Application of panax japonicus polysaccharide in preparation of medicine for resisting porcine reproductive and respiratory syndrome virus

Abstract

The invention discloses application of panax japonicus polysaccharide in preparation of drugs for resisting porcine reproductive and respiratory syndrome virus. According to the application disclosed by the invention, the regulation and control effect of the panax japonicus polysaccharide on the replication of the porcine reproductive and respiratory syndrome virus (PRRSV) is studied, and the fact that the panax japonicus polysaccharide can effectively inhibit the replication of the PRRSV on a Marc-145 cell is found for the first time. On the basis, the invention provides a new application of the panax japonicus polysaccharide in preparation of the medicine for resisting the porcine reproductive and respiratory syndrome virus, and preferably, the concentration of the panax japonicus polysaccharide is 12.5 mu g / mL-100 mu g / mL. The research on the antiviral activity of the panax japonicus polysaccharide is enriched, and meanwhile, a new technical means is provided for research and clinical application of anti-PRRSV drugs.

Description

TECHNICAL FIELD

[0001] The application relates to a new anti-porcine reproductive and respiratory syndrome virus drug, in particular to application of Panax japonicus polysaccharide in preparation of the anti-porcine reproductive and respiratory syndrome virus drug. BACKGROUND

[0002] Porcine reproductive and respiratory syndrome (PRRS) is an acute infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV) infection, which is distributed worldwide and has caused great losses to the global pig industry. The main symptoms are reproductive disorders in sows, such as abortion, stillbirth and mummification, and severe respiratory diseases and slow growth in piglets. PRRSV belongs to the order Nidovirales, family Arteriviridae and genus Arterivirus, and is a single-stranded positive-sense RNA virus. PRRSV has the characteristics of rapid variation, multiple lineages and easy recombination of strains, and the cross protection between different lineages is not strong, which makes the prevention and control of vaccine immunization increasingly difficult. It is of great significance to develop safe and effective anti-PRRSV drugs for epidemic prevention and control.

[0003] Chinese herbal medicine has many advantages in antiviral aspects, such as good effect, low toxicity and low cost. Panax japonicus (scientific name: Panax japonicus (T.Nees) C.A.Mey.), also known as "bamboo joint ginseng", "seven leaf" and "white three seven" in folk, belongs to the Araliaceae Panax genus and was first recorded in Zhao Xuekai's book "Bai Cao Jing". Panax japonicus tastes sweet and slightly bitter, and is warm in nature. It has a wide range of pharmacological activities and multiple effects, such as preventing ulcer, protecting liver, promoting gastrointestinal peristalsis, sedation and sleep, anticonvulsant, fatigue elimination, blood lipid reduction, cardiovascular and cerebrovascular protection, immune enhancement, analgesia, anti-inflammatory, antitumor and anti-aging. Panax japonicus contains saponins, sugar components, amino acids, volatile oils, nucleosides and inorganic salts. Phytochemical studies have shown that the main active ingredients of Panax japonicus are saponins from Panax japonicus and Panax japonicus polysaccharide (PSPJ). The total saponin content in the root of Panax japonicus is as high as 15%, which is 2 to 7 times that of ordinary ginseng. The efficacy of Panax japonicus mainly comes from the four or five ring triterpene saponins it contains, and polysaccharide is a complex macromolecular sugar substance with the general formula (C6H 12 O6) x . SUMMARY

[0004] The present application aims to provide a new use of polysaccharide from Panax japonicus (PSPJ) in preparing a medicine for resisting porcine reproductive and respiratory syndrome virus (PRRSV).

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical means

[0006] The present application explores the regulation of PRRSV replication by polysaccharide from Panax japonicus (PSPJ) at different concentrations (12.5 μg / mL-100 μg / mL). The results show that PSPJ at the measured concentration range can inhibit PRRSV replication, and the inhibitory effect of the drug on the virus gradually increases with the increase of the concentration of PSPJ. This shows that PSPJ can effectively inhibit the proliferation of PRRSV, and the inhibitory effect is concentration-dependent, and the antiviral effect of 100 μg / mL PSPJ is the best. Marc-145 cells inoculated with LN1701 are treated with 50 μg / mL and 100 μg / mL PSPJ with high concentration. After 48 hours of culture, the effect of the drug on the expression amount of PRRSV N protein is detected by IFA test. The results show that compared with the control group, the expression amount of N protein gradually decreases with the increase of the concentration of PSPJ, and the gradually decreasing trend is positively correlated with the concentration of PSPJ. This shows that PSPJ can inhibit the proliferation of PRRSV in Marc-145 cells in a concentration-dependent manner. The effect of PSPJ on the proliferation curve of PRRSV is further explored. Marc-145 cells inoculated with LN1701 are treated with 100 μg / mL PSPJ, and the cells are collected at 12 h, 24 h, 36 h and 48 h after infection for TCID 50 Test. The results show that PSPJ treatment can significantly inhibit virus replication at 24 h, 36 h and 48 h after infection, with a 10-fold decrease in virus titer at 24 h, an 8.91-fold decrease at 36 h, and a 3.76-fold decrease at 48 h.

[0007] On the basis of the above research, the present application proposes the use of polysaccharide from Panax japonicus in preparing a medicine for resisting porcine reproductive and respiratory syndrome virus.

[0008] Preferably, the polysaccharide from Panax japonicus can significantly inhibit the replication of PRRSV in Marc-145 cells.

[0009] Preferably, the concentration of the polysaccharide from Panax japonicus is 12.5 μg / mL-100 μg / mL.

[0010] Compared with the prior art, the present application has the following beneficial effects:

[0011] The application explores the regulation effect of PSPJ on PRRSV replication, and for the first time finds that PSPJ can effectively inhibit the replication of PRRSV on Marc-145 cells. On this basis, the application proposes a new application of PSPJ in preparing an anti-porcine reproductive and respiratory syndrome virus drug. The application enriches the research on the antiviral activity of PSPJ, and also provides a new technical means for the research and clinical application of anti-PRRSV drugs. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 For comparison of the viability of Marc-145 cells added with different concentrations of PSPJ;

[0013] Figure 2 For detecting the influence of different doses of PSPJ on PRRSV replication (ORF7 mRNA level);

[0014] Figure 3 For detecting the influence of different doses of PSPJ on PRRSV replication (N protein expression amount);

[0015] Among them, A: IFA identifies the influence of different concentrations of PSPJ on the expression of PRRSV N protein; B: fluorescence quantity statistics result;

[0016] Figure 4 For detecting the influence of PSPJ on the proliferation curve of PRRSV. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the present application will be described clearly and completely below in combination with the drawings in the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0018] The experimental methods involved in the following embodiments are all conventional methods in the art if not specifically mentioned, for example, the experimental manuals in the art can be referred to, or the conditions suggested in the manufacturer's instructions can be followed.

[0019] The experimental materials and reagents involved in the following embodiments can be obtained from commercial channels if not specifically mentioned.

[0020] Example 1 Application of PSPJ in Anti-porcine reproductive and respiratory syndrome virus

[0021] 1 Material

[0022] 1.1 Source of cells and viruses for test

[0023] Marc-145 cells were preserved in the laboratory and cultured with DMEM medium (10% FBS); PRRSV-LN1701 strain was isolated by the laboratory.

[0024] 1.2 Main instruments

[0025] Table 1 Main instruments

[0026]

[0027]

[0028] 1.3 Main reagents and their preparation

[0029] 1.3.1 Main reagents

[0030] Table 2 Main reagents

[0031]

[0032] 1.3.2 Preparation of reagents

[0033] (1) 10% DMEM cell culture solution: add 10% fetal bovine serum to DMEM cell culture solution, mix well, and store at 4°C.

[0034] (2) 5 mg / mL MTT developing solution: dissolve 0.5 g MTT in 100 mL PBS buffer under light protection, filter sterilization, and store in the dark.

[0035] (3) PBS phosphate buffer: weigh 7.9 g NaCl, 0.2 g KCl, 0.24 g KH2PO4 (or 1.44 g Na2HPO4), and 1.8 g K2HPO4, and dissolve them in 800 mL distilled water. Adjust the pH of the solution to 7.4, and add distilled water to make up to 1 L. After making up, perform dispensing and high-pressure sterilization. Store the sterilized solution at 4°C, and the longest storage period is one month.

[0036] 2 Methods

[0037] 2.1 Culture of Marc-145 cells

[0038] 2.1.1 Thawing of Marc-145 cells

[0039] (1) Take Marc-145 cells out of liquid nitrogen and place them in a 37°C constant temperature water bath for rapid thawing.

[0040] (2) After complete thawing, centrifuge in a low-speed centrifuge at 1000 rpm for 5 min, and discard the supernatant.

[0041] (3) Add about 1 mL of 10% cell culture solution to resuspend the cells and transfer them to a 25T cell bottle, and then add the culture medium to 5 mL. Place the cells in the incubator and culture them at 37°C and 5% CO2.

[0042] 2.1.2 Subculture of Marc-145 cells

[0043] (1) Observe the cell growth state every day, and when the Marc-145 cells cultured in the cell bottle grow into a monolayer, subculture them.

[0044] (2) Use a pipette to aspirate and discard the culture solution in the bottle, and add 1 mL of trypsin to the 25T cell bottle to digest the cells for 1-3 min. When the cells become spherical in shape and the gap between the cells widens, add 1 mL of growth medium to terminate the digestion process.

[0045] (3) After the digestion is terminated, use a pipette to blow the cells thoroughly until they are completely detached, then add an appropriate amount of 10% cell culture solution to resuspend the cells and continue the culture.

[0046] 2.1.3 Cryopreservation of Marc-145 cells

[0047] Subculture the Marc-145 cells that have grown into a monolayer by trypsinization, and after the digestion is completed, terminate the digestion by adding growth medium. Transfer the cell-containing solution to a low-speed centrifuge and centrifuge it at 1000 rpm for 5 min, then discard the supernatant. After adding the cryopreservation solution, dispense it carefully and label it. First, place the cryopreservation tube in a -80°C freezer overnight, and then transfer it to liquid nitrogen for long-term storage.

[0048] 2.2 Cytotoxicity assay of PSPJ

[0049] 2.2.1 Dissolution of PSPJ

[0050] Dissolve PSPJ in DMSO to achieve an initial concentration of 100 mg / mL, and then perform serial dilutions for the determination of the cytotoxicity of the drug.

[0051] 2.2.2 Cytotoxicity assay of PSPJ

[0052] (1) Cell inoculation and treatment: evenly distribute Marc-145 cells in a 96-well plate, and after they adhere, add 6 concentration gradients of PSPJ solution (100 μL / well), and the control group (Mock) is added with an equal amount of cell maintenance solution (100 μL / well), with 4 replicate wells for each concentration.

[0053] (2) Culture and observation: place the culture plate in a 37°C, 5% CO2 incubator for 72 h, and observe the cell state (such as whether it detaches or disintegrates) daily.

[0054] (3) MTT detection: Discard the culture medium, add 25 μL MTT solution to each well, and continue to incubate for 4 h. Discard the MTT solution, add 150 μL DMSO to dissolve the formazan crystals, and stand at 37 °C for 30 min.

[0055] (4) Data analysis: Determine the absorbance (OD value) at 490 nm using a microplate reader. Cell viability (%) = (OD value of the test group / OD value of the control group) x 100%. Maximum safe concentration (MNTC): the highest drug concentration with a cell viability of ≥ 90%.

[0056] 2.3 Determination of the effect of different doses of polysaccharides from Panax japonicus on PRRSV

[0057] (1) Indirect immunofluorescence (IFA)

[0058] a. Virus inoculation and infection

[0059] Inoculate the 5th passage virus supernatant (1 MOI) of LN1701 strain to a 24-well plate with Marc-145 cells, and incubate for 2 h, shaking gently every 15 min. Discard the supernatant, wash twice with PBS, and replace the maintenance medium. Continue to incubate for 48 h until the cytopathic effect (CPE) is obvious.

[0060] b. Cell fixation

[0061] Discard the culture medium, and wash the cells with pre-cooled PBS 3 times. Add 200 μL of cell fixation solution to each well, and fix for 10 min. Discard the fixation solution, and wash with PBS 3 times (5 min each time). Add 0.1% Triton X-100 (400 μL / well), and incubate at room temperature for 10 min (on a shaker).

[0062] c. Blocking and antibody incubation

[0063] Block with 2% BSA for 30 min (at room temperature), and wash with PBS 3 times. Add PRRSV N protein monoclonal antibody (1:1000), and incubate at 4 °C for 12 h. Wash with PBS 3 times. Add FITC-labeled secondary antibody (1:200, 150 μL / well), and incubate in the dark for 1 h. Wash with PBS 3 times.

[0064] d. Result observation

[0065] Place the culture plate under an inverted fluorescence microscope to observe the fluorescence, and calculate the virus titer.

[0066] (2) Fluorescence quantitative PCR (RT-qPCR)

[0067] After freeze-thawing 3 times, the sample to be tested was centrifuged to obtain the supernatant, and the Trizol method was used for RNA extraction. Then, RT-qPCR detection was performed, and the required system components (Table 4) were added, mixed, centrifuged briefly, and placed in a fluorescence quantitative PCR instrument. The program was set according to Table 5, and the CT value was exported after the test was completed. The data were analyzed by 2 －△△CT method.

[0068] Result determination: the internal parameter value is uniform, and the detection sample curve is a standard S-shaped curve. The detection result CT value ≤ 38 is reliable.

[0069] Table 3 Primers required for RT-qPCR reaction

[0070]

[0071] Table 4 RT-qPCR reaction system

[0072]

[0073] Table 5 Fluorescence quantitative PCR reaction conditions

[0074]

[0075] (3) TCID 50 Test

[0076] a. Virus dilution and inoculation

[0077] When the cell density of the 96-well plate reached 70-80%, the -80℃ frozen virus solution was thawed in an ice bath, and 10-fold gradient dilution method was used for continuous dilution of 7 concentrations (100 μL virus solution + 900 μL maintenance solution). Each dilution was set with 8 repeated wells, and each well was inoculated with 100 μL of diluted virus solution. Negative control wells were also set up.

[0078] b. Culture and fixation

[0079] 37℃, 5% CO2 culture for 48h, discard the culture medium, wash with PBS for 3 times, add 50 μL of fixing solution to each well for 10 min.

[0080] c. Immunofluorescence staining

[0081] After washing with PBS, 0.1% Triton X-100 was treated for 10 min, 2% BSA was blocked for 30 min, primary antibody (1:1000) was incubated at 4℃ for 12h, and FITC-labeled secondary antibody (1:200) was incubated in the dark for 1h.

[0082] d. Result determination

[0083] Place the culture plate under an inverted fluorescence microscope to observe fluorescence, and calculate the virus titer.

[0084] 3 Results and analysis

[0085] 3.1 Maximum safe concentration of PSPJ on Marc-145 cells

[0086] To explore the effect of different concentrations of PSPJ on the activity of Marc-145 cells, PSPJ was diluted to 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL and 200 μg / mL, respectively. The MTT method was used to detect the activity of cells treated with the above concentrations to determine the maximum safe concentration of PSPJ on Marc-145 cells. The results are shown in Table 1. Figure 1 As shown in the table, there was no difference in cell activity between the 100 μg / mL and below concentrations of PSPJ and the control group after acting on Marc-145 cells. When the concentration of PSPJ increased to 200 μg / mL, the cell activity was less than 90%, which indicated that 100 μg / mL and below concentrations of PSPJ had no significant effect on the activity of Marc-145 cells.

[0087] 3.2 Effect of different doses of PSPJ on PRRSV replication

[0088] To evaluate the effect of PSPJ on PRRSV replication, RT-qPCR, IFA and TCID 50 The effect of PSPJ on the level of PRRSV ORF7 gene, the expression of N protein and the viral titer was analyzed, as follows:

[0089] Marc-145 cells were inoculated with LN1701 and treated with 12.5 μg / mL, 25 μg / mL, 50 μg / mL and 100 μg / mL of PSPJ. After 48 h, cell samples were collected and the level of PRRSV ORF7 gene in each group was detected by RT-qPCR. The results are shown in Table 2. Figure 2 As shown in Table 2, different concentrations of PSPJ could inhibit the level of PRRSV ORF7 gene, and the inhibitory effect of the drug on the virus gradually increased with the increase of the concentration of PSPJ. This indicated that PSPJ could effectively inhibit the proliferation of PRRSV, and the inhibitory effect was concentration-dependent, with the best inhibitory effect at 100 μg / mL of PSPJ.

[0090] Marc-145 cells inoculated with LN1701 were treated with 50 μg / mL and 100 μg / mL of PSPJ. After 48 h of culture, the effect of the drug on the expression of PRRSV N protein was detected by IFA. The results are shown in Table 3. Figure 3As shown, compared with the control group, the expression of N protein gradually decreased with the increase of the concentration of PSPJ, and this gradually decreasing trend was positively correlated with the concentration of PSPJ. This further confirmed that PSPJ could inhibit the proliferation of PRRSV in Marc-145 cells, and this inhibitory effect was concentration-dependent.

[0091] Marc-145 cells were inoculated with LN1701 and then treated with 100 μg / mL of PSPJ, and the cells were collected at 12 h, 24 h, 36 h and 48 h after infection to detect the effect of PSPJ on the proliferation curve of the virus. The results are shown in Figure 4 As shown, the PSPJ treatment group could significantly inhibit virus replication at 24 h, 36 h and 48 h after infection, with a 10-fold decrease in virus titer at 24 h, an 8.91-fold decrease at 36 h, and a 3.76-fold decrease at 48 h.

[0092] 4 Summary

[0093] 4.1 The maximum safe concentration of PSPJ on Marc-145 cells is 100 μg / mL.

[0094] 4.2 PSPJ can significantly inhibit the replication of PRRSV in Marc-145 cells, and the inhibitory effect shows a clear concentration gradient dependence (12.5 μg / mL-100 μg / mL).

Claims

1. The use of polysaccharides from Panax japonicus in the preparation of a medicine for resisting Porcine reproductive and respire tory syndrome virus (PRRSV).

2. Use according to claim 1, wherein The polysaccharides from Panax japonicus can significantly inhibit the replication of PRRSV in Marc-145 cells.

3. The use according to claim 1, wherein The concentration of the polysaccharides from Panax japonicus is 12.5 μg / mL-100 μg / mL.

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