New application of compound Lanolin in resisting foot-and-mouth disease virus

Abstract

The invention belongs to the field of biology, and particularly relates to novel application of a compound Lanolin in resisting foot and mouth disease viruses. The invention accidentally discovers that the compound Lanolin can obviously inhibit the expression of foot and mouth disease virus RNA polymerase 3D protein, reduce the half histiocyte infection dose (TCID50) of the foot and mouth disease virus and obviously reduce the replication capacity of the foot and mouth disease virus, has the effect of antagonizing foot and mouth disease virus infection, can be used for preparing the anti-foot and mouth disease virus medicine, and can be used for preparing the anti-foot and mouth disease virus medicine. Good application prospects are realized.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a novel use of the compound Lanolin for combating foot-and-mouth disease virus. Background Technology

[0002] Foot-and-mouth disease (FMD) is an acute, highly virulent, and contagious animal disease that primarily infects cloven-hoofed animals such as cattle, pigs, sheep, and goats. In my country, it is classified as a Class A animal disease and falls under the category of key animal infectious diseases requiring prevention and control. The causative agent of this disease is the foot-and-mouth disease virus (FMDV), which belongs to the Picornaviridae family. Its structural characteristics include a lack of envelope and a single-stranded positive-sense RNA genome.

[0003] From a serotype perspective, FMDV has seven different serotypes, including O, A, C, Asia1, and SAT1, SAT2, and SAT3. Notably, each serotype has a large number of subtypes, and there is no cross-protection between different serotypes. This characteristic poses a significant challenge to disease control.

[0004] Currently, vaccination is the mainstream and effective means of controlling foot-and-mouth disease (FMD). However, FMDV possesses strong immunosuppressive capabilities, enabling it to rapidly breach the host animal's immune system barrier, thereby achieving efficient viral replication and spread. Therefore, relying solely on vaccines to control FMD is insufficient to completely address the viral threat. Consequently, simultaneously developing drugs to combat FMD virus infection is of paramount practical significance and necessity.

[0005] Lanolin is a natural lipid extracted from wool, with cholesterol, lanosterol, and fatty acid esters as its main components. It has wide applications in the medical and skincare fields, with core functions including moisturizing and locking in moisture, repairing the skin barrier, and promoting drug absorption. It is suitable for applications such as dry, cracked skin, eczema care, and post-operative wound protection. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention unexpectedly discovered that the compound Lanolin can significantly reduce the replication level of foot-and-mouth disease virus (FMDV), thus blocking FMDV infection and serving as an inhibitor of FMDV for anti-FMDV purposes. Specifically, it includes the following: In a first aspect, the present invention provides the use of the compound Lanolin in the preparation of antiviral drugs, wherein the structural formula of the compound Lanolin is shown in the following formula (Ⅰ):

[0007] Equation (Ⅰ).

[0008] Preferably, the virus is foot-and-mouth disease virus.

[0009] Preferably, the compound Lanolin inhibits the replication of foot-and-mouth disease virus.

[0010] Preferably, the compound Lanolin inhibits the expression of foot-and-mouth disease virus RNA polymerase 3D protein.

[0011] Preferably, the compound Lanolin reduces the TCID of foot-and-mouth disease virus. 50 .

[0012] Preferably, the compound Lanolin is formulated into a pharmaceutically acceptable dosage form by adding pharmaceutically acceptable excipients.

[0013] The beneficial effects of this invention are: This invention unexpectedly discovered that the compound Lanolin can significantly inhibit the expression of foot-and-mouth disease virus RNA polymerase 3D protein, thereby reducing the TCID of foot-and-mouth disease virus. 50 It can significantly reduce the replication ability of foot-and-mouth disease virus and has the effect of antagonizing foot-and-mouth disease virus infection. It can be used to prepare drugs against foot-and-mouth disease virus and has good application prospects. Attached Figure Description

[0014] Figure 1 The effect of compound Lanolin on the activity of BHK21 cells is shown in the figure.

[0015] Figure 2 Figure 1 shows the results of a real-time quantitative PCR (RT-qPCR) experiment in which the compound Lanolin inhibits the replication of foot-and-mouth disease virus.

[0016] Figure 3 The results of Western blot (WB) experiments showing the inhibition of foot-and-mouth disease virus 3D protein expression by the compound Lanolin.

[0017] Figure 4 The compound Lanolin reduces the median cytotoxic dose (TCID) of foot-and-mouth disease virus. 50 The experimental results are shown in the figure.

[0018] Figure 5 Figure 1. Indirect immunofluorescence (IFA) experiment results of the compound Lanolin inhibiting the expression of foot-and-mouth disease virus 3D protein. Detailed Implementation

[0019] To make the technical means, creative features, objectives, and effects of this invention easier to understand, the invention is further described below in conjunction with specific embodiments. However, the scope of protection of this invention is not limited to the embodiments described below.

[0020] DMEM medium, trypsin, fetal bovine serum, goat anti-mouse IgG antibody, Alexa Fluor 488, and DAPI nuclear staining solution were purchased from Thermofisher. β-Tubulin monoclonal antibody was purchased from Sigma-Aldrich; RNA extraction TRIzol and reverse transcription kit were purchased from Takara; quantitative real-time PCR kit was purchased from Nanjing Novizan; BHK21 cells were purchased from ATCC; FMDV strain (FMDV O strain O-BY-CHA-2010) and FMDV-3D protein monoclonal antibody were preserved by Professor Pei Jingjing's research group of the Animal Immunology and Metabolism Team, Research Center for Animal Disease Pathogen Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0021] The compound Lanolin described in the following examples has the following structural formula (Ⅰ), CAS No.: 8006-54-0, purchased from Selleckchem, Houston, TX, USA (S 5106).

[0022]

[0023] Equation (Ⅰ).

[0024] Unless otherwise specified, all cells and reagents described in the following examples are commercially available.

[0025] This invention uses BHK21 host cells as an example. The study has demonstrated that Lanolin can inhibit the replication of FMDV in BHK21 host cells, indicating that the Lanolin described in this invention can inhibit the replication of FMDV and can be used to prepare drugs or adjuvants against FMDV infection.

[0026] Example 1: Effect of compound Lanolin on the viability of BHK21 cells 1. Preparation of Lanolin Compound Formulation Take 10 μL of 10 mM Lanolin stock solution and add it to 190 μL of serum-free DMEM medium for serial dilution to obtain a 500 μM Lanolin working solution. Store the working solution in a sealed container at -80℃ for later use.

[0027] BHK21 cell suspension was seeded at 100 μL / well into 96-well cell culture plates and incubated at 37°C with 5% CO2 for 24 h. After cell attachment, the original culture medium in each well was discarded, and 5% DMEM medium containing different concentrations of Lanolin (final volume 100 μL / well) was added, corresponding to Lanolin working solution volumes of 0.5, 1, 2, 4, 6, 8, and 10 μL, resulting in a final Lanolin concentration gradient of 2.5, 5, 10, 20, 30, 40, and 50 μM. Each concentration gradient was set up in triplicate, and the plates were incubated at 37°C with 5% CO2 for another 24 h. After treatment, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated in the dark for 30 min. Subsequently, the absorbance (OD value) of each well was measured using a microplate reader at 450 nm.

[0028] Cell viability test results as follows Figure 1 As shown, when the Lanolin concentration is ≤30 μM, the viability of BHK21 cells is greater than 90%. This indicates that Lanolin has no significant toxicity to BHK21 cells at this concentration, meaning that the compound Lanolin described in this application has good safety.

[0029] Example 2: Inhibitory effect of compound Lanolin on foot-and-mouth disease virus replication 1. Viral infection and compound handling Cellular inoculation and the use of the compound Lanolin Cell plating: BHK21 cells were plated at a density of 4 × 10⁶ cells / cm². 5 The cells were seeded at a density of cells / well in 12-well cell plates and cultured in a cell culture incubator containing 5% CO2 at 37°C. Inoculation: When cell confluence reaches approximately 80%, inoculate with a 0.1 MOI dose of foot-and-mouth disease virus (FMDV O strain O-BY-CHA-2010). The dosage is calculated using the formula: PFU = number of cells × MOI = 0.7 × TCID. 50 Calculate the required viral volume. Dilute the required viral volume to 940 μL of 2% DMEM medium and inoculate into cells in 12-well plates. Incubate at 37°C in a 5% CO2 incubator.

[0030] Lanolin treatment: 60 μL of Lanolin working solution was added to the cells in the 12-well plates simultaneously with the addition of the virus solution, bringing the final concentration of Lanolin in the wells to 30 μM. The solution was then mixed with the culture medium. A solvent group (DMSO+FMDV group) was set up as a control group. The cells were incubated at 37℃ in a 5% CO2 incubator for 12 h and 24 h. Once significant cytopathic effects were observed in the cells, samples were collected for testing.

[0031] 2. Extract total RNA Discard the culture medium, wash once with pre-cooled PBS, add 1 mL of RNA extraction reagent (Trizol), mix well by pipetting, transfer to a 1.5 mL EP tube, and incubate at room temperature for 3-5 min. Add 200 μL of chloroform, vortex vigorously for 15 sec, and incubate at room temperature for 5 min. Centrifuge at 12,000 rpm (approximately 14,000 × g) at 4°C for 10 min. The sample will separate into three layers. Transfer the upper aqueous phase to a new EP tube, add an equal volume of isopropanol, and invert to mix 10-20 times. Centrifuge at 12,000 rpm (approximately 14,000 × g) at 4°C for 10 min, and discard the supernatant to obtain the RNA precipitate. Add 1 mL of 75% ethanol, vortex gently for 15 sec, and wash the RNA precipitate. Centrifuge at 12,000 rpm (approximately 14,000 × g) at 4°C for 5 min. The RNA precipitate will reach the bottom of the tube; discard the supernatant. Add 100-200 μL of RNA dissolving solution or RNase-free ddH2O to dissolve the RNA.

[0032] 3. Reverse transcription Take an equal amount of extracted RNA for reverse transcription, and perform the reverse transcription step according to PrimeScript™ RT Master Mix (Perfect Real Time) (Takara).

[0033] 4. RT-qPCR Prepare sterile, RNase-free PCR tubes and place them on an ice box. First, add 2 μL of 5X PrimeScriptRT Master Mix (Perfect Real Time), then add an appropriate amount of Total RNA, and finally add RNase-free ddH2O to bring the total volume of the reaction system to 10 μL.

[0034] Place the PCR tubes containing the prepared reaction solution into a real-time quantitative PCR instrument (or a regular PCR instrument), and set the reaction program: react at 37°C for 15 min, raise the reaction temperature to 85°C and maintain it for 5 sec, and after the reaction is complete, lower the temperature to 4°C.

[0035] Real-time quantitative PCR results are as follows Figure 2 As shown, compared to the solvent control group (DMSO+FMDV group), the compound Lanolin described in this application can significantly inhibit the replication of foot-and-mouth disease virus.

[0036] Example 3: Immunoblot verification of the inhibitory effect of compound Lanolin on foot-and-mouth disease virus replication. Repeat the experimental method in Example 2. When significant cytopathic effects appear, collect the cells, discard the supernatant, add 150 μL / well of NP40 (containing protease and phosphatase inhibitors), lyse on ice for 30 min, centrifuge at 13000 rpm for 10 min at 4 °C, and discard the precipitate. Add 50 μL of 4× protein loading buffer to the supernatant, vortex to mix, centrifuge at 8000 rpm for 5 min at 100 °C, perform SDS-PAGE (20 μL loading), incubate at 80 V for 30 min, then at 120 V for 60 min; transfer to a membrane, incubate at 230 mA for 2 h, block with 5% skim milk powder for 2 h, incubate overnight at 4 °C with RNA polymerase 3D protein antibody, wash 3 times for 10 min each time, incubate with secondary antibody for 1 h, wash 3 times for 10 min each time, and expose to sunlight.

[0037] The results are as follows Figure 3 As shown, compared with the solvent control group (DMSO+FMDV group), the compound Lanolin described in this application can significantly inhibit the expression level of foot-and-mouth disease virus RNA polymerase 3D protein.

[0038] Example 4: Compound Lanolin against Foot-and-Mouth Disease Virus TCID 50 Impact Repeat the experimental method in Example 2. When significant cytopathic effects appeared, collect the supernatant, centrifuge at 3000 rpm for 5 min, and then seed it into a 96-well cell plate. Incubate at 37 ℃ in a 5% CO2 incubator until the cell confluence reaches 80%-90%. Then, divide the cells into three groups for treatment, with three biological replicates for each group: Pure cell group (blank control group): The original culture medium in the culture plate was removed, and only 100 μL of 5% DMEM culture medium was added. No virus infection or drug treatment was performed. Solvent control group (DMSO+FMDV group): Remove the original culture medium from the culture plate, add 100 μL of 5% DMEM medium containing DMSO and foot-and-mouth disease virus (MOI=0.1), and incubate at 37℃ in a 5% CO2 incubator for 12 h and 24 h (to ensure that the final concentration of DMSO is consistent with that of the drug treatment group and to eliminate solvent interference). Drug treatment group (Lanolin+FMDV group): Remove the original culture medium from the culture plate, add 100 μL of a mixture of 30 μM Lanolin and foot-and-mouth disease virus in 5% DMEM medium (MOI=0.1) to each well, and incubate at 37℃ in a 5% CO2 incubator for 12h and 24h, and then collect the samples for testing. The results are as follows Figure 4 As shown, compared to the solvent control group (DMSO+FMDV group), the compound Lanolin described in this application significantly reduced the TCID of foot-and-mouth disease virus in BHK21 cells at both 12 h and 24 h. 50 This indicates that Lanolin can significantly inhibit the replication of foot-and-mouth disease virus.

[0039] Example 5: Indirect immunofluorescence (IFA) experiment on the effect of Lanolin on the expression level of foot-and-mouth disease virus 3D protein. BHK21 cells in logarithmic growth phase were adjusted to a suitable concentration using 5% DMEM complete medium and seeded into 96-well cell culture plates at a volume of 100 μL per well (ensuring uniform cell distribution). The 96-well plates were then incubated at 37°C for 12 h.

[0040] Pure cell group (blank control group): The original culture medium in the culture plate was removed, and only 100 μL of 5% DMEM culture medium was added. No virus infection or drug treatment was performed. Solvent control group (DMSO+FMDV group): Remove the original culture medium from the culture plate, add 100 μL of 5% DMEM culture medium containing DMSO and foot-and-mouth disease virus (MOI=0.1), and incubate at 37℃ in a 5% CO2 incubator for 12 h and 24 h (to ensure that the final concentration of DMSO is consistent with that of the drug treatment group and to eliminate solvent interference). Drug treatment group (Lanolin+FMDV group): Remove the original culture medium from the culture plate, add 100 μL of a mixture of 30 μM Lanolin and foot-and-mouth disease virus in 5% DMEM medium (MOI=0.1) to each well, and incubate at 37℃ in a 5% CO2 incubator for 12h and 24h, and then collect the samples for testing. Discard the culture supernatant from each well of the 96-well plate. Add 100 μL of 4% paraformaldehyde fixative to each well and incubate at room temperature for 30 min. Add 100 μL of 0.2% Triton X-100 permeation buffer to each well and incubate at room temperature for 10 min. Wash each well three times for 5 min each time. Discard the PBS wash buffer. Add 100 μL of 5% BSA blocking buffer to each well and incubate at 37 ℃ for 1 h. Discard the blocking buffer. Add 100 μL of primary antibody against foot-and-mouth disease virus RNA polymerase 3D protein diluted 1:1000 with 5% BSA to each well and incubate overnight at 4 ℃. After incubation, discard the primary antibody solution and wash each well three times with enzyme-free PBS buffer for 10 min each time. Add 100 μL of Alexa Fluor 488-labeled mouse anti-secondary antibody diluted 1:1000 with 5% BSA to each well. Incubate at 37 °C for 1 h in the dark. Wash each well three times for 10 min each time. Discard the PBS wash buffer. Add 100 μL of DAPI staining solution diluted 1:5000 with enzyme-free PBS to each well. Incubate at room temperature in the dark for 5 min. Wash each well three times with PBS buffer in the dark for 5 min each time. Then take pictures using an inverted fluorescence microscope.

[0041] The results are as follows Figure 5 As shown, compared with the solvent control group (DMSO+FMDV group), the compound Lanolin described in this application can significantly reduce the expression level of foot-and-mouth disease virus 3D protein in BHK21 cells, further demonstrating that the compound Lanolin can significantly inhibit the replication of foot-and-mouth disease virus.

[0042] In summary, the compound Lanolin described in this application can significantly inhibit the replication of foot-and-mouth disease virus. This compound can be used as a candidate drug or in combination with other inhibitors for the treatment of foot-and-mouth disease virus.

[0043] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Therefore, all equivalent changes or modifications made in accordance with the structure, features and principles of the present invention should be included in the scope of the present invention.

Claims

1. Use of a compound Lanolin in the preparation of an antiviral drug, the structural formula of the compound Lanolin is as follows formula (I): Formula (I).

2. Use according to claim 1, wherein The virus is foot-and-mouth disease virus.

3. Use according to claim 2, wherein the compound is ###0002### The compound Lanolin inhibits the replication of foot-and-mouth disease virus.

4. The use according to claim 3, wherein the compound is ###0002### The compound Lanolin inhibits the expression of foot-and-mouth disease virus RNA polymerase 3D protein.

5. The use according to claim 3, wherein the compound is ###0002### The compound Lanolin reduces the TCID of foot-and-mouth disease virus 50 .

6. Use according to any one of claims 1 to 5, wherein the compound is ###0002### The compound Lanolin is added to pharmaceutically acceptable adjuvant to prepare pharmaceutically acceptable dosage form.

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