CN117959313A - Application of corilagin in preparation of rotavirus resistant medicines - Google Patents

Abstract

The invention discloses an application of corilagin in preparing an anti-rotavirus medicament, and belongs to the technical field of biology. The Cor has the effect of resisting RV biosynthesis and can play the role of resisting RV by inhibiting the expression of VP6 genes.

Description

Application of corilagin in preparation of rotavirus resistant medicines

Technical Field

The invention relates to the technical field of biology, in particular to application of corilagin in preparation of anti-rotavirus medicines.

Background

Rotavirus (Rotavirus, RV), belonging to the genus rotavirus of the reoviridae, is one of the major pathogens responsible for acute gastroenteritis diarrhea in infants under 5 years old, especially in hospitalization of infants in developing countries. The highly contagious and pathogenic nature of RV poses a major hazard to the entire society, with about 1.3 hundred million people infected each year worldwide, and about 20 ten thousand deaths. At present, no specific medicine for treating RV infection is clinically available, and meanwhile, the range of RV vaccine pre-defense strains is limited due to variability and diversity of RV strains, so that how to prevent RV infection is a serious clinical problem.

The 1-acyl-3, 6-hexahydroxybiphenyl diformyl glucose (corilagin, cor) is polyphenol tannic acid compound extracted from phyllanthus urinaria, geranium, citrus reticulata, trifolium pratense and other plants, and has wide biological activities of resisting tumor, resisting oxidation, resisting atherosclerosis, resisting fibrinolysis, reducing blood pressure, inhibiting viruses, resisting bacteria, resisting inflammation and the like. At present, no effect of Cor anti-RV is reported.

Therefore, providing the application of corilagin in preparing anti-rotavirus drugs is a problem that needs to be solved by the skilled person.

Disclosure of Invention

In view of this, the present invention provides the use of corilagin in the preparation of anti-rotavirus drugs.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

The application of corilagin in preparing anti-rotavirus medicine is shown in figure 1.

Further, the application of the corilagin in preparing anti-rotavirus biosynthesis medicines.

Further, the rotavirus is RV-WA strain and RV-SA-11 strain.

Compared with the prior art, the application of corilagin in preparing anti-rotavirus medicines is disclosed, cor has the effect of resisting RV biosynthesis, has no obvious RV adsorption resistance and direct inhibition effect, and can play the role of resisting RV by inhibiting VP6 gene expression.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.

FIG. 1 is a diagram of the chemical formula of Cor;

FIG. 2 is a drawing of MA104 cells of the invention before and after RV virus infection;

wherein A: normal MA104 cells; b: MA104 cells 48h after RV infection;

FIG. 3 is a graph showing the toxicity results of Cor on cells (Control group represents Control group, ^**P＜0.05,^*** P < 0.001 compared with Control group);

FIG. 4 is a graph of Cor anti-RV adsorption of the present invention;

wherein A: RV-WA strain; b: RV-SA-11 strain;

FIG. 5 is a graph showing the effect of Cor on RV-resistance synthesis according to the present invention;

Wherein A: RV-WA strain; b: RV-SA-11 strain; ^*P＜0.05,^**P＜0.05,^*** P < 0.001 compared to Ribavirin group;

FIG. 6 is a graph of the direct RV inhibition by Cor of the present invention;

Wherein A: RV-WA strain; b: RV-SA-11 strain; ^**** P < 0.0001 compared with Ribavirin group;

FIG. 7 is a graph showing the relative expression level of RV-VP6 gene of the present invention;

wherein A: RV-WA strain; b: RV-SA-11 strain; control group represents normal Control group of cells, ^**** P <0.0001 compared to RV group).

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Cell grade dimethyl sulfoxide, fetal bovine serum, high sugar DMEM broth from GIBCO company, usa; 0.25% trypsin digest, 0.25% EDTA-containing trypsin digest, anti-green streptomycin (diabody) was purchased from beijing solebao corporation; MA104 cell lines were from the university of zhongshan cell bank; RV-Wa strain and RV-SA-11 strain are from third army medical university immune institute; 1 XPBS phosphate buffer was purchased from Jiangsu Biyun biotechnology institute.

Cor mother liquor: 9.8mg of Cor standard product is weighed, the purity is more than or equal to 98 percent, and the batch number is as follows: o26IB230022 (Shanghai Yuan Yes Biotechnology Co., ltd.) was dissolved in 154.46. Mu.L of cell-grade DMSO in a super clean bench, and the solution was sterilized by filtration through a 0.22. Mu.M filter using a disposable sterile syringe to prepare a mother liquor of 100mM.

Ribavirin solution: 100mg/mL Ribavirin stock solution was stored at 4℃and diluted to 1mg/mL with high-sugar DMEM medium as a positive control (as prepared).

DMEM medium containing 10% fetal bovine serum: sequentially adding 5mL of fetal bovine serum and 0.5mL of double antibody into 45mL of high-sugar DMEM culture solution in an ultra-clean workbench, fully and uniformly mixing, placing into a marked 50mL centrifuge tube, sealing, and preserving at 4 ℃.

Pancreatin without EDTA at 10 μg/mL: 0.25% trypsin digest (0.25 g pancreatin/100 mL, 2500. Mu.g/mL) was diluted to 10. Mu.g/mL with high sugar DMEM medium.

RV growth maintenance solution: 0.25% trypsin digest (0.25 g pancreatin/100 mL, 2500. Mu.g/mL) was diluted to 1. Mu.g/mL with high sugar DMEM medium.

And (3) statistical data analysis:

All experiments were repeated three times. Experimental data are expressed in mean ± standard deviation (x ± s). SPSS software statistical data is adopted, t-test is adopted for comparison between two groups, and single-factor variance analysis is adopted for comparison of mean values between multiple groups. The data obtained are expressed as mean ± standard deviation. Data analysis Mann-Whitney statistics were performed using statistical analysis software Graphpapdprism 8.01. P <0.05 indicates that the difference is statistically significant.

EXAMPLE 1RV infection of MA104 cells

After MA104 cells are digested and passaged, a new cell culture bottle is taken out, 1mL of cell suspension and 3mL of DMEM culture solution containing 10% fetal bovine serum are added, the cells are incubated for 48 hours at 37 ℃ and 5% CO ₂, and after the cells grow to a monolayer, the cells can be used for RV amplification. Firstly, respectively placing RV-Wa strain and RV-SA-11 strain in a water bath kettle at 37 ℃ for melting, taking 500 mu L of RV virus liquid and 500 mu L of pancreatin without EDTA at 10 mu g/mL, fully and uniformly mixing, and incubating for 30min at 37 ℃ with 5% CO ₂. The MA104 cell flask grown to a monolayer was then removed, washed once with phosphate buffer (PBS, pH=7), rinsed twice with high-sugar DMEM medium, then added with 1mL of the incubated virus solution, and then added with 3mL of RV growth maintaining solution. Cytopathy CPE (Cytopathic effect) can occur in MA104 cells after virus infection, and when the pathological change degree reaches 75%, the MA104 cells are frozen in a refrigerator at minus 20 ℃. Repeating the freezing and thawing process for 3 times, centrifuging at low temperature, and collecting supernatant to obtain virus liquid. The above procedure was repeated to amplify RV.

MA104 cells before and after infection of RV-Wa strain virus are shown in figure 2, the shape of normal MA104 cells is triangle or fusiform, and the cell outline is obvious and clear; after infection of MA104 cells of RV-Wa strain, obvious lesions of the cells occur, cell boundaries become blurred, intercellular spaces are increased, black particles in the cells are increased, and finally the cells completely fall off and float.

Example 2 CCK8 assay for toxicity of Cor to MA104 cells

The cytotoxicity pre-experiment was performed between 3.9 and 1000. Mu.M according to the concentration range of the common drug for Cor. Taking MA104 cells in logarithmic growth phase, observing under microscope, digesting, centrifuging and re-suspending when the cell morphology is uniform and full, the edge is clear and the number reaches 80%, further diluting the suspension, taking 10 μl to count on a blood cell counting plate, and calculating the required cell volume. Cells were then plated in 96-well plates with 100 μl of cell suspension per well at a cell density of 8×10 ⁴/mL. When the cells grow to a monolayer, a series of concentration liquid medicine is added, and only the same volume of high-sugar DMEM culture solution is added into the control group. After 48h incubation, the CCK-8 kit detects Cor cytotoxicity. 1/10 volume of CCK-8 solution was added to each well, incubated in an incubator, and absorbance was measured and recorded at a wavelength of 450nm after 1 h. The relative viability of the cells was formulated as:

{(A _{experimental group} -A _{Blank space} )/(A _{Control group} -A _{Blank space} )×100％}

as shown in FIG. 3, the safety concentration of Cor drug is 3.9-125. Mu.M, and the cell survival rate is above 90%.

Example 3 CCK8 method for detecting three effects of Cor on RV

In order to study whether Cor has the effect of resisting RV infection in vitro, three effects of adsorption, direct inhibition and biosynthesis of Cor on MA104 cell model infected by RV-WA strain and RV-SA-11 strain are carried out.

(1) Cor anti RV adsorption

The liquid was added to a 96-well plate of MA104 cells grown to a monolayer, and 6 wells were repeated for each liquid, 100. Mu.L per well. An equal volume of Ribavirin (Ribavirin) was added to the positive control, and only an equal volume of high-sugar DMEM broth was added to the normal cell control and the virus control. Incubation was carried out at 37℃for 2h with 5% CO ₂. The solution was then aspirated, and 100. Mu.L of 100TCID50 virus (virus was allowed to react with 10. Mu.g/mL pancreatin at 37℃for 30 min) was added to each of the drug, positive control and virus control. Normal cell control groups were added with only an equal volume of high-sugar DMEM medium. Incubation was carried out at 37℃for 2h with 5% CO ₂. The virus solution was aspirated, and the drug, positive control and virus control groups were each added with RV growth maintaining solution, 100. Mu.L per well, 37℃and 5% CO ₂ were incubated for continuous observation, except for the normal cell control group. After 48h incubation, the cells were assayed using the CCK-8 kit. 1/10 volume of CCK-8 solution was added to each well, incubated in an incubator, and absorbance was measured and recorded at a wavelength of 450nm after 1 h. Experiments were repeated 3 times.

The results are shown in FIG. 4, on MA104 cell models infected by RV-WA strain and RV-SA-11 strain, when Cor is between 7.8 and 125 mu M, the inhibition rate of RV is not obviously increased, the highest inhibition rate is only about 8 percent, and compared with Ribavirin group, the results have no statistically significant difference, so that Cor has no obvious effect of resisting RV adsorption.

(2) Anti-RV synthesis by Cor

100TCID50 virus (virus acting 30mm with 10. Mu.g/mL pancreatin) was added to a 96-well plate of MA104 cells grown to a monolayer, 100. Mu.L per well, and the cells were washed 2 times with PBS before. Setting a normal control group of cells, and adding an equal volume of high-sugar DMEM culture solution. Incubating at 37deg.C with 5% CO ₂ for 2 hr, sucking out virus solution, adding different concentration medicinal liquid and 1mg/mL Ribavirin, respectively, and 100 μl per well; a virus control group was set, and only RV growth maintaining solution was added, 100. Mu.L per well. Incubation at 37℃with 5% CO ₂ was followed. After 48 hours of continuous culture, the cells were assayed using the CCK-8 kit. 1/10 volume of CCK-8 solution was added to each well, incubated in an incubator, and absorbance was measured and recorded at a wavelength of 450nm after 1 h. The virus inhibition rate of the drug was calculated and the experiment was repeated 3 times.

As a result, FIG. 5 shows that on MA104 cell models infected with RV-WA strain and RV-SA-11 strain, the inhibition rates of Cor on RV-WA strain are 68.52%, 53.29% and 50.22% respectively at 15.625-62.5. Mu.M; the inhibition rates of RV-SA-11 strain viruses are 57.38%, 74% and 72.26%, respectively. The inhibition rates of Ribavirin on RV-WA strain and RV-SA-11 virus are 67.42% and 70.64%, respectively, which shows that Cor has the effect of resisting RV biosynthesis.

(3) Direct inhibition of RV by Cor

The drug was mixed with an equal volume of 100TCID50 virus solution (virus was allowed to react with 10. Mu.g/mL pancreatin for 30 min) for 2h. Cells were washed 2 times with PBS and added to 96-well plates grown to monolayer MA104 cells. In the positive control group, ribavirin and RV were subjected to the same procedure as described above, and only the same volume of high-sugar DMEM medium was added to both the normal cell control group and the virus control group. Incubation was performed at 37℃for 2h with 5% CO ₂, then the mixture was aspirated, 100. Mu.L of RV growth maintaining solution was added per well, incubation was continued at 37℃with 5% CO ₂, and after 48h incubation was performed, detection was performed using CCK-8 kit. 1/10 volume of CCK-8 solution was added to each well, incubated in an incubator, and absorbance was measured and recorded at a wavelength of 450nm after 1 h. The virus inhibition rate of the drug was calculated and the experiment was repeated 3 times.

As a result, in the MA104 cell model infected by RV-WA strain and RV-SA-11 strain, the inhibition rate of Cor is not obviously increased at 7.8-125 mu M, the highest inhibition rate of each concentration group of the medicine is only about 10%, and compared with the Ribavirin group, the statistical significance difference exists, so that the Cor has no obvious effect of directly inhibiting RV.

Example 4qPCR detection of expression level of RV structural protein VP6 Gene

(1) Extraction and quantification of Total RNA

① To further verify whether Cor has anti-RV biosynthesis, cell plating was performed in 6-well plates, with 2mL of cell suspension added per well, with a cell density of 1X 10 ⁵ cells/mL. Cor anti-RV biosynthesis is carried out when the cells grow to a monolayer. The incubator was continuously observed, and total RNA was extracted from 16. Mu.M, 32. Mu.M, 64. Mu.M drug group, ribavirin group, control group and RV group according to the Trizol method after 24 hours of incubation. The supernatant was removed first, washed twice with PBS, 1mL of Trizol reagent was added, allowed to stand for 5min, and collected in a 1.5mL enzyme-free EP tube. 200. Mu.L of chloroform was added to the tube, vortexed for 15s, then allowed to stand at room temperature for 3min, centrifuged (4 ℃,12000r/min,15 min), and the centrifuged sample was divided into three layers, i.e., a colorless upper layer, a white middle layer and a red lower layer.

② Carefully aspirate the supernatant into a fresh 1.5mL enzyme-free EP tube (volume about 500. Mu.L), add an equal volume of pre-chilled isopropyl alcohol, shake mix vigorously up and down, stand for 10min at 4deg.C, centrifuge (4deg.C, 12000r/min,10 min).

③ After centrifugation, the bottom of the EP tube is subjected to white precipitation, the supernatant is removed, the precipitation is reserved, 1mL of prepared 75% ethanol solution (prepared by absolute ethanol and enzyme-free water according to the ratio of 3:1) is added, shaking and mixing are carried out uniformly, centrifugation (4 ℃,12000r/min,5 min) is carried out, the supernatant is removed, and the mixture is left at room temperature for 15min-20min and dried.

④ After air-drying, 20. Mu.L of DEPC water was added to the EP tube, and the wall of the tube was gently blown to dissolve RNA. After the RNA concentration of the sample is measured by the NanoDrop micro ultraviolet spectrophotometer, the sample can be directly used for experiments or stored at the temperature of minus 80 ℃ for standby.

(2) Reverse transcription of mRNA

① Genomic DNA removal reaction

A reaction mixture was prepared on ice at a reaction volume of 20. Mu.L according to Evo M-MLV RT KIT WITH GDNA CLEAN for qPCR II instructions. The consumables used in the experiment are all Axygen enzyme-free consumables.

The reaction system for removing genome DNA is shown in Table 1.

TABLE 1 reaction System for removing genomic DNA

Reaction conditions: 42 ℃ for 2min;4 ℃.

*1: The amount of RNA may be added as needed. In a 20. Mu.L reverse transcription system, at most 1. Mu.g total RNA was used; when the probe method is used, at most 2. Mu.g total RNA is used.

② Reverse transcription reaction

The reaction solution was prepared in accordance with Table 2, and the reverse transcription reaction was performed.

TABLE 2 reverse transcription reaction system

Reaction conditions: 15min at 37 ℃; 5sec at 85 ℃;4 ℃.

(3) REAL TIME PCR reaction

The real-time quantitative PCR adopts SYBR Green I fluorescent label to detect VP6 expression levels of a drug group, a Ribavirin group, an N group and an RV group, adoptsThe Green Premix Pro TAQ HS QPCR KIT II kit adopts GAPDH as an internal reference. Real-time PCR amplification reaction system for the experimental Axygen special purpose was prepared according to Table 3, and the reaction solution was prepared on ice for the operation (10. Mu.L of the total reaction system). qPCR reaction conditions are shown in Table 4; the primer sequences are shown in Table 5.

TABLE 3 PCR reaction system

TABLE 4qPCR reaction conditions

TABLE 5 primer sequences

The results are shown in FIG. 7, on MA104 cell models infected by RV-WA strain and RV-SA-11 strain, VP6 is not expressed in the Control group, and compared with the RV group, VP6 gene expression of the Ribavirin group is obviously reduced, and statistically significant difference exists, so that the Ribavirin has the effect of resisting RV. Compared with RV group, the expression level of VP6 is obviously reduced in Cor group at the concentration of 16 mu M,32 mu M and 64 mu M, and statistically significant difference exists, wherein the inhibition of VP6 expression by Cor at 64 mu M is most significant. Cor was shown to exert an anti-RV effect by inhibiting the expression of the RV-VP6 gene.

Conclusion: cor has the effect of resisting RV biosynthesis, has no obvious RV adsorption resistance and direct inhibition effect, and can play the role of resisting RV by inhibiting VP6 gene expression.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended 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 (3)

1. Application of corilagin in preparing medicine for resisting rotavirus is provided.

2. Application of corilagin in preparing medicine for resisting rotavirus biosynthesis is provided.

3. The use according to claim 1 or 2, wherein the rotavirus is RV-WA strain and RV-SA-11 strain.