Application of curcumin in resisting tembusu virus
Curcumin solves the inhibition and inactivation of Tambsuvir by applying it in vitro and in vivo, significantly improves the survival rate of avians and reduces the copy number of viruses, alleviates body damage, and has significant antiviral effects.
Patent Information
- Application Number
- CN202510433715.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-08
- Publication Date
- 2025-07-04
AI Technical Summary
The prior art lacks effective drugs to prevent, control and treat the Tambusu virus disease, especially in vitro and in vivo, and the inhibition and inactivation effect of Tambusu virus is not significant.
Curcumin is used as an active ingredient to prepare products through different concentrations and doses of curcumin, which are used to inhibit the replication of Tambusu virus in in vitro cells, reduce the level of viral E protein, reduce the number of virus copies in biological tissues after infection, and improve the survival rate of infected birds, and alleviate body damage.
Curcumin significantly inhibits the post-adsorption period of Tambusu virus without harming cells, and can directly inactivate the virus in vitro. In vivo tests show good therapeutic effects, which improves the survival rate of infected birds and reduces the copy number of tissue viruses, alleviates body damage.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biomedicine, and particularly to the application of curcumin in the prevention and treatment of duck tembusu virus disease. Background Art
[0002] Tembusu virus disease (TMUVD) is an acute and highly contagious infectious disease caused by duck tembusu virus (TMUV). The main clinical features are a significant decrease in egg production in laying ducks, and neurological symptoms in ducklings or growing ducks. This disease was first discovered in East China in 2010 and then quickly spread across China. The infection range of this virus shows an expanding trend, from initially infecting laying ducks to infecting meat ducks, geese, and even poultry such as chickens and sparrows. Currently, the prevention and control of TMUV mainly rely on inoculation with attenuated vaccines and inactivated vaccines, which have to some extent controlled the prevalence and spread of the virus. However, sporadic cases of TMUV infection still occur in the main duck-raising areas across the country. Relevant research shows that various active substances in some traditional Chinese medicines can inhibit the replication of TMUV, improve the body's functions, and are not prone to drug resistance. Traditional Chinese medicines have significant curative effects in the treatment of flaviviruses. Therefore, developing an effective antiviral drug is of great significance for the prevention, control, and treatment of TMUV.
[0003] Curcumin is a component extracted from the rhizomes of Curcuma longa. It is a natural phenolic antioxidant with therapeutic potential both in vitro and in vivo, and has antiviral, antibacterial, anti-inflammatory, antioxidant, and anticancer activities. It has a wide range of antiviral effects against coronaviruses, influenza viruses, rotaviruses, flaviviruses, and arteriviruses. Summary of the Invention
[0004] The purpose of the present invention is to solve the deficiencies in the prior art, and to propose the application of curcumin in the prevention and treatment of duck tembusu virus disease.
[0005] The purpose of the present invention is to propose the application of curcumin in any one of the following (1)-(5):
[0006] (1) Preparing a product for inhibiting the replication of tembusu virus in vitro cells;
[0007] (2) Preparing a product for reducing the level of tembusu virus E protein in vitro cells;
[0008] (3) Preparing a product for anti-tembusu virus in vitro;
[0009] (4) Preparing a product for reducing the virus copy number in biological tissues after infection with tembusu virus;
[0010] (5) Preparing a product for increasing the survival rate of poultry infected with tembusu virus;
[0011] (6) Preparing a product for relieving the body damage caused by infection with Tembusu virus;
[0012] (7) Preparing a product for preventing or treating Tembusu virus.
[0013] Preferably, in item (1), (2) or (3), the concentration of curcumin is 22.5 - 30 μM.
[0014] Preferably, in item (4), (5) or (6), the dose of curcumin is 200 - 400 mg of curcumin per kilogram of body weight.
[0015] Preferably, in item (4), the biological tissue is at least one of avian liver tissue, avian spleen tissue, and avian brain tissue.
[0016] Preferably, in item (4) or (5), the avian is at least one of duck, goose, chicken, and sparrow.
[0017] Preferably, in item (6), the body damage is that at least one of avian liver tissue, avian spleen tissue, and avian brain tissue has lesions or pathological changes.
[0018] Preferably, in item (7), the product is a drug, a feed additive, or / and feed.
[0019] Beneficial effects:
[0020] The present invention has been confirmed by cell experiments that curcumin has a significant inhibitory effect on TMUV without damaging cells, and through in vivo experiments, it is found that curcumin has a good therapeutic effect on Tembusu virus. It can significantly inhibit the post-attachment period of the virus in different life cycles of TMUV, and can also directly inactivate TMUV in vitro. Therefore, curcumin can be used to prepare a product for preventing or treating Tembusu virus.
[0021] The present invention has been confirmed in an animal model that curcumin can resist the infection of TMUV, has a good therapeutic effect, and has certain practical value. Therefore, the present invention discloses for the first time that curcumin has an anti-TMUV effect and has important application value. Description of the drawings
[0022] Figure 1 It is a graph showing the toxicity test results of curcumin at different concentrations in the CCK8 experiment of the BHK-21 cell line in Example 1.
[0023] Figure 2 It is a graph showing the inhibitory effect of curcumin at different concentrations on the replication of TMUV in infected cells detected by RT-qPCR in Example 1.
[0024] Figure 3For the inhibitory effect diagram of curcumin at different concentrations on TMUV replication in infected cells detected by the median tissue culture infective dose (TCID 50 ).
[0025] Figure 4 For the inhibitory effect diagram of curcumin at different concentrations on TMUV replication in infected cells detected by indirect immunofluorescence in Example 1.
[0026] Figure 5 For the diagram of the effect of curcumin at different concentrations on E protein expression detected by immunoblotting in Example 1
[0027] Figure 6 For the inhibitory effect diagram of curcumin at different life cycles of TMUV detected by RT-qPCR in Example 2.
[0028] Figure 7 For the inhibitory effect diagram of curcumin at different life cycles of TMUV detected by TCID50 in Example 2.
[0029] Figure 8 For the inhibitory effect diagram of curcumin at different life cycles of TMUV detected by indirect immunofluorescence in Example 2.
[0030] Figure 9 For the inhibitory effect diagram of curcumin at different life cycles of TMUV detected by immunoblotting in Example 2.
[0031] Figure 10 For the effect diagram of the survival rate of ducklings in each group in Example 3.
[0032] Figure 11 For the virus copy number diagram detected by RTq-PCR in the spleen tissue, liver tissue and brain tissue of ducklings in each group in Example 3.
[0033] Figure 12 For the effect diagram of the necropsy lesions of TMUV-infected ducklings in each group in Example 3.
[0034] Figure 13 For the histopathological changes diagram of the brain, spleen and liver of TMUV-infected ducklings in each group in Example 3. Detailed implementation manners
[0035] The present invention will be further explained below with reference to specific embodiments.
[0036] In Example 1, the inhibition of TMUV replication by curcumin at the cellular level is dose-dependent
[0037] (1) Determination of TCID50
[0038] Step 1: When BHK-21 cells reach 85% confluence, discard the culture medium and rinse 1-2 times with PBS.
[0039] Step 2: Dilute the virus solution 10-fold serially (10 -1 ~10 -8 ) with DMEM medium without fetal bovine serum. Inoculate 8 wells for each dilution, with 100 μL inoculated into each well. At the same time, set up a cell control group (add 100 μL of DMEM medium per well).
[0040] Step 3: Add 100 μL of 1% DMEM to each well, place it in the incubator for culture, observe and record the number of CPE wells daily. Generally, it needs to be observed for 3-5 days.
[0041] Step 4: Calculate the TCID 50 of the virus according to the Reed-Muench method, and repeat the experiment three times and take the average value.
[0042] Table 1 Results of the determination of the TCID 50 value of TMUVAQ-19 on BHK-21 cells
[0043]
[0044] Using the Reed-Muench method, according to the formula, the TCID 50 value of the TMUVAQ-19 strain on BHK-21 cells was calculated to be 10 -5.19 / 0.1 mL.
[0045] (2) Cytotoxicity detection of curcumin
[0046] The cytotoxicity test was detected using Cell Counting Kit-8 (Jumei, Beijing). On a 96-well cell plate of BHK-21 cells with a cell density of 80-90%, different concentrations of curcumin (10-50 μM) were added. Each concentration was set with 6 replicates. At the same time, a cell control group (BHK-21 + 100 μL of 2% DMEM per well), a drug control group (add 100 μL of the drug per well in the blank well), and 0.5% DMSO was used as a negative control.
[0047] After incubating in a 37°C 5% CO2 incubator for 48 h, discard the culture supernatant, wash 2 times with PBS, add 100 μL of DMEM culture medium and 10 μL of CCK-8 reagent, incubate in a 37°C 5% CO2 incubator for 2 h, and then detect on an enzyme-linked immunosorbent assay (ELISA) reader to read OD 450 . The data was processed using Graphpad.
[0048] As Figure 1As shown, when the concentration of curcumin is less than 30 μM, the effect on cell viability is relatively small. Therefore, in the subsequent in vitro antiviral experiments, the applicant selected 30 μM as the maximum concentration for the experiments.
[0049] (3) Verification of the in vitro anti-TMUV effect of different concentrations of curcumin
[0050] Inoculate BHK-21 cells in a 12-well plate and incubate them in an incubator at 37°C with 5% CO2. When the cell density reaches 80-90%, discard the culture supernatant, wash twice with PBS, infect with DTMUV at a multiplicity of infection (MOI) of 0.1, and incubate in an incubator at 37°C with 5% CO2 for 2 h. Remove the supernatant, wash three times with PBS, add 7.5-30 μM curcumin and 1% DMEM, and incubate in an incubator at 37°C with 5% CO2 for 48 h. The control group does not add curcumin, and the blank group is not infected with TMUV and does not add curcumin.
[0051] Use indirect immunofluorescence and Western blot to detect the expression of TMUV E protein, use RT-qPCR to detect the virus copy number, and use the TCID 50 assay to evaluate the virus titer.
[0052] (4) Detection of TMUV virus load using real-time quantitative PCR technology
[0053] RNA extraction steps: Freeze and thaw the sample three times, centrifuge to obtain the supernatant, and extract total RNA from BHK-21 cells according to the virus RNA extraction kit (Beyotime, R0035L).
[0054] Step 1: Add 100 μL of cell supernatant to 300 μL of lysis buffer and gently invert and mix 3-5 times;
[0055] Step 2: Add 400 μL of binding buffer and gently invert and mix 3-5 times;
[0056] Step 3: Transfer the mixture to a purification column and centrifuge at 12,000 g for 30 s in two batches, and discard the liquid in the collection tube;
[0057] Step 4: Add 600 μL of Wash Buffer I and centrifuge at 12,000 g for 30 s, and discard the liquid in the collection tube;
[0058] Step 5: Add 600 μL of Wash Buffer II and centrifuge at 12,000 g for 30 s, and discard the liquid in the collection tube;
[0059] Step 6: Repeat Step 5 once.
[0060] Step 7: Centrifuge at 14,000 g for 2 min to remove the residual liquid;
[0061] Step 8: Place the RNA purification column in the elution tube, add 30 μL of elution buffer, incubate at room temperature for 3 min, and centrifuge at 14,000 g for 30 s.
[0062] Step 9: Repeat Step 8 once.
[0063] cDNA synthesis: Reverse transcribe to obtain cDNA according to the operation of the All-in-one 1st Strand cDNA Synthesis SuperMix (gDNAPurge) kit (Novoprotein, E047). The reaction system is shown in Table 2. All-in-one 1st Strand cDNA SynthesisSuperMix(gDNAPurge) kit (Novoprotein, E047) to perform reverse transcription to obtain cDNA, and the reaction system is shown in Table 2.
[0064] Table 2 cDNA synthesis reaction system
[0065]
[0066] Set the program: Incubate at 50 °C for 15 min and heat at 85 °C for 5 s. The obtained cDNA should be placed on ice for subsequent experiments or stored at -20 °C.
[0067] qPCR detection: Use the 2X M5 HiPer Realtime PCR Super mix (SYBRgreen, with anti-Taq) kit from Polymerase Company to perform detection in a fluorescence PCR instrument. The primers are as follows:
[0068] TMUV-F: 5’-AGGAAGTGGAGCAATCAGGAA-3’;
[0069] TMUV-R: 5’-TAACAAGTGGCAGAGCAAAGG-3’.
[0070] The length of the target fragment is 178 bp. The reaction system for fluorescence quantitative PCR is shown in Table 3 (for 1 sample).
[0071] Table 3 Reaction system for fluorescence quantitative PCR
[0072]
[0073] Calculate the volume of each reagent added according to the number of reactions. Pay attention to light protection. Add 18 μL of the mixed reagent to each well of a 0.1 ml eight-well strip tube, and finally add 2 μL of cDNA. Mix well, centrifuge, and amplify in a fluorescence PCR instrument. The reaction program is shown in Table 4.
[0074] Table 4 Fluorescence PCR amplification reaction program
[0075]
[0076]
[0077] (5) The experimental procedure of indirect immunofluorescence staining is as follows:
[0078] Seed the cells at an appropriate density in a 12-well plate. After the treatment is completed, wash twice with PBS, fix with 4% paraformaldehyde for 10 min, wash three times with PBS, permeabilize with 0.3% Triton X for 10 min, wash three times with PBS, block with 5% BSA for 30 min. Then incubate the slides with the primary antibody overnight at 4 °C, wash three times with PBS, incubate the sections with the secondary antibody at 37 °C for 1 h, wash three times with PBS. All slides are stained with DAPI for 10 min, and finally photographed with a confocal microscope.
[0079] (6) Western blot experiment
[0080] Wash three times with PBS, add RIPA lysis buffer, and lyse on ice for 30 min. Centrifuge at 12000 rpm for 10 min at 4 °C, and collect the supernatant, which is the cell protein lysate.
[0081] Step 1: Prepare the gel: Prepare 10% separating gel and stacking gel according to the instructions of Servicebio company;
[0082] Step 2: SDS-PAGE: Take 10 μL of the sample and add it to the gel well. Run at 80 V for 30 min in the stacking gel; run at 120 V for 90 min in the separating gel;
[0083] Step 3: Transfer the membrane: Soak the PVDF membrane in methanol for 30 s on both sides, and perform electrotransfer using the wet transfer method at 220 mA for 90 min;
[0084] Step 4: Block and incubate with antibodies: Place the PVDF membrane in a TBST solution containing 5% non-fat milk powder and gently shake to block at room temperature for 1 h; Incubate with mouse anti-E protein antibody and mouse anti-β-actin antibody at 4 °C overnight for the primary antibody, wash three times with TBST, 5 min each time; Incubate with HRP-labeled goat anti-mouse antibody at 37 °C for 1 h for the secondary antibody, wash three times with TBST, 5 min each time;
[0085] Step 5: Develop the color: Prepare the luminescent solution according to the instructions of BeyoECL Plus (Super-sensitive ECL Chemiluminescence Kit) of Beyotime at a 1:1 ratio, add it to the PVDF membrane for 1 min, and then collect and analyze the image with a gel imager.
[0086] Analyze the gray value of the bands using Image J software, and repeat each experiment three times.
[0087] (7) Detection of the effect of the drug on TCID 50 Detection of the effect
[0088] The obtained groups of samples were repeatedly frozen and thawed 3 times, centrifuged at 12,000 rpm for 5 min at 4°C, the cell pellet was discarded, and the TCID of the supernatant of each group was measured with reference to the method in (1) of Example 1. 50 .
[0089] The results are as Figure 2 and Figure 3 shown. In BHK-21 cells, after treatment with curcumin, the copy number and titer of DTMUV decreased significantly in a dose-dependent manner.
[0090] As Figure 4 and Figure 5 shown, the level of viral E protein decreased after treatment with curcumin. These results indicate that curcumin can inhibit the replication of TMUV in BHK-21 cells.
[0091] Example 2 Adsorption and post-adsorption experiments
[0092] To determine the stage at which the TMUV life cycle is specifically affected by curcumin, viral adsorption and post-adsorption detection were performed in BHK-21 cells.
[0093] BHK-21 cells were seeded in 12-well plates and incubated in a 37°C, 5% CO2 incubator. When the cell density reached 80-90%, the culture supernatant was discarded and the cells were washed twice with PBS.
[0094] Adsorption group: TMUV was incubated with 30 μM curcumin at 4°C for 1 h at an MOI of 0.1. After discarding the supernatant, the cells were washed twice with PBS, and then 1% DMEM was added and the cells were cultured in a 37°C, 5% CO2 incubator for 48 h.
[0095] Post-adsorption group: TMUV was pre-incubated at 4°C for 1 h at an MOI of 0.1. After discarding the supernatant, the cells were washed twice with PBS, and then 1% DMEM containing 30 μM curcumin was added and the cells were cultured in a 37°C, 5% CO2 incubator for 48 h.
[0096] Simultaneous group: TMUV was cultured with 30 μM curcumin at an MOI of 0.1 in a 37°C, 5% CO2 incubator for 48 h.
[0097] Infection group: TMUV AQ-19 was cultured at an MOI of 0.1 in a 37°C, 5% CO2 incubator for 48 h.
[0098] (1) RT-qPCR detection was the same as in (4) of Example 1.
[0099] (2) The procedure for indirect immunofluorescence assay was the same as in (5) of Example 1.
[0100] (3) The procedure for Western blot was the same as in (6) of Example 1.
[0101] (4) TCID 50 The measurement experimental procedure was the same as that in (7) of Example 1.
[0102] The results were as Figure 6 and Figure 7 shown. Compared with the TMUV-infected group, the virus copy numbers and titers in the simultaneous group, adsorption group, and post-adsorption group were all reduced to varying degrees. Compared with the TMUV group, the simultaneous group and post-adsorption group showed a strong antiviral effect, while the inhibitory effect of the adsorption group was weak.
[0103] As Figure 8 and Figure 9 shown. Compared with the TMUV group, the detection results of viral E protein in the three groups were similar to the above results.
[0104] Therefore, curcumin mainly inhibits TMUV infection by blocking the post-adsorption stage.
[0105] Example 3 Study on the Intervention Effect of Curcumin on TMUV-Infected Ducklings
[0106] (1) Grouping and Treatment of Experimental Animals
[0107] Forty ducklings were adaptively raised for 7 days and then the experiment was carried out. They were randomly divided into 4 groups, namely the blank control group, virus control group, low-concentration curcumin group, and high-concentration curcumin group, with 10 ducklings in each group. They were isolated and raised under the same environmental conditions, fed with full-price duckling feed, and allowed to eat and drink freely. The experiment started when the ducklings were 15 days old. The virus control group, low-concentration curcumin group, and high-concentration curcumin group were injected with 0.5 mL / duckling of virus solution into the leg muscle, and the blank control group was injected with 0.5 mL / duckling of physiological saline into the leg muscle.
[0108] From the inoculation day, the administration groups continuously orally administered curcumin (the dose of the low-concentration curcumin group was 200 mg curcumin / kg body weight, and the dose of the high-concentration curcumin group was 400 mg curcumin / kg body weight). The survival rates of ducklings in each group were observed and recorded daily during the experiment.
[0109] Fourteen days later, all ducklings were euthanized, the pathological changes of each organ tissue were observed and recorded, and partial tissues of the heart, liver, spleen, and brain were taken and stored in a -80 °C refrigerator for standby.
[0110] (2) Detection of Tissue Viral Load
[0111] RNA Extraction:
[0112] Step 1, Weigh 50 mg of tissue and add it to a sterile centrifuge tube containing grinding beads, and add 1 mL of Trizol;
[0113] Step 2: Add 200 μL of chloroform, mix well, let stand at room temperature for 5 min, centrifuge at 12,000 rpm at 4°C for 15 min, and take the supernatant.
[0114] Step 3: Add an equal volume of isopropanol, invert and mix well, let stand at 4°C for 10 min, and centrifuge at 12,000 rpm for 10 min.
[0115] Step 4: Add 1 mL of pre-cooled 75% ethanol, centrifuge at 12,000 rpm at 4°C for 5 min, discard the supernatant, and dry at room temperature for 5 min.
[0116] Step 5: Add 50 μL of RNase-free deionized water, dissolve, and store at -80°C.
[0117] The subsequent RT-qPCR operation is carried out according to step (4) in Example 1.
[0118] (3) Histopathological examination
[0119] The brain, liver, and spleen tissues fixed in 10% formalin solution are dehydrated in sequence, embedded in paraffin, cut into 5-μm-thick sections, and the sections are placed in xylene I for 20 min - xylene II for 20 min - absolute ethanol I for 5 min - absolute ethanol II for 5 min - 75% ethanol for 5 min in sequence, washed with tap water for 5 min, stained with hematoxylin solution for 3 min, washed with tap water for 5 min, differentiated with differentiating solution for 5 s, blued with tap water, rinsed with running water, the sections are dehydrated in gradient ethanol of 85% and 95% for 5 min each in sequence, stained with eosin staining solution for 5 min. Then the sections are placed in absolute ethanol I for 5 min - absolute ethanol II for 5 min - absolute ethanol III for 5 min - xylene I for 5 min - xylene II for 5 min in sequence, made transparent, and sealed with neutral gum, and the tissue lesions are observed under an optical microscope.
[0120] As Figure 10 shown, 15 days after artificial challenge with TMUV and treatment with drugs, compared with the control group, the survival rates of ducklings infected with low-concentration and high-concentration curcumin were significantly increased.
[0121] As Figure 11 shown, compared with the TMUV group, the virus copy numbers in different tissues of ducklings were significantly reduced after curcumin treatment.
[0122] As Figure 12 shown, there were no tissue lesions in the blank control group, the liver in the virus control group was yellowish-brown, the spleen was enlarged, and the meninges were severely congested and hemorrhaged. Compared with the virus control group, there were no obvious changes in the livers of the low-concentration curcumin group and the high-concentration curcumin group, the spleens were slightly enlarged, and the meninges were slightly hemorrhaged.
[0123] As Figure 13As shown, in the virus challenge group, vacuolar degeneration and necrosis of hepatocytes were visible; capillary congestion in the brain tissue; necrosis of splenocytes. After treatment with curcumin, there was a small amount of inflammatory cell infiltration in the liver of the low-concentration curcumin group, and no obvious pathological changes were observed in the others.
[0124] Therefore, curcumin intervention in TMUV-infected ducklings can alleviate the body damage caused by TMUV infection and reduce the virus-carrying amount in tissues and the mortality rate of ducklings.
[0125] As mentioned above, the above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes should be covered within the protection scope of the present invention.
Claims
1. The application of curcumin in any one of the following (1)-(5): (1) Preparing a product for inhibiting the replication of Tembusu virus in vitro cells; (2) Preparing a product for reducing the level of Tembusu virus E protein in vitro cells; (3) Preparing a product for anti-Tembusu virus in vitro; (4) Preparing a product for reducing the virus copy number in biological tissues after infection with Tembusu virus; (5) Preparing a product for increasing the survival rate of poultry infected with Tembusu virus; (6) Preparing a product for alleviating the body damage caused by infection with Tembusu virus; (7) Preparing a product for preventing or treating Tembusu virus.
2. The application according to claim 1, wherein (1), (2) or (3), the concentration of curcumin is 22.5 - 30 μM.
3. The application according to claim 1, characterized in that, (4), (5) or (6), the dose of curcumin is 200 - 400 mg of curcumin per kilogram of body weight.
4. The application according to claim 1, wherein (4), the biological tissue is at least one of avian liver tissue, avian spleen tissue, and avian brain tissue.
5. The application according to claim 1 or 4, characterized in that, (4) or (5), the poultry is at least one of duck, goose, chicken, and sparrow.
6. The application according to claim 1, characterized in that (6), the body damage is that at least one of avian liver tissue, avian spleen tissue, and avian brain tissue has lesions or pathological changes.
7. The application according to claim 1, wherein (7), the product is a drug, a feed additive or / and feed.