Application of harringtonine in preparation of anti-rotavirus drugs

Abstract

The invention discloses application of harringtonine in preparation of anti-rotavirus drugs, and belongs to the technical field of biology. According to the application of the harringtonine in preparation of the anti-rotavirus medicine, the harringtonine has a certain direct inhibition effect and an anti-biosynthesis effect on RV, and the anti-RV effect can be achieved by inhibiting expression of a structural protein VP6 gene.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to the application of harringtonine in the preparation of anti-rotavirus drugs. Background Art

[0002] Rotavirus (RV) belongs to the genus Rotavirus of the family Reoviridae. It is a non-enveloped double-stranded RNA (dsRNA) virus, consisting of an inner capsid (viral protein VP2), a middle capsid (VP6), and an outer capsid (VP7 and spike protein VP4). Three concentric capsids surround a genome of 11 dsRNA segments. RV infection is the leading cause of severe dehydrating gastroenteritis in children under 5 years old. In 2016, RV infection caused more than 258 million cases of diarrheal episodes in children, an estimated 128,500 deaths, and 1.537 million hospitalizations for RV infection. RV is mainly transmitted through the fecal-oral route and is highly contagious. Currently, four group A rotavirus vaccines, Rotarix, Rotasiil, RotaTeq, and Rotavac, have been pre-certified by the World Health Organization (WHO) for marketing. However, anti-RV vaccines have the potential risk of intussusception and inclusion of infectious PCV genes, and their effectiveness in developing countries and low-income areas is not ideal. Therefore, the discovery of new compounds for the prevention and treatment of RV diarrhea is of great significance.

[0003] Harringtonine (molecule formula: C 28 H 37 O9N, chemical structure see Figure 1 ) is an alkaloid extracted from Cephalotaxus or its cognate plants of the Cephalotaxaceae family. Studies have shown that harringtonine has a strong anti-tumor effect and also has an inhibitory effect on herpes zoster virus and Zika virus. However, there is currently a lack of in-depth research on the pharmacological effects of harringtonine in terms of its antiviral effect, and there is no report on the anti-RV effect of harringtonine.

[0004] Therefore, providing the use of harringtonine in the preparation of anti-rotavirus drugs is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention provides the use of harringtonine in the preparation of anti-rotavirus drugs.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] Application of harringtonine in the preparation of anti-rotavirus drugs.

[0008] Furthermore, the invention relates to the application of harringtonine in the preparation of anti-rotavirus biosynthetic drugs.

[0009] Furthermore, the application of harringtonine in the preparation of drugs for directly inhibiting rotavirus.

[0010] Furthermore, the rotavirus is RV-WA strain.

[0011] It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses the use of harringtonine in the preparation of anti-rotavirus drugs. Harringtonine has a certain direct inhibitory effect and anti-biosynthetic effect on RV, and can exert its anti-RV effect by inhibiting the expression of the structural protein VP6 gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0013] Figure 1 The accompanying drawing is the chemical structural formula of Harringtonine of the present invention;

[0014] Figure 2 The accompanying drawings show MA104 cells before and after infection with the RV virus of the present invention;

[0015] Among them, A: normal MA104 cells; B: MA104 cells infected with RV for 48 hours;

[0016] Figure 3 The attached figure shows the toxicity results of Harringtonine of the present invention on cells; N is the control group, i.e., the group without drug administration;

[0017] Figure 4 The attached figure shows the anti-RV adsorption effect of Harringtonine of the present invention; compared with the Ribavirin group, *p<0.1, **p<0.01, ****p<0.0001;

[0018] Figure 5 The attached figure shows the anti-biosynthesis effect of Harringtonine of the present invention on RV; compared with the Ribavirin group, *p<0.1, **p<0.01, ***p<0.001, ****p<0.0001;

[0019] Figure 6The attached figure shows the direct inhibitory effect of Harringtonine of the present invention on RV; compared with the Ribavirin group, **p<0.01, ***p<0.001, ****p<0.0001;

[0020] Figure 7 The attached figure shows the effect of Harringtonine of the present invention on the gene expression of viral structural protein VP6; compared with the RV group, ****p<0.0001. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] The experimental cell lines and reagents are shown in Table 1.

[0023] Table 1

[0024]

[0025] The main instruments are shown in Table 2.

[0026] Table 2

[0027]

[0028]

[0029] Preparation method of main reagents:

[0030] Harringtonine mother solution: Weigh an appropriate amount of Harringtonine standard (purity ≥ 98%, item number: ab-M3964-5mg), dissolve it in 1 ml of cell-grade DMSO in a clean bench, filter the solution through a 0.22 μM filter membrane with a disposable sterile syringe to sterilize, and prepare a 10 mM mother solution.

[0031] DMEM medium containing 10% fetal bovine serum: Add 50 ml of fetal bovine serum and 5 ml of penicillin-streptomycin double antibody to 445 ml of high-glucose DMEM medium in an ultra-clean workbench, mix thoroughly, and then dispense into labeled 50 ml centrifuge tubes, seal, and store at 4°C.

[0032] 10 μg / mL EDTA-free trypsin: 0.25% trypsin digestion solution (0.25 g trypsin / 100 mL, 2500 μg / mL) was diluted to 10 μg / mL with high-glucose DMEM medium.

[0033] Ribavirin solution: 100 mg / mL Ribavirin stock solution was stored at 4°C and diluted to 1 mg / mL with high-glucose DMEM medium as a positive control group (prepare and use immediately).

[0034] RV growth maintenance medium: 10 μg / mL EDTA-free trypsin and high-glucose DMEM medium were diluted to 1 μg / mL at a ratio of 1:10.

[0035] Statistical data analysis

[0036] All experiments were repeated three times. The experimental data are expressed as mean ± standard deviation. The data were analyzed by SPSS software, the t-test was used for comparison between the two groups, and the one-way analysis of variance was used for comparison between the means of multiple groups. P<0.05 indicated that the difference was statistically significant. The obtained data results were expressed as mean ± standard deviation. The data were analyzed by Harringtoninenn-Whitney statistics using statistical analysis software Graphpadprism9.5.0.

[0037] Example 1RV infection of MA104 cells

[0038] After the MA104 cells were subcultured, a new cell culture bottle was taken out, 1 mL of cell suspension and 3 mL of DMEM medium containing 10% fetal bovine serum were added, and the cells were incubated at 37°C and 5% CO2 for 48 hours. When the cells grew to a monolayer, they could be used for RV amplification. First, the RV-Wa strain was placed in a 37°C water bath to melt, 500 μL of RV virus solution was taken and mixed with 500 μL of 10 μg / mL EDTA-free trypsin, and incubated at 37°C and 5% CO2 for 30 minutes. Then, the MA104 cells grown to a monolayer were taken out, rinsed once with phosphate buffered saline (PBS, pH=7), and then rinsed twice with high-glucose DMEM medium, and then 1 mL of incubated virus solution was added, and then 3 mL of RV growth maintenance solution was added to the cell culture bottle. After virus infection, MA104 cells will develop cytopathic CPE (Cytopathiceffect). When the degree of lesions reaches about 75%, they are frozen in a -20°C refrigerator. After repeating the freezing and thawing process three times, centrifuge at low temperature and collect the supernatant, which is the virus liquid. Repeat the above process to amplify RV.

[0039] MA104 cells before and after RV-Wa virus infection Figure 2 Normal MA104 cells are triangular or spindle-shaped, with clear cell outlines; after being infected with RV, MA104 cells undergo obvious lesions, cell boundaries become blurred, the distance between cells increases, and finally the cells completely fall off and float.

[0040] Example 2 Detection of Harringtonine's toxicity to MA104 cells by CCK8 assay

[0041] According to the commonly used drug concentration range of Harringtonine, this experiment conducted a preliminary cytotoxicity experiment between 0.125-160μM. MA104 cells in the logarithmic growth phase were observed under a microscope. The cell morphology was uniform and full, the edges were clear, and the number reached about 80%. Digestion, centrifugation and resuspending were performed. After the suspension was further diluted, 10μl was taken on a hemocytometer to count and calculate the required cell volume. The cells were then plated in a 96-well plate, and 100μl of cell suspension was added to each well. The cell density was 8×10 4 / mL. When the cells adhered to the wall and formed a monolayer, the drug was administered, and only an equal volume of high-glucose DMEM medium was added to the control group. After incubation for 48 hours, CCK8 reagent was used to detect the cytotoxicity of Harringtonine. 1 / 10 volume of CCK8 solution was added to each well and incubated in an incubator. After 90 minutes, the absorbance was detected and recorded under oscillation for 10 seconds and a wavelength of 450nm. The relative survival rate of cells is:

[0042] {(A 实验组 -A 空白 ) / (A 对照组 -A 空白 )×100%}

[0043] Results Figure 3 , Harringtonine drug concentration below 4μM, no drug toxicity to cells and the average survival rate was above 90%. Compared with the control group, there was no significant difference in cell viability between the drug group and the control group when Harringtonine was 0.125-4μM. Therefore, the experimental drug concentration of Harringtonine was selected to be 0.125-4μM.

[0044] Example 3 CCK8 method to detect three effects of Harringtonine against RV

[0045] In order to study whether Harringtonine has an in vitro anti-RV infection effect, experiments were conducted on the MA104 cell model from three aspects: Harringtonine's anti-RV virus adsorption, direct inactivation and biosynthesis.

[0046] (1) Harringtonine anti-RV adsorption effect

[0047] Add the drug solution to the 96-well culture plate of MA104 cells grown to a monolayer, and repeat 6 wells for each drug solution, 100 μL per well. Add an equal volume of 1 mg / mL ribavirin to the positive control group, and only add an equal volume of high-glucose DMEM medium to the normal cell control group and the virus control group. Incubate for 2 hours at 37°C and 5% CO2. Aspirate the drug solution, and add 100 TCID50 of virus (virus and 10 μg / mL pancreatin at 37°C for 30 minutes) to the normal cell control group, 100 μL per well, incubate for 2 hours at 37°C and 5% CO2, and only add an equal volume of high-glucose DMEM medium to the normal cell control group. Aspirate the virus, and add RV growth maintenance solution to the drug group, positive control group, and virus control group, 100 μL per well, incubate at 37°C and 5% CO2, and observe continuously. After 48 hours of culture, detect with CCK-8 kit. Add 1 / 10 volume of CCK-8 solution to each well, incubate in an incubator, and detect and record the absorbance at 450 nm after 90 min. Repeat the experiment 3 times.

[0048] Results Figure 4 Compared with the Ribavirin group, the anti-RV inhibition rates of the Harringtonine group at 0.125μM, 0.25μM, 0.5μM, and 4μM were about 2.47%, -0.88%, 4.39%, and 4.05%, respectively, which were all lower than those of the Ribavirin group, with statistical differences; the anti-RV inhibition rates of Harringtonine at 1μM and 2μM were about 9.76% and 8.44%, respectively, which were not statistically different from those of the Ribavirin group. Therefore, the experimental results showed that Harringtonine had no anti-RV adsorption effect.

[0049] (2) Harringtonine's anti-RV synthesis effect

[0050] 100TCID50 virus solution (virus and 10μg / mL trypsin for 30min) was added to a 96-well culture plate of MA104 cells grown to a monolayer, 100μL per well, and the cells were rinsed twice with PBS before. Set up a normal cell control and add an equal volume of high-glucose DMEM medium. Incubate at 37℃, 5% CO2 for 2h, then aspirate the virus solution, add different concentrations of drug solution and 1mg / mL Ribavirin, 100μL per well; set up a virus control group, only add RV growth maintenance solution, 100μL per well. Incubate at 37℃, 5% CO2, and observe continuously. After continuous culture for 48h, detect with CCK-8 kit. Add 1 / 10 volume of CCK-8 solution to each well, incubate in an incubator, and detect and record the absorbance at a wavelength of 450nm after 90min. Calculate the virus inhibition rate of the drug, and repeat the experiment 3 times.

[0051] Results Figure 5 Compared with the Ribavirin group, the inhibition rates of 0.5μM, 1μM, and 2μM Harringtonine groups on RV were 78.58%, 73.33%, and 75.74%, respectively, which were statistically significant (p values ​​were 0.0006, 0.0105, and 0.0026, respectively); the inhibition rates of 0.25μM and 4μM Harringtonine groups on RV were 47.05% and 68.10%, respectively, which were not statistically significant compared with the Ribavirin group. The experimental results showed that Harringtonine had an anti-biosynthesis effect on RV at 0.25μM, 0.5μM, 1μM, 2μM, and 4μM.

[0052] (3) Direct killing effect of Harringtonine on RV

[0053] The drug was mixed with an equal volume of 100TCID50 virus solution (virus and 10μg / mL pancreatin for 30min) for 2h. The cells were rinsed twice with PBS and added to a 96-well culture plate grown to a monolayer of MA104 cells. In the positive control group, Ribavirin and RV were subjected to the same operation as above, and only an equal volume of high-glucose DMEM medium was added to the normal cell control group and the virus control group. Incubate at 37℃, 5% CO2 for 2h, then aspirate the mixed solution, add RV growth maintenance solution, 100μL per well, incubate at 37℃, 5% CO2, and observe continuously. After 48h of incubation, use CCK-8 kit to detect. Add 1 / 10 volume of CCK-8 solution to each well, incubate in an incubator, and detect and record the absorbance at a wavelength of 450nm after 90min. Calculate the virus inhibition rate of the drug, and repeat the experiment 3 times.

[0054] Results Figure 6Compared with the Ribavirin group, the inhibition rates of Harringtonine on RV at 0.125μM, 0.25μM, and 0.5μM were 13.12%, 38.38%, and 35.36%, respectively, which were all lower than those in the Ribavirin group, with statistical differences (p values ​​were 0.0001, 0.0024, and 0.0004, respectively), indicating that Harringtonine had no direct inhibitory effect on RV at 0.125μM, 0.25μM, and 0.5μM; the inhibition rates of Harringtonine on RV at 1μM, 2μM, and 4μM were about 44.26%, 47.14%, and 52.79%, respectively, which were not statistically different from those in the Ribavirin group, indicating that the direct inhibitory effect of Harringtonine on RV at 1μM, 2μM, and 4μM was equivalent to that of Ribavirin, and Harringtonine had a direct inhibitory effect on RV at 1μM, 2μM, and 4μM.

[0055] Example 4 qPCR detection of the expression level of RV structural protein VP6 gene

[0056] (1) Extraction and quantification of total RNA

[0057] ① In order to further verify whether Harringtonine has an anti-biosynthetic effect on RV, after 48 hours of Harringtonine's anti-biosynthetic effect on RV, each drug group (1μM, 2μM, 4μM), Ribavirin group and RV group (virus control group) were selected, the supernatant was removed, PBS was rinsed twice, 1mL Trizol reagent was added, and it was allowed to stand for 5min, and collected in a 1.5mL enzyme-free EP tube. 200μL of chloroform was added to the tube, vortexed for 15s, and allowed to stand at room temperature for 3min, and centrifuged (4℃, 12000rpm / min, 15min). The sample after centrifugation was divided into three layers, namely, a colorless upper layer, a white middle layer and a red lower layer.

[0058] ② Carefully pipette the colorless upper layer into a new 1.5 mL enzyme-free EP tube (volume about 500 μL), add an equal volume of pre-cooled isopropanol, shake vigorously up and down to mix, let stand at 4°C for 10 min, and centrifuge (4°C, 12000 rpm / min, 10 min);

[0059] ③ After centrifugation, white precipitate can be seen at the bottom of the EP tube. Remove the supernatant and keep the precipitate. Add 1 mL of prepared 75% ethanol solution (anhydrous ethanol and enzyme-free sterile water in a ratio of 3:1), shake to mix, centrifuge (4°C, 12000 rpm / min, 5 min), discard the supernatant, leave at room temperature for 15 min to 20 min, and dry.

[0060] ④ After drying, add 20 μL of DEPC water to the EP tube and gently blow the tube wall to dissolve the RNA. After the RNA concentration of the sample is measured by NanoDrop micro-volume UV spectrophotometer, it can be directly used for subsequent experiments, or stored at -80℃ for future use.

[0061] (2) Reverse transcription of mRNA

[0062] ① Removal of genomic DNA

[0063] Prepare the reaction mixture on ice according to the ingredients in Table 3, with a reaction volume of 20 μL, and perform the operation according to the instructions of Evo M-MLV RTKit with gDNA Clean for qPCR II. All consumables used in this experiment are Axygen enzyme-free consumables.

[0064] Table 3 Genomic DNA removal reaction system

[0065]

[0066] Reaction conditions: 42°C for 2 min; 4°C.

[0067] *1: The amount of RNA added can be adjusted according to the needs. In a 20 μL reverse transcription system, a maximum of 1 μg total RNA can be used; when using the probe method, a maximum of 2 μg total RNA can be used.

[0068] ②Reverse transcription reaction

[0069] Prepare the reaction solution according to Table 4 and perform reverse transcription reaction.

[0070] Table 4 Reverse transcription reaction system

[0071]

[0072] Reaction conditions: 37℃15min; 85℃5sec; 4℃.

[0073] (3) Real-Time PCR reaction

[0074] Real-time quantitative PCR was performed using SYBR Green I fluorescent labeling to detect the expression levels of VP6 in each drug group (1 μM, 2 μM, 4 μM), Ribavirin group, and RV group. GreenPremixPro TaqHSqPCRKitII kit, GAPDH was selected as the internal reference. The experiment used Axygen's dedicated Real-time eight-row PCR tube, and the real-time fluorescence quantitative PCR amplification reaction system was prepared according to Table 5. The reaction solution was prepared on ice for operation (the total reaction system was 10 μL). The qPCR reaction conditions are shown in Table 6; the primer sequences are shown in Table 7.

[0075] Table 5 PCR reaction system

[0076]

[0077]

[0078] Table 6 qPCR reaction conditions

[0079]

[0080] Table 7 Primer sequences

[0081]

[0082] like Figure 7 As shown, compared with the RV group, the VP6 expression levels in the Harringtonine group at concentrations of 1 μM, 2 μM, and 4 μM were significantly reduced, with statistically significant differences (p < 0.0001), indicating that Harringtonine can exert its anti-RV effect by inhibiting the gene expression of VP6 through anti-RV biosynthesis.

[0083] Conclusion: Harringtonine has a certain direct inhibitory effect and anti-biosynthetic effect on RV, and can exert its anti-RV effect by inhibiting the expression of structural protein VP6 gene.

[0084] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of harringtonine in the preparation of anti-rotavirus drugs.

2. Application of harringtonine in the preparation of anti-rotavirus biosynthetic drugs.

3. Application of harringtonine in the preparation of drugs that directly inhibit rotavirus.

4. The use according to any one of claims 1 to 3, characterized in that: The rotavirus is RV-WA strain.

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