Application of bufotalin derivatives in the preparation of anti-IHNV products

Abstract

This invention discloses the application of bufotoxin lactones in the preparation of anti-IHNV products, belonging to the field of biomedical technology. This invention is the first to discover and confirm that four bufotalin-based drugs (bufotalin, bufotalin ester, bufotalin, and bufotalin) have significant anti-IHNV effects: their IC50 values ​​against IHNV in vitro range from 0.0879 to 0.1809 μM, with selectivity indices all greater than 110. After 48 hours of treatment, they can reduce viral RNA expression by 110 to 173 times and viral titer by 4.36 to 5.15 lg; they also show broad-spectrum inhibitory effects against different genotypes of IHNV, such as LN-15, QH-17, and Blk94; in a rainbow trout in vivo model, after administration of a 0.5 mg / kg dose, the cumulative mortality rate decreased from 95% in the control group to 13.3% to 21.7%, with the highest relative protection rate reaching 85.96% (bufotalin), indicating that these compounds have highly efficient, broad-spectrum, and low-toxicity anti-IHNV activity and have excellent application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of bufotoxin lactones in the preparation of anti-IHNV products. Background Technology

[0002] Infectious hematopoietic necrosis (IHN) is one of the major diseases threatening salmon and trout farming worldwide. It is an acute infectious disease caused by infectious hematopoietic necrosis virus (IHNV), which seriously threatens salmon and trout. Depending on the species, age of the fish, virus strain, and rearing environment, it can typically cause a mortality rate of 80%-100%. Therefore, IHN is listed as a reportable animal disease by the World Organisation for Animal Health (WOAH), and my country classifies it as a Class II infectious disease requiring mandatory reporting. In recent years, IHN has frequently broken out in my country, causing severe economic losses to salmon farming and seriously hindering the healthy and sustainable development of my country's fish farming industry. Despite the enormous economic losses caused by IHN to the salmon and trout farming industry, there is only one commercially available DNA vaccine against IHN globally, and my country currently has no access to this vaccine.

[0003] Therefore, in order to solve the serious problem of IHN disease, it is necessary to develop more effective antiviral drugs or vaccines to protect salmon and trout from the harm of IHN. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide the application of bufotoxin lactone drugs in the preparation of anti-IHNV products.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the application of bufotoxin lactones in the preparation of anti-IHNV products.

[0006] This invention provides the application of bufotoxin lactone drugs in inhibiting the replication of IHNV in EPC cells.

[0007] This invention provides the application of bufotoxin lactones in the preparation of products that inhibit IHNV infection in vivo.

[0008] Preferably, the bufotalin drugs include bufotalin, bufotalin ester, bufotalin, and bufotalin from China.

[0009] Preferably, the IHNV strains include Blk94, LN-15, and QH-17.

[0010] It contains at least the following beneficial technical effects: This invention is the first to discover and confirm that four bufotalin-based drugs (bufotalin, bufotalin ester, bufotalin, and bufotalin) have significant anti-IHNV effects: their IC50 values ​​against IHNV in vitro range from 0.0879 to 0.1809 μM, with selectivity indices all greater than 110. After 48 hours of treatment, they can reduce viral RNA expression by 110 to 173 times and viral titer by 4.36 to 5.15 lg; they also show broad-spectrum inhibitory effects against different genotypes of IHNV, such as LN-15, QH-17, and Blk94; in a rainbow trout in vivo model, after administration of a 0.5 mg / kg dose, the cumulative mortality rate decreased from 95% in the control group to 13.3% to 21.7%, with the highest relative protection rate reaching 85.96% (bufotalin), indicating that these compounds have highly efficient, broad-spectrum, and low-toxicity anti-IHNV activity and have excellent application prospects. Attached Figure Description

[0011] Figure 1 Screening process for anti-IHNV drugs and structural formulas of bufotalin drugs; where A is the screening process for anti-IHNV drugs, B is the structural formula of bufotalin, C is the structural formula of bufotalin ester, D is the structural formula of bufotalin, and E is the structural formula of bufotalin. Figure 2 cytotoxic CC of bufotalin derivatives 50 and antiviral active IC 50 Detection; where AD represents the concentrations of bufotoxin, bufotoxin ester, bufotoxin ether, and bufotoxin on EPC cells, respectively. 50 Detection results: IC50 of EH (bufotoxin), bufotoxin ester, bufotoxin ether, and bufotoxin ether on IHNV in EPC cells. 50 Test results.

[0012] Figure 3 The study aimed to detect the inhibitory effect of bufotoxin lactones on the IHNV-Sn1203 strain; where A represents the inhibitory effect on the mRNA of the IHNV-Sn1203 strain, and B represents the inhibitory effect on the viral titer of the IHNV-Sn1203 strain.

[0013] Figure 4 This study aims to detect the inhibitory effects of bufotoxin lactones on different IHNV strains; where A represents the inhibitory effect on mRNA of different IHNV strains, and B represents the inhibitory effect on viral titer of different IHNV strains.

[0014] Figure 5 Detection of the antiviral efficacy of bufotalin drugs in rainbow trout. Detailed Implementation

[0015] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0016] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0017] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0018] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0019] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0020] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0021] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0022] Example 1. Screening of anti-IHNV drugs The Chinese herbal monomer database (HY-L065) was purchased from MedChemExpress.

[0023] First, EPC cells (ATCC number CRL-2872) were screened for anti-IHNV activity using the Cell Counting Kit-8 (CCK8, B34304, Bimake, Shanghai, China). EPC cells were seeded in 96-well plates and co-incubated with different drugs at 10 μM. After 6 h of incubation, IHNV virus (Genbank accession number: KC660147.1) was introduced at 15°C for 1 h at a viral infection concentration (MOI) of 0.1, followed by 7 days of co-culture with the drugs. 10 μl of CCK8 solution was added to each well, and the cells were incubated at 15°C for 2 h to observe antiviral activity. Cell viability was detected using a microplate reader at an optical density (OD) of 450 nm. The results showed that bufotalin-based drugs—Bufo bufotoxin, Bufotoxin ester, Bufotoxin succinate, and Bufo bufotoxin—exhibited significant anti-IHNV activity. The specific screening scheme and drug structures are shown in Figure 1.

[0024] 2. Cytotoxic CC of bufotalin derivatives 50 and antiviral active IC 50 Detection EPC cells were seeded in 96-well plates and incubated with bufotalin at concentrations of 0.01 μM, 0.02 μM, 0.05 μM, 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM, and 20 μM. Cells treated with 0.1% DMSO served as a control. After 6 days of culture, the cytotoxicity of the bufotalin was assessed using a CCK8 assay. The OD of cells treated with bufotalin was... 450 The drug concentration at which the value decreases to 50% of the untreated cell concentration is defined as the 50% cytotoxic concentration (CC) of bufotalin drugs. 50 The results showed that bufotalin-based drugs inhibited C-cell activity in EPC cells. 50 >20 μM (AD in Figure 2). EPC cells in 96-well plates were also treated with different concentrations of bufotoxin. After 6 h of culture, they were infected at 15°C for 1 h at an IHNV MOI of 0.1. After 6 days of culture, the antiviral activity of the bufotoxin was detected using a CCK8 assay. The inhibition rate was calculated as [(drug OD]]. 450 -Virus control OD 450 ) / (Control cell OD 450 -Virus control OD 450The 50% inhibitory concentration (IC50) of bufotalin against IHNV was calculated using regression analysis, with the result set at 100%. The results showed that the IC50 values ​​for bufotalin were 0.0879 μM, 0.1247 μM for bufotalin ester, 0.1337 μM for bufotalin, and 0.1809 μM for bufotalin (EH in Figure 2). The selectivity index of bufotalin against IHNV was consistently high (SI = CC). 50 / IC 50 The result above indicates that the antiviral effect of bufotalin drugs against IHNV is not due to their toxicity.

[0025] 3. Bufotenoids inhibit the replication of IHNV in vivo and in vitro. 3.1 Bufotalin derivatives can inhibit the replication of IHNV in EPC cells. To further evaluate the anti-IHNV activity of bufotalin drugs, we examined the replication capacity of the IHNV-Sn1203 strain (Genbank accession number: KC660147.1) after 24 and 48 h of treatment with bufotalin drugs. RT-qPCR results showed that bufotalin drugs significantly inhibited the replication of the IHNV-Sn1203 strain in cells. Compared with the dimethyl sulfoxide control group, the relative RNA expression levels after 24 h of treatment with bufotalin drugs decreased by 12-fold (Bufo bufotoxin), 13-fold (Bufo bufotoxin ligand), 27-fold (Bufo bufotoxin), and 13-fold (Bufo bufotoxin from the far-China region), respectively. After 48 h of treatment, the relative RNA expression levels decreased by 114-fold (Bufo bufotoxin), 110-fold (Bufo bufotoxin ligand), 173-fold (Bufo bufotoxin from the far-China region), and 129-fold (Bufo bufotoxin from the far-China region), respectively (Figure 3A). Compared with the dimethyl sulfoxide control group, the extracellular viral titer of the IHNV-Sn1203 strain was also significantly inhibited. After 24 h of treatment with bufotalin, the relative RNA expression levels decreased by 1.97 lg (bufotalin), 2.09 lg (bufotalin ester), 2.44 lg (bufotalin stomatine), and 2.25 lg (bufotalin far-China), respectively. After 48 h of treatment, the relative RNA expression levels decreased by 4.36 lg (bufotalin), 4.47 lg (bufotalin ester), 5.15 lg (bufotalin stomatine), and 4.70 lg (bufotalin far-China) (Figure 3B). These results indicate that bufotalin can significantly inhibit the replication of IHNV-Sn1203 in EPC cells, but bufotalin stomatine has a more significant effect.

[0026] 3.2 Bufotenoids can inhibit different IHNV virus strains To demonstrate that bufotalin can inhibit infection by different IHNV virus strains, we evaluated the inhibitory effects of bufotalin on different IHNV strains Blk94 (Genbank accession number: DQ164100), LN-15 (Genbank accession number: MH170315.1), and QH-17 (Genbank accession number: MH170343.1) (Figure 4). RT-qPCR results showed that bufotoxin lactones significantly inhibited intracellular viral replication of all viruses. Compared with the dimethyl sulfoxide control group, the relative RNA expression levels of LN-15, QH-17, and Blk94 decreased by 30-fold, 19-fold, and 60-fold, respectively, after 48 h of treatment with bufotoxin. After 48 h of treatment with bufotoxin ligand, the relative RNA expression levels of LN-15, QH-17, and Blk94 decreased by 30-fold, 20-fold, and 58-fold, respectively. After 48 h of treatment with bufotoxin, the relative RNA expression levels of LN-15, QH-17, and Blk94 decreased by 67-fold, 93-fold, and 333-fold, respectively. After 48 h of treatment with far-China bufotoxin, the relative RNA expression levels of LN-15, QH-17, and Blk94 decreased by 36-fold, 38-fold, and 162-fold, respectively (Figure 4A). Compared with the dimethyl sulfoxide control group, the extracellular viral titer was also significantly inhibited. After 48 h of treatment with bufotoxin, the relative RNA expression levels of LN-15, QH-17, and Blk94 decreased by 3.76 lg, 3.81 lg, and 4.1 lg, respectively. After 48 h of treatment with bufotoxin ligand, the relative RNA expression levels of LN-15, QH-17, and Blk94 decreased by 3.85 lg, 3.82 lg, and 4.16 lg, respectively. After 48 h of treatment with bufotoxin, the relative RNA expression levels of LN-15, QH-17, and Blk94 decreased by 4.58 lg, 4.44 lg, and 4.62 lg, respectively. After 48 h of treatment with far-China bufotoxin, the relative RNA expression levels of LN-15, QH-17, and Blk94 decreased by 3.87 lg, 3.96 lg, and 4.03 lg, respectively (Figure 4B). The above results indicate that bufotalin drugs can significantly inhibit IHNV infection of EPC cells, and the inhibitory effect is a random phenomenon targeting all strains rather than a single strain, with bufotalin showing a more significant effect.

[0027] 3.3 Bufotalin derivatives can inhibit IHNV infection in vivo. To evaluate the protective effect of bufotalin against IHNV infection in rainbow trout, 1 μL (10 7 TCID 50Rainbow trout were treated with IHNV (in vitro venom) at a concentration of 0.5 mg / kg and bufotalin (0.5 mg / kg). In this experiment, the bufotalin was dissolved in corn oil, thus a corn oil-based control group was used as a mock treatment group. The results showed that the cumulative mortality rate after IHNV infection in the corn oil-based control group was 95%, while the cumulative mortality rate in the bufotalin-based control group was significantly reduced. The cumulative mortality rates were 16.7% in the bufotalin-based control group, 21.7% in the bufotalin-based control group, 13.3% in the bufotalin-based control group, and 18.3% in the bufotalin-based control group. These results indicate that bufotalin has a significant inhibitory effect on IHNV infection in rainbow trout, with the bufotalin-based control group showing the most significant protective effect, with a relative protection rate (RPS) of 85.96%. Figure 5 ).

[0028] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of bufotalin drugs in the preparation of anti-IHNV products.

2. Application of bufotalin drugs in inhibiting IHNV replication in EPC cells.

3. Application of bufotalin drugs in the preparation of products that inhibit IHNV infection in vivo.

4. The application according to any one of claims 1-3, characterized in that, The bufotalin drugs include bufotalin, bufotalin ester, bufotalin, and bufotalin from China.

5. The application according to any one of claims 1-3, characterized in that, The IHNV strains include Blk94, LN-15, and QH-17.

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