Use of hyperoside against infection with equid herpesvirus 8

Abstract

The application discloses a use of hyperoside in resisting equid herpesvirus 8 (EHV-8) infection. Through qPCR, Western Blot and other experimental techniques, it is revealed on multiple levels that the hyperoside can significantly inhibit the replication of EHV-8, and the mechanism of the hyperoside in resisting the EHV-8 infection is clarified from the aspect of blocking the internalization of the EHV-8, so that the hyperoside can be applied in preparing an anti-EHV-8 medicine.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology and relates to drugs for the prevention and treatment of Equine Herpesvirus 8 (EHV-8) infection, specifically the application of hyperoside in the preparation of anti-EHV-8 drugs. Background Technology

[0002] EHV-8 is an enveloped double-stranded DNA virus belonging to the Alphaherpesvirus subfamily. The EHV-8 genome is approximately 150 kb in length, containing 80 open reading frames and encoding 76 proteins. Studies have found that EHV-8 infection can cause respiratory diseases, abortion, and viral encephalitis in equines (horses and donkeys), seriously endangering their health. EHV-8 was first isolated from the nasal cavity of donkeys and named Asinine herpesviruses 3 (AHV-3); subsequently, the EHV-8wh strain was isolated from horses; and later, the EHV-8351076 / 2019 strain was isolated from adult male donkeys.

[0003] In recent years, with the rapid development of the donkey industry, diseases in large-scale donkey farms have gradually increased. Among them, abortion and respiratory diseases seriously hinder the sustainable development of the donkey industry, causing huge economic losses every year. Previous studies have found that EHV-8 is the main pathogen causing abortion and respiratory diseases in donkeys. Currently, there are no effective vaccines or drugs available for EHV-8, and there is an urgent need to develop economical and effective antiviral drugs to control EHV-8.

[0004] Hyperoside (Hyp) is a flavonoid compound with antiviral activity extracted from traditional Chinese medicine. Its structure is quercetin-3-O-galactoside, and it is widely distributed in the fruits and whole herbs of plants belonging to the Hypericaceae, Campanulaceae, Lamiaceae, Rosaceae, Ericaceae, Malvaceae, Berberidaceae, Clusiaceae, Fabaceae, and Celastraceae families. Hyperoside possesses various effects including anticancer, anti-inflammatory, antidepressant, lipid-lowering, anti-hepatitis B virus, and hepatocyte protection. Currently, there are no reports on the use of hyperoside for the prevention and treatment of EHV-8 infection.

[0005] Although literature reports the antiviral efficacy of quercetin-3-O-glycosides, including hyperoside, drugs developed using these compounds, such as rutin and troxerutin, have not been directly used in clinical antiviral treatment. Furthermore, Chinese patent CN101744829A, given the low antiviral index of hyperoside, describes the use of hyperoside gel in combination with antiviral drugs ribavirin, acyclovir, polyinosinic-polycytidylic acid, and B vitamins to treat human herpes zoster. Currently, there are no reports of hyperoside being used in the development and application of antiviral drugs related to EHV-8. Summary of the Invention

[0006] The purpose of this invention is to provide the use of hyperoside against equine herpesvirus type 8 infection.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] Application of hyperoside in the preparation of anti-EHV-8 drugs.

[0009] Preferably, the maximum safe concentration of hyperoside for EHV-8 susceptible cells (such as RK-13 ​​and NBL-6) is 80 μM (μmol / L), meaning that hyperoside is non-toxic to EHV-8 susceptible cells.

[0010] Preferably, the hyperoside significantly inhibits the replication of EHV-8.

[0011] Preferably, the hyperoside inhibits the replication of EHV-8 strains EHV-8SDLC66, EHV-8SD2020113, and EHV-8donkey / Shandong / 10 / 2021, indicating that hyperoside has a broad-spectrum anti-infective effect against different EHV-8 strains isolated from diseased animals (respiratory diseases, abortions, viral encephalitis, etc.).

[0012] Preferably, the hyperoside blocks the internalization process of EHV-8.

[0013] Preferably, the hyperoside is used to prevent and treat viremia caused by EHV-8 infection, for example, it can significantly reduce the viral load in the blood.

[0014] Application of plant extracts containing hyperoside in the preparation of anti-EHV-8 drugs.

[0015] Application of traditional Chinese medicine compound containing hyperoside in the preparation of anti-EHV-8 drugs.

[0016] Application of hyperoside in the preparation of drugs for the prevention and treatment of respiratory diseases, miscarriage and viral encephalitis.

[0017] Preferably, the treatment of respiratory diseases, miscarriages, and viral encephalitis is caused by infection with EHV-8 in an organism (such as an equine).

[0018] The beneficial effects of this invention are reflected in:

[0019] This invention, through pharmacological experiments on hyperoside, reveals that hyperoside can significantly inhibit the replication of EHV-8 and can be used as an effective drug component for the prevention and treatment of EHV-8 infection. It also fully leverages the advantage of plant-derived natural compounds having fewer toxic side effects, providing valuable theoretical basis and reference for the clinical use of EHV-8. Attached Figure Description

[0020] Figure 1 The results are from a toxicity test of hyperoside on EHV-8 susceptible cells.

[0021] Figure 2 The results show the effects of hyperoside on gD gene transcription (A) and gD protein expression (B) during EHV-8 strain replication; where: **P<0.01, ***P<0.001.

[0022] Figure 3 The results show the effects of hyperoside on the infection titer (A) and gD protein expression (B) of different EHV-8 strains; where: *P<0.05, **P<0.01, ***P<0.001.

[0023] Figure 4 A schematic diagram illustrating the experimental setup for different hyperoside treatments and EHV-8 infection.

[0024] Figure 5 Results of the inhibitory effects of different treatments of hyperoside on EHV-8 replication: (A) Changes in gD gene transcription level; (B) Changes in gD protein expression level; **P<0.01, ***P<0.001.

[0025] Figure 6 Results show the effect of hyperoside on viremia induced by EHV-8 infection; where: *P<0.05, **P<0.01. Detailed Implementation

[0026] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0027] (I) Toxicity test of hyperoside on EHV-8 susceptible cells

[0028] Rabbit kidney cells RK-13 ​​and horse dermal cells NBL-6 were seeded into 96-well culture plates (1×10⁻⁶). 4Cells were cultured in microplates (wells per cell). When the cells reached approximately 70%-80% confluence, 100 μL of hyperoside solution (0, 10 μM, 20 μM, 40 μM, 80 μM, 160 μM, DMSO) at different concentrations were added to each well in the experimental groups. A negative control (DMSO treatment group) and a blank control were also included, with three replicates per group. After incubation at 37℃ and 5% CO2 for 24 h, 10 μL of CCK-8 solution was added to each well, and the cells were cultured for another 2 h. The absorbance at 450 nm was read using a microplate reader. The effect of different concentrations of hyperoside on the viability of RK-13 ​​and NBL-6 cells was calculated using the following formula, and the maximum safe concentration of hyperoside was estimated.

[0029] Cell viability (%) = [A1 - A2] / [A3 - A2] × 100%

[0030] A1: Absorbance of the experimental group (wells containing cells, culture medium, CCK-8 solution, and drug solutions of corresponding concentrations).

[0031] A2: Absorbance of blank control (wells containing culture medium and CCK-8 solution but without cells, and without drug solution)

[0032] A3: Absorbance of negative control (wells containing cells, culture medium, and CCK-8 solution but without drug)

[0033] The results are as follows Figure 1 As shown, different concentrations of hyperoside had no significant effect on the viability of susceptible cells RK-13 ​​and NBL-6, and 80 μM was the maximum safe concentration of hyperoside.

[0034] (II) Inhibitory effect of hyperoside on EHV-8 replication

[0035] 1. Analysis of EHV-8gD gene expression using qPCR and Western blot methods

[0036] RK-13 ​​and NBL-6 cells were seeded into 6-well plates (2×10⁻⁶ cells per well). 5Cells were cultured in wells (number per well). When the cells reached 80% confluence, different concentrations of hyperoside (0, 10 μM, 20 μM, 40 μM, 80 μM) were added to the experimental groups for pretreatment for 2 hours (incubated together at 37°C, 5% CO2). Then, 0.1 MOI EHV-8SDLC66 (GenBank accession number: MW816102.1) was added. After 1 hour of infection, the supernatant containing the drug and virus was discarded, and cell maintenance medium containing the corresponding concentration (as per pretreatment) of hyperoside (mixed with DMEM containing 3% FBS and hyperoside solution) was added. The cells were then incubated at 37°C, 5% CO2. Simultaneously, a DMSO-treated group was set up as a negative control, and a normal untreated group (untreated, uninfected, and without cell maintenance medium) was set up as a blank control. After 24 hours, cells were collected, total RNA was extracted using a kit, and reverse transcribed. Changes in the transcription level of the EHV-8gD gene were measured using qPCR. The results are as follows: Figure 2 As shown in Figure A, hyperoside significantly inhibits the transcription of the gD gene in a dose-dependent manner.

[0037] RK-13 ​​and NBL-6 cells were seeded into 6-well culture plates (2×10⁻⁶ cells each). 5 Cells were cultured in wells (number per well). When the cells reached 80% confluence, they were pretreated with different concentrations of hyperoside solution (0, 10 μM, 20 μM, 40 μM, 80 μM) for 2 h. Then, 0.1 MOI EHV-8SDLC66 was added for infection. After 1 h, the supernatant containing the drug and virus was discarded, and cell maintenance medium containing the corresponding concentration (as per pretreatment) of hyperoside was added. The cells were then incubated at 37°C in a 5% CO2 incubator. A DMSO-treated group was used as a negative control, and an untreated group of normal cells was used as a blank control. After 24 h, cells were collected, and total cellular protein was extracted using a kit. Changes in EHV-8gD protein expression levels were detected using Western blot. Results are as follows: Figure 2 As shown in Figure B, hyperoside significantly inhibits the expression of gD protein in a dose-dependent manner.

[0038] 2. Hyperoside in vitro anti-EHV-8 infection test

[0039] RK-13 ​​and NBL-6 cells were seeded into 6-well culture plates (2×10⁶ cells per well). 5Cells were cultured in wells (number per well) until they reached 80% confluence. They were then pretreated with hyperoside (80 μM) for 2 hours, followed by the addition of 0.1 MOI EHV-8SDLC66, EHV-8SD2020113 (GenBank accession number: MW822570.1), and EHV-8donkey / Shandong / 10 / 2021 (GenBank accession number: OL856098.1). After 1 hour of infection, the supernatant containing the drug and virus was discarded, and cell maintenance medium containing 80 μM hyperoside was added. The cells were then incubated at 37°C in a 5% CO2 incubator. A DMSO treatment group was set up as a negative control. After 36 hours, the cell supernatant was collected and analyzed using TCID50. 50 The assay method calculated the progeny virus titer, and the results showed that hyperoside significantly reduced the infection titers of different EHV-8 strains. Figure 3 A); Simultaneously, cells were collected and total cellular protein was extracted. Western blot analysis was used to detect changes in EHV-8 gD protein expression. The results showed that hyperoside significantly reduced the expression level of gD protein in different EHV-8 strains. Figure 3 B).

[0040] The above TCID 50 The specific procedure for the assay is as follows: New RK-13 ​​cells or NBL-6 cells are seeded into 96-well plates (1×10⁻⁶ cells per well). 4 (cells / well), when the cells have grown to 80% confluence, the collected EHV-8 infection supernatant is serially diluted 10-fold to 10⁻⁶. -8 Gradient (other gradients are 10) -7 10 -6 10 -5 10 -4 10 -3 10 -2 10 -1 Eight parallel wells were set up for each dilution. After adding the corresponding supernatant to the cells for 1 hour, the supernatant was discarded, and DMEM containing 3% FBS was added. The cells were then incubated at 37°C and 5% CO2 for 3-5 days. Cytopathic effects were observed daily, and TCID was calculated using the Reed-Muench formula. 50 value:

[0041] lgTCID 50 = Difference between the distance and the logarithm of the dilution + Logarithm of the dilution with a lesion rate higher than 50%

[0042] In the formula, the ratio = (percentage of lesions with a rate higher than 50% - 50%) / (percentage of lesions with a rate higher than 50% - percentage of lesions with a rate lower than 50%).

[0043] 3. Determining the effect of hyperoside on the EHV-8 replication cycle.

[0044] See Figure 4 To investigate the mechanism by which hyperoside affects EHV-8 infection, RK-13 ​​and NBL6 cells were seeded into 6-well culture plates (2 × 10⁶ cells per well). 5 Cells were collected in DMSO-treated groups (number per well) until they reached 70%-80% confluence. EHV-8 SDLC66 was added to each of the following groups: pretreatment group (Pre), co-treatment group (Co), post-infection group (Post), and all-stages-treatment group (All stages-treatment). The EHV-8 infection dose was controlled at 0.1 MOI. Cells were collected after 24 hours. The transcriptional and protein levels of the EHV-8 gD gene were measured using qPCR and Western blotting. Results showed that the expression levels of the EHV-8 gD gene in the Pre and Co groups were significantly lower than those in the DMSO-treated group, while there was no significant difference between the Post and DMSO-treated groups. This suggests that hypericin can reduce EHV-8 infection by inhibiting EHV-8 internalization (blocking EHV-8 internalization). Figure 5 A, Figure 5 B).

[0045] 4. Hyperoside in vivo anti-EHV-8 infection test

[0046] Nine male EHV-8 negative BALB / c mice were randomly divided into three groups (n=3 per group): a hyperoside treatment group (100 mg / kg / d), a DMSO treatment control group, and an EHV-8 infection control group. Mice in the hyperoside treatment group received intraperitoneal injections of the appropriate dose of hyperoside 24 h and 12 h before infection, while mice in the DMSO treatment control group received the same dose of DMSO as the drug treatment group. Then, all mice in each group were intranasally infected with 100 μL of EHV-8SDLC66 (1×10⁻⁶). 5 PFU / mouse). Twelve hours post-infection, mice were intraperitoneally injected with the corresponding dose of the drug and DMSO once daily for seven days. Serum samples were collected before infection and at 3, 5, and 7 days post-infection. qPCR was used to determine changes in serum EHV-8 viral copy number (represented by gD gene copy number). Results showed that the EHV-8 viral copy number in the drug-treated group was significantly lower than that in the EHV-8 infected control group (P<0.05), while there was no significant difference in EHV-8 viral copy number between the DMSO-treated control group and the EHV-8 infected control group. Figure 6 The results suggest that hyperoside can significantly inhibit viremia caused by EHV-8 infection.

[0047] In summary, this invention, based on traditional Chinese medicine techniques, seeks effective drug components to combat EHV-8 infection. Through treatment of EHV-8 susceptible cells RK-13 ​​and NBL-6 with hyperoside and in vivo experiments, it was determined that hyperoside has a significant inhibitory effect on EHV-8 replication. This inhibitory effect was further verified in different EHV-8 strains tested. In-depth analysis revealed that hyperoside primarily affects the internalization process of EHV-8, thus demonstrating that hyperoside can be used to prepare drugs for the prevention and treatment of EHV-8 infection.

Claims

1. Application of hyperoside as the sole active ingredient in the preparation of anti-EHV-8 drugs.

2. The application according to claim 1, characterized in that: The maximum safe mass concentration of hyperoside is 80 µM.

3. The application according to claim 1, characterized in that: Hypericin inhibits the replication of EHV-8.

4. The application according to claim 1, characterized in that: The hyperoside described herein has an inhibitory effect on the replication of EHV-8 strains EHV-8SDLC66, EHV-8 SD2020113, and EHV-8 donkey / Shandong / 10 / 2021.

5. The application according to claim 1, characterized in that: Hypericin blocks the internalization process of EHV-8.

6. The application according to claim 1, characterized in that: Hyperoside is used to prevent and treat viremia caused by EHV-8 infection.

7. The application of hyperoside as the sole active ingredient in the preparation of drugs for the prevention and treatment of respiratory diseases, miscarriage, and viral encephalitis, characterized in that: The respiratory illnesses, miscarriages, and viral encephalitis mentioned above are caused by infection with EHV-8.

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