Use of coenzyme Q10 in preparation of medicine for resisting new duck reovirus

Abstract

The application discloses application of coenzyme Q10 in preparation of a medicine for resisting new duck reovirus, and belongs to the technical field of veterinary biological products. The application locks a key pathogenic protein sigma C of the new duck reovirus and a host protein COQ6 interacting with the key pathogenic protein sigma C by using a cell model infected by the NDRV and a duckling model; further combining the core role of the COQ6 in a coenzyme Q10 synthesis path, it is found that the coenzyme Q10 can resist the new duck reovirus infection, and a new prevention and treatment strategy is provided for prevention and treatment of the new duck reovirus disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of veterinary biological products, and particularly relates to application of coenzyme Q10 in preparation of a medicine for resisting new duck reovirus. BACKGROUND

[0002] New duck reovirus (NDRV) belongs to the family of Reoviridae, is a double-stranded, segmented RNA virus, and mainly prevails in China and some Southeast Asian regions, and is mostly found in ducklings (1-3 weeks old) and can be transmitted through the digestive tract, respiratory tract or vertically. Infected ducks often show depression, decreased appetite, diarrhea, emaciation, ruffled feathers, and obvious splenomegaly with hemorrhagic necrosis, and some cases can show liver spot-like necrosis and intestinal mucosal congestion. Infection can weaken the immune system, making the duck population more susceptible to mixed infections of bacteria, viruses, etc., and the mortality rate can reach 10%-50%. The disease is more likely to occur in spring and autumn, but it can also break out throughout the year under intensive breeding conditions. New duck reovirus disease has rapid transmission and high mortality, and is difficult to prevent and control, causing significant economic losses to breeders.

[0003] Most vaccines for new duck reovirus are still in the laboratory research stage, and there are few vaccines for new duck reovirus disease on the market. There is also a lack of specific treatment drugs for new duck reovirus infection, and currently, the main intervention method is to add Huangqin polysaccharide and antibiotics to the duck feed, but the treatment effect on new duck reovirus infection is limited. Therefore, there is an urgent need to develop new drugs for preventing and treating new duck reovirus infection.

[0004] Pyroptosis is a form of programmed cell death executed by Gasdermin family proteins, characterized by cell membrane perforation, leakage of cellular contents, and significant secretion of inflammatory factors (such as IL-1β and IL-18), often accompanied by a strong inflammatory response. Studies have shown that pyroptosis plays a crucial role in viral-induced tissue lesions. Furthermore, evidence indicates that viruses, bacteria, or other stressors can induce mitochondrial damage, manifested as decreased membrane potential (ΔΨm loss), excessive production of reactive oxygen species (ROS), and release of mitochondrial DNA (mtDNA). These changes can activate the NLRP3 inflammasome as damage-associated molecular patterns (DAMPs), further amplifying the pyroptotic response and exacerbating tissue damage. Therefore, drug intervention targeting mitochondrial dysfunction is considered an effective strategy to alleviate inflammatory pyroptosis and related tissue damage. By restoring mitochondrial membrane potential, reducing ROS production, and inhibiting mitochondrial DNA release, these drugs can block the excessive activation of the NLRP3 inflammasome, thereby reducing pyroptosis and inflammation, offering new avenues and strategies for the treatment of viral diseases. However, current research mainly focuses on broad-spectrum antiviral strategies, lacking a systematic exploration of the specific molecular mechanisms mediating the pathogenesis of NDRV infection, and the relevant targets remain unclear. Therefore, in-depth and systematic analysis of the molecular mechanisms by which NDRV infection induces spleen damage provides important theoretical basis and technical reserves for revealing the interaction network between NDRV pathogenesis and host defense, and for developing precise prevention and control measures. Summary of the Invention

[0005] The purpose of this invention is to provide the application of coenzyme Q10 in the preparation of drugs against novel duck reovirus (DRV). This invention utilizes NDRV-infected cell models and duckling models to identify the key pathogenic protein σC of NDRV and its interacting host protein CoQ6. Furthermore, by combining the core role of CoQ6 in the coenzyme Q10 synthesis pathway, it was discovered that coenzyme Q10 can resist DRV infection, providing a new prevention and control strategy for DRV disease.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A first aspect of the present invention provides the use of coenzyme Q10 in the preparation of a medicament against a novel duck reovirus.

[0008] In the above applications, the coenzyme Q10 resists novel duck reovirus infection through at least one of the following pathways (1)-(3):

[0009] (1) Inhibit mitochondrial dysfunction induced by novel duck reovirus;

[0010] (2) Reduce the inflammatory response induced by novel duck reovirus;

[0011] (3) Inhibit excessive pyroptosis.

[0012] In the above applications, the drug contains a therapeutically effective amount of coenzyme Q10. In this invention, "therapeutically effective amount" refers to the amount of coenzyme Q10 required to inhibit the replication of novel duck reovirus or to treat novel duck reovirus infection. The effective level may depend on factors such as the severity of the disease, drug sensitivity, timing of administration, route of administration, and duration of treatment.

[0013] In a preferred embodiment of the invention, the drug contains 50-100 μM coenzyme Q10.

[0014] In the above applications, the drug may also contain a pharmaceutically acceptable carrier; the pharmaceutically acceptable carrier includes one or more of the following: diluent, binder, disintegrant, lubricant, adsorbent, and humectant. For example, materials that can be used as binders include gelatin, gum arabic, carboxymethyl cellulose, hydroxypropyl cellulose, polyvinyl alcohol, etc.; materials that can be used as disintegrants include corn starch, agar powder, bentonite, sodium alginate, calcium citrate, etc.; materials that can be used as lubricants include calcium stearate, magnesium stearate, talc, liquid paraffin, petrolatum, etc.

[0015] In the above applications, the drug can be formulated into various dosage forms according to conventional methods, such as granules, powders, capsules, tablets, sprays, inhalers, injections, etc.

[0016] In a second aspect, the present invention provides a drug for combating a novel duck reovirus, wherein the drug comprises coenzyme Q10 and antiviral transfer factor as active ingredients.

[0017] Preferably, the antiviral transfer factor (TF) is a transfer factor oral solution (trade name: Symbit).

[0018] Preferably, in the drug, the ratio of coenzyme Q10 to antiviral transfer factor is (1-10) mg: (0.1-1) mL.

[0019] The beneficial effects of this invention are:

[0020] (1) The present invention found that coenzyme Q10 can inhibit mitochondrial dysfunction induced by novel duck reovirus, reduce the inflammatory response induced by novel duck reovirus, inhibit the excessive occurrence of pyroptosis, and thus achieve anti-infection of novel duck reovirus, providing a new prevention and control strategy for the prevention and control of novel duck reovirus disease.

[0021] (2) Further research in this invention found that the combined use of coenzyme Q10 antiviral transfer factor can synergistically improve the relief effect on the inflammatory response induced by novel duck reovirus and reduce the viral load of NDRV in the spleen of ducklings. Attached Figure Description

[0022] Figure 1 Photographs of spleens from NDRV-infected ducklings after autopsy; the control group in the image shows the results measured at 72 hours.

[0023] Figure 2 Detection of serum IL-1β and IL-6 levels in ducklings infected with NDRV at different time points.

[0024] Figure 3 HE staining results of spleens of ducklings infected with NDRV at different time points; the scale bar in the figure is 150 μm.

[0025] Figure 4 Transmission electron micrographs of spleen tissue in ducklings infected with NDRV at different time points; scale bar in the figure is 2 μm.

[0026] Figure 5 Immunohistochemistry was used to detect the expression of pyroptosis-related proteins in the spleen of ducklings infected with NDRV 48 hpi.

[0027] Figure 6 The activity of PBMCs in NDRV-infected and control groups was detected using a CCK-8 assay kit.

[0028] Figure 7 The detection results of lactate dehydrogenase content in the supernatant of PBMCs infected with NDRV and in the control group.

[0029] Figure 8 The levels of IL-6, IL-1β, and TNF-α in the supernatant of PBMCs infected with NDRV and in the control group were detected by ELISA.

[0030] Figure 9 Mitochondrial damage detection results: Figure A shows the detection results of mitochondrial reactive oxygen species in NDRV-infected and control PBMCs cells. The Mitosox detection kit was used for detection by flow cytometry; Figure B is the statistical analysis of the Mitosox positivity of each group of cells in Figure A; Figure C shows the detection results of mitochondrial membrane potential in NDRV-infected and control PBMCs cells.

[0031] Figure 10Screening of key viral proteins for NDRV-induced pyroptosis; Figure A shows the IL-1β content in cell supernatant after DEF transfection with NDRV overexpression plasmids; Figure B shows the lactate dehydrogenase content in cell supernatant after DEF transfection with NDRV overexpression plasmids.

[0032] Figure 11 Screening of host proteins that interact with σC; Figure A shows the host proteins screened by a combination of immunoprecipitation and mass spectrometry; Figure B shows the Western blot results of knocking down COQ6 protein in DEF cells using small interfering RNA (siRNA).

[0033] Figure 12 Effects of COQ6 deficiency on mitochondrial reactive oxygen species (ROS) levels, mitochondrial membrane potential, and intracellular coenzyme Q10; Figure A shows the increase in mitochondrial ROS levels in DEF cells after COQ6 knockdown; Figure B shows the decrease in mitochondrial membrane potential in DEF cells after COQ6 knockdown; Figure C shows the decrease in DEF cell viability after COQ6 knockdown; Figure D shows the decrease in CoQ10 content in DEF cells after COQ6 knockdown.

[0034] Figure 13 Figure 1 shows the regulatory role of coenzyme Q10 in NDRV-infected PBMCs; Figure A shows the detection results of mitochondrial reactive oxygen species in PBMCs of each group; Figure B shows the detection results of mitochondrial membrane potential in PBMCs of each group; Figure C shows the detection results of PBMC activity in each group; Figure D shows the bright field and PI staining results of PBMCs of each group; Figure E shows the IL-1β content in the supernatant of PBMCs of each group; Figure F shows the release of lactate dehydrogenase in PBMCs of each group.

[0035] Figure 14 The therapeutic effects of coenzyme Q10 and transfer factor on NDRV-infected ducklings; Figures A, B, and C show the levels of IL-1β, IL-6, and TNF-α in the serum of ducklings in each group, respectively; Figure D shows the detection results of novel duck reovirus load in the spleen of ducklings in each group. Detailed Implementation

[0036] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0037] As mentioned earlier, the novel duck reovirus disease (NDRV) is characterized by rapid onset, rapid spread, and high mortality, making prevention and control difficult and causing significant economic losses to poultry farmers. Currently, there is a lack of systematic research into the specific molecular mechanisms underlying the disease process mediated by NDRV infection, and the relevant target molecules remain unclear.

[0038] In light of this, this invention systematically reveals the crucial role of pyroptosis in driving splenic inflammatory responses during novel duck reovirus (NDRV) infection. In vivo experiments showed that NDRV infection in ducklings resulted in significant congestion, swelling, and necrosis of the spleen, accompanied by a significant increase in serum levels of inflammatory factors such as IL-6 and IL-1β, suggesting that the infection induced a strong systemic inflammatory response. Histological and transmission electron microscopy observations further confirmed that viral infection led to cell membrane damage, nuclear membrane abnormalities, and mitochondrial structural impairment in splenic immune cells and vascular endothelial cells, indicating cellular energy metabolism disorders and programmed cell death. Through immunohistochemistry and an in vitro PBMCs model, this study, for the first time, elucidated the molecular mechanism by which NDRV infection activates the NLRP3 inflammasome, promotes Caspase-1-dependent GSDME cleavage, and subsequently induces pyroptosis of inflammatory cells.

[0039] Furthermore, this invention identifies the key NDRV viral protein σC as an important inducing factor of pyroptosis. The σC protein interacts specifically with the host mitochondrial protein COQ6, interfering with its mitochondrial localization and coenzyme Q10 synthesis, leading to mitochondrial dysfunction. Silencing of COQ6 exacerbates this process.

[0040] Therefore, it is inferred that coenzyme Q10 may affect the NDRV infection process. Experimental verification has shown that coenzyme Q10 can inhibit the inflammatory response during NDRV infection. Furthermore, combining coenzyme Q10 with antiviral transfer factor can synergistically reduce NDRV viral infection levels. Therefore, this invention provides a new therapeutic agent for the prevention and treatment of NDRV. This invention is thus proposed.

[0041] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0042] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions. Wherein:

[0043] The CAS number of the coenzyme Q10 used in this invention is 303-98-0. The antiviral transfer factor used is a transfer factor oral solution (trade name: Symbit; approval number: Veterinary Drug Approval No. 150439001).

[0044] The NDRV strain N19 used is described in the journal article "Yan H, Xu G, Zhu Y, Xie Z, Zhang R, Jiang S. Isolation and characterization of a naturally attenuated novel duckreovirus strain as a live vaccine candidate. Vet Microbiol. 2021 Oct;261:109214", and is available to the public from the applicant for use in replicating this invention.

[0045] Three-day-old SPF ducklings were purchased from Shandong Haotai Experimental Animal Breeding Co., Ltd.; 293T cells were purchased from ATCC; duck embryo fibroblasts (DEFs) were obtained from 10-day-old specific pathogen-free (SPF) duck embryos and purchased from Shandong Haotai Experimental Animal Breeding Co., Ltd.; the anti-NDRV-σC antibody was prepared in our laboratory. anti-duGSDME and anti-ducaspase-1 were prepared by Abclonal; anti-NLRP3 and anti-COQ6 were purchased from Wuhan Sanying; anti-ASC, anti-IL-1β, HRP-labeled goat anti-mouse secondary antibody, and HRP-labeled goat anti-rabbit secondary antibody; propidium iodide (PI) was purchased from Beyotime; Mitosox and TRMR were purchased from Thermo Fisher Scientific; siRNA:siCOQ6 was synthesized by Gemma Gene.

[0046] Example 1: Investigation of the effects of NDRV infection on ducklings

[0047] 1. Test method:

[0048] Three-day-old SPF ducklings were randomly divided into a control group and an NDRV-infected group. Ducklings in the NDRV-infected group received an intramuscular injection of 0.5 ml of NDRV virus solution (MOI=2) in their legs, while the control group received an equal volume of 0.5 ml of sterile PBS in their legs. All ducklings had free access to food and water during the rearing period. After specific time points (24 hours, 48 ​​hours, and 72 hours) following infection, the ducklings were euthanized, and serum and spleen tissue were collected. Animal experiments were conducted in accordance with the institutional guidelines issued by the Animal Care and Use Committee of Shandong Agricultural University.

[0049] (1) Determination of serum IL-6 and IL-1β levels:

[0050] The levels of IL-6 and IL-1β in duck serum were detected using an ELISA kit (Mlbio, Shanghai, China). 50 μL of standard and sample were added to the corresponding wells, followed by 100 μL of enzyme conjugate. The plates were sealed and incubated at 37°C for 1 hour. After washing, 50 μL of substrate A and substrate B were added to each well, and the reaction was carried out at 37°C for 15 minutes. Then, 50 μL of stop solution was added, and the optical density (OD) was measured at 450 nm using a microplate reader within 15 minutes. The sample concentrations were calculated based on the standard curve.

[0051] (2) Hematoxylin-Eosin Staining:

[0052] After the ducks were euthanized, the spleens were removed and fixed in 4% neutral paraformaldehyde for at least 24 hours. The tissue blocks were then dehydrated using a gradient of ethanol. After dehydration, the tissues were embedded in paraffin and sectioned. Paraffin sections were cut into 4μm thick sections using a paraffin microtome. After dewaxing and rehydration, the sections were stained with hematoxylin for 3–5 minutes, washed with water, differentiated in 1% hydrochloric acid ethanol for 5–10 seconds, washed with water, counterstained with eosin, dehydrated using a gradient method, mounted with neutral resin, and the pathological changes in the spleen were observed under a microscope.

[0053] (3) Transmission electron microscopy observation:

[0054] Transmission electron microscopy was used to observe the spleen tissues of the NDRV infection group and the control group to detect the changes in the ultrastructure of the spleen tissues after NDRV infection.

[0055] (4) Immunohistochemical detection:

[0056] After dewaxing and rehydration of 4 μm paraffin sections, antigen retrieval was performed. The tissue was circled with an immunohistochemical pen, and the membrane was perforated with 0.3% Triton. Endogenous peroxidase inhibitor was added, and the sections were incubated at room temperature. After washing three times with PBS, 10% BSA was added, and the sections were blocked at 37°C for 30 min. 100 μL of primary antibody (NLRP3, IL-1β, NDRV-σC, caspase-1, GSDME) diluted with 1% BSA was added, and the sections were incubated overnight at 4°C. After washing three times with PBS, 150 μL of reaction enhancement solution was added, and the sections were incubated at room temperature for 20 min, followed by washing with PBS for 5 min. This process was repeated three times. 150 μL of enzyme-labeled goat anti-mouse or rabbit IgG polymer was added, and the sections were incubated at room temperature for 20 min, followed by washing with PBS for 5 min. This process was repeated three times. A suitable amount of freshly prepared DAB chromogenic solution was added, and the sections were stained with hematoxylin and incubated. The sections were dehydrated stepwise with ethanol, cleared with xylene, mounted with neutral resin, and observed under an inverted microscope.

[0057] 2. Test Results:

[0058] Post-mortem examination of ducklings infected with NDRV 24 hours later revealed spleen congestion and swelling; 48 hours after infection, the inflammatory response, including spleen congestion and swelling, further intensified; by 72 hours after infection, necrotic foci began to appear in the spleen on the basis of congestion and swelling, thus exacerbating the secondary inflammatory response. Figure 1 ).

[0059] Serum levels of IL-6 and IL-1β in infected ducklings increased as early as 24 hours post-infection; concentrations of IL-6 and IL-1β further increased at 48 and 72 hours. Figure 2 This suggests that NDRV infection induces a systemic inflammatory response.

[0060] HE staining results showed that the spleen structure of infected ducklings was disordered, with significant proliferation in the white pulp area accompanied by a large number of inflammatory cell infiltrations. Hemorrhage and necrosis of varying degrees were also observed in the red pulp area. By 72 hours post-infection, with the increase of necrotic foci, the inflammatory cell infiltration further intensified, and local tissue damage was obvious. Figure 3 This suggests that the viral infection caused severe damage to the spleen's immune function area.

[0061] We then used transmission electron microscopy to observe the ultrastructural changes in spleen tissue after NDRV infection. The results showed that, compared with the control group, the immune cells and vascular endothelial cells of the infected spleen exhibited cell membrane damage and irregular swelling; the nuclear membranes of some cells showed rupture or irregular indentation; mitochondrial morphology was severely disrupted; mitochondrial function was severely impaired; and cellular energy metabolism was significantly interfered with. Figure 4 This suggests that viral infection may have induced programmed cell death.

[0062] To further investigate the specific mechanisms by which NDRV infection drives splenic inflammation, we focused on the role of inflammatory programmed cell death—pyroptosis—in this process. Using immunohistochemistry (IHC), we detected the expression of pyroptosis-related proteins (NLRP3, caspase-1, GSDME, IL-1β) in the spleen 48 hpi after viral infection. The results showed that the expression of pyroptosis-related proteins in the spleen was significantly enhanced after NDRV infection. Figure 5 This suggests that pyroptosis may play a key role in NDRV-induced inflammatory responses in the spleen of ducklings.

[0063] Example 2: Investigation of the molecular mechanism of NDRV infection-induced pyroptosis

[0064] 1. Test method:

[0065] Fresh duck blood samples were obtained using the jugular vein sampling method, and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque (Cytiva) gradient centrifugation. PBMCs were cultured in DMEM medium supplemented with 10% fetal bovine serum (BI), 100 U / mL penicillin, and 100 mg / mL streptomycin (Solarbio). Cells were cultured in a 37°C, 5% CO2 incubator.

[0066] After culture, PBMCs were divided into a control group and an NDRV-infected group. NDRV-infected PBMCs were incubated with 1 MOI of NDRV virus solution at 37°C for 1 hour, while control group cells were treated with DMEM medium containing 2% fetal bovine serum for 1 hour. After 1 hour, both groups were cultured in DMEM medium containing 2% fetal bovine serum for further processing. Samples were collected at different time points for subsequent treatment.

[0067] (1) Cell viability assay:

[0068] Cell viability after NDRV infection was assessed using a CCK8 assay kit (Beyotime). 10 μL of CCK8 solution was added to a 96-well plate containing the cells, and the plates were incubated for 3 hours. The optical density (OD) of each well was measured at 450 nm using a Biotek microplate reader. 450 nm ).

[0069] (2) Lactate dehydrogenase (LDH) detection:

[0070] After collecting the culture medium, it was added to 96-well plates, and LDH release was detected using the LDH release assay kit (Beyotime Biotechnology Co., Ltd., Beijing, China) according to the manufacturer's instructions. After incubation at 37°C in the dark for 20 minutes, the reaction was terminated by adding stop solution. The luminescence intensity was then measured at 450 nm using a BioTek Synergy H1 microplate reader.

[0071] (3) ELISA detection:

[0072] The IL-1β content in cell supernatant was detected using an ELISA kit (Mlbio, Shanghai, China).

[0073] (4) Detection of mitochondrial damage:

[0074] Mitochondrial reactive oxygen species were detected using MitoSOX Red (invitrogen). The cells to be tested were washed twice with PBS, and 2 μM of MitoSOX working solution diluted with serum-free medium was added. The cells were incubated at 37°C for 20 min, then washed three times with PBS and detected by flow cytometry.

[0075] Mitochondrial membrane potential was measured using tetramethylrhodamine methyl ester (TMRM) according to the manufacturer's instructions. Cells were incubated with 200 nM TMRM at 37 °C for 30 min. After washing twice with PBS, fluorescence intensity was measured using a Biotek multimode microplate reader according to the manufacturer's instructions.

[0076] 2. Test Results:

[0077] Given that peripheral blood mononuclear cells (PBMCs) are rich in various immune-related cells, and that some cell types are functionally similar to spleen immune cells, PBMCs were chosen as an in vitro model to simulate the stress state of immune cells under NDRV infection conditions. After infection, PBMC cell viability significantly decreased ( Figure 6 Increased LDH release levels in cell supernatant Figure 7 Meanwhile, ELISA analysis showed a significant upregulation of IL-6, IL-1β, and TNF-α levels in the supernatant. Figure 8 This suggests that NDRV infection can induce inflammatory pyroptosis in PBMCs.

[0078] Subsequently, we assessed whether NDRV infection could cause mitochondrial damage in PBMCs. A decrease in mitochondrial membrane potential (ΔΨm) and accumulation of mtROS are considered hallmarks of mitochondrial damage. Fluorescent staining results showed a significant decrease in the fluorescence intensity of tetramethylrhodamine methyl ester (TMRM) in NDRV-infected PBMCs, suggesting mitochondrial membrane potential (ΔΨm) depolarization; enhanced MitoSOX fluorescence signal indicated a large accumulation of mitochondrial reactive oxygen species (mtROS). Figure 9 The above results demonstrate that NDRV infection can induce significant mitochondrial damage.

[0079] Example 3: Screening of key viral proteins and their interacting host proteins for NDRV-induced pyroptosis

[0080] 1. Screening of key viral proteins for NDRV-induced pyroptosis:

[0081] (1) Construction of overexpression plasmid:

[0082] The viral proteins of NDRV (λA, λB, λC, μA, μB, μNS, σA, σB, σC, σNS, P10, P18) were amplified from NDRV cDNA by PCR and ligated into the pEGFP-C3 vector. The results were verified by sequencing, and 12 overexpression plasmids of NDRV viral proteins were constructed.

[0083] The sequence information of the 12 NDRV viral proteins in NCBI is shown in Table 1.

[0084] Table 1: Sequence information of NDRV viral proteins in NCBI

[0085]

[0086] (2) Transfection:

[0087] The 12 NDRV viral protein overexpression plasmids were transfected into duck embryonic fibroblasts (DEF cells), and the levels of lactate dehydrogenase (LDH) and interleukin-1β (IL-1β) in the cell supernatant were detected 48 hours after transfection.

[0088] The results showed that the LDH release level in the σC protein transfection group was significantly higher than that in other groups, and the secretion of IL-1β was also significantly increased. Figure 10 This suggests that σC may play a key role in NDRV-mediated pyroptosis.

[0089] 2. Screening of host proteins that interact with σC:

[0090] Overexpression plasmids of σC were transfected into DEF cells to construct a DEF cell model with σC protein overexpression. Immunoprecipitation (IP) was used to enrich σC and its potential interacting proteins. Combined with high-throughput mass spectrometry (MS), host factors interacting with σC were systematically screened and identified. Figure 11 -A). Among numerous candidate interacting proteins, COQ6 (Coenzyme Q6 monooxygenase) attracted our attention. COQ6 is a key enzyme in the coenzyme Q (also known as ubiquinone) biosynthesis pathway, mainly located in the inner mitochondrial membrane, and participates in the regulation of cellular energy metabolism and redox homeostasis. To further clarify the role of COQ6 in σC regulation, we used siRNA to specifically silence COQ6 expression in DEF cells. Figure 11 -B). The results showed that COQ6 deficiency led to increased mitochondrial reactive oxygen species (ROS) levels, mitochondrial membrane potential depolarization, and decreased intracellular coenzyme Q10 (CoQ10) levels. Figure 12 ).

[0091] Example 4: Effect of Coenzyme Q10 on NDRV Infection

[0092] 1. Test method:

[0093] PBMCs were randomly divided into four groups: control group (NC), NDRV infection group (NDRV), and NDRV-infected group supplemented with CoQ10 at two concentration gradients: 50 μM and 100 μM. The specific treatment methods are as follows:

[0094] NC group: PBMCs were treated with DMEM medium containing 2% fetal bovine serum for 1 hour, and then cultured in DMEM medium containing 2% fetal bovine serum for another hour.

[0095] NDRV group: PBMCs cells were incubated with 1 MOI of NDRV virus solution at 37°C for 1 hour, and then cultured in DMEM medium containing 2% fetal bovine serum.

[0096] NDRV+ CoQ10-50μM group: PBMCs were incubated with 1 MOI of NDRV virus solution at 37°C for 1 hour, and then cultured in DMEM medium containing 2% fetal bovine serum and 50μM CoQ10.

[0097] NDRV+ CoQ10-100μM group: PBMCs were incubated with 1 MOI of NDRV virus solution at 37°C for 1 hour, and then cultured in DMEM medium containing 2% fetal bovine serum and 100μM CoQ10.

[0098] Cell samples were collected from each group 24 hours after treatment for testing.

[0099] (1) Propidium iodide (PI) staining:

[0100] PI staining solution (Beyotime Biotechnology Co., Ltd., Beijing, China) was added to the culture medium at a final concentration of 1.0 μg / mL and incubated for 20 minutes in the dark. After incubation, bright-field and fluorescence images of pyroptosis cells were acquired using a fluorescence microscope (Nikon, ECLIPSE Ts2R).

[0101] 2. Test Results:

[0102] The results showed that, compared with the NDRV infection group, the CoQ10 treatment group exhibited significantly reduced cell morphological damage and significantly improved cell viability. Flow cytometry analysis revealed that CoQ10 intervention significantly decreased intracellular mitochondrial ROS levels, mitochondrial membrane potential, and cytoplasmic ox-mtDNA levels. Figure 13 In summary, these results indicate that exogenous CoQ10 supplementation can effectively inhibit NDRV-induced mitochondrial dysfunction, thereby significantly alleviating virus-induced pyroptosis.

[0103] Example 5: Animal Experiment

[0104] 1. Test method:

[0105] In the NDRV-infected cell model of Example 4, we verified that CoQ10 can inhibit excessive pyroptosis by alleviating mitochondrial damage and reducing inflammatory response. Building on this, we further investigated the anti-inflammatory effects of CoQ10 in an NDRV-infected duckling model and combined it with transfer factor, which has antiviral activity, to verify the synergistic therapeutic effect of the two in NDRV-infected ducklings. Details are as follows:

[0106] Five-day-old ducklings were randomly divided into five groups: a control group (0.5 ml PBS intramuscularly), an NDRV infection group (0.5 ml 2 MOI virus solution intramuscularly), an NDRV+CoQ10 group (0.5 ml 2 MOI virus solution intramuscularly, and CoQ10 administered orally at a dose of 30 mg / kg), an NDRV+transfer factor group (0.5 ml 2 MOI virus solution intramuscularly, and antiviral transfer factor administered at the recommended dose of 0.3 ml / duckling / day for 3 consecutive days), and an NDRV+CoQ10+transfer factor group (0.5 ml 2 MOI virus solution intramuscularly, and CoQ10 administered orally at a dose of 30 mg / kg, and antiviral transfer factor administered at the recommended dose of 0.3 ml / duckling / day for 3 consecutive days; the CoQ10 to antiviral transfer factor ratio was 6 mg:0.9 mL). Serum and tissue samples were collected on days 3, 6, and 9 post-infection to detect inflammatory factor levels and viral load. The results showed that the combined use of transfer factor and CoQ10 could significantly reduce tissue damage and inflammatory response in NDRV-infected ducklings, inhibit NDRV proliferation, and exhibit synergistic anti-inflammatory and immunoprotective effects.

[0107] (1) qPCR detection:

[0108] The transcriptional level of NDRV was detected using real-time quantitative PCR. After obtaining the Ct value, the viral copy number was calculated using a standard curve. All RT-qPCR experiments were performed on a Light Cycler 96 instrument (Roche).

[0109] 2. Test Results:

[0110] The results showed that, compared with the NDRV infection group, CoQ10 intervention significantly reduced the serum levels of IL-1β, IL-6, and TNF-α, indicating that CoQ10 could alleviate the inflammatory response caused by NDRV infection; CoQ10 intervention also reduced the viral load of NDRV in the spleen of ducklings to a certain extent. Figure 14 ).

[0111] The combined use of CoQ10 and transfer factor TF has a synergistic effect in alleviating the inflammatory response caused by NDRV infection and reducing the viral load of NDRV in the spleen of ducklings, achieving a synergistic effect of 1+1>2.

[0112] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. Application of Coenzyme Q10 in the preparation of drugs against novel duck reovirus.

2. The application according to claim 1, characterized in that, The coenzyme Q10 resists novel duck reovirus infection through at least one of the following pathways (1)-(3): (1) Inhibit mitochondrial dysfunction induced by novel duck reovirus; (2) Reduce the inflammatory response induced by novel duck reovirus; (3) Inhibit excessive pyroptosis.

3. The application according to claim 1, characterized in that, The drug contains 50-100 μM coenzyme Q10.

4. The application according to any one of claims 1-3, characterized in that, The drug also contains a pharmaceutically acceptable carrier.

5. The application according to claim 4, characterized in that, The pharmaceutically acceptable carriers include one or more of the following: diluents, binders, disintegrants, lubricants, adsorbents, and humectants.

6. The application according to claim 4, characterized in that, The dosage form of the drug is granules, powders, capsules, tablets, sprays, inhalers, or injections.

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