Application of manna disinfection pill in resisting feline infectious peritonitis virus

Abstract

The invention discloses application of manna disinfection pills in resisting feline infectious peritonitis virus. The effective concentration of the manna disinfection pills is 120 micrograms per milliliter. According to the invention, it is found for the first time that the manna disinfection pill has very small toxicity to cells, can effectively inhibit feline infectious peritonitis virus infection, and has a significant effect in inhibiting the replication of feline infectious peritonitis virus to cells.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to the application of Ganlu Xiaodudan in preparing medicines for resisting feline infectious peritonitis virus. Background Art

[0002] Feline infectious peritonitis (FIP) is a highly contagious disease in cats. It is a fatal, immune-mediated disease in kittens and intact male cats caused by a variant strain of feline coronavirus (FCoV), feline infectious peritonitis virus (FIPV). The most common clinical symptoms are lethargy, loss of appetite, abdominal distension, weight loss, fever, and rough fur. The typical pathological features are fibrinous and granulomatous serositis, protein-rich serosal effusions or granulomatous lesions (pyogranulomas) in the body cavity. FIP can be divided into wet (exudative, non-solid), dry (non-exudative, solid) and mixed types according to whether there is protein effusion in the abdominal and thoracic cavities. Among them, wet FIP ​​is more prevalent than dry and mixed types.

[0003] FCoV belongs to the Coronavirus family and the Coronavirus genus. It is a single-stranded positive-strand RNA virus characterized by a protruding rod-shaped spike on the surface. It mainly encodes four structural proteins (spike protein S, envelope protein E, membrane protein M, and nucleocapsid protein N) and seven non-structural proteins (ORF1a, 1b, 3a, 3b, 3c, 7a, and 7b). FCoV is divided into feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV) according to its pathogenicity. It is generally believed that FIPV is produced by internal mutations of FECV; according to its serotype, it is divided into type I (common in clinical practice, but difficult to culture cells in vitro) and type II (easy to culture cells in vitro). Monocytes and macrophages infected by FIPV have been identified as the main target cells. FIPV can effectively infect and replicate in them, and trigger the activation of these cells to produce a large number of cytokines. Relevant literature reports that the immunity of FIPV is mainly mediated by cell immunity. During FIPV infection, the reduction of peripheral blood lymphocytes plays an important role in the pathogenesis of the virus, and the infection of macrophages enhances systemic symptoms. The treatment of FIP remains a challenge. Currently, there are no approved antiviral drugs or recommended vaccines to treat or prevent infection. Therefore, there is an urgent need to develop effective, broad-spectrum anti-FIPV drugs for the treatment of FIP.

[0004] Traditional Chinese medicine has become a hot topic of research at home and abroad in recent years due to its advantages such as low price, low toxicity and side effects, no drug resistance and broad antiviral spectrum. Chinese herbal polysaccharides are a type of active polysaccharides extracted from Chinese herbal medicines. A large number of domestic and foreign studies have shown that active polysaccharides can stimulate the proliferation of the reticuloendothelial system, macrophages and lymphocytes, and play an important role in immune function regulation and cancer diagnosis and treatment. Licorice, as a traditional Chinese herbal medicine, has high medicinal value. It is ranked first in the herbal medicine section in the Compendium of Materia Medica, before astragalus and ginseng. Ganlu Xiaodudan (GPS) is a water-soluble active polysaccharide isolated and extracted from licorice, and is one of the most important active ingredients in licorice. Current pharmacological studies have shown that Ganlu Xiaodudan has immunomodulatory, antioxidant, anti-tumor, antiviral, anti-cancer, antibacterial and anti-inflammatory activities.

[0005] The antiviral effect of Ganlu Xiaodu Dan is mainly against bovine viral diarrhea virus, bovine HIV virus, adenovirus, coxsackie virus, pseudorabies virus and swine influenza virus. Studies have also confirmed that although Ganlu Xiaodu Dan has a certain inhibitory effect on some viruses, it is relatively weak and cannot achieve an effective inhibitory effect. In addition, Ganlu Xiaodu Dan does not even have an inhibitory effect on some viruses. There are no reports on Ganlu Xiaodu Dan against feline infectious peritonitis virus, and its antiviral effect is still unclear. Therefore, studying the antiviral effect and mechanism of Ganlu Xiaodu Dan against feline infectious peritonitis virus is of great significance for the development of effective anti-feline infectious peritonitis virus drugs. Summary of the invention

[0006] The purpose of the present invention is to provide an application of Ganlu Xiaodu Dan to resist feline infectious peritonitis virus, so as to solve the problems existing in the above-mentioned prior art. The present invention firstly finds that Ganlu Xiaodu Dan has little toxicity to cells, can effectively inhibit feline infectious peritonitis virus infection, and has a significant effect in inhibiting the replication of feline infectious peritonitis virus to cells.

[0007] This solution is achieved through the following technical measures:

[0008] Application of Ganlu Xiaodu Dan in combating feline infectious peritonitis virus.

[0009] The use according to claim 1 is characterized in that the effective concentration of Ganlu Xiaodu Dan is 120 μg / mL.

[0010] Beneficial effects of the present invention:

[0011] The present invention discovers for the first time that the toxicity of Ganlu Xiaodu Dan to cells is very low, and that it can effectively inhibit the infection of feline infectious peritonitis virus, and has a significant effect in inhibiting the replication of feline infectious peritonitis virus to cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1The distribution of APN receptor protein in different tissues of cats with FIP;

[0013] Figure 2 Distribution of ACE2 receptor protein in different tissues of cats with FIP;

[0014] Figure 3 Distribution of DC-SIGN receptor protein in different tissues of cats with FIP;

[0015] Figure 4 Changes in the expression of APN receptors in different tissues of cats before and after FIPV infection;

[0016] Figure 5 Changes in the expression of ACE2 receptors in different tissues of cats before and after FIPV infection;

[0017] Figure 6 Differences in the expression of DC-SIGN receptors in different tissues of cats before and after FIPV infection. DETAILED DESCRIPTION

[0018] In order to clearly illustrate the technical features of this solution, this solution is described below through specific implementation methods and in conjunction with the accompanying drawings.

[0019] 1.ELIS detection of receptor protein content

[0020] 1) Sample addition: Set up blank wells, standard wells, and sample wells. Accurately add 50μL of the standard sample to the ELISA coated plate, add 40μL of sample diluent to the sample well, and then add 10μL of the sample to be tested. Add the sample to the bottom of the ELISA plate well, try not to touch the well wall, and gently shake to mix.

[0021] 2) Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 minutes.

[0022] 3) Liquid preparation: Dilute the 30-fold concentrated washing solution with 30-fold distilled water for later use.

[0023] 4) Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times and pat dry.

[0024] 5) Add enzyme: Add 50 μL of enzyme-labeled reagent to each well, except for the blank well.

[0025] 6) Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 minutes.

[0026] 7) Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times and pat dry.

[0027] 8) Color development: First add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake to mix, and incubate at 37°C in the dark for 10 minutes.

[0028] 9) Termination: Add 50 μL of stop solution to each well. When the blue color turns to yellow, the termination is complete.

[0029] 10) Measurement: Use the blank well as zero setting and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm.

[0030] 11) Calculation: Draw a standard curve with the concentration of the standard as the horizontal axis and the OD value as the vertical axis, and calculate the concentration of the sample.

[0031] 12) Data analysis: The receptor content in the intestine was used as the control group, and GraphpadPrism8 was used for statistical analysis.

[0032] 2. Determination of key receptors for FIPV

[0033] 1) Passaging of FCWF-4 macrophages

[0034] Take a T25 bottle of laboratory fcwf-4 cells, bring it into the clean bench, wash it three times with preheated PBS, add 2ml of trypsin, put it in the incubator for digestion for 3 minutes, take it out, and observe under an inverted microscope that all the adherent cells have fallen off and become round, add 1ml of DMEM containing 2% FBS to stop digestion, transfer the cell suspension to a 15ml centrifuge tube, centrifuge at 800r for 8min, discard the supernatant, add fresh DMEM containing 10% FBS to resuspend, and transfer it to a new T25 cell culture bottle at a subculture ratio of 1:3, put it in a cell culture incubator for culture, and set aside.

[0035] 2) Dissolution of Ubenimex

[0036] Ubenimex was prepared into 0.5, 1, 1.5, 2 and 3 nM solutions according to the instructions and kept in a refrigerator at 4°C until use.

[0037] 3) Ubenimex-treated cells

[0038] Ubenimex was processed into a solution according to the previous step and added to the fcwf-4 cells that had grown into a monolayer in a six-well plate, 200 μL per well, and incubated in the incubator for different times (20 min, 40 min, 60 min and 120 min). The control group added an equal amount of DMEM. Then it was washed three times with PBS, inoculated with FIPV, and the virus solution was discarded after incubation for 90 min. It was washed three times, and the cell maintenance solution was added and cultured for 48 hours. It was placed in a -80 ° C refrigerator and repeatedly frozen and thawed three times for use.

[0039] 4) Primer design

[0040] The APN primer is the same as 3.2.2, and the FCOV primer sequence is as follows:

[0041]

[0042] 5) Extraction of total RNA

[0043] Grinding tissue samples with liquid nitrogen: Write the name of the tissue sample on the cap and wall of a 1.5 ml centrifuge tube, and pre-cool the centrifuge tube, collection spoon, and mortar in liquid nitrogen; take out the mortar, put in the tissue, and pour in about 2 / 3 of the liquid nitrogen to ensure that the tissue is completely immersed in the liquid nitrogen; repeatedly beat the tissue, and crush it in one direction after breaking it up; add liquid nitrogen to gather the powder, stir, and when the liquid nitrogen evaporates completely and the powder turns white, transfer the powder into a pre-cooled centrifuge tube.

[0044] Add 500 μL Trizol to the centrifuge tube, mix thoroughly, and place on ice for 5 min;

[0045] Centrifuge at 12000r in a 4 degree centrifuge for 5 minutes, collect the supernatant, add 1 / 5 Trizol volume (100μL) of chloroform, mix vigorously, and let stand on ice for 15min;

[0046] The upper aqueous phase was drawn into another centrifuge tube (labeled with the sample name in advance), 200 μL of isopropanol was added, mixed, placed on ice for 30 min, and centrifuged at 12,000 r in a 4-degree centrifuge for 10 min;

[0047] A white precipitate can be seen at the bottom of the tube. Discard the supernatant, add 1 ml of pre-cooled 75% ethanol, and centrifuge at 12,000 r / min in a 4-degree centrifuge.

[0048] Discard the supernatant, dry and add 20 μL DEPC water to dissolve

[0049] 6) Reverse transcription

[0050] According to the instructions of the EvoM-MLV Reverse Transcription Reagent Premix (for qPCR) kit, the extracted RNA was reverse transcribed as follows (performed on ice):

[0051] Centrifuge 5×EvoMMLVRTMasterMix and add 2 μL of the reagent to the eight-tube strip.

[0052] Then add 1 μL of extracted RNA.

[0053] Add 7 μL RNase free water to make the total system 10 μL, vortex and centrifuge in a handheld centrifuge to collect all the solution at the bottom of the centrifuge tube.

[0054] Reverse transcription was performed according to the program of 37°C, 15 min; 85°C, 5 s, and finally stored at 4°C.

[0055] 7) Fluorescence quantitative PCR detection of FIPV proliferation under different treatment conditions

[0056] Preparation of fluorescent quantitative PCR system:

[0057]

[0058] Mix well, centrifuge, and divide into 8-well tube strips or 96-well PCR plates. Perform 3 parallel PCR reactions for each gene in each sample.

[0059] Set up the reaction program

[0060]

[0061]

[0062] The data were analyzed by comparative Ct method and Graphpad Prism 8 statistical analysis software was used for data statistical analysis.

[0063] According to the effect of Ganlu Xiaodudan on the viability of fcwf-4 cells detected by CCK8 in the laboratory in the early stage, 120 μg / ml was selected and added to the cells 2 hours after infection. Total RNA was then extracted to detect the expression of APN after treatment with Ganlu Xiaodudan.

[0064] Differences in the distribution and expression of APN, ACE2, and DC-SIGN receptor proteins in different tissues of cats after FIPV infection

[0065] The results are as follows Figures 1 to 3 The results showed that the expression of APN, ACE2 and DC-SIGN receptors could be found in nine tissues of sick cats, including heart, liver, spleen, lung and kidney. APN was highest in the stomach and lowest in the peritoneum. ACE2 was highest in the heart and lowest in the lung. DC-SIGN was highest in the liver and lowest in the spleen.

[0066] Depend on Figure 4 It can be seen that after infection, except for the spleen, the expression of APN increased, and the expression of heart, brain and stomach increased significantly; Figure 5 It can be seen that, like APN, ACE2 expression increased in all organs except the spleen after infection, and the expression in the heart, brain, and stomach increased significantly. Figure 6 It can be seen that the content of DC-SIGN in the heart, liver, stomach, kidney and intestine increased after infection, and the content in the heart, liver and intestine increased significantly, while the expression in other tissues decreased. This indicates that FIPV infection may have some kind of stimulating effect on the body's receptors.

[0067] Technical features not described in the present invention can be implemented by existing technologies and will not be described in detail here. The present invention is not limited to the above specific embodiments, and changes, modifications, additions or substitutions made by ordinary technicians in the field within the essential scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. Application of Ganlu Xiaodu Dan in preventing and treating feline infectious peritonitis virus.

2. The use according to claim 1, characterized in that: The effective concentration of the Ganlu Xiaodu Dan is 120 μg / mL.

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