the inoculation solution, rinse with PBS, and then add fresh L-15 medium (containing 10% FBS) and incubate at 25°C for 96 h. Premixed drug delivery Mix 1 mL of L-15 medium (containing 10 μmol / L ER and 10% FBS) with 1 mL of 1 MOI MRV dilution buffer, add the mixture to cell wells, and incubate for 4 h. After washing with PBS, add fresh L-15 medium (containing 10% FBS) and incubate at 25°C for 96 h.

[0049] Cells were cultured at a constant temperature of 25°C. Cells were observed under a microscope and photographed every 12 h to record their state. After 96 h, the cell plate was placed in a freezer at -80°C and subjected to three freeze-thaw cycles. Cells and supernatant were collected, nucleic acid was extracted using a nucleic acid extractor, and the MRV virus copy number was detected by RT-qPCR.

[0050] The test results for 1.1 to 1.3 are as follows: Figure 1 As shown, the results indicate that the maximum safe concentration of ER in SCB3 cells is 12.5 μmol / L, and the CC... 50 The concentration was 43.76 μmol / L; the inhibition of MRV replication by ER was dose-dependent, and ER concentrations >3.125 μmol / L in the culture medium effectively inhibited viral proliferation, with an EC50 of 43.76 μmol / L. 50 The concentration was 8.522 μmol / L; among the three administration methods of prophylaxis, treatment, and premixing, ER prophylaxis showed the best anti-MRV effect.

[0051] 1.4 Indirect Immunofluorescence Assay (IFA) of SCB3 Cells

[0052] Place the cell spreaders in 24-well cell culture dishes until SCB3 cells have filled the spreaders, then group the cell wells:

[0053] (1) ER group: Add 1 mL of L-15 diluted ER (10 μmol / L) to each well and incubate at 25℃;

[0054] (2) ER+MRV group: Add 1 mL of L-15 diluted ER (10 μmol / L) to each well, incubate for 24 h, then add 1 mL of MRV diluted solution (1 MOI) to each well, incubate for 4 h, then replace with fresh L-15 medium and incubate at 25℃;

[0055] (3) MRV group: Add 1 mL of MRV dilution buffer (1 MOI) to each well, incubate for 4 h, then replace with fresh L-15 medium and incubate at 25℃;

[0056] (4) Mock group: Add 1 mL of fresh L-15 medium to each well and incubate at 25°C.

[0057] After incubation at 25℃ for 96 h, the slides from each treatment group were removed, fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.3% Triton-100 for 5 min, washed with PBS, and blocked with 5% bovine serum albumin (BSA) for 1 h. After spin-drying, rabbit anti-MRV-MCP antibody (primary antibody, 1:1000) was added, and the slides were incubated at 4℃ for 12 h. After warming for 30 min, the slides were washed with PBS, and Alexa Fluor 647-labeled goat anti-rabbit IgG fluorescent secondary antibody (Abcam, UK) diluted 1:1000 was added. The slides were incubated at room temperature in the dark for 1 h. After washing with PBS, DAPI (Sigma, USA) diluted 1:1000 was added and incubated for 5 min. An autofluorescence quencher was added for 5 min, followed by washing. The slides were then mounted with an antifluorescence quenching mounting medium and observed under a fluorescence microscope.

[0058] 1.5 qPCR detection of Nrf2-Keap1 / SOD pathway genes in SCB3 cells

[0059] SCB3 cells were seeded into 24-well plates and cultured at 25°C until 90% confluence. Cells were then grouped according to section 1.4. (1) After 24 hours of treatment, RNA was extracted using TRIzol (Thermo Fisher Scientific, USA), and cDNA was prepared using a cDNA synthesis kit (Novizan, China) as a template. 18S rRNA was used as an internal control. The relative expression levels of Nrf2, SOD, and GPX4a were detected using a qPCR kit (Novizan, China) (primers are shown in Table 1). The amplification program was: 95°C pre-denaturation for 3 min; 95°C denaturation for 10 s, 63°C annealing for 30 s, and 72°C extension for 15 s, for a total of 40 cycles. 2 -ΔΔCt (1) Calculate the relative expression level of the gene using the method; (2) After 96 h of treatment, use a DNA extraction kit (Tianneng, China) to extract DNA from the cells and supernatant, and detect the MRV virus copy number according to the method in the literature [Jia M, Ma Y, Hao L, et al. Lycorine provides antiviral activities against mandarin fish ranavirus via suppressing apoptosis and activating type Iinterferon pathway[J]. Aquaculture, 2025, 598: 742030. DOI:10.1016 / j.aquaculture.2024.742030.].

[0060] The test results for 1.4 and 1.5 are as follows: Figure 2 As shown, the results indicate that ER can significantly activate the gene expression of Nrf2, SOD, and GPX4a (P<0.001). Figure 2 After ER treatment for 24 h, compared with the Mock group, the Nrf2 expression levels in the ER group and the ER+MRV group were significantly increased by 7.99 and 12.85 times, respectively (P<0.001). Figure 2 (a) MRV nucleic acid quantification analysis showed that ER could significantly inhibit MRV proliferation in SCB3 cells (P<0.001). Figure 2 (d) The IFA also confirmed this regarding MRV-MCP. Figure 2 (e) ER treatment significantly inhibited viral MCP protein replication (P<0.001), and the fluorescence intensity in cells decreased by 27.68% ( Figure 2 f in the middle.

[0061] Part Two: Protective Experiment and Analysis of Saponin Against Largemouth Bass Challenge

[0062] Grouping: The 200 largemouth bass were randomly divided into 4 groups:

[0063] (1) ER group: ER was administered via gavage at a dose of 21 mg / Kg;

[0064] (2) ER+MRV group: ER was administered via gavage at a dose of 21 mg / kg. 48 h after gavage, each animal was intraperitoneally injected with 100 μL of MRV diluted in PBS (10 mg / kg). 8 TCID 50 / mL);

[0065] (3) MRV group: No ER was administered, and each fish was intraperitoneally injected with 100 μL of MRV diluted in PBS (10 8 TCID 50 / mL);

[0066] (4) Mock group: No ER was administered, and each tail was injected intraperitoneally with 100 μL of PBS.

[0067] 2.1 Sampling: 24 h after challenge, liver tissue was randomly collected from 10 largemouth bass in each group for Nrf2 / Keap1 pathway gene expression (2.1.1), ROS detection (2.1.2), and antioxidant enzyme activity detection (2.1.3). 120 h after challenge, liver tissue was randomly collected from 10 largemouth bass in each group for HE staining (2.1.4), IFA (2.1.5), IHC (2.1.6), Western blot, and viral load detection (2.1.7). Survival rate was analyzed using GraphPad Prism 9.0.

[0068] 2.1.1 RT-qPCR detection of Nrf2 / Keap1 pathway gene expression

[0069] Following the method described in section 1.5 of Part 1, cDNA was prepared from the liver tissue of largemouth bass to detect the gene expression of Keap1, Nrf2, and SOD. The qPCR reaction conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 10 s, 63℃ annealing for 30 s, and 72℃ extension for 15 s, for a total of 40 cycles. Two... -ΔΔCt The relative expression level of a gene can be calculated.

[0070] The results are as follows Figure 3 As shown in the results, after ER gavage, the expression of antioxidant genes Nrf2 and SOD in the liver of largemouth bass was significantly increased by 12.45-fold and 14.13-fold, respectively, compared with the Mock group (P<0.001), while the expression of Keap1 gene was significantly decreased (P<0.01), proving that ER can induce the expression of antioxidant genes. After ER gavage and MRV challenge, compared with the MRV-infected group, the expression of Nrf2 and SOD genes was significantly increased by 19.46-fold and 5.93-fold, respectively (P<0.001), while the expression of Keap1 gene was significantly decreased (P<0.01). This indicates that ER can activate the Nrf2-Keap1 / SOD antioxidant pathway in largemouth bass and increase the expression of antioxidant genes in largemouth bass after MRV infection.

[0071] 2.1.2 ROS FCM Detection

[0072] Single cells from liver tissue were prepared by mechanical separation. 0.1 g of tissue was minced in L-15 medium using ophthalmic surgical scissors, passed through a 70 μm sieve, and the filtered cells were collected and centrifuged at 400×g for 5 min. The cell pellet was washed with PBS, resuspended, centrifuged again, and the cells were collected. A 1:1000 diluted DCFH-DA fluorescent probe (Beyotime, China) was added, and the cells were incubated at 37℃ for 30 min. The fluorescent probe was washed off, and the ROS level of largemouth bass liver tissue cells in each group was detected by flow cytometry.

[0073] The results are as follows Figure 4 As shown, flow cytometry analysis of ROS revealed that ER administration reduced the ROS level in largemouth bass liver cells after MRV challenge from 49.7% (mean of the three experimental groups) to 12.4% (mean of the three experimental groups), effectively reducing oxidative stress caused by MRV infection.

[0074] 2.1.3 Antioxidant enzyme activity detection

[0075] Weigh an appropriate amount of largemouth bass liver tissue and prepare a 10% tissue homogenate. According to the kit instructions from Nanjing Jiancheng Biotechnology Co., Ltd., the SOD, MDA, and T-AOC of the liver tissue were detected. The reaction solution was added to a 96-well plate as required by the instructions, and the absorbance was measured using an ELISA reader and a spectrophotometer. The results were calculated according to the kit instructions.

[0076] The effect of antioxidant enzyme (ER) on the antioxidant capacity of largemouth bass was analyzed by measuring the activity of antioxidant enzymes in the liver tissue. Results are as follows: Figure 5 As shown, the results indicated that ER gavage significantly increased the T-AOC and SOD enzyme activities in the liver of largemouth bass 24 h after MRV challenge, with increases of 21.40% and 12.74% respectively compared to the MRV group (P<0.01); simultaneously, it reduced the MDA level in the liver tissue of largemouth bass. This suggests that ER gavage can increase the activity of the antioxidant enzyme SOD and the total antioxidant capacity of the system in largemouth bass, while reducing MDA levels, thus mitigating oxidative stress and cell damage caused by MRV infection.

[0077] 2.1.4 H&E staining

[0078] Liver tissue was fixed in 4% PFA fixative. After 48 h of fixation, the tissue was trimmed into 5 mm × 5 mm pieces and placed in an embedding cassette. The pieces were rinsed under running water overnight, dehydrated using a dehydrator, embedded in paraffin, frozen at -20 °C, and then cut into 5 μm continuous thin sections. The sections were spread on glass slides, baked at 37 °C overnight, and then baked at 55 °C for 1 h. After baking, the sections were dewaxed with xylene and ethanol, stained with hematoxylin and eosin (H&E), mounted, and observed under an optical microscope.

[0079] The results are as follows Figure 6As shown, the results indicated that MRV infection caused significant histopathological changes in liver tissue. Extensive congestion and necrosis were observed around the central vein, with severe hepatocyte degeneration, lack of normal lobular structure, and hepatocyte swelling and liquefaction. Simultaneously, hepatic sinusoidal dilation was observed, with a small amount of erythrocyte exudation in some areas. Hepatocyte cytoplasm was condensed, and cell volume decreased; nuclei were pyknoid and deeply stained, with obvious nuclear pyknosis and fragmentation. Compared to the MRV group, the ER+MRV group showed significantly reduced liver tissue lesions under a light microscope, although some necrotic areas remained; cell structure was relatively intact, with fewer nuclear changes and fewer cell morphological alterations.

[0080] 2.1.5 IFA Analysis

[0081] Liver tissue was sectioned according to 2.1.4. The slides were placed in boiling sodium citrate buffer (pH=6.0) for antigen retrieval for 10 min, blocked with 0.3% hydrogen peroxide for 30 min, permeabilized with 0.3% Triton-100 for 8 min, rinsed with PBS, and then blocked with 5% BSA at 37°C for 1 h. Subsequent steps were performed according to the method described in Part 1.4.

[0082] The results are as follows Figure 7 As shown, the results indicate that strong MRV-positive fluorescence signals were observed in the MRV group sections under a fluorescence microscope. Figure 7 (a) Compared to the abundant red fluorescence signal in the MRV group, MRV-positive red fluorescence signal could also be detected in liver sections of the ER+MRV group. Figure 7 (a) but its fluorescence intensity is significantly reduced ( Figure 7 (b) in the middle.

[0083] 2.1.6 Immunohistochemical (IHC) staining analysis

[0084] Liver tissue sections were prepared and antigen retrieval was performed according to section 2.1.5. After blocking with 5% BSA, MRV-MCP antibody diluted 1:1000 was added, and the sections were incubated at 4°C for 12-16 h. After washing with PBS, HRP-labeled IgG secondary antibody diluted 1:1000 was added. After washing with PBS, the tissue was stained with DAB staining kit for 15 min, and then rinsed with tap water to stop staining. The sections were counterstained with hematoxylin, treated with hydrochloric acid for 8 s, dehydrated and cleared, treated with anhydrous ethanol for 2 min, mounted with resin, and observed under a microscope after drying.

[0085] The results are as follows Figure 8 As shown, the results indicated that the MRV group liver group contained a large number of cells deeply stained with MCP antibody ( Figure 8 (The white arrow in the image) indicates that MCP synthesis was significantly reduced in the ER+MRV group. Figure 6 , Figure 7 and Figure 8 The results showed that ER treatment could inhibit the replication of MRV virus particles in largemouth bass, reduce MRV proliferation in largemouth bass, and alleviate MRV-induced liver tissue damage.

[0086] 2.1.7 Western blot and qPCR detection of MCP

[0087] cDNA and total protein samples were prepared from liver tissue for qPCR and Western blot analysis to assess viral proliferation. RT-qPCR was performed according to the method described in the literature [Jia M, Ma Y, Hao L, et al. Lycorine provides antiviral activities against mandarin fish ranavirus via suppressing apoptosis and activating type I interferon pathway[J]. Aquaculture, 2025, 598: 742030. DOI:10.1016 / j.aquaculture.2024.742030.]. Western blot experiments were performed using a mixture of RIPA lysis buffer and protease inhibitor to extract proteins. After SDS-PAGE gel electrophoresis, the membrane was transferred at 100 V for 90 min, blocked with 5% BSA for 2 h, and incubated at 4 ℃ for 12 h with rabbit anti-MRV-MCP polyclonal antibody (primary antibody, 1:1000) diluted in PBST. Mouse anti-β-actin (Abcam, UK) polyclonal antibody was used as an internal control antibody and incubated under the same conditions. The membrane was washed three times with PBST and then incubated with a 1:2... HRP-labeled goat anti-rabbit IgG (secondary antibody) and HRP-labeled goat anti-mouse IgG (internal control secondary antibody) were diluted 0.00 and incubated at room temperature for 2 h. The membranes were washed three times with PBST and the protein band gray values ​​were analyzed using an ECL chemiluminescence kit (Thermo Fisher Scientific, USA) after exposure and development (Image J).

[0088] The copy number of MRV virus at 120 hpi in liver tissue of each treatment group was detected by qPCR, and the relative expression level of MRV virus MCP protein was detected by Western blot. The results are as follows: Figure 9 As shown, the results indicate that, compared to the MRV group, ER treatment significantly reduced the MRV virus copy number in the liver tissue of largemouth bass. Figure 9 (a) (P<0.001), while significantly reducing the expression of MRV-MCP protein in the liver ( Figure 9The results show that ER can reduce MRV replication and inhibit MRV proliferation in largemouth bass, and ER exhibits in vivo anti-MRV activity.

[0089] 2.1.8 Survival Rate Statistics

[0090] The mortality rate of largemouth bass in each group was recorded during the 21-day experiment, and the survival rate was analyzed. The results are as follows: Figure 10 As shown in the results, the survival rate of largemouth bass in the ER+MRV group was 63% 21 days after MRV infection, which was significantly higher than the 27% survival rate in the MRV group (P<0.001). This indicates that ER can significantly improve the survival rate of largemouth bass infected with MRV.

[0091] In conclusion, SOD activation plays a crucial role in MRV resistance. ER can activate the Nrf2-Keap1 / SOD pathway in vitro and in vivo, enhancing antioxidant capacity, clearing ROS accumulation caused by MRV infection, inhibiting MRV proliferation, reducing tissue damage caused by MRV infection, and improving the survival rate of largemouth bass, thus exerting an anti-MRV effect.

[0092] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. Use of sennaol in the preparation of products for the prevention and / or treatment of fish infected with mandarin frog iridovirus.

2. The use according to claim 1, characterized in that, The product in question is a drug whose active ingredient is sennaol.

3. The use according to claim 2, characterized in that, The drug is available in oral or injectable form.

4. The use according to claim 1, characterized in that, The product in question is fish feed or fish feed additive containing sennaol.

5. A drug for treating mandarin frog iridovirus, characterized in that, The drug contains sennaol.

6. A fish feed resistant to mandarin frog iridovirus, characterized in that, The fish feed contains succinyl phenol.

7. A fish feed additive resistant to mandarin frog iridovirus, characterized in that, The fish feed additive mentioned above contains succinyl phenol.

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