Application of curcumin in preparation of inhibitor of carp herpesvirus type II

Abstract

The present application relates to the fields of biotechnology and agricultural fishery, and discloses application of curcumin in preparation of an inhibitor of CyHV-2, and finds that curcumin can effectively inhibit the infection and replication of CyHV-2 virus in GiCF cells.The present application first applies curcumin to the prevention and treatment of CyHV-2 infection, thereby providing a new treatment scheme for CyHV-2 virus.Curcumin can effectively reduce the copy number of CyHV-2 virus, and curcumin can be used to prepare a drug for preventing and treating CyHV-2 infection, and a drug for treating viral hematopoietic organ necrosis in fish.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and agriculture and fisheries, specifically to aquaculture disease prevention and control technology. Background Technology

[0002] Cyprinid herpesvirus 2 (CyHV-2) is a double-stranded DNA virus, approximately 290,304 bp in length. This virus induces herpesviral hematopoietic necrosis (HVHN) in crucian carp and goldfish. Infected fish primarily exhibit extensive hemorrhage on the body, gills, and fins, as well as spleen enlargement. CyHV-2 is characterized by horizontal and vertical transmission and latent infection, seriously jeopardizing the development of crucian carp and goldfish aquaculture. However, current research on CyHV-2 mainly focuses on virus identification and detection; there are no effective commercial vaccines or drugs to control its spread. Therefore, measures are urgently needed to reduce the economic losses caused by CyHV-2.

[0003] Curcumin, also known as turmeric pigment or acid yellow, is a natural phenolic antioxidant extracted from the roots and rhizomes of plants in the ginger family, such as turmeric, turmeric zedoaria, mustard, curry, and turmeric root. It possesses excellent anti-inflammatory and anti-cancer properties. Its CAS number is 458-37-7, and its chemical formula is C2. 21 H 20 O6, the structural formula is:

[0004]

[0005] Recent studies have shown that natural compounds are a sustainable source of novel antiviral drugs. Curcumin, as a plant-derived compound, not only possesses various immunomodulatory properties such as antitumor, antibacterial, and antiviral effects, but also exhibits good safety and tolerability. In antiviral research, curcumin has demonstrated antiviral activity against various viruses, including hepatitis viruses, influenza viruses, human cytomegalovirus, and Epstein-Barr virus.

[0006] There are currently no reports on curcumin inhibiting CyHV-2. Summary of the Invention

[0007] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide the application of curcumin in the preparation of inhibitors of carp herpesvirus type II.

[0008] To achieve the above objectives, this invention proposes the application of curcumin in the preparation of inhibitors of carp herpesvirus type II.

[0009] On the other hand, this invention proposes the application of curcumin in the preparation of drugs for the prevention or treatment of carp herpesvirus type II infection.

[0010] In another aspect, the present invention also proposes the application of curcumin in the preparation of drugs for the prevention or treatment of viral hematopoietic organ necrosis in fish.

[0011] Preferably, the fish is a crucian carp or a goldfish.

[0012] It should be noted that the fish mentioned can be crucian carp, goldfish, or other fish susceptible to carp herpesvirus.

[0013] Preferably, the curcumin is an antiviral active ingredient, and its application dose is 250 nM to 10 μM.

[0014] It should be noted that in the technical solution described in this invention, the dosage is the final concentration of curcumin when it is added to the culture medium. However, as those skilled in the art will know, when it is used as a fish medicine, those skilled in the art can also adjust it based on this concentration condition, for example, by mixing it into feed.

[0015] Furthermore, the present invention proposes a drug for the prevention or treatment of carp herpesvirus type II infection, wherein the active component of the drug against carp herpesvirus type II is curcumin.

[0016] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0017] More preferably, the dosage form of the anti-carp herpesvirus type II drug includes tablets, powders, granules, capsules, or sustained-release formulations.

[0018] It should be noted that, in the technical solution described in this invention, curcumin is used in the preparation of carp herpesvirus type II (i.e., CyHV-2) inhibitor drugs, which aims to provide an alternative solution for the preparation of drugs for the prevention and treatment of crucian carp hematopoietic organ necrosis.

[0019] This is because curcumin can effectively inhibit the replication of CyHV-2 virus in GiCF cells, reducing the copy number and titer of CyHV-2 virus. Since curcumin can effectively reduce the titer of carp herpesvirus type II in GiCF cells and supernatant, it indicates that curcumin can effectively inhibit the infection and replication of carp herpesvirus type II in GiCF cells, achieving the effect of inhibiting viral replication. Therefore, curcumin can effectively prevent and treat carp herpesvirus type II infection and the diseases it causes, and can be used to prepare drugs for the prevention and treatment of carp herpesvirus type II infection, as well as drugs for viral hematopoietic organ necrosis in fish.

[0020] The technical solution of this application has the following beneficial effects:

[0021] 1. This invention is the first to apply curcumin to the prevention and treatment of carp herpesvirus type II (CyHV-2) infection. At concentrations of 250 nM to 10 μM (especially 10 μM), it can significantly reduce the intracellular titer of CyHV-2 virus, thereby inhibiting viral replication. This provides a new solution for the prevention and treatment of CyHV-2 virus infection and viral hematopoietic organ necrosis in fish.

[0022] 2. Curcumin can produce significant effects at low application concentrations and commonly used dosages. It has low cytotoxicity and is expected to become a candidate drug for the prevention and treatment of carp herpesvirus type II. Attached Figure Description

[0023] Figure 1 The results of intracellular virus titer detection after curcumin treatment in Example 1 of the present invention are shown.

[0024] Figure 2 The results of virus titer detection in cell supernatant after curcumin treatment in Example 1 of the present invention are shown.

[0025] Figure 3 The results of intracellular virus titer detection after resveratrol treatment in Example 1 of the present invention are shown.

[0026] Figure 4 The results of intracellular viral titer detection after THZ1 treatment in Example 1 of this invention are shown. Detailed Implementation

[0027] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0028] The present invention will be further described below with reference to the embodiments:

[0029] Experimental materials

[0030] (I) Experimental strains and cells

[0031] The caudal fin cell line GiCF (The caudal fin of Carassius auratus gibelio) was constructed in our laboratory. The construction method can be found in Lu, JF; Xu, D; Lu, LQ. A novel cell line established from caudal fin tissue of Carassius auratus gibelio is susceptible to cyprinid herpesvirus 2 infection with the induction of apoptosis[J]. Virus Research. 2018; 258:19-27. The cells were cryopreserved in liquid nitrogen. The cell resuscitation medium was M199 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and the cells were cultured in a constant temperature incubator at 27℃. CyHV-2 isolate YC-01 (hereinafter referred to as CyHV-2, GenBank: MN593216.1) was used to infect cells at 25°C. After all cells died, the cell supernatant was collected and filtered through a 0.22μm syringe filter to obtain pure virus solution, which was then stored at -80°C for later use.

[0032] (II) Experimental Reagents

[0033] M199 culture medium, fetal bovine serum, and penicillin-streptomycin 100× were purchased from Gibco; curcumin, resveratrol, and THZ1 were purchased from Selleck; DMSO was purchased from Sigma-Aldrich; Trizol was purchased from Invitrogen; chloroform was purchased from Shanghai Kelings Reagent Co., Ltd.; anhydrous ethanol and isopropanol were purchased from Sinopharm Chemical Reagent Co., Ltd.; DEPC-treated water and PBS were purchased from Sangon Biotech (Shanghai) Co., Ltd.; TB Premix Ex Taq TM II (RR820A), PrimeScript TM RT Master Mix (RR036A) was purchased from TaKaRa; Viral Genomic DNA / RNA Extraction Kit (DP315) was purchased from Tiangen.

[0034] Example

[0035] 1. In vitro inhibition experiments were conducted using curcumin as an inhibitor. The control examples in this case were resveratrol and THZ1 as inhibitors in in vitro inhibition experiments.

[0036] Curcumin was dissolved in DMSO at 100 mM and diluted to the specified concentration with 10% M199 medium before administration.

[0037] Resveratrol was dissolved in DMSO at 100 mM and diluted to the specified concentration with 10% M199 medium before administration.

[0038] THZ1 was dissolved in DMSO at 10 mM and diluted to the specified concentration with 10% M199 medium before administration.

[0039] The in vitro inhibition experiment was divided into three groups: Group 1 - Curcumin: experimental concentrations of 250 nM, 500 nM, 1 μM, or 10 μM, with the DMSO group serving as a control; Group 2 - Resveratrol: experimental concentrations of 250 nM, 500 nM, 1 μM, or 10 μM, with the DMSO group serving as a control; Group 3 - THZ1: experimental concentrations of 25 nM and 100 nM, with the DMSO group serving as a control. GiCF cells were first pretreated with the inhibitor for 1 h, and then infected with CyHV-2 (MOI = 1). 72 h after infection, cells were collected, and the CyHV-2 viral titer was measured.

[0040] 2. RNA extraction

[0041] Collect the drug-treated cells and add 1 ml of Trizol, mixing thoroughly by pipetting. Add 200 μL of chloroform, vortex for 15 seconds, incubate for 3 minutes, and centrifuge at 12000 × g for 15 minutes at 4°C. Collect the supernatant, add an equal volume of isopropanol, incubate for 10 minutes, and centrifuge at 12000 × g for 10 minutes at 4°C. Discard the supernatant, add 1 mL of 75% ethanol (prepared with DEPC-treated water), and centrifuge at 7500 × g for 5 minutes at 4°C. Add 20 μL of DEPC-treated water to dissolve the RNA.

[0042] 3. DNA extraction

[0043] Collect the cell supernatant after curcumin treatment and transfer it to a 1.5 mL centrifuge tube. Centrifuge at 1000 rpm for 5 min at room temperature. Transfer 200 μL of the supernatant to a new centrifuge tube and extract viral DNA from the cell supernatant using a viral genomic DNA / RNA extraction kit (DP315). Dissolve the DNA in 30 μL of Nase-free double-distilled water.

[0044] It should be noted that in this embodiment, RNA extraction was used to observe changes in intracellular viral titer. When it was found that the intracellular viral titer was significantly inhibited in the experiment using curcumin, DNA extraction was used on the cell supernatant to better observe changes in viral titer in the cell supernatant after curcumin treatment.

[0045] 4. Quantitative Real-Time PCR (RT-qPCR) detection

[0046] The RT-qPCR reaction system followed the TB Green Premix Ex Taq II (Tli RNaseH Plus) Kit. The primer sequences used for RT-qPCR are shown in Table 1. The reaction system and reaction conditions are shown in Tables 2 and 3.

[0047] Table 1 RT-qPCR primers

[0048]

[0049] Table 2 RT-qPCR reaction system

[0050]

[0051] Table 3 RT-qPCR reaction conditions

[0052]

[0053] 5. Virus titer detection

[0054] The recombinant plasmid pDsRed-express-N1-ORF121 was constructed in our laboratory, following the construction method described in Chinese patent document CN116064511A, published on May 5, 2023, entitled "An RNA interference sequence for inhibiting the immediate early gene ORF121 of carp herpesvirus type II and its application." The plasmid concentration was measured using NanoDrop 2000. The copy number (copies / μL) was 6.02 × 10⁻⁶. 23 (copies / mol) × plasmid concentration (ng / μL) × 10 -9 / (recombinant plasmid base number × 660 g / mol). RT-qPCR analysis was performed using plasmids serially diluted 10-fold as templates. A standard curve was constructed with cycle number (Ct) and logarithm of copy number as the x and y axes, respectively. The equation of the curve is y = -0.2968x + 11.3163R. 2 =0.996, and the primers used are listed in Table 1. Using cellular cDNA as a template, RT-qPCR was performed using the same primers as the standard curve. The Ct value was substituted into the standard curve to obtain the viral copy number, and finally, the viral copy number was substituted into the formula to calculate the viral titer.

[0055] The formula for calculating titer (integration units per mL, IU / mL) is as follows:

[0056] IU / mL=(C×N×D×1000) / V

[0057] Where: C = average number of viral copies integrated per genome; N = number of cells at infection (approximately 1 × 100,000); D = dilution factor of the viral vector; V = volume of diluted virus added. For example... Figure 1 , Figure 2 As shown in Tables 4 and 5, compared with the control group DMSO, curcumin treatment significantly reduced the intracellular and supernatant titers of CyHV-2 cells. At a concentration of 10 μM, it significantly inhibited CyHV-2 virus replication within cells, with statistical significance. Resveratrol and THZ1, after 72 hours of treatment, showed similar results. Figure 3 , Figure 4 As shown in Tables 6 and 7, resveratrol and THZ1 did not reduce the intracellular CyHV-2 viral titer. This indicates that curcumin can effectively inhibit CyHV-2 viral replication. Resveratrol and THZ1, however, had no inhibitory effect on CyHV-2.

[0058] It should be noted that resveratrol is a flavonoid polyphenol compound that naturally exists in a variety of plants. Resveratrol has anti-inflammatory, antibacterial, and antiviral effects, and has a strong inhibitory effect on herpes simplex virus. Curcumin is also a flavonoid polyphenol compound that exists in a variety of plants. Therefore, resveratrol was chosen as the control group for curcumin.

[0059] In addition, for experimental technical comparison, this case used a THZ1 inhibitor for comparison with curcumin. The CAS number for THZ1 is 1604810-83-4, and its chemical formula is C2. 31 H 28 ClN7O 2.

[0060] Tables 4 to 7 show the virus titer detection results for cells treated with curcumin, the supernatant of cells treated with curcumin, cells treated with resveratrol, and cells treated with THZ1, respectively.

[0061] Each experiment was conducted in parallel with three sets of measurements.

[0062] Table 4

[0063]

[0064] Table 5

[0065]

[0066]

[0067] Table 6

[0068]

[0069]

[0070] Table 7

[0071]

[0072] It should be noted that the scope of protection of the prior art in this invention is not limited to the embodiments given in this application. All prior art that does not contradict the solution of this invention, including but not limited to prior patent documents, prior publications, prior public uses, etc., can be included in the scope of protection of this invention.

[0073] Furthermore, the combination of the technical features in this case is not limited to the combination methods described in the claims of this case or the combination methods described in the specific embodiments. All technical features described in this case can be freely combined or combined in any way, unless they contradict each other.

[0074] Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. Unless otherwise specified, the embodiments and features described in this application can be arbitrarily combined with each other.

Claims

1. Application of curcumin in the preparation of inhibitors of carp herpesvirus type II.

2. Application of curcumin in the preparation of drugs for the prevention or treatment of carp herpesvirus type II infection.

3. Application of curcumin in the preparation of drugs for the prevention or treatment of herpesvirus hematopoietic necrosis in crucian carp or goldfish.

4. The application according to claim 2 or 3, characterized in that, The drug also includes pharmaceutically acceptable excipients.

5. The application according to claim 2 or 3, characterized in that, The dosage forms of the drug include tablets, powders, granules, capsules, or sustained-release formulations.

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