Application of emetine in the preparation of anti-rotavirus drugs

Abstract

This invention discloses the application of emetine in the preparation of anti-rotavirus drugs, belonging to the field of biotechnology. The application of emetine in the preparation of anti-rotavirus drugs disclosed in this invention demonstrates that emetine has an anti-biosynthetic effect on RV, exerting its anti-RV effect by inhibiting the expression of the structural protein VP6 gene.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to the application of emetine in the preparation of anti-rotavirus drugs. Background Technology

[0002] Rotavirus (RV) belongs to the genus Rotavirus in the family Reoviridae. It is a non-enveloped double-stranded RNA (dsRNA) virus with an icosahedral structure. Its genome contains 11 double-stranded RNAs encoding six structural proteins (VP1–VP4, VP6, and VP7) and six non-structural proteins (NSP1–NSP6). RV is a leading cause of severe dehydrating gastroenteritis in children under five years of age. In 2016, more than 258 million children under five years of age worldwide suffered from diarrhea due to RV infection, resulting in an estimated 128,500 deaths and approximately 1.537 million hospitalizations. RV is transmitted via the fecal-oral route and is easily spread through contact with contaminated surfaces or direct contact with infected individuals, making it highly contagious. Currently, there are no specific drugs for treating RV enteritis, and anti-RV vaccines carry the potential risks of intussusception and the presence of infectious PCV genes. Their effectiveness has been reported poor in developing countries and low-income areas, with limited accessibility and efficacy. Therefore, effective prevention and treatment of RV infection is of great significance.

[0003] Emetine (molecular formula C) 29 H 40 N2O4, chemical structural formula see [see details] Figure 1 Imetidine, also known as emetine, is an isoquinoline alkaloid derived from the rhizome of the Rubiaceae plant *Imetidine*. It possesses various pharmacological activities, including antitumor, antiviral, antiparasitic, and emetic activities. Studies have shown that emetine can effectively inhibit cytomegalovirus (CMV) replication and also exerts an inhibitory effect during the early replication stage of dengue virus type 2 (DNV) entering cells. Furthermore, emetine can exert antiviral effects by blocking retrograde transport of rabies virus through nerve axons. However, there are no reports of emetine's anti-RV activity.

[0004] Therefore, providing the application of emetine in the preparation of anti-rotavirus drugs is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

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

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

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

[0008] Furthermore, the application of emetine in the preparation of anti-rotavirus biosynthetic drugs.

[0009] Furthermore, the rotavirus is the RV-WA strain.

[0010] As can be seen from the above technical solution, compared with the prior art, the present invention discloses the application of emetine in the preparation of anti-rotavirus drugs. Emetine has an anti-biosynthetic effect on RV and can exert its anti-RV effect by inhibiting the expression of the structural protein VP6 gene. Attached Figure Description

[0011] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0012] Figure 1 The attached figure shows the chemical structural formula of the emeticine of the present invention;

[0013] Figure 2 The attached figure shows MA104 cells before and after RV virus infection according to the present invention;

[0014] Where A: normal MA104 cells; B: MA104 cells 48 hours after RV infection;

[0015] Figure 3 The attached figure shows the cytotoxicity results of the emeticine of the present invention on cells; N is the control group, i.e., the untreated group;

[0016] Figure 4 The attached figure illustrates the anti-RV adsorption effect of ipecacine of the present invention;

[0017] Figure 5 The attached figure shows the anti-biosynthesis effect of emetine on RV in this invention; compared with the Ribavirin group, *p<0.1, ****p<0.0001;

[0018] Figure 6 The attached figure shows the direct inhibitory effect of emetine on RV in this invention; compared with the Ribavirin group, ****p < 0.0001;

[0019] Figure 7 The attached figure shows the effect of the emeticine of the present invention on the expression of the viral structural protein VP6 gene; compared with the RV group, ****p<0.0001. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] The experimental cell lines and reagents are shown in Table 1.

[0022] Table 1

[0023]

[0024] The main instruments are listed in Table 2.

[0025] Table 2

[0026]

[0027]

[0028] Main reagent preparation methods:

[0029] Emetine stock solution: Weigh an appropriate amount of etanerine standard (purity ≥98%, batch number: AFCB1415), add 1 mL of cell-grade DMSO to dissolve it in a clean bench, and filter the solution through a 0.22 μM filter membrane using a disposable sterile syringe to prepare a 10 mM stock solution.

[0030] DMEM medium containing 10% fetal bovine serum: In a clean bench, add 50 mL of fetal bovine serum and 5 mL of penicillin-streptomycin antibiotics to 445 mL of high-glucose DMEM medium, mix thoroughly, aliquot into labeled 50 mL centrifuge tubes, seal, and store at 4°C.

[0031] 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.

[0032] 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 (prepared fresh for immediate use).

[0033] 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.

[0034] Statistical data analysis

[0035] All experiments were repeated three times. Experimental data are expressed as mean ± standard deviation (ˉx±s). SPSS software was used for statistical analysis. The t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons of means among multiple groups. P < 0.05 was considered statistically significant. The results are expressed as mean ± standard deviation. Mann-Whitney statistics were performed using Graphpad Prism 9.5.0.

[0036] Example 1: RV infection of MA104 cells

[0037] After passage of MA104 cells, a new cell culture flask was taken out, and 1 mL of cell suspension and 3 mL of DMEM medium containing 10% fetal bovine serum were added. The cells were incubated at 37°C and 5% CO2 for 48 h until they grew to a monolayer, at which point they could be used for RV expansion. The RV-Wa strain was first thawed in a 37°C water bath. 500 μL of RV virus solution was mixed thoroughly with 500 μL of 10 μg / mL EDTA-free trypsin and incubated at 37°C and 5% CO2 for 30 min. Then, the MA104 cells that had 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. 1 mL of the incubated virus solution was then added, followed by 3 mL of RV growth maintenance medium. MA104 cells infected with the virus will develop cytopathic effect (CPE). When the degree of cytopathic effect reaches approximately 75%, the cells were frozen at -20°C. After repeating the freezing and thawing process three times, centrifuge at low temperature and collect the supernatant, which is the viral fluid. Repeat the above process to amplify RV.

[0038] MA104 cells before and after infection with RV-Wa strain virus were observed. Figure 2 Normal MA104 cells are triangular or spindle-shaped with a clear and distinct cell outline. After infection with RV, MA104 cells show obvious pathological changes, with blurred cell boundaries, increased intercellular distance, and eventually complete cell detachment and floating.

[0039] Example 2: Detection of the toxicity of emetine to MA104 cells using the CCK8 assay.

[0040] Based on the commonly used concentration range of emetine, this experiment conducted a preliminary cytotoxicity test between 0.125-16 μM. MA104 cells in the logarithmic growth phase were observed under a microscope. The cells were uniform in morphology, plump, with clear edges, and approximately 80% cell number. After digestion, centrifugation, and resuspending, the suspension was further diluted, and 10 μL was used on a hemocytometer to count the cells and calculate the required cell volume. Subsequently, cells were seeded in 96-well plates, with 100 μL of cell suspension added to each well, resulting in a cell density of 8 × 10⁶ cells / well. 4Cells / mL. Drug administration was initiated when cells adhered to the culture medium to form a monolayer, while the control group received only an equal volume of high-glucose DMEM medium. After 48 h of incubation, the cytotoxicity of emetine was assessed using the CCK8 reagent. 1 / 10 volume of CCK8 solution was added to each well, and the cells were incubated for 90 min. The absorbance was then measured and recorded at 450 nm with shaking for 10 s. The relative cell viability formula is:

[0041] {(A 实验组 -A 空白 ) / (A 对照组 -A 空白 )×100%}

[0042] See results Figure 3 Emetine, at concentrations between 0.125 and 1 μM, showed no drug toxicity to cells and maintained an average cell viability of over 90%. Compared to the control group, the cell viability of the drug group at 0.125–1 μM was not significantly different; at 2 μM, the cell viability of the drug group was approximately 89.3%. Therefore, the experimental drug concentration of emetine was selected as 0.125–2 μM.

[0043] Example 3: Detection of three effects of emetical against RV using the CCK8 method

[0044] To investigate whether emetic has an in vitro anti-RV infection effect, experiments were conducted on the MA104 cell model to examine the adsorption, direct inhibition, and biosynthesis of emetic against RV virus.

[0045] (1) Anti-RV adsorption effect of ipecacine

[0046] Drug solution was added to 96-well plates containing monolayered MA104 cells, with 6 replicates per well, 100 μL per well. The positive control group received an equal volume of 1 mg / mL ribavirin, while the normal cell control and virus control groups received only an equal volume of high-glucose DMEM medium. Incubation was performed at 37°C and 5% CO2 for 2 h. Different concentrations of emetine solutions were then aspirated, and 100 TCID50 of virus (incubated with 10 μg / mL trypsin at 37°C for 30 min) was added to each well (except for the normal cell control group), 100 μL per well. Incubation was performed at 37°C and 5% CO2 for 2 h. The normal cell control group received only an equal volume of high-glucose DMEM medium. The virus was then aspirated, and RV growth maintenance medium (100 μL per well) was added to all groups except the normal cell control group (drug group, positive control group, and virus control group), 100 μL per well. Incubation was performed at 37°C and 5% CO2, with continuous observation. After 48 h of culture, the results were analyzed using a CCK-8 assay 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 450 nm after 90 min. Repeat the experiment 3 times.

[0047] See results Figure 4 The inhibition rate of emetics against RV was less than 0 at 0.125 μM, 0.25 μM, 0.5 μM, 1 μM and 2 μM, indicating that emetics has no adsorption effect against RV.

[0048] (2) Anti-RV synthesis effect of ipecacine

[0049] 100 μL of TCID50 virus solution (virus reacted with 10 μg / mL trypsin for 30 min) was added to 96-well plates of MA104 cells grown into a monolayer, 100 μL per well. Cells were washed twice with PBS beforehand. A normal control group was established, with an equal volume of high-glucose DMEM medium added. The cells were incubated at 37°C and 5% CO2 for 2 h. Then, the virus solution was aspirated, and different concentrations of emetine and 1 mg / mL Ribavirin were added, 100 μL per well. A virus control group was established, with only RV growth maintenance medium added, 100 μL per well. The cells were incubated at 37°C and 5% CO2, and continuously observed. After 48 h of continuous culture, the CCK-8 assay was performed. 1 / 10 volume of CCK-8 solution was added to each well, and the cells were incubated for 90 min. The absorbance was measured and recorded at 450 nm. The viral inhibition rate of the drug was calculated, and the experiment was repeated three times.

[0050] See results Figure 5 Compared with the Ribavirin group, the inhibition rates of RV by emetine at 0.125 μM, 0.25 μM, and 0.5 μM were 20.00%, 26.81%, and 38.02%, respectively, showing statistically significant differences (p < 0.0001). The inhibition rates of RV by emetine at 1 μM and 2 μM reached 63.76% and 63.88%, respectively, showing statistically significant differences (p < 0.05). These results indicate that emetine has a good anti-biosynthetic effect on RV.

[0051] (3) Direct inhibitory effect of emetine on RV

[0052] Different concentrations of emetine were mixed with an equal volume of 100 TCID50 viral solution (virus incubated with 10 μg / mL trypsin for 30 min) and incubated for 2 h. Cells were washed twice with PBS and then added to 96-well culture plates containing monolayers of MA104 cells. In the positive control group, ribavirin and RV were treated in the same manner. The normal cell control and virus control groups were incubated with an equal volume of high-glucose DMEM medium. The mixture was incubated at 37°C and 5% CO2 for 2 h. The mixture was then aspirated, and 100 μL of RV growth maintenance medium was added to each well. The mixture was incubated at 37°C and 5% CO2, and continuously observed. After 48 h of culture, the CCK-8 assay was performed. 1 / 10 volume of CCK-8 solution was added to each well, and the mixture was incubated for 90 min. The absorbance was measured and recorded at 450 nm. The viral inhibition rate of the drug was calculated, and the experiment was repeated three times.

[0053] See results Figure 6 Compared with the Ribavirin group, the inhibition rates of RV in each drug group were lower, and the differences were statistically significant, indicating that emetine has no significant direct inhibitory effect on RV.

[0054] Example 4: qPCR detection of the expression level of the RV structural protein VP6 gene.

[0055] (1) Extraction and quantification of total RNA

[0056] ① To further verify whether emetine has an anti-biosynthetic effect on RV, after 48 h of emetine's anti-biosynthetic effect on RV, samples from each drug group (0.5 μM, 1 μM, 2 μM), Ribavirin group, N group (normal cell control group), and RV group (virus control group) were selected. The supernatant was discarded, the samples were washed twice with PBS, 1 mL of Trizol reagent was added, and the samples were allowed to stand for 5 min and collected in 1.5 mL enzyme-free EP tubes. 200 μL of chloroform was added to the tubes, the samples were vortexed for 15 s, allowed to stand at room temperature for 3 min, and then centrifuged (4℃, 12000 rpm / min, 15 min). The centrifuged samples separated into three layers: a colorless upper layer, a white middle layer, and a red lower layer.

[0057] ② Carefully aspirate the colorless upper layer into a new 1.5 mL enzyme-free EP tube (approximately 500 μL), add an equal volume of pre-chilled isopropanol, vortex vigorously to mix, incubate at 4°C for 10 min, and centrifuge (4°C, 12000 rpm / min, 10 min).

[0058] ③ After centrifugation, a white precipitate can be seen at the bottom of the EP tube. Remove the supernatant and keep the precipitate. Add 1 mL of the prepared 75% ethanol solution (prepared by mixing anhydrous ethanol and enzyme-free sterile water in a 3:1 ratio), shake to mix, centrifuge (4℃, 12000 rpm / min, 5 min), discard the supernatant, place at room temperature for 15 min to 20 min, and air dry.

[0059] ④ After drying, add 20 μL of DEPC water to the EP tube and gently blow on the tube wall to dissolve the RNA. After measuring the RNA concentration of the sample with a NanoDrop micro-ultraviolet spectrophotometer, it can be used directly for subsequent experiments, or the sample can be stored at -80℃ for later use.

[0060] (2) Reverse transcription of mRNA

[0061] ① Genomic DNA removal reaction

[0062] Prepare the reaction mixture on ice according to the components in Table 3, with a reaction volume of 20 μL. Follow the instructions for the Evo M-MLV RTKit with gDNA Clean for qPCR II. All consumables used in this experiment were Axygen enzyme-free consumables.

[0063] Table 3 Genomic DNA Reaction System

[0064]

[0065] Reaction conditions: 42℃ for 2 min; 4℃.

[0066] *1: The amount of RNA can be added as needed. In a 20 μL reverse transcription system, use a maximum of 1 μg total RNA; when using the probe method, use a maximum of 2 μg total RNA.

[0067] ②Reverse transcription reaction

[0068] Prepare the reaction solution according to the contents of Table 4 and carry out the reverse transcription reaction.

[0069] Table 4 Reverse transcription reaction system

[0070]

[0071] Reaction conditions: 37℃ for 15 min; 85℃ for 5 sec; 4℃.

[0072] (3) Real-Time PCR reaction

[0073] Real-time quantitative PCR was performed using SYBR Green I fluorescent labeling to detect VP6 expression levels in each drug group (0.5 μM, 1 μM, 2 μM), the Ribavirin group, and the RV group. The GreenPremix Pro Taq HS qPCR Kit2II was used, with GAPDH as the internal control. Axygen's dedicated Real-time 8-tube PCR system was used. The real-time quantitative PCR amplification reaction system was prepared according to Table 5, and the reaction solution was prepared on ice (total reaction volume: 10 μL). qPCR reaction conditions are shown in Table 6; primer sequences are shown in Table 7.

[0074] Table 5 PCR reaction system

[0075]

[0076] Table 6 qPCR reaction conditions

[0077]

[0078] Table 7 Primer sequences

[0079]

[0080] like Figure 7 As shown, compared with the RV group, the expression level of VP6 in the ipecac group was significantly reduced at concentrations of 0.5 μM, 1 μM, and 2 μM, with statistically significant differences. This indicates that ipecac can exert its anti-RV effect by inhibiting the gene expression of VP6 through anti-RV biosynthesis.

[0081] Conclusion: Emetine has an anti-RV biosynthesis effect, but no obvious anti-RV adsorption or direct RV inhibition effect. It exerts its anti-RV effect by inhibiting the gene expression of VP6.

[0082] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The use of emetine in the preparation of anti-rotavirus drugs.

2. The use of emetine in the preparation of anti-rotavirus biosynthesis drugs.

3. Use according to claim 1 or 2, characterized in that, The rotavirus is RV-WA strain.

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