Application of methyl protocatechuate in preparation of product for treating and / or preventing respiratory syncytial virus infection

Abstract

The invention discloses an application of methyl protocatechuate in preparation of a product for treating and / or preventing respiratory syncytial virus infection. The methyl protocatechuate is a natural product, is good in permeability, small in toxic and side effects and simple in structure, can effectively inhibit replication and infection of the respiratory syncytial virus and reduce inflammatory response of a body, can target FTO and inhibit FTO protein expression, and provides a new direction for treating infection of the respiratory syncytial virus.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology, and particularly relates to application of protocatechuic acid methyl ester in preparation of a product for treating and / or preventing respiratory syncytial virus infection. BACKGROUND

[0002] Respiratory syncytial virus (RSV) is an enveloped single-stranded negative-sense RNA virus belonging to the family Paramyxoviridae and the subfamily Pneumovirinae, and is divided into two subtypes A and B. At present, RSV is one of the most common causes of severe lower respiratory tract infection in infants and young children worldwide, and is also an important pathogen of lower respiratory tract infection in the elderly and immunocompromised population, causing more deaths than influenza each year, and bringing a huge disease burden to patients.

[0003] In 2019, an estimated 4.1 million children worldwide were hospitalized due to RSV infection, of which about 105,000 children under the age of 5 died, accounting for 2.0% of all causes of death. Studies have shown that the first infection with RSV in infancy may trigger or exacerbate adult asthma, although antibodies exist after infection with RSV, due to the short duration, half of the infants will have a second infection during this period. RSV is one of the most common pathogens of acute respiratory tract infection in adults, and the proportion of people over 65 years of age hospitalized due to RSV infection is about 10.6%. However, there is currently a lack of effective and specific RSV treatment drugs in clinical practice. Therefore, there is an urgent need to explore or develop efficient and safe drugs for treating RSV infection. SUMMARY

[0004] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application proposes the use of protocatechuic acid methyl ester in the preparation of a product for treating and / or preventing respiratory syncytial virus infection.

[0005] The present application also proposes a product.

[0006] According to a first aspect of the present application, the use of protocatechuic acid methyl ester in the preparation of a product for treating and / or preventing respiratory syncytial virus infection is proposed.

[0007] According to some embodiments of the present application, the product has at least one of the following functions:

[0008] A1) inhibiting the replication of respiratory syncytial virus;

[0009] A2) reducing the inflammatory response;

[0010] A3) inhibiting the glycolysis caused by respiratory syncytial virus infection of Beas2b cells;

[0011] A4) Inhibits death induced by respiratory syncytial virus infection of Beas2b cells.

[0012] In some embodiments of the present invention, the reduction of inflammatory response includes reducing the expression levels of inflammatory factors.

[0013] In some embodiments of the present invention, the inflammatory factors include IL-6, IL1α, and IL1β.

[0014] In some embodiments of the present invention, the product for treating and / or preventing respiratory syncytial virus is a product that can inhibit FTO protein expression.

[0015] In some embodiments of the present invention, the content of protocatechuic acid methyl ester in the product is a therapeutically effective amount.

[0016] In some embodiments of the present invention, the content of protocatechuic acid methyl ester in the product can be 0.1%-100%, for example 0.1%, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 100%.

[0017] In some embodiments of the present invention, the product is at least one of pharmaceuticals, health products, and functional foods.

[0018] In some embodiments of the present invention, the pharmaceutical product further includes a pharmaceutically acceptable carrier.

[0019] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of diluents, excipients, fillers, binders, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, sweeteners, and flavorings.

[0020] In some embodiments of the invention, the excipient comprises water.

[0021] In some embodiments of the present invention, the filler includes at least one of starch and sucrose.

[0022] In some embodiments of the present invention, the adhesive includes at least one of cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone.

[0023] In some embodiments of the present invention, the wetting agent includes glycerin.

[0024] In some embodiments of the present invention, the disintegrant includes at least one of agar, calcium carbonate, and sodium bicarbonate.

[0025] In some embodiments of the present invention, the absorption enhancer includes a quaternary ammonium compound.

[0026] In some embodiments of the present invention, the surfactant includes hexadecyl alcohol.

[0027] In some embodiments of the present invention, the adsorbent carrier includes at least one of kaolin and soap clay.

[0028] In some embodiments of the present invention, the lubricant includes at least one of talc, calcium stearate, magnesium stearate, and polyethylene glycol.

[0029] In some embodiments of the present invention, the administration methods of the drug include, but are not limited to, oral administration, enteral administration, subcutaneous injection, intramuscular injection, intravenous injection, nasal administration, transdermal administration, subconjunctival administration, intraocular administration, orbital administration, retroocular administration, retinal administration, choroidal administration, intrathecal injection, etc.

[0030] In some embodiments of the present invention, the dosage forms of the medicine include, but are not limited to, tablets, capsules, pills, injections, inhalers, lozenges, suppositories, emulsions, microemulsions, submicroemulsions, nanoparticles, gels, powders, suspensions, creams, gels, sprays, etc. The various dosage forms of the medicine can be prepared according to conventional pharmaceutical manufacturing methods.

[0031] In some embodiments of the present invention, the protocatechuic acid methyl ester is the sole active ingredient of the product, or it may be used as an active ingredient in combination with other drugs for treating respiratory syncytial virus.

[0032] In some embodiments of the present invention, the product is intended for use on mammals.

[0033] In some embodiments of the present invention, the mammal is at least one of a human, a mouse, a rabbit, and a dog.

[0034] According to a second aspect of the invention, the use of methyl protocatechuate in any one of B1) to B2) is proposed:

[0035] B1) Reduce the expression level of FTO protein;

[0036] B2) Prepare products that reduce the expression level of FTO protein.

[0037] According to a third aspect of the invention, a product for treating and / or preventing respiratory syncytial virus infection is provided, said product comprising methyl protocatechuate.

[0038] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the protocatechuic acid methyl ester of the present invention is a natural product with good permeability, low toxicity and side effects, and simple structure. It can effectively inhibit respiratory syncytial virus replication and infection, reduce the body's inflammatory response, and can also target FTO and inhibit FTO protein expression, providing a new direction for the treatment of respiratory syncytial virus infection. Attached Figure Description

[0039] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0040] Figure 1 This is a diagram showing the results of screening natural product drugs targeting FTO expression using an existing small molecule drug library through virtual screening in an embodiment of the present invention.

[0041] Figure 2 This is a graph showing the results of treating Beas2b cells with methyl protocatechuate at a concentration gradient in an embodiment of the present invention, and detecting cell viability and screening for the optimal concentration of the drug to inhibit the virus using the CCK8 assay. In the graph, A represents the results of detecting cell viability and screening for the optimal concentration of the drug to inhibit the virus, and B represents the results of detecting cell viability and screening for the optimal concentration of the drug to inhibit the virus.

[0042] Figure 3 The figures shown are from an embodiment of the present invention, illustrating the detection of respiratory syncytial virus (RSV) viral load and FTO expression levels by QPCR assay after treating Beas2b cells with a TCID500 titer for 24 hours and then with methyl protocatechuate 3 μg / mL for 24 hours. In the figures, A represents the RSV RNA expression level, B represents the FTO expression level, C represents the IL-6 expression level, D represents the IL1α expression level, and E represents the IL1β expression level. "*" indicates p < 0.05, "**" indicates p < 0.01, and "***" indicates p < 0.001.

[0043] Figure 4 This is a graph showing the detection results of FTO protein expression level by WB in an embodiment of the present invention;

[0044] Figure 5 The image shows the detection results of QPCR detection of respiratory syncytial virus RNA expression in mouse lungs in this embodiment of the invention, where "*" indicates p<0.05, "**" indicates p<0.01, "***" indicates p<0.001, and "****" indicates p<0.0001.

[0045] Figure 6 This is a graph showing the detection results of FTO protein expression level by WB in an embodiment of the present invention;

[0046] Figure 7 The image shows the results of HE staining to detect respiratory syncytial virus infection in the lungs of mice. Detailed Implementation

[0047] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0048] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0049] The reagents and materials used in this invention are as follows:

[0050] 1. Cell line

[0051] Normal human bronchial epithelial cells (Beas2b) were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0052] RSV A2 strain was purchased from the American Cell Culture Collection Center (ATCC). Cells were cultured in RPMI 1640 DMEM medium containing 10% fetal bovine serum, passaged by digestion with 0.25% trypsin-EDTA, and cultured at 37°C in a 5% CO2 incubator.

[0053] 2. Cell culture materials

[0054] DMEM culture medium, 0.01 mol / L PBS (1×), and trypsin-EDTA solution were all purchased from Solarbio Science & Technology Co., Ltd. (Beijing), China, and stored at 4°C. Fetal bovine serum was purchased from Zhejiang Tianhang Biotechnology Co., Ltd., China, and stored at 20°C.

[0055] 3. Medicines

[0056] Methyl protocatechuate (PACAME) was purchased from Medchemexpress, with a molecular weight of 168.15 and a purity of 98.93%, in 50 mg / vial. After dissolving in DMSO solution, the stock solution concentration was 10 mg / mL, and it was stored at -20°C.

[0057] 4. Main reagents and antibodies

[0058] The CCK8 kit was purchased from Shanghai Haoyuan Biotechnology Co., Ltd., China; DMSO was domestically produced analytical grade; Giemsa staining solution was purchased from Beijing Bio-Rad Biotechnology Co., Ltd.; the GSH kit was purchased from Abbkine; the protein chemiluminescence solution was purchased from Shanghai Yamei Biomedical Technology Co., Ltd.; the FTO antibody was purchased from Abcam, USA; and the β-actin antibody was purchased from Wuhan Sanying Biotechnology Co., Ltd., China.

[0059] Example 1: Application of protocatechuic acid methyl ester in the preparation of medicaments for the prevention and / or treatment of respiratory syncytial virus infection.

[0060] 1. Drug screening

[0061] Using Auto DCOK software, 2541 natural products were docked with FTO, and the binding free energy of the conjugates was calculated using the GROMACS tool (g_mmpbsa) based on the MM-PBSA method. This included electrostatic energy and van der Waals energy, reflecting the interaction energy between the drug and FTO. Through extensive screening, methyl protocatechuate was identified as a potential treatment for respiratory syncytial virus (RSV).

[0062] Furthermore, based on this, a 10 ns molecular dynamics simulation was performed on the docking complex of protocatechuic acid methyl ester and FTO. In addition, the average structural RMSD of the main chain atoms on each simulated trajectory was calculated to study the positional stability of the conformation, such as... Figure 1 As shown.

[0063] The chemical structure of protocatechuic acid methyl ester is shown in formula (1) below:

[0064]

[0065] 2. CCK8 assay for cell viability of Beas2b cells treated with methyl protocatechuate and determination of the optimal concentration of methyl protocatechuate for inhibiting respiratory syncytial virus infection.

[0066] (1) Detection of cell viability of Beas2b cells after treatment with methyl protocatechuate by CCK8 assay

[0067] Besa2b cells were loaded at 5 × 10 3Cells were seeded at a density of cells / well in 96-well plates containing DMEM medium. The cells were incubated overnight at 37°C, 5% CO2, and 95% humidity. Subsequently, cells were treated for 24 hours with different concentrations of protocatechuic acid methyl ester (1, 2, 3, 4, and 5 μg / mL) and 0.05% DMSO (control group), with DMEM medium serving as a blank control. 10 μL of CCK8 reagent was added to each well, and the cells were incubated for 2 hours. Optical density (OD) was measured at 450 nm using a microplate reader (FLUOstar Omega-ACU, USA). Cell viability of lung epithelial cells treated with protocatechuic acid methyl ester was determined using the following formula: Cell viability (%) = (OD treatment - OD blank) / (OD control - OD blank) × 100.

[0068] (2) Determination of the optimal concentration of protocatechuic acid methyl ester for inhibiting respiratory syncytial virus infection using the CCK8 assay.

[0069] RSV infection group: Besa2b cells were infected at a rate of 5 × 10⁶ 3 Cells were seeded at a density of 1 cell / well in 96-well plates containing DMEM medium. When the cell confluence reached 60%–70%, the original medium was discarded using a pipette, and the cells were washed 2–3 times with pre-cooled 2–3 mL of PBS. Subsequently, 1 mL of RSV suspension (concentration 1×10⁻⁶) was taken from the liquid nitrogen tank. -8.130.1 ml of the solution was added to the cell culture flask. A normal control group was also established, differing from the RSV infection group only in that 1 ml of cell maintenance medium was added instead of 1 ml of RSV suspension to the control group. The culture flasks were placed in a 37°C, 5% CO2 incubator for virus adsorption. Every 15 minutes, the cells were gently agitated several times using a "cross" motion to ensure even virus adsorption. After 90 minutes, 4 ml of cell maintenance medium was added to each flask, and the cells were cultured overnight in a 37°C, 5% CO2, and 95% humidity incubator. Different concentrations of protocatechuic acid methyl ester were prepared in advance. Different concentrations of protocatechuic acid methyl ester (final concentrations of 1, 2, 3, 4, and 5 μg / ml) were added to each well of the normal control group and the RSV infection group. 100 μL of cell maintenance medium (2% cell maintenance medium preparation: 100 mL glass bottles were sterilized at high temperature and dried beforehand. After irradiation with UV lamp for 30 minutes, the operation was performed in a sterile laminar flow hood. 1 mL of fetal bovine serum and 1 mL of penicillin / streptomycin mixture were added to the glass bottle, and then DMEM high-glucose medium was added to make up to 100 mL. After thorough mixing, it was stored in a 4℃ refrigerator for later use; the preparation method for 0% medium was the same.) After 24 hours, the culture medium was removed, and the OD450nm value was measured after 1-2 hours in the dark according to the CCK8 detection method. Virus inhibition rate (%) = (OD450nm of experimental group - OD450nm of RSV infection group) / (OD450nm of normal control group - OD450nm of RSV infection group). The half-maximal effective concentration (EC50) was calculated using GraphPad Prism 9.0.

[0070] The results are as follows Figure 2 As shown, after treating Beas2b cells with methyl protocatechuate at concentration gradients of 1, 2, 3, 4 and 5 μg / mL for 24 hours, Beas2b activity and respiratory syncytial virus replication were inhibited in a dose-dependent manner, with IC50 and EC50 values ​​of 391.9 μg / mL and 1.535 μg / mL, respectively.

[0071] 3. qRCR assay to detect viral RNA expression in Beas2b cells infected with respiratory syncytial virus 24 hours after treatment with methyl protocatechuate.

[0072] Experimental methods:

[0073] Experimental group treatment: according to RNAeasy TMAnimal RNA Extraction Kit (Centrifuge Column) Instructions: Aspirate the culture medium from the six-well plate containing Beas2b cells infected with respiratory syncytial virus (RSV) at a final concentration of 3 μg / mL protocatechuate (the preparation of RSV-infected Beas2b cells is the same as in section 2, CCK8 assay for detecting cell viability of Beas2b cells treated with protocatechuate and the optimal concentration of protocatechuate for inhibiting RSV infection). After 24 hours, wash the remaining cells twice with PBS solution, remove the liquid, add 300 μL of lysis buffer, and gently pipette 6-10 times. Then transfer the supernatant to a sterile 1.5 mL... Add an equal volume of binding buffer to the EP tube and gently invert to mix 5 times. Transfer the mixture to the purification column provided with the reagent kit, centrifuge at 12,000 rpm for 30 seconds at 4°C, and discard the liquid in the lower collection tube. Add 600 μL of wash buffer I to the purification column, centrifuge at 12,000 rpm for 30 seconds at 4°C, and discard the liquid in the collection tube. Add 600 μL of wash buffer II to the purification column, centrifuge at 12,000 rpm for 30 seconds at 4°C, and discard the liquid in the collection tube. Repeat once. Centrifuge at 14,000 rpm for 3 minutes at 4°C. Discard the lower tube and place the RNA purification column into the elution tube provided with the kit. Add 40 μL of elution buffer, incubate at room temperature for 2 minutes, and centrifuge at 14,000 rpm for 1 minute at 4°C. To obtain a larger amount of RNA, the liquid in the elution tube can be added back to the purification column, and the process can be repeated once more. The resulting solution is the purified RNA. Label the cap with the date and name.

[0074] The positive control group was treated with RSV: the only difference from the experimental group was that methyl protocatechuate was replaced with an equal volume of PBS solution.

[0075] Negative control group (NC): The difference between this group and the experimental group is that it consists only of normal Beas2b cells.

[0076] PACAME group: The only difference from the experimental group was that the Beas2b cells infected with respiratory syncytial virus were replaced with normal Beas2b cells after treatment with methyl protocatechuate at a final concentration of 3 μg / mL.

[0077] According to the operating rules of the micro-volume nucleic acid analyzer, the sample was cleaned in advance. Using RNase-free water as a control, 1 μL of the RNA sample to be tested was taken and measured. The RNA concentration and the 260 / 280 ratio were recorded (a 260 / 280 ratio between 1.8 and 2 indicates good RNA purity). The reverse transcription reaction solution was prepared in the PCR reaction tube (following the instructions of the RNA reverse transcription kit (Genestar). The detailed reaction system was: RNA template ≤ 1 μg total RNA, StarScript III All-in-one RT Mix 1 μL, 5×StarScript III All-in-one RT Buffer μL, Nuclease-free Water (DEPC-treated) to a final volume of 20 μL). After briefly centrifuging to remove air bubbles, the prepared solution was used for reverse transcription on the PCR instrument. The reaction was carried out at 37°C for 2 minutes, then extended to 15 minutes at 55°C, followed by 2 minutes at 85°C, and finally cooled to 4°C before removal. After extracting the cDNA, 40 μL LEPC water was added to each sample to dilute the cDNA to 3-fold, and then stored in a -20°C freezer for subsequent quantitative analysis.

[0078] Prepare the following reaction system for real-time PCR: 1 μL cDNA template, 0.5 μL forward primer (10 μM), 0.5 μL reverse primer (10 μM), 10 μL 2×RealStar Fast SYBR qPCR Mix, and Nuclease-free Water (DEPC-treated) to a final volume of 20 μL. The primer sequences are shown in Table 1.

[0079] Table 1

[0080]

[0081]

[0082] Four replicates were set up. After a brief centrifugation to remove air bubbles, the samples were analyzed using the following reaction conditions: pre-denaturation at 95℃ for 2 min, denaturation at 95℃ for 15 s, annealing at 60℃ for 15 s, and extension at 72℃ for 30 s. The denaturation, annealing, and extension cycles were repeated 40 times. Fluorescence signals were collected from the amplified samples, and finally analyzed using 2... -△△CT The expression of the target gene FTO was analyzed using a method.

[0083] Simultaneously, the expression levels of inflammatory factors IL-6, IL1α, and IL1β, as well as the expression level of respiratory syncytial virus RNA, were detected.

[0084] The results are as follows Figure 3As shown in the figure, compared with the control group, the FTO gene expression in the experimental group decreased, and the replication of respiratory syncytial virus was inhibited; the expression levels of inflammatory factors IL-6, IL1α, and IL1β decreased.

[0085] Remove the cells from the 6-well plate and place them on ice. Discard the supernatant and wash twice with pre-chilled PBS (2 mL). Aspirate the liquid and add 200 μL / well or 100 μL / well of protein lysis buffer (RIPA: phosphatase inhibitor: protease inhibitor = 50:1:1). Incubate on ice for 5 minutes to ensure the protein lysis buffer completely covers the cell surface. Use a cell scraper to scrape the cell layer from top to bottom and from left to right to fully lyse the cells. Transfer the cells to 1.5 mL EP tubes and incubate on ice for 25 minutes (vortexing for 20 seconds every 5 minutes). Centrifuge the EP tubes at 14000 rpm for 20 minutes at 4°C. Transfer the supernatant to a new 1.5 mL EP tube and store at -80°C for long-term storage. Use a pipette to add 2.5 μL / well of color marker and the corresponding volume of denatured protein sample in sequence, avoiding protein sample overflow and air bubble formation. Fill both glass plates with electrophoresis buffer. Add the electrophoresis buffer outside the tank according to the Bio-Rad electrophoresis tank markings for 2 / 4 gels. Cover the electrophoresis tank, insert the electrodes (red to red, black to black), and set a constant voltage of 80V to begin electrophoresis. Once bromophenol blue enters the separating gel, increase the voltage to 110V and continue electrophoresis. Stop electrophoresis when the bottom layer of bromophenol blue is approximately 2cm from the bottom edge of the separating gel. After Western blotting, wash the membrane with TBST buffer for 7 minutes, blot dry with filter paper, add 5mL of stripping buffer, and gently shake and rinse on a shaker for 15 minutes. The white bands will become transparent. Remove the bands and wash the membrane again with TBST buffer for 7 minutes. Blot dry with absorbent filter paper and drop ECL luminescent solution onto the membrane to detect antibody stripping effectiveness. If no exposed bands are found, wash the membrane again with TBST buffer for 5 minutes and then reblock with rapid blocking buffer for 20 minutes. Repeat this process to expose other molecular weight proteins.

[0086] The results are as follows Figure 4 As shown in the figure, the expression level of FTO in the drug-treated group was significantly lower than that in the respiratory syncytial virus infection group, indicating that the expression level of FTO protein can be inhibited by protocatechuic acid methyl ester.

[0087] 4. Animal experiments

[0088] SPF-grade healthy female BALB / c mice aged 6-8 weeks were provided by the Zhuhai Baishitong Animal Experiment Center in Guangdong Province. The animals had free access to food and water, were housed in an isolated environment at a temperature of 23±2℃ and a relative humidity of 60-70%, and were kept under a 12-hour light / 12-hour dark cycle. Based on a review of literature, 25 mg / kg / d and 12.5 mg / kg / d of methyl protocatechuate were selected as the high-dose and low-dose groups for animal experiments. Mice were divided into four groups: normal control (NC), RSV infection group, RSV infection + 25 mg / kg / d methyl protocatechuate group, RSV infection + 12.5 mg / kg / d methyl protocatechuate group, RSV infection + 40 mg / kg / d ribavirin positive control group, and PACAME group. Six mice were assigned to each group, and they were numbered using picric acid.

[0089] After 4 days of acclimatization, the experiment began. The treatment of RSV infection + 25 mg / kg / d protocatechuate methyl ester group and RSV infection + 12.5 mg / kg / d protocatechuate methyl ester group was as follows: After the mice were anesthetized with ether, the respiratory syncytial virus TCID400 titer was administered into BALB / c mice once a day for a total of three times. Then, protocatechuate methyl ester was injected intraperitoneally once a day at concentrations of 12.5 mg / kg and 25 mg / kg.

[0090] The positive control group of 40 mg / kg / d ribavirin was treated as follows: Mice were anesthetized with ether and nasal instillation was started. BALB / c mice were infected with respiratory syncytial virus TCID400 titer once a day for a total of three times. Ribavirin was then injected intraperitoneally once a day at a concentration of 40 mg / kg / d.

[0091] The treatment of the control group in the PCAME (25 mg / kg / d) group was as follows: After the mice were anesthetized with ether, nasal instillation was started. PBS was instilled into BALB / c mice once a day for a total of three times. Then, protocatechuic acid methyl ester was injected intraperitoneally once a day at a concentration of 3 mg / kg / d.

[0092] The normal control group received no treatment.

[0093] RSV infection group: Mice were anesthetized with ether and nasal instillation was initiated. BALB / c mice were then infected with respiratory syncytial virus (RSV) TCID400 titers once daily for a total of three times and then fed normally.

[0094] Mice were treated 3 days after administration (day 7). During the experiment, mouse weight was measured at 9:00 AM every day, and the mice's mental state, feeding, and activity status (coughing, arching back, ruffled fur, etc.) were closely observed.

[0095] (1) Detection of respiratory syncytial virus RNA expression in mouse lungs using QPCR

[0096] According to RNAeasy TM Animal RNA extraction kit (centrifuge column type) instructions. The real-time PCR reaction system and procedure are the same as those in step 3, qCR experiment to detect viral RNA expression in Beas2b cells infected with respiratory syncytial virus 24 hours after treatment with methyl protocatechuate. Primer sequences are shown in Table 2.

[0097] Table 2

[0098]

[0099] Four replicates were set up. After a brief centrifugation to remove air bubbles, the samples were analyzed using a three-step method: amplification of the samples, acquisition of fluorescence signals, and finally, 2-ΔΔ. CT The expression of the target gene was analyzed.

[0100] The results are as follows Figure 5 As shown in the figure, compared with the RSV group, respiratory syncytial virus replication and inflammatory factors can be inhibited by methyl protocatechuate at concentrations of 25 mg / kg / d and 12.5 mg / kg / d.

[0101] (2) FTO protein expression in mouse lung tissue

[0102] When processing animal samples, 20 mg of mouse lung tissue was placed in a pre-chilled homogenizer, liquid nitrogen was added, and the frozen tissue was quickly pulverized using a homogenizing column. Then, 125 μL of lysis buffer was added for every 10 mg of tissue for lysis, and the mixture was thoroughly mixed by pipetting. The mixture was then transferred to a 1.5 mL EP tube. The lysed sample was vortexed (30 seconds each time, with 5-minute intervals, repeated 5 times), followed by centrifugation at 12000 rpm for 20 minutes in a pre-chilled 4°C centrifuge. Finally, the supernatant obtained after centrifugation was collected in a new EP tube, which is the total protein extract. Western blotting was used to detect FTO protein expression in mouse lung tissue.

[0103] The results are as follows Figure 6 As shown, PCAME can reduce the increase in FTO caused by RSV infection, and its effect is concentration-dependent and superior to ribavirin.

[0104] (3) Pathological examination of mouse lung tissue

[0105] Lung tissue samples from mice in the RSV-infected group, normal control group, and 12.5 mg / kg / d protocatechuate methyl ester group were fixed in 4% paraformaldehyde for 24-48 hours. After dehydration, clearing, and paraffin embedding using an automated dehydrator, the tissues were embedded in paraffin (dehydration completed in approximately 16-18 hours). The dehydrated tissues were then placed in paraffin at 60°C and quickly solidified on an ice plate to prevent cracking, and stored overnight at 4°C. After a 20 μm coarse cut to expose the tissue, the paraffin block was placed in a -20°C freezer for at least 4 hours, then cut into 3 μm thick sections. The sections were flattened with 40°C warm water and then retrieved using a glass slide, ensuring the tissue sections were positioned in the lower center of the slide. The slides were marked with a pencil and then dried in a 60°C oven for 1 hour. The sections were sequentially placed into various staining jars for HE staining: xylene I 10 min – xylene II 10 min – anhydrous ethanol I 5 min – anhydrous ethanol II 5 min – 95% ethanol 5 min – 90% ethanol 5 min – 80% ethanol 5 min – rinse with tap water for 2 min – hematoxylin staining for 6-10 min – rinse with tap water for 1 min – 1% hydrochloric acid ethanol for 3 seconds – eosin for 5-7 min – rinse with tap water for a few seconds – anhydrous ethanol I 1 min – xylene I 2 min – xylene II 2 min. After air-drying, the sections were mounted with neutral resin and observed under a pathological microscope (nuclei were blue, cytoplasm was red).

[0106] The results are as follows Figure 7 As shown in the figure, compared with the respiratory syncytial virus (RSV) infection group, mice treated with methyl protocatechuate at a concentration of 12.5 mg / kg / d showed a significant decrease in inflammatory cells, suggesting that the drug may alleviate RSV infection by inhibiting FTO.

[0107] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Use of methyl protocatechuate in the preparation of products for the treatment and / or prevention of respiratory syncytial virus infection.

2. The application according to claim 1, characterized in that, The product has at least one of the following functions: A1) Inhibits respiratory syncytial virus replication; A2) Reduces inflammatory response; A3) Inhibits glycolysis induced by respiratory syncytial virus infection of Beas2b cells; A4) Inhibits death induced by respiratory syncytial virus infection of Beas2b cells.

3. The application according to claim 2, characterized in that, The reduction of the inflammatory response includes lowering the expression levels of inflammatory factors; Preferably, the inflammatory factors include IL-6, IL1α, and IL1β.

4. The application according to claim 1, characterized in that, The content of protocatechuic acid methyl ester in the product is the content that achieves a therapeutically effective amount; Preferably, the content of protocatechuic acid methyl ester in the product can be 0.1%-100%.

5. The application according to claim 1, characterized in that, The product is at least one of pharmaceuticals, health products, and functional foods.

6. The application according to claim 5, characterized in that, The medicine also includes a pharmaceutically acceptable carrier; Preferably, the pharmaceutically acceptable excipients include at least one of diluents, excipients, fillers, binders, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, sweeteners, and flavorings.

7. The application according to claim 5, characterized in that, The dosage forms of the medicines include tablets, capsules, pills, injections, inhalers, lozenges, suppositories, emulsions, microemulsions, submicroemulsions, nanoparticles, gels, powders, suspensions, creams, gels, and sprays. And / or, the administration routes of the drug include oral, enteral, subcutaneous, intramuscular, intravenous, nasal, transdermal, subconjunctival, intraocular, orbital, retroocular, retinal, choroidal, and intrathecal injection.

8. The product according to claim 1, characterized in that, The methyl protocatechuate is the sole active ingredient of the product, or it may be used in combination with other drugs for treating respiratory syncytial virus as active ingredients.

9. Application of methyl protocatechuate in any of B1) to B2): B1) Reduce the expression level of FTO protein; B2) Prepare products that reduce the expression level of FTO protein.

10. A product for treating and / or preventing respiratory syncytial virus infection, characterized in that, The product contains methyl protocatechuate.

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