Use of sulforaphane in the preparation of an ant-enterovirus drug

Abstract

The application discloses application of sulforaphane in preparation of an antienterovirus drug and belongs to the technical field of medicines, wherein the sulforaphane can inhibit the expression of viral proteins, inhibit viral genome replication and inhibit the production of virus particles to achieve the purpose of resisting enteroviruses; specifically, the sulforaphane can inhibit the replication of enteroviruses by up-regulating the content of p53 proteins. In addition, the sulforaphane can also be used for reducing the cell morphological damage caused by enteroviruses.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically the application of sulforaphane in the preparation of anti-enterovirus drugs. Background Technology

[0002] Sulforaphane is mainly derived from cruciferous vegetables such as cauliflower, cabbage, and Chinese cabbage. It is an isothiocyanate obtained by the hydrolysis of glucosinolates by myrosinase enzymes within the plant. Glucosinolates are abundant in cruciferous vegetables such as broccoli, kale, and northern round red radishes. Studies have shown that consuming cruciferous vegetables can reduce the risk of breast cancer, lung cancer, and prostate cancer. In anti-tumor research, sulforaphane has been found to induce cell cycle arrest in the G2 / M or G1 / S phases, suggesting that sulforaphane can inhibit tumor cell growth through cell cycle arrest. Sulforaphane can also induce apoptosis in host cells, inhibiting cancer cell proliferation through apoptosis. Sulforaphane can also exert antioxidant activity by upregulating transcription factors to regulate oxidative stress processes; and it can inhibit viral replication by blocking dengue virus protease activity through NRF-2-HO-1. In addition, studies have found that sulforaphane has a variety of pharmacological effects, including antiviral effects; it has a good inhibitory effect on dengue virus, SARS-CoV-2, human immunodeficiency virus (HIV), EB virus, hepatitis C virus, etc., but its inhibitory effect on enteroviruses is still unknown.

[0003] Enterovirus EVD68, belonging to the genus Enterovirus in the family Picornaviridae, replicates rapidly and primarily causes respiratory symptoms. Patients are mostly newborns and infants. Mild symptoms are similar to those caused by influenza, such as fatigue and cough, while severe cases include acute flaccid myelitis (AFM). Currently, there are no targeted drugs or vaccines, making research into antiviral drugs for EVD68 particularly important. Summary of the Invention

[0004] The purpose of this invention is to provide an application of sulforaphane in the preparation of anti-enterovirus drugs, so as to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0006] Application of sulforaphane in the preparation of anti-enterovirus drugs.

[0007] Furthermore, the sulforaphane achieves its anti-enterovirus effect by inhibiting the expression of viral proteins, inhibiting viral genome replication, and inhibiting the production of viral particles.

[0008] Furthermore, the sulforaphane is used to reduce cell morphological damage caused by enteroviruses.

[0009] Furthermore, the sulforaphane inhibits the replication of enteroviruses by upregulating the content of p53 protein.

[0010] Furthermore, the enterovirus is enterovirus EVD68.

[0011] Furthermore, the anti-enterovirus drug is in powder or solution form.

[0012] Furthermore, the anti-enterovirus drug is a solution-based drug that includes at least sulforaphane and saline solution.

[0013] Furthermore, the concentration of sulforaphane in the anti-enterovirus drug is 2-6 μM.

[0014] This invention provides the application of sulforaphane in the preparation of anti-enterovirus drugs. Sulforaphane achieves its anti-enterovirus effect by inhibiting viral protein expression, viral genome replication, and viral particle production. Specifically, sulforaphane inhibits enterovirus replication by upregulating the content of p53 protein; that is, when the cell does not contain the p53 gene, sulforaphane cannot exert its antiviral effect. In addition, sulforaphane can also be used to alleviate cellular morphological damage caused by enteroviruses. Attached Figure Description

[0015] Figure 1 Figure showing the effect of different concentrations of sulforaphane on cell viability;

[0016] Figure 2 The figure shows the effect of sulforaphane on viral protein expression.

[0017] Figure 3 Figure showing the effect of sulforaphane on viral genome levels;

[0018] Figure 4 The graph shows the effect of sulforaphane on the number of virus particles.

[0019] Figure 5 Figure showing the experimental results of different concentrations of sulforaphane protecting host cells from damage;

[0020] Figure 6 The graph shows the effect of sulforaphane on the expression of different proteins.

[0021] Figure 7 Figure showing the experimental results of P53 inhibiting viral replication;

[0022] Figure 8 Figure showing experimental results to explore the mechanism by which sulforaphane inhibits viral replication and amplification. Detailed Implementation

[0023] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0024] In one embodiment of the present invention, an application of sulforaphane in the preparation of anti-enterovirus drugs is provided; specifically, sulforaphane achieves the purpose of anti-enterovirus by inhibiting the expression of viral proteins, inhibiting viral genome replication, and inhibiting the production of viral particles.

[0025] In a preferred embodiment of the present invention, sulforaphane is used to reduce cell morphological damage caused by enteroviruses.

[0026] In a preferred embodiment of the present invention, sulforaphane inhibits the replication of enteroviruses by upregulating the content of p53 protein; when the cell does not contain the p53 gene, sulforaphane cannot exert its antiviral effect.

[0027] In a preferred embodiment of the present invention, the enterovirus is enterovirus EVD68. It should be noted that this embodiment of the present invention uses enterovirus EVD68 as only one example for research; similarly, sulforaphane can also inhibit other types of enteroviruses.

[0028] In a preferred embodiment of the present invention, the anti-enterovirus drug is in powder or solution form, but is not limited thereto; that is, in addition to sulforaphane, the anti-enterovirus drug mentioned above may also include a pharmaceutically acceptable carrier.

[0029] In a preferred embodiment of the present invention, the anti-enterovirus drug is a drug in solution form, which includes at least sulforaphane and physiological saline.

[0030] In a preferred embodiment of the present invention, the concentration of sulforaphane in the anti-enterovirus drug is 2-6 μM.

[0031] The following embodiments are some specific implementation examples of the present invention in practical applications, but are not limited thereto.

[0032] Example 1

[0033] This example is for determining the safe dosage of sulforaphane, and the specific method is as follows:

[0034] 1. Preparation of sulforaphane:

[0035] Sulforaphane (Lot#113975) was purchased from MedChem Express. It was dissolved and diluted with PBS buffer, and the concentration used was 0-20 μM. It was prepared as needed.

[0036] 2. CCK8 assay for cell proliferation

[0037] Appropriate amounts of host cells (RD) were seeded into 96-well plates, with four replicates per concentration. A control group (no drug, no cells) and a control group were also included. When the cell density reached approximately 80%, the diluted sulforaphane drug was added, and the plates were incubated. After 24 hours, the supernatant was discarded, and the cells were washed with 100 μL of pure DMEM. 100 μL of the prepared CCK8 mixture (90 μL of 10% DMEM + 10 μL of CCK8) was added to each well, and the plates were incubated. After 24 hours of culture, the liquid in the 96-well plates gradually turned orange. The absorbance of the solution was measured using a microplate reader at 450 nm monochromatic light. Cell viability was calculated based on the absorbance.

[0038] The CCK8 assay revealed that sulforaphane at concentrations below 6 μM (dissolved in PBS) had no significant inhibitory effect on RD in host cells (e.g., Figure 1 As shown in the figure, this also indicates that the antiviral effect of sulforaphane is not due to its inhibition of cell proliferation.

[0039] Example 1

[0040] This embodiment aims to verify the mechanism of sulforaphane against enteroviruses. The specific method is as follows:

[0041] 1. Western Blot

[0042] 1.1. Preparation of SDS-PAGE protein gel

[0043] 1.1.1 Select an appropriate gel concentration for the formulation based on the molecular weight of the protein.

[0044] 1.1.2 In the following order, add pure water, 30% acrylamide, pH 8.8 solution, SDS solution, 10% APS solution, and TEMED. After mixing thoroughly, add the mixture to the gap between the clamped glass plates. Add an appropriate amount of isopropanol to the liquid surface, let stand for 30 minutes, pour out the isopropanol, and use filter paper to absorb the liquid.

[0045] 1.1.3 Prepare the reagents for the lower gel in the same order as the upper gel (replace the pH 8.8 solution with a pH 6.8 solution). After adding the upper gel liquid, insert the comb, let it stand for 30 minutes, collect the gel plate, store it in pure water, and then place it in a refrigerator at 4°C.

[0046] 1.2. Preparation of protein samples

[0047] Cell collection: Discard the cell supernatant, add an appropriate amount of 0.25% EDTA trypsin to digest the cells. When the cells become round and detach from the bottom of the culture dish, add 1 mL of culture medium (containing 10% fetal bovine serum) to stop the digestion. Collect the trypsin, culture medium, and cells into a 1.5 mL EP tube and centrifuge at 12000 rpm for 2 min 30 s. Discard the supernatant, add 200 μL of PBS to wash the cells, centrifuge again under the above conditions, and discard the supernatant to obtain the cell sample.

[0048] Depending on the cell quantity, add an appropriate amount of lysis buffer (60 μL–100 μL), mix the cell clusters thoroughly, and place the sample on ice for lysis for 30 min. Add 1 / 3 volume of 4× Loading buffer, vortex to mix, and heat on a 100°C heating module for 30 min. During this period, open the cap of the EP tube once to prevent the cap from being blown open by the gas flow and causing sample loss. After 30 min, remove the sample, centrifuge at 12000 rpm for 5 min, and collect the sample for later use.

[0049] 1.3. Gel electrophoresis

[0050] 1.3.1 Set up the pre-prepared SDS-PAGE gel, fill the center of the gel plate with 1×Running, and add an appropriate amount of Running Buffer to the electrophoresis tank.

[0051] 1.3.2 Take an appropriate amount of sample and transfer it to the wells of a pre-prepared 12% protein gel for gel electrophoresis.

[0052] 1.3.3 After electrophoresis, a PVDF membrane is used for membrane transfer.

[0053] 1.3.4 After the transfer is complete, remove the PVDF membrane and immediately place it in the blocking solution for 1 hour at room temperature.

[0054] 1.3.5 After the sealing is completed, rinse with 1×TBST solution, and then cut the membrane.

[0055] 1.3.6 Place the membrane in the primary antibody solution and incubate overnight at 4°C. The next day, recover the primary antibody and wash three times with 1×TBST solution for 5 min each time.

[0056] 1.3.7 Select a suitable HRP secondary antibody based on antibody characteristics and instructions. Incubate on a shaker at room temperature for 2 hours, then discard the secondary antibody. Add TBST buffer to the antibody cassette and wash three times on a shaker for 10 minutes each time.

[0057] 1.3.8 After cleaning, mix ECL color development solutions A and B in a 1:1 ratio, develop the color and record the data.

[0058] 2. Real-time quantitative PCR

[0059] 2.1. RNA extraction:

[0060] 2.1.1 Remove the supernatant, add 1 mL of PBS to wash the cell surface, add 500 μL of lysis buffer, let stand for 2 min, blow well and transfer to a 1.5 mL EP tube.

[0061] 2.1.2 Add an equal volume of binding solution to the EP tube and invert it 3-5 times.

[0062] 2.1.3 Transfer the above liquid to a purification column, centrifuge at 12000 rpm for 30 s, and discard the liquid in the collection tube.

[0063] 2.1.4 Add 600 μL of washing buffer I, centrifuge at 12000 rpm for 30 s, and discard the liquid in the collection tube.

[0064] 2.1.5 Add 600 μL of washing solution II, centrifuge at 12000 rpm for 30 s, and discard the liquid in the collection tube.

[0065] 2.1.6 Repeat step 2.1.5 once.

[0066] 2.1.7 Centrifuge at 14000-16000 rpm for 2 min to remove residual liquid.

[0067] 2.1.8 Place the purification column in an elution tube, add 30-50 μL of elution buffer, incubate at room temperature for 2-3 minutes, and centrifuge at the highest speed for 30 seconds to obtain purified RNA.

[0068] 2.2. cDNA Synthesis: cDNA synthesis was performed using a Monad kit.

[0069] 2.2.1 Reverse transcription system

[0070] Template: 50 ng - 1 μg;

[0071] MonScriptTM5*RTIII All-in-One Mix: 4μL;

[0072] MonScript™ dsDNase: 1 μL;

[0073] Nuclease-Free Water:To20μL;

[0074] 2.2.2 Reverse transcription conditions

[0075] 37℃, 2 min;

[0076] 55℃, 15min;

[0077] 85℃, 5 min;

[0078] After removal from the container, store at -20℃.

[0079] 2.3. Real-time PCR Quantitative Analysis

[0080] The target gene was amplified according to the Beijing Monad RT-qPCR kit.

[0081] 2.3.1 Target gene amplification system

[0082] Each sample was set up with 3 replicates, and each primer pair was used as a negative control. No template was added.

[0083] MonAmpTM ChemoHS qPCR Mix: 10μL;

[0084] Forward primer (10 μM): 0.5 μL;

[0085] Reverse primer (10 μM): 0.5 μL;

[0086] Low ROX Dye(100x)or High ROX Dye(100x):0.2μL;

[0087] Nuclease-Free Water:To20μL;

[0088] cDNA template: 10-200 ng.

[0089] 2.3.2 Target gene amplification conditions (as shown in Table 1)

[0090] Table 1

[0091]

[0092] 2.3.3 Use the ΔΔCt method to calculate data changes.

[0093] 3. Virus amplification and titer determination

[0094] Viral titer assay: A suitable amount of RD cells were seeded into 96-well plates. The next day, when the cell density reached 70%–80%, the cell surface was washed with PBS. The virus was then serially diluted to (10⁻⁶)⁻¹. -1 ~10 -8 Afterwards, the cells were added to a 96-well plate, with four parallel replicates for each concentration. Normal cells were used as the control group. The cell state was observed under a microscope. The cell CPE phenomenon was observed and recorded after 72 hours. The experimental results were substituted into the Reed-Muench formula to calculate the viral titer.

[0095] 4. Cell morphology

[0096] Following the cell culture method in Example 1, cells were seeded in six-well plates. Once the cell density reached 70%, the next step was performed. The cell surface was washed with an appropriate amount of sterile PBS, and after 2 hours of viral infection, the culture medium or drug was replaced, and the cells were placed in a cell culture incubator for further culture. When relevant experimental phenomena occurred, the cells were observed under a microscope and photographed for recording.

[0097] 5. Plasmid transfection

[0098] 5.1 Seed cells in culture dishes and conduct experiments when the density is 60-70%.

[0099] 5.2 Transfect with plasmid, prepare two 1.5mL tubes:

[0100] Tube A: DNA mixture. Tube B: PEI dilution solution. Mix gently and let stand for five minutes.

[0101] 5.3 Add the PEI dilution to the DNA mixture and incubate at room temperature for 20 min.

[0102] 5.4 Remove the cells, discard the supernatant, and slowly add the mixture into the culture dish.

[0103] 5.5 After 4-6 hours, replace with a solution containing 10% FBS, and after 24 hours of incubation, perform the corresponding experimental procedures.

[0104] 6. Experimental Results

[0105] 6.1 Determining the antiviral activity of sulforaphane

[0106] 6.1.1 Sulforaphane inhibits viral protein expression

[0107] With increasing sulforaphane dosage, Western blot analysis showed that the expression of VP1, a protein of EV D68 virus, gradually decreased 24 hours after viral infection (e.g., ...). Figure 2 (as shown in the image); therefore, sulforaphane can inhibit viral protein expression.

[0108] 6.1.2 Sulforaphane inhibits viral genome replication

[0109] Treatment of virus-infected cells with 4 μM sulforaphane resulted in a decrease in EVD68 viral genome levels using real-time PCR 24 h after infection (e.g., Figure 3 (As shown); therefore, sulforaphane can inhibit viral genome replication.

[0110] 6.1.3 Sulforaphane inhibits virus particle production

[0111] Treatment of virus-infected cells with 4 μM sulforaphane resulted in a decrease in EVD68 viral particle count as observed by Real-time PCR 24 h after infection (e.g., Figure 4 (As shown); therefore, sulforaphane can inhibit the generation of virus particles.

[0112] 6.1.3 Sulforaphane protects host cells from damage.

[0113] With increasing dose (concentration) of sulforaphane, cell morphology showed that sulforaphane inhibited virus-induced cell death (e.g., 24 hours after viral infection). Figure 5 (as shown); therefore, sulforaphane can protect host cells from damage.

[0114] 6.2 Determining the antiviral mechanism of sulforaphane

[0115] 6.2.1 Sulforaphane upregulates P53 protein levels

[0116] Sulforaphane time-dependently upregulates p53 protein levels (e.g. Figure 6 (as shown), but had no significant effect on the expression of BAX, BC1-2, LC3, Beclin1, CDK1, and CyclinB1 (as shown). Figure 6 (As shown).

[0117] 6.2.2 P53 inhibits viral replication

[0118] In H1299 cells lacking p53, after transfection with a control vector and a p53 plasmid, morphological changes were analyzed 24 hours after EVD68 virus infection. The results showed that viral infection induced cell death, but p53 protein expression inhibited cell death (e.g., ...). Figure 7 (As shown in A). In P53-deficient H1299 cells, after transfection with control vector and P53 plasmid, viral protein expression was analyzed 24 h after viral infection. The presence of P53 was found to inhibit viral protein expression (e.g., ...). Figure 7 (As shown in B). In P53-deficient H1299 cells, transfection with the control vector and P53 plasmid significantly reduced the number of viral particles (as shown in B). Figure 7 (As shown in C); therefore, P53 protein expression can inhibit viral replication and amplification.

[0119] 6.2.3 Sulforaphane inhibits viral replication and amplification through the P53 protein.

[0120] In p53-deficient cells, sulforaphane did not significantly inhibit the expression of the viral protein VP1, but after re-expression of p53 protein, sulforaphane significantly reduced the expression of the viral protein VP1 (e.g., Figure 8As shown in A); Real-time PCR experiments showed that sulforaphane did not inhibit viral genome replication in P53-deficient cells, but after re-expression of P53 protein, sulforaphane inhibited enterovirus replication (as shown in A). Figure 8 As shown in B). Regarding virus particle production, sulforaphane also inhibits virus particle production when p53 protein is expressed (e.g., ...). Figure 8 (As shown in C); therefore, sulforaphane inhibits enterovirus replication by upregulating the P53 protein.

[0121] In summary, 2-6 μM sulforaphane has the effect of inhibiting the replication and amplification of enterovirus EVD68. Based on the mechanism by which sulforaphane inhibits the replication and amplification of enteroviruses, it is not difficult to infer that sulforaphane also has an inhibitory effect on other types of enteroviruses.

[0122] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. The application of sulforaphane in the preparation of anti-enterovirus drugs, characterized in that, The sulforaphane inhibits the replication of enteroviruses by upregulating the content of p53 protein; the enterovirus is enterovirus EVD68.

2. The application of sulforaphane according to claim 1 in the preparation of anti-enterovirus drugs, characterized in that, The sulforaphane achieves its anti-enterovirus effect by inhibiting the expression of viral proteins, inhibiting viral genome replication, and inhibiting the production of viral particles.

3. The application of sulforaphane according to claim 1 in the preparation of anti-enterovirus drugs, characterized in that, The sulforaphane is used to reduce cellular morphological damage caused by enteroviruses.

4. The use of sulforaphane according to any one of claims 1-3 in the preparation of anti-enterovirus drugs, characterized in that, The anti-enterovirus drug is in powder or solution form.

5. The application of sulforaphane according to claim 4 in the preparation of anti-enterovirus drugs, characterized in that, The anti-enterovirus drug is a solution-based drug that includes at least sulforaphane and saline solution.

6. The application of sulforaphane according to claim 5 in the preparation of anti-enterovirus drugs, characterized in that, The concentration of sulforaphane in the anti-enterovirus drug is 2-6 μM.

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