Application of tupistra chinensis in preparation of anti-HSV-1 virus medicine

Abstract

The invention provides application of tupistra chinensis or an extract thereof in preparation of drugs for preventing and / or inhibiting and / or inactivating HSV-1 viruses. In-vivo and in-vitro experiments prove that tupistra chinensis has an anti-HSV-1 virus effect, can inactivate the HSV-1 virus or inhibit the adhesion of the HSV-1 virus to cells, and has an unobvious replication effect on virus infection; besides, tupistra chinensis can inhibit virus splitting infection and latent reactivation infection, has good antiviral activity, good curative effect, high safety and wide application, and has comprehensive development and application prospects of new drugs for preventing and treating viral infection diseases such as HSV-1 and the like.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to the use of Rohdea chinensis in the preparation of drugs against HSV-1 virus. Background Art

[0002] HSV-1 (Herpes Simplex Virus Type 1) is a neurotropic virus widely prevalent in humans, mainly transmitted through the oral cavity. After HSV-1 infection, it can cause blisters or ulcers on the skin or mucosa, usually manifested as herpes in the oral cavity or lips. This virus is highly contagious and can persist in the host for a long time. After primary infection, HSV-1 enters a dormant state in the ganglia. When the host's immunity declines or is stimulated by other factors, the virus will reactivate, resulting in recurrent infections. HSV-1 infection not only brings physical pain to patients, but may also cause psychological stress, seriously affecting the quality of life of patients.

[0003] Currently, the first-line clinical treatment plan for HSV-1 infection mainly relies on acyclovir drugs. These drugs play an antiviral role by inhibiting the viral DNA polymerase, thereby preventing virus replication. However, acyclovir drugs have the following limitations: (1) Drug resistance problem: Long-term use of acyclovir may lead to drug resistance of HSV-1 virus, resulting in a decline in the treatment effect, increasing the treatment cost and difficulty; (2) Toxic and side effects: Long-term taking of acyclovir may cause a series of adverse reactions, including joint pain, diarrhea, headache, nausea, vomiting, dizziness, etc. In addition, rapid intravenous injection of a large amount of acyclovir may cause nephrotoxicity, manifested as increased levels of blood urea nitrogen and creatinine, crystal precipitation in the renal tubules, hematuria and other symptoms; (3) Unable to eliminate latent virus: Acyclovir drugs are ineffective against HSV-1 virus that has already latent in the ganglia, and can only relieve herpes skin lesions, and cannot cure HSV-1 infection.

[0004] Rohdea chinensis (Baker) N. Tanaka is a plant with a long medicinal history. Its rhizomes and leaves are widely used in traditional Chinese medicine for clearing heat and detoxifying, expelling wind and removing dampness, dispersing stasis and relieving pain, etc. In recent years, modern pharmacological studies have found that the extracts of Rohdea chinensis contain a variety of bioactive substances, such as steroidal saponin compounds, polysaccharides, etc. These components show significant activities in anti-inflammatory, antibacterial, antitumor and other aspects. For example, the extracts of Rohdea chinensis have good inhibitory effects on Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, etc. In addition, Rohdea chinensis polysaccharide also has antioxidant activity and strong antibacterial and bactericidal abilities. However, there is currently no research report on the application of Rohdea chinensis in the treatment of HSV-1 virus. Summary of the Invention

[0005] The purpose of the present invention is to provide the use of open-mouthed arrow in the preparation of anti-HSV-1 virus drugs. Open-mouthed arrow increases the maturation of DC cells in mice, thereby enhancing the DC cell antigen presentation ability, promoting CD8+T cell differentiation, promoting the secretion of IFN-γ and CXCL1, and inhibiting the immune escape of HSV-1 virus to exert an antiviral effect.

[0006] In a first aspect, the present invention provides the use of Radix Eupatorii or an extract thereof in the preparation of a drug for preventing and / or inhibiting and / or inactivating HSV-1 virus.

[0007] In some embodiments of the present invention, the extract is selected from water extracts.

[0008] In the present invention, the opening arrow is selected from the group consisting of opening arrow, umbrella-column opening arrow, long-column opening arrow, long-stem opening arrow, Yunnan opening arrow, tube-flower opening arrow, tooth-petal opening arrow, disc-flower opening arrow, Emei opening arrow, long-spike opening arrow, Liangshan opening arrow, sword-leaf opening arrow and Mabian opening arrow.

[0009] There are about 12 species of Tupistra in China, including Tupistra chinensis, Tupistra fungilliformis, Tupistra grandistigma, Tupistra longipedunculata, Tupistra yunnanensis, Tupistra delavayi, Tupistra fimbriata, Tupistra tui, Tupistra omeiensis, Tupistra longispica, Tupistra liangshanensis and Tupistra ensifolia. These species are mostly distributed in provinces and regions south of the Yangtze River in China, and are commonly found in the shade and wet places under forests, beside streams or roadsides at an altitude of 1000-2000 meters.

[0010] In some specific embodiments of the present invention, the open arrow is a horse-edge open arrow.

[0011] Rohdea mabianensis H.X.Yan & J.C.Guo is a plant belonging to the genus Rohdea of the family Asparagaceae. This plant was initially accidentally discovered during a survey of folk antiviral drugs in Mabian, Sichuan Province, China. After detailed morphological comparison and phylogenetic analysis, it was confirmed as a new species.

[0012] In the present invention, the water extract of Rohdea japonica refers to the extract obtained by extracting Rohdea japonica with water, and the extraction methods include but are not limited to conventional extraction methods such as leaching, heat reflux, and ultrasonic extraction.

[0013] In a specific embodiment of the present invention, the method of leaching is used to extract the anti-HSV-1 virus active ingredient from Rohdea japonica, and the extraction conditions are a liquid-to-solid ratio of 0.1 - 10 mL / g. -1 。

[0014] In the present invention, the medicinal concentration of the water extract of Rohdea japonica is 0.01 - 1000 μg / mL.

[0015] In some specific embodiments of the present invention, the medicinal concentration of the water extract of Rohdea japonica is 7.5 - 500 μg / mL.

[0016] In the present invention, the drug is a drug that regulates the expression of at least one gene or protein among ICP0, VP16, gD, IKKβ, and NF-κB.

[0017] In some specific embodiments of the present invention, the drug is a drug that inhibits the expression of at least one gene or protein among ICP0, VP16, gD, IKKβ, and NF-κB.

[0018] In a second aspect, the present invention provides an immunomodulator using the Rohdea japonica or its extract as an active substance.

[0019] In the present invention, the immunomodulator can increase the number of at least one type of immune cell among phagocytes, lymphocytes, eosinophils, basophils, natural killer cells, and mast cells.

[0020] In some specific embodiments of the present invention, the immunomodulator can increase the number of phagocytes and lymphocytes.

[0021] In some specific embodiments of the present invention, the immunomodulator can increase the number of DC cells, CD4 + 、CD8 + T cells.

[0022] In the present invention, the protection of the host (an individual infected with HSV-1 virus) against infection is mainly mediated by the immune system; the skin, as a barrier organ, is one of the first lines of defense against pathogens. Under steady state, the skin-resident immune system includes macrophages, dendritic cells (DCs), mast cells, and subsets of T cells. These cells act as sentinels and respond to body injury or pathogens by inducing the recruitment of additional immune cells through the production of a strong inflammatory response. Skin DCs absorb foreign antigens and present them to naive T cells in the skin-draining lymph nodes (dLNs), thereby initiating an adaptive T cell response, promoting the differentiation of CD8+ T cells, promoting the secretion of IFN-γ and CXCL1, and inhibiting the immune escape of HSV-1 virus to exert an antiviral effect.

[0023] In a third aspect, the present invention provides a pharmaceutical composition comprising the Tupistra chinensis or its extract and a pharmaceutically acceptable excipient.

[0024] In a fourth aspect, the present invention provides a method for preparing the above-mentioned drug, which comprises the following steps: extracting the Tupistra chinensis by water extraction method, and adding pharmaceutically common excipients or auxiliary components to the extract to obtain the drug.

[0025] In the present invention, "pharmaceutically acceptable excipient" refers to a material, composition or medium, such as a liquid or solid filler, diluent, carrier, solvent or coating material, which can be used to carry or transport the target component from one organ or part of the body to another organ or part of the body. An "acceptable" excipient means that it is compatible with other components in the composition. The pharmaceutical preparation comprises the Tupistra chinensis or the aqueous extract of Tupistra chinensis described in the present invention, and a pharmaceutically acceptable carrier. The above excipients can be solid, semi-solid or liquid diluents, creams or capsules.

[0026] In some specific embodiments of the present invention, the preparation is an oral preparation, an injection or a topical preparation.

[0027] In some specific embodiments of the present invention, the oral preparation is an ointment, a pill, an oral liquid, a powder, a tablet, a granule or a capsule.

[0028] In some specific embodiments of the present invention, the topical preparation is a solution, a lotion, a liniment, an ointment, a plaster, a paste or a patch.

[0029] In some specific embodiments of the present invention, the injection is a solution, a powder or a tablet.

[0030] In a fifth aspect, the present invention further provides a gel for preventing and / or inhibiting and / or inactivating HSV-1 virus, with the Tupistra chinensis or its extract as the active substance.

[0031] In the present invention, the inhibition includes but is not limited to: inhibiting the adsorption of the virus, inhibiting the replication of the virus, inhibiting the activity of the virus, inhibiting the immune escape of HSV-1 virus, and the like.

[0032] In a sixth aspect, the present invention also provides a method for preventing and / or treating HSV-1 virus infection, comprising: administering / dispensing an effective amount of Tupistra chinensis Bak. and / or an extract of Tupistra chinensis Bak. to an individual infected with HSV-1 virus; the administering / dispensing can be by systemic administration or by local administration.

[0033] In some specific embodiments of the present invention, the pharmaceutical mode at least comprises the following steps: spraying an effective concentration of Tupistra chinensis Bak. or an aqueous extract of Tupistra chinensis Bak. on the air or an individual to be used as a disinfectant to prevent or treat virus infection. The concentration of Tupistra chinensis Bak. or the aqueous extract of Tupistra chinensis Bak. in the preparation used is 0.01 - 100000 μg / mL.

[0034] In some specific embodiments of the present invention, the pharmaceutical mode at least comprises the following steps: locally spraying or using a dressing on an individual to administer an effective concentration of Tupistra chinensis Bak. or an aqueous extract of Tupistra chinensis Bak. to treat HSV-1 virus infection or a disease associated with HSV-1 virus infection. The preparation used can be a biocompatible material, which comprises a biocompatible matrix, and the gel or ointment-like Tupistra chinensis Bak. or the aqueous extract of Tupistra chinensis Bak. is distributed in or on the surface of the matrix. In the preparation used, the concentration of Tupistra chinensis Bak. or the aqueous extract of Tupistra chinensis Bak. can be 0.01 - 100000 μg / mL.

[0035] In the present invention, the term "treatment" refers to a preventive (e.g., prophylactic medication), curative or palliative treatment. Specifically, treatment herein refers to administering or dispensing the Tupistra chinensis Bak. / aqueous extract of Tupistra chinensis Bak. described in the present invention or a pharmaceutical composition comprising this Tupistra chinensis Bak. or the aqueous extract of Tupistra chinensis Bak. to an individual who may have a medical disorder, symptoms associated with the disorder, secondary diseases or abnormalities of the disorder, or is prone to suffering from the above-mentioned disorder, in order to partially or completely relieve, improve, alleviate one or more symptoms or characteristics of a specific abnormality and / or disease condition, or delay its occurrence, hinder its progression, reduce its severity and / or reduce the incidence rate. Treatment can also be carried out on an individual who has not yet shown signs of a disease, abnormality and / or disease condition and / or an individual who shows early signs, in order to reduce the risk of developing pathological changes associated with the disease, abnormality and / or disease condition. In the present disclosure, the signs of the disease, abnormality and / or disease condition refer to virus infection caused by the virus, and signs of diseases, abnormalities and / or disease conditions associated with or derived from the virus infection. In a preferred embodiment, the Tupistra chinensis Bak. / aqueous extract of Tupistra chinensis Bak. proposed herein can be used to inhibit virus replication in vitro and in vivo. Therefore, the treatment method proposed herein can substantially eradicate the pathogen from the host organism, such that the virus cannot be detected in the host body.

[0036] In the present invention, an "individual" refers to a mammal, including a human, who can receive the Tupistra chinensis or the aqueous extract of Tupistra chinensis or the pharmaceutical composition described in the present invention. Unless otherwise specified, the terms "administer" and "administered" can be used interchangeably herein, and they refer to providing the Tupistra chinensis, the aqueous extract of Tupistra chinensis or the pharmaceutical composition of the present invention to an individual in need of treatment.

[0037] In the present invention, an "effective amount" refers to an amount of Tupistra chinensis and / or the aqueous extract of Tupistra chinensis and / or the pharmaceutical composition sufficient to elicit the desired therapeutic response. The effective amount of the agent does not have to be capable of curing the disease or disorder, but can delay, impede or prevent the occurrence of the disease or disorder, or can alleviate the symptoms associated with the disease or disorder. The therapeutically effective amount can be divided into one, two or more doses and administered one, two or more times in a suitable dosage form within a specified period. The specific therapeutically effective amount or sufficient dose depends on various factors, such as the specific condition to be treated, the physiological condition of the individual (e.g., the weight, age or sex of the individual), the mammalian or animal type being treated, the duration of treatment, the nature of concurrent treatments (if any), and the specific formulation and structure of the active ingredient used. The effective amount can be expressed in any suitable manner, for example, it can be expressed as the total weight of the aqueous extract of Tupistra chinensis (e.g., expressed in grams, milligrams or micrograms) or as the ratio of the aqueous extract of Tupistra chinensis to body weight (e.g., expressed as milligrams per kilogram of body weight mg / kg).

[0038] In the present invention, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range. Or, the term "about" represents that the actual value falls within the acceptable standard error of the mean, depending on the consideration of those with ordinary knowledge in the technical field to which the present invention pertains. Except for experimental examples, or unless otherwise clearly stated, it is understood that all ranges, amounts, values and percentages used herein (e.g., used to describe the amount of materials, the length of time, temperature, operating conditions, quantity ratios and others similar) are modified by "about". Therefore, unless otherwise stated to the contrary, the numerical parameters disclosed in this specification and the appended patent claims are approximate values and can be changed as needed. At least these numerical parameters should be understood as the values indicated by the significant digits and obtained by applying the general rounding method. Here, a numerical range is expressed as from one endpoint to the other endpoint or between two endpoints; unless otherwise stated, the numerical ranges described herein all include the endpoints.

[0039] The beneficial effects of the present invention are as follows:

[0040] (1) The aqueous extract of Tupistra chinensis in the present invention can inhibit the lytic infection and latent reactivation infection of viruses in vitro and has good antiviral activity;

[0041] (2) The extract of Tupistra chinensis in the present invention can enhance the antigen presentation ability of DC cells by increasing the maturation of DC cells in mice, promote the differentiation of CD8 + T cells, promote the secretion of IFN-γ and CXCL1, and inhibit the immune escape of HSV-1 virus to play an antiviral role. Description of the Drawings

[0042] Figure 1 T lymphocyte gating strategy;

[0043] Figure 2 DC cell gating strategy;

[0044] Figure 3 Total ion chromatogram of the sample solution: (A) Positive ion mode; (B) Negative ion mode;

[0045] Figure 4 RCAE inhibits HSV-1 infection in Vero cells: (A) Effect of RCAE on the viability of Vero cells; (B) Dose-dependent inhibitory effect of RCAE on HSV-1-infected Vero cells; (C) Plaque number; (D) Relative fluorescence expression rate; (E) Effect of RCAE on different stages of HSV-1-infected Vero cells;

[0046] Figure 5 RCAE alleviates HSV-1-induced viral infection and inflammation in mouse skin: (A) Skin lesions and histopathological changes in HSV-1-infected mice; (B) Lesion score; (C-F) Effect of RCAE on the expression of ICP0, VP16, gD and ICP27 mRNA in the skin lesions of infected mice; (G-I) Effect of RCAE on the expression of TNF-α, IL-6 and IL-1β mRNA in the skin of HSV-1-infected mice;

[0047] Figure 6 RCAE alleviates viral infection and inflammation in HaCaT cells stimulated by HSV-1: (A) Effect of RCAE on the viability of HaCaT cells; (B-D) Effect of RCAE on the expression of ICP0, ICP27 and gD; (E-G) Effect of RCAE on the expression of TNF-α, IL-6 and IL-1β mRNA; (H) Effect of RCAE on the expression of IKKβ, NF-κB p65 proteins and the phosphorylation level of NF-κB p65;

[0048] Figure 7RACE blocks the immune escape of HSV-1 virus: (A) Effect of RCAE on the number of mature DCs in the skin of HSV-1-infected mice; (B) Effect of RCAE on the expression of gD protein and CD11c protein in HSV-1-infected mice; (C-D) Effect of RCAE on the expression of CXCL1 and IFN-γ in the skin tissues of HSV-1-infected mice; (E) Effect of RCAE on the number of mature DCs in the draining lymph nodes of HSV-1-infected mice; (F) Draining lymph node index of mice; (G-H) Effect of RCAE on the expression of CXCL1 and IFN-γ in the lymph node tissues of HSV-1-infected mice; (I) Effect of RCAE on CD8 + T and CD4 + T lymphocytes;

[0049] Figure 8 RCAE can reduce HSV-1 infection in the brains and dorsal root ganglia of mice: (A) H&E staining pictures; (B-D) Effect of RCAE on the expression of ICP0, ICP27 and gD mRNA in the brains of HSV-1-infected mice; (E-G) Effect of RCAE on the expression of ICP0, gD and LAT mRNA in the DRGs of HSV-1-infected mice;

[0050] Figure 9 RCAE prevents the reactivation of HSV-1 in neuronal cells: (A) Effect of RCAE on the activity of N2a cells; (B) Timeline for the establishment of the HSV-1 latency model in N2a cells; (C) Fluorescence expression pictures; (D-F) Effect of RCAE on the expression of ICP0, gD and LAT mRNA; (G) Timeline for RCAE to affect the latency reactivation of HSV-1 in N2a cells; (H) Fluorescence expression and cell changes after the reactivation of latent HSV-1; (I-K) Fluorescence expression of the virus and the expression of ICP0 and gD mRNA. Detailed implementation manners

[0051] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0052] Example 1

[0053] 1 Experimental methods

[0054] 1.1 Preparation and analysis of drugs

[0055] 1.1.1 Preparation of Tupistra wattii drugs

[0056] Take 100 g of the root of Tupistra fimbriata Hand.-Mazz., add 10 ml of distilled water (distilled water: crude drug = 1:10), fully mash it into a paste, freeze-dry and powder it, and pass through a No. 9 pharmacopoeia sieve to obtain the water extract of Tupistra fimbriata Hand.-Mazz. (RCAE).

[0057] 1.1.2 High-resolution mass spectrometry LC / Q-TOF / MS

[0058] An electrospray ionization source was used, with positive and negative ion detection modes. The sheath gas pressure was 206.8 kPa, the ion transfer tube temperature was 320 °C, the auxiliary gas flow rate was 10 L / min, the spray voltage was 2.0 kV, and the auxiliary gas temperature was 350 °C. The scanning mode was Full MS / dd-MS2, the Full MS resolution was 70,000, the dd-MS2 resolution was 17,500, and the scanning range was m / z 80 - 1200. In the MS / MS mode, the collision energy was stepped energy 30, 40 eV.

[0059] 1.1.3 Preparation of gel

[0060] Take 0.1 g of carbomer 940 powder, 0.5 g of glycerol, and 0.5 g of ethylparaben, and add water to 10 mL to prepare a 1% carbomer gel matrix. Dissolve 5 mg of freeze-dried powder (RCAE) in 5 mL of distilled water and mix it evenly with 5 mL of carbomer gel matrix, and gradually add triethanolamine dropwise to adjust the pH to 7 to prepare the freeze-dried powder gel of Tupistra fimbriata Hand.-Mazz.

[0061] 1.2 Drug toxicity experiment

[0062] The CCK8 method was used to detect the toxicity of RCAE and ACV to Vero, HaCaT, and N2a cells respectively. Vero, HaCaT, and N2a cells were evenly plated in 96-well plates (100 μL / well) at a cell density of 1×10 5 cells / mL. Dilute RCAE and ACV serially with culture medium to the corresponding concentrations. After culturing Vero, HaCaT, and N2a cells overnight, use a pipette to aspirate the remaining culture medium, wash once with PBS, and then add different concentrations of RCAE and ACV to the 96-well plates. Set 3 replicates for each concentration gradient, and at the same time set a cell control group and a blank well. After culturing for 24 h, take out the CCK-8 reagent from the 4 °C refrigerator and operate in a biosafety cabinet. Add 10 μL of CCK-8 reagent to each well and protect from light, and then put the 96-well plates into the incubator for 1 - 2 h. Preheat the microplate reader, take out the 96-well plates, set the wavelength of 450 nm on the microplate reader to detect the absorption light, and calculate the cell survival rate.

[0063] 1.3 Antiviral activity experiment

[0064] 1.3.1 RCAE against HSV-1 infection of Vero cells

[0065] Study the inhibitory effect of RCAE on different stages of HSV-1 (MOI = 0.1) infection in Vero cells.

[0066] 1.3.1.1 Fluorescence experiment

[0067] (1) Experimental grouping: blank control group (without virus), Model group (HSV-1 infection group), ACV (3 μg / mL), RCAE-L group (7.5 μg / mL), RCAE-M group (15 μg / mL), RCAE-H group (30 μg / mL). Treat Vero cells with drugs at various stages of HSV-1 infection.

[0068] (2) Antiviral experiment: Prepare drug-containing media with different high, medium, and low concentrations. Remove the original medium in the 24-well plate, wash it twice with PBS, and simultaneously add HSV-1-Mcherry virus and the drug-containing medium. Incubate the three in an incubator at 37°C and 5% CO 2 for 2 h. After removing the virus and drug culture solution, wash the cells twice with PBS, add 1 mL of the drug-containing medium, and observe the change in virus fluorescence intensity under an inverted fluorescence microscope after 72 h.

[0069] (3) Preventive effect against virus (Phase I): Prepare drug-containing media with different high, medium, and low concentrations. Remove the original medium in the 24-well plate, wash it twice with PBS, add the drug-containing medium, and incubate it in an incubator at 37°C and 5% CO 2 for 1 h. Remove the drug-containing medium, wash the cells twice with PBS. Add HSV-1-Mcherry virus to the cells and incubate them in an incubator at 37°C and 5% CO2 for 1 h. After removing the virus solution, wash it twice with PBS. Add 1 mL of maintenance solution and observe the change in virus fluorescence intensity under an inverted fluorescence microscope after 24 h.

[0070] (4) Inactivation effect on virus (Phase II): Prepare drug-containing media with different high, medium, and low concentrations. Add HSV-1-Mcherry virus to the drug-containing medium and incubate it in an incubator at 37°C and 5% CO 2 for 1 h. Remove the original medium in the 24-well plate, wash it twice with PBS, add the virus and drug-containing medium mixture, and incubate it in an incubator at 37°C and 5% CO 2 for 1 h. After removing the mixture, wash the cells twice with PBS, add 1 mL of maintenance solution, and observe the change in virus fluorescence intensity under an inverted fluorescence microscope after 24 h.

[0071] (5) Anti-viral adsorption effect (Phase III): Prepare culture media containing drugs at different high, medium, and low concentrations. Remove the original culture medium in the 24-well plate, wash it twice with PBS, and simultaneously add the HSV-1-Mcherry virus and the culture medium containing the drug. Incubate the three in an incubator at 37°C and 5% CO 2 for 1 h. After removing the virus and the drug culture solution, wash the cells twice with PBS, add 1 mL of maintenance solution, and observe the change in the fluorescence intensity of the virus under an inverted fluorescence microscope after 24 h.

[0072] (6) Anti-viral replication effect (Phase IV): Prepare maintenance solutions containing drugs at different high, medium, and low concentrations. Remove the original culture medium in the 24-well plate, wash it twice with PBS, add the HSV-1-Mcherry virus, and incubate it in an incubator at 37°C and 5% CO 2 for 1 h. After removing the virus solution, wash it twice with PBS and then add 1 mL of the maintenance solution containing the drug. Observe the change in the fluorescence intensity of the virus under an inverted fluorescence microscope after 24 h.

[0073] 1.3.1.2 Plaque assay

[0074] (1) Experimental grouping: The experimental grouping is the same as in 1.3.1.1.

[0075] (2) Anti-viral experiment: Prepare culture media containing drugs at different high, medium, and low concentrations. Remove the original culture medium in the 24-well plate, wash it twice with PBS, and simultaneously add the HSV-1-Mcherry virus and the culture medium containing the drug. Incubate the three in an incubator at 37°C and 5% CO 2 for 2 h. After removing the virus and the drug culture solution, wash the cells twice with PBS, add 1 mL of the culture medium containing the drug. After 72 h, remove the culture medium, add 1 mL of plaque overlay solution, fix the cells after 3 to 4 days, and count the plaques after crystal violet staining.

[0076] (3) Virus prevention effect (Phase I): Prepare culture media containing drugs at different high, medium, and low concentrations. Remove the original culture medium in the 24-well plate, wash it twice with PBS, add the culture medium containing the drug, and incubate it in an incubator at 37°C and 5% CO 2 for 1 h. Remove the culture medium containing the drug and wash the cells twice with PBS. Add 100 PFU of the virus to the cells and incubate them in an incubator at 37°C and 5% CO 2 for 1 h. After removing the virus solution, wash it twice with PBS. Add 1 mL of plaque overlay solution, fix the cells after 3 to 4 days, and count the plaques after crystal violet staining.

[0077] (4) Virus inactivation effect (Phase II): Prepare culture media containing drugs at different high, medium, and low concentrations. Add 100 PFU of the virus to the culture medium containing the drug and incubate it at 37°C and 5% CO2 Incubate in an incubator for 1 h. Remove the original culture medium in the 24-well plate, wash twice with PBS, add a mixture of virus and drug-containing culture medium, and place it at 37 °C and 5% CO 2 Incubate in an incubator for 1 h, remove the mixture, wash the cells twice with PBS, add 1 mL of plaque overlay fluid, and fix the cells after 3 to 4 days, then count the plaques after crystal violet staining.

[0078] (5) Anti-viral adsorption effect (Phase III): Prepare drug-containing culture media with high, medium, and low different concentrations. Remove the original culture medium in the 24-well plate, wash twice with PBS, and simultaneously add 100 PFU of virus and the drug-containing culture medium, and place all three at 37 °C and 5% CO 2 Incubate in an incubator for 1 h, remove the virus and drug culture solution, wash the cells twice with PBS, add 1 mL of plaque overlay fluid, and fix the cells after 3 to 4 days, then count the plaques after crystal violet staining.

[0079] (6) Anti-viral replication effect (Phase IV): Prepare drug-containing overlay fluids with high, medium, and low different concentrations. Remove the original culture medium in the 24-well plate, wash twice with PBS, add 100 PFU of virus, and place it at 37 °C and 5% CO 2 Incubate in an incubator for 1 h, remove the virus solution, wash twice with PBS, then add 1 mL of drug-containing overlay fluid, and fix the cells after 3 to 4 days, then count the plaques after crystal violet staining.

[0080] 1.3.2 RCAE against HSV-1 infection in HaCaT cells

[0081] After infecting HaCaT cells with HSV-1 (MOI = 0.1) for 2 h, treat HaCaT cells with ACV (3 μg / mL) or RCAE (7.5, 15, 30 μg / mL) for 48 h, and collect cell RNA and protein for subsequent experimental analysis.

[0082] 1.3.3 RCAE against HSV-1 latent reactivation in N2a cells

[0083] Establishment of an HSV-1 latent infection cell model: Seed N2a cells at a density of 4×10 5 cells / well in a 24-well plate, and place it at 37 °C and 5% CO 2Cultivate until a monolayer is formed in an incubator at a constant temperature. Subsequently, replace the original medium with a medium containing ACV (3 μg / mL) (1 mL / well). After treating with ACV for 24 h, discard the medium, and add HSV-1 virus solution expressing red fluorescent protein mCherry (MOI = 1) to each well to allow the virus to adsorb for 1 - 2 h. After adsorption is complete, remove the virus inoculum, and add 1 mL of fresh maintenance medium without ACV to each well. To maintain the latent state of the virus, replace the maintenance medium every two days. From the 4th day post-infection (4 dpi), the maintenance medium no longer contains any antiviral drugs.

[0084] Establishment of the HSV-1 latent reactivation cell model: To induce HSV-1 reactivation, incubate the cells at 42 °C for 30 min. On the 4th day post-infection (4 dpi), discard the medium containing ACV, and add a medium containing ACV (3 μg / mL) and different concentrations of RCAE (1.5, 3, and 6 μg / mL) to each well. Observe the change in the expression of red fluorescent protein mCherry by fluorescence microscopy at the designated time points to monitor virus reactivation. After virus reactivation, extract cellular RNA for subsequent experimental analysis.

[0085] 1.4 Establishment of the HSV-1-infected murine herpes zoster model

[0086] Randomly cage 6 - 8-week-old BALB / c mice and divide them into a control (Control) group, a model (HSV-1) group, an acyclovir (ACV at 0.01 g / mL) group, and low, medium, and high Tupistra chinensis (RCAE at 0.2, 0.4, and 0.6 g / mL) groups, with 6 mice in each group. After scratching the flanks of the mice to infect them with HSV-1 (1×10 6 PFU) for 3 h, apply gels of Tupistra leptophylla and acyclovir (100 μL / cm 2 ) twice a day (with a 12-h interval) for 6 consecutive days.

[0087] 1.5 Histopathological analysis (HE staining)

[0088] Prepare paraffin sections of the skin lesion tissues, perform HE staining, and observe the skin pathological changes under a light microscope.

[0089] 1.6 Scoring of murine skin lesions

[0090] The skin lesion scoring criteria are as follows: 0 = no skin lesions; 2 = one or two blisters on the back; 4 = multiple blisters on the back, surrounding the inoculation site, or both; 6 = mild herpes zoster-like skin lesions; 8 = obvious herpes zoster-like skin lesions, or both; 10 = severe herpes zoster-like skin lesions. Sacrifice the mice 6 days after inoculation.

[0091] 1.7 RT-qPCR

[0092] (1) Total RNA extraction

[0093] Use a sterile ophthalmic scissors to cut the skin tissue into small pieces in a 1.5 mL enzyme-free EP tube, add zirconium beads and 1 mL of RNAisoPlus, and grind under high pressure for 15 cycles until there are no visible tissue chunks in the tube. Centrifuge the homogenate at 12,000 g for 5 min at 4°C. Carefully aspirate the supernatant and transfer it to a new enzyme-free EP tube (do not aspirate the precipitate). Add 200 μL of chloroform to the supernatant, tightly cap the centrifuge tube, and mix until the solution emulsifies into a milky white color. Let it stand at room temperature for 5 min. Centrifuge at 12,000 g for 15 min at 4°C. Carefully take out the centrifuge tube from the centrifuge. At this time, the homogenate is divided into three layers, namely: a colorless supernatant (containing RNA), a white protein layer in the middle (mostly DNA), and a colored lower organic phase. Aspirate the supernatant and transfer it to another new enzyme-free EP tube (do not aspirate the white middle layer). Add 1 mL of isopropanol to the supernatant, invert the centrifuge tube up and down to mix well, and then let it stand at room temperature for 10 min. After standing, centrifuge at 12,000 g for 10 min at 4°C. After centrifugation, an RNA precipitate will appear at the bottom of the test tube. Carefully discard the supernatant without touching the precipitate, add 1 mL of 75% ethanol (prepared with enzyme-free water and used freshly), and gently invert the centrifuge tube up and down to wash the inner wall of the centrifuge tube. After centrifuging at 7,500 g for 5 min at 4°C, carefully discard the supernatant without touching the precipitate. Open the centrifuge tube cap and dry the precipitate at room temperature for 5 min. After the precipitate is dried, add an appropriate amount of RNase-free water to dissolve the precipitate. Do not centrifuge or heat-dry, otherwise the RNA will be difficult to dissolve. Detect the concentration and purity of RNA. Take 1 μL of the RNA solution to be tested on a nucleic acid analyzer for absorbance detection; if the RNA concentration is too high, it can be appropriately diluted.

[0094] (2) Reverse transcription

[0095] Add 2 μg of RNA solution to a PCR tube, add 2 μL of 5×gDNA Eraser Buffer, 1 μL, 6 μL of RNaseFree dH 2 O, mix well, let it stand at room temperature for 5 min, and then add 10 μL of Master Mix for a total of 20 μL. Reverse transcribe to cDNA on a PCR instrument with a program of 37°C for 15 min and 85°C for 5 s, and then cool on ice after completion.

[0096] (3) PCR reaction

[0097] Prepare the PCR reaction solution according to Table 1 and Table 3, set the qRT-PCR reaction program according to Table 3, and then start the detection. The relative expression level of the target gene in the sample is calculated using the 2 -△△CT method.

[0098] Table 1 Proportion of PCR reaction solution preparation

[0099]

[0100]

[0101] Table 2 Primer sequence list

[0102]

[0103] Table 3 RT-qPCR reaction procedure

[0104]

[0105]

[0106] 1.8ELISA test

[0107] Skin and lymph node tissue homogenate preparation, use pre-cooled PBS (0.01M, pH = 7.4) to rinse the tissue, remove residual blood, weigh and cut the tissue. Add the cut tissue and the corresponding volume of PBS (1:9) and zirconium beads into a low-temperature high-pressure tissue homogenizer. After fully circulating 15 times, place the homogenate in a refrigerated centrifuge at 5000rpm, centrifuge for 10 minutes, and take the supernatant for detection. Strictly follow the instructions of the ELISA kit to add the sample reaction and measure the OD value of each well at a wavelength of 450nm.

[0108] 1.9 Protein expression analysis (Western Blot)

[0109] (1) Protein extraction: Rinse the cells with PBS buffer for 2-3 times. Aspirate the waste liquid as cleanly as possible for the last time, add an appropriate volume of RIPA lysis buffer (containing protease inhibitors) to the 24-well plate for 3-5 minutes, and shake repeatedly during the period to ensure that the reagent and cells are in full contact. Scrape the cells with a cell scraper, collect the cell and reagent suspension into a 1.5mL centrifuge tube, and place it in an ice bath for 30 minutes. During this period, use a pipette to repeatedly blow and beat to ensure that the cells are completely lysed. Then centrifuge at 12000g in a refrigerated centrifuge for 5 minutes, and collect the supernatant as the total protein solution.

[0110] (2) Protein concentration determination: The BCA method was used to detect the protein concentration. Prepare an appropriate amount of BCA working solution as needed. The working solution can be stably stored at room temperature for one week. Mix BCA reagent A and BCA reagent B in a volume ratio of 50:1 and mix well. Prepare a standard protein solution, and use distilled water as a blank control. Dilute the sample appropriately. The diluent should be the same as that of the standard. Dilute with 1×PBS or distilled water. Using the microplate method, take 20 μL of standard protein solutions with different concentrations, the sample to be tested, and the blank control (distilled water) and place them in each well respectively. Add 200 μL of BCA working solution to each well, gently shake and mix, seal the microplate, and place it at 37 °C for 30 minutes. Then take out the microplate and let it return to room temperature. Use a microplate reader with a wavelength of 562 nm to detect the absorbance value, draw a standard protein curve, and calculate the protein concentration of the sample to be tested through the standard curve regression formula.

[0111] (3) Preparation of protein samples: Add the corresponding amount of 5×SDS loading buffer according to the ratio of the total protein solution volume: 5×SDS loading buffer volume of 4:1. Mix well by vortex and then put it into a metal bath and boil at 95 °C for 5 minutes.

[0112] (4) SDS-PAGE electrophoresis: First, clean the glass plates, and then pour the gel and load the samples. Align the glass plates and clamp them in the holder. During the operation, make sure the two glasses are aligned to avoid gel leakage. Prepare the separating gel according to the experimental arrangement, shake well immediately after adding TEMED and then pour the gel. After about 45 minutes, pour off the water on the upper layer of the gel and blot the remaining water with absorbent paper. Prepare 5% stacking gel, shake well immediately after adding TEMED and then pour the gel. Fill the remaining space with stacking gel and then insert the comb into the stacking gel. After adding enough electrophoresis buffer, load the samples. Add the samples into the electrophoresis wells and then perform electrophoresis. The voltage of the stacking gel is 80 V, and the voltage of the separating gel is 120 V.

[0113] (5) Transfer: Cut the PVDF membrane according to the size of the gel, and activate the cut PVDF membrane in methanol. Then make a transfer "sandwich". Take out the "sandwich" clamp in the transfer tank and open it. Place the black plate downwards, and place the sponge pad → transfer filter paper → gel → PVDF membrane → transfer filter paper → sponge pad in sequence (pay attention to avoiding air bubbles between the gel and the PVDF membrane). Then cover the red plate and clamp the whole "sandwich". Place the made "sandwich" into the transfer tank in the way of black to black and red to red. Put an ice box into the transfer tank and then pour an appropriate amount of transfer buffer (the transfer buffer should basically submerge the whole "sandwich"). Cover the transfer tank cover and connect the instrument power supply in the way of black to black and red to red. Then set the transfer parameters, constant voltage 100 V, current 230 mA. The specific transfer time depends on the molecular weight of the target protein.

[0114] (6) Blocking: Phosphorylated proteins are blocked with 5% BSA, and the rest of the proteins are blocked with 5% skim milk at room temperature for 1 h.

[0115] (7) Primary antibody incubation: Dilute the primary antibody with TBST according to the antibody instruction manual and the specific experimental concentration. Prepare an appropriate amount of primary antibody solution (3 mL / grid) according to the size of the antibody incubation box, and mark the name of the antibody (protein) placed in each grid on the lid of the incubation box. After washing the blocked PVDF membrane 3 times with TBST (to wash away the residual blocking solution to avoid affecting the primary antibody incubation effect and reuse), place it in the corresponding grid of the antibody incubation box and incubate overnight at 4°C.

[0116] (8) Secondary antibody incubation: Dilute the secondary antibody according to the antibody instruction manual. Pour out and aspirate the TBST in the incubation box, add the prepared secondary antibody solution (3 mL / grid), and incubate at room temperature for 1 h. Discard the secondary antibody solution, and wash the PVDF membrane with TBST, 8 min × 4 times.

[0117] (9) Develop the film and analyze the results: Take out the PVDF membrane, blot off the excess liquid with filter paper, then add an appropriate amount (about 200 μL / membrane) of ECL luminescent solution, place it in the Bio-Rad gel imaging system for development, and save all the developed results. Use Image Lab software to perform gray-scale analysis on the developed results and calculate the relative expression level of the target protein.

[0118] 1.10 Flow cytometry

[0119] (1) Preparation of skin single-cell suspension

[0120] Use a sterile ophthalmic scissors to cut the skin tissue into minced pieces, place it in DMEM medium containing collagenase IV (1 mg / ML), and incubate it in a 37°C, 5% CO 2 cell incubator for 90 min. Collect the cell suspension and filter it through a 40-μm cell strainer, then transfer the cell suspension to the corresponding flow tube. Centrifuge at 1200 rpm for 3 min, discard the supernatant; add 3 volumes of red blood cell lysis buffer, let it stand at room temperature for 5 min, and centrifuge at 1200 rpm for 3 min. Wash twice with pre-cooled PBS, each time centrifuge at 1200 rpm for 3 min, and discard the supernatant. Resuspend the cells in 100 μL PBS for later use.

[0121] (2) Preparation of lymph node single-cell suspension

[0122] Use a 1-mL sterile syringe needle to crush the lymph node until there is no visible tissue fluid, collect the suspension and filter it through a 40-μm cell strainer, then transfer the cell suspension to the corresponding flow tube. Centrifuge at 1200 rpm for 3 min. Wash twice with pre-cooled PBS, centrifuge at 1200 rpm for 3 min, and discard the supernatant. Resuspend in 100 μL PBS for later use.

[0123] (3) Flow antibody staining

[0124] Prepare skin cells and stain them with FITC-CD11c, PE-cyanine5.5-CD83, APC-CD80, and PE-HVEM fluorescent antibodies.

[0125] Divide the prepared lymphocytes into two groups. The first group is stained with FITC-CD4 and PE-CD8 fluorescent antibodies; the second group is stained with FITC-CD11c, PE-cyanine5.5-CD83, APC-CD80, and PE-HVEM fluorescent antibodies.

[0126] After staining, place the cells in an ice box at 4°C and incubate them in the dark for 30 min. Wash the cells twice with PBS, centrifuge them at 1200 rpm for 3 min, and discard the supernatant. Resuspend the cells in 500 μL of PBS and detect them using a flow cytometer.

[0127] (4) Flow cytometry data analysis

[0128] Export the FSC file of the original data from the BD FACSDiva software and analyze it using the NovoExpress software. The gating strategy is as follows:

[0129] 1) CD4 + T lymphocytes, CD8 + Analysis method for T lymphocytes: Gate with FSC-H / SSC-H to remove cell debris and dead cells; gate with SSC-H / SSC-A to remove adherent cells. Use CD4-H as the abscissa and CD8-H as the ordinate to create a density plot, draw a cross gate, and circle the CD4 + CD8 - cells and CD4 - CD8 + cells (see Figure 1 ).

[0130] 2) Analysis method for DC cells: Gate with FSC-H / SSC-H to remove cell debris and dead cells; gate with SSC-H / SSC-A to remove adherent cells. Use CD11c-H as the abscissa and HVEM-H, CD83-H, and CD80-H as the ordinates respectively to create density plots, draw cross gates, and circle the CD11c + HVEM + DC cells, CD11c + CD80 + DC cells, CD11c + CD83 + DC cells (see Figure 2 ).

[0131] 1.11 Immunofluorescence experiment

[0132] (1) Dewaxing of paraffin sections: sequentially place the sections in xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% alcohol for 5 min, 75% alcohol for 5 min, and wash with distilled water.

[0133] (2) Antigen repair: Add appropriate repair buffer (citrate buffer) to a microwave oven container and heat to boiling on high heat. Take out and put in glass slides, boil on medium heat for 8 minutes, stop heating for 8 minutes, and switch to medium-low heat for 7 minutes. During this process, excessive evaporation of the buffer should be prevented and the slides should not be dried. After natural cooling, place the slides in PBS (pH 7.4) and shake on a decolorizing shaker to wash 3 times, 5 minutes each time.

[0134] (3) Blocking endogenous peroxidase: After the slices are slightly dried, use an immunohistochemistry pen to draw a circle around the tissue. Place the slices in a 3% hydrogen peroxide solution and incubate at room temperature in the dark for 15 minutes. Place the slides in PBS and wash them three times on a decolorizing shaker, each time for 5 minutes.

[0135] (4) Serum blocking: Add 5% goat serum into the circle and incubate for 30 min.

[0136] (5) Primary antibody incubation: Gently shake off the blocking solution on the slices, add the prepared antibody (1:100) to the tissue, and place the slices flat in a humidified box and incubate at 4°C overnight. (Add a small amount of water to the humidified box to prevent the antibody from evaporating).

[0137] (6) Secondary antibody incubation: Place the slides in a wet box containing PBS and wash three times, each wash for 5 minutes. After drying the sections, add fluorescent secondary antibody to the circle so that the antibody completely covers the tissue and incubate at 37° for 40 minutes in the dark.

[0138] (7) DAPI counterstaining of cell nuclei: Place the slide in PBS and wash on a decolorizing shaker three times, 5 min each time. After the slices are slightly dried, add DAPI staining solution in the circle and incubate at room temperature for 10 min away from light.

[0139] (8) Sealing: Place the slides in PBS and wash them on a decolorizing shaker three times, 5 min each time. After the sections are slightly dried, seal them with an anti-fluorescence quenching sealing medium.

[0140] (9) Photography: Observe the slices and collect images under a fluorescence microscope.

[0141] 1.12 Statistical analysis

[0142] With health statistics as the reference, the measurement data were expressed as mean and standard deviation. Multiple sample means were compared to calculate the mean and standard deviation of each group. The t-test was used for comparison between two groups, and one-way analysis of variance was used for comparison between multiple groups. P<0.05 was considered statistically significant.

[0143] 2 Results Analysis

[0144] 2.1 Chemical Composition Analysis of RCAE

[0145] Nine compounds were preliminarily identified as the main components of RCAE by UPLC-Q-TQF-MS / MS. According to the mass spectrometry identification, seven compounds matched with the SIRIUS online database all contained a cyclopentane polyhydrophenol nucleus, belonging to the steroid nucleus. Through database comparison, it was found that except for compound 6, podocarpus sterone C, which was a known component, the structures of the remaining eight components had not been reported ( Figure 3 , Table 4).

[0146] Table 4 Chemical Composition Analysis of RCAE by UPLC-Q-TQF-MS / MS

[0147]

[0148]

[0149] 2.2 RCAE Inhibits HSV-1 Infection in Vero Cells

[0150] First, the toxicity of RCAE to Vero cells was detected. The results showed that RCAE at 25 μg / mL reduced the survival rate of Vero cells by no more than 15%. Therefore, 7.5, 15, and 30 μg / mL were selected as the low, medium, and high dose concentrations of RCAE for subsequent experiments ( Figure 4 A). At the same time, the cytotoxicity of ACV to Vero cells was analyzed, and it was found that ACV ≤ 100 μg / mL had no obvious toxic effect on Vero cells, and the cell survival rate was above 95% ( Figure 4 A).

[0151] The anti-HSV-1 activity of RCAE against Vero cells was detected by plaque assay and mCherry-labeled HSV-1. As shown in Figure 4 B-D, RCAE at 30 μg / ml significantly reduced the number of virus plaques and the number of cells infected with mCherry-HSV-1, and decreased the immunofluorescence intensity of mCherry. In addition, to determine the effect of RCAE on different stages of HSV-1 infection, RCAE was added at different stages of the virus life cycle to observe its anti-HSV-1 effect. The plaque assay and fluorescence results showed that RCAE had the best effect in stages II and III, indicating that RCAE could directly kill HSV-1 and inhibit the adhesion of HSV-1; among them, RCAE inhibited the adhesion of HSV-1 better than ACV ( Figure 4 E).

[0152] 2.3 RCAE alleviates HSV-1-induced viral infection and inflammation in mouse skin

[0153] To further investigate the anti-HSV-1 effect of RCAE, a flank scratch model of HSV-1 virus in mice was established to observe the antiviral effect of RCAE in vivo. As a barrier organ, the skin is one of the first lines of defense against pathogens. Therefore, it was first found that RCAE could significantly reduce the skin damage area and lesion score, and improve the histological damage of the skin ( Figure 5 A-B), and it was further observed that RCAE could significantly inhibit the expression of virus-related genes ICP0, VP16, gD, and ICP27 in the skin ( Figure 5 C-F). At the same time, RCAE could reduce the expression of TNF-α, IL-6, and IL-1β mRNA in the skin of HSV-1-infected mice ( Figure 5 G-I).

[0154] 2.4 RCAE alleviates viral infection and inflammation in HaCaT cells stimulated by HSV-1

[0155] To further clarify the antiviral and anti-inflammatory effects of RCAE, the immortalized human skin keratinocyte HaCaT was studied in vitro. First, the toxicity of RCAE and ACV to HaCaT cells was detected in this invention. The results showed that 30 μg / mL of RCAE and ACV had no obvious effect on the viability of HaCaT cells ( Figure 6 A), so subsequent experiments were carried out within this concentration range. Next, it was observed that RCAE significantly down-regulated the expression of virus-related genes ICP0, ICP27, and gD ( Figure 6 B-D), reduced the expression of TNF-α, IL-6, and IL-1β mRNA in HSV-1-infected HaCaT cells ( Figure 6 E-G) and the expression levels of IKKβ, NF-κB p65 protein, and NF-κB p65 phosphorylation level ( Figure 6 H).

[0156] 2.5 RACE blocks the immune escape of HSV-1 virus

[0157] Host protection against infection is mainly mediated by the immune system. DCs are powerful antigen-presenting cells in the immune response. HSV-1 inhibits the maturation of DCs by degrading the mature markers on DCs and hindering antigen presentation. Studies have shown that after HSV-1 infection, skin DCs migrate from the skin to the draining lymph nodes, where they cross-present viral antigens to CD8 T cells. Therefore, the effect of RACE on the immune system of HSV-1-infected mice was further detected. The results showed that RCAE increased the number of mature DCs in the skin of HSV-1-infected mice, reduced the expression of gD protein, and increased the expression of CD11c proteinFigure 7 A - B). In addition, RCAE increased the levels of CXCL1 and IFN - γ in the skin of HSV - 1 - induced mice ( Figure 7 C - D). Flow cytometry results showed that high - concentration RCAE increased the number of mature DCs in the draining lymph nodes of mice ( Figure 7 E). RCAE decreased the lymph node index of HSV - 1 - infected mice ( Figure 7 F), up - regulated the expression of CXCL1 and IFN - γ in the lymph nodes of mice ( Figure 7 G - H) and the number of CD4 + and CD8 + T cells ( Figure 7 I). The above results indicate that RCAE may enhance the antigen - presenting ability of DCs by increasing the maturation of DCs in mice, promote the differentiation of CD8 + T cells, promote the secretion of CXCL1 and IFN - γ, and block the immune escape of HSV - 1 virus, thereby exerting an antiviral effect.

[0158] 2.6 RCAE can alleviate HSV - 1 infection in the brains and dorsal root ganglia of mice

[0159] HSV - 1 is a neurotropic virus that can cause lifelong latent infection. Therefore, the effect of RCAE on HSV - 1 infection in the brains and dorsal root ganglia of mice was further observed. HE staining results showed that RCAE could alleviate the pathological damage of the brain tissue of HSV - 1 - infected mice ( Figure 8 A), down - regulate the expression of ICP0, ICP27, and gD mRNA in the brain tissue of mice ( Figure 8 B - D), and down - regulate the expression of ICP0, gD, and LAT mRNA in the dorsal root ganglia of mice ( Figure 8 E - G).

[0160] 2.7 RCAE blocks the re - activation of HSV - 1 in neuronal cells

[0161] To further clarify the anti - HSV - 1 infection effect of RCAE in the brain tissue and nerves, N2a cells were used as the research object, and an HSV - 1 latent infection and re - activation model was constructed in vitro. The toxicity of RCAE and ACV to N2a cells was detected, and it was found that RCAE and ACV had no effect on the viability of N2a cells at concentrations below 6 μg / mL ( Figure 9 A).

[0162] As Figure 9As shown in B-F, under the action of ACV, with the increase in the number of days of HSV-1 infection, the viral fluorescence expression, as well as the expression levels of ICP0 and gD mRNA, decreased, while the relative expression level of LAT mRNA remained stable compared with the HSV-1 group, indicating that the virus entered the latent period. When the virus entered the latent period, external stimuli activated the virus, resulting in lytic infection. The latency model was activated at 42°C for 30 min at 4 dpi, and obvious lesions appeared in N2a cells infected with HSV-1( Figure 9 G-H), indicating that HSV-1 was reactivated. Next, the effect of RCAE on the latent reactivation of HSV-1 virus in N2a cells was further studied, and the results showed that RCAE significantly inhibited the production of HSV-1 virus fluorescence and down-regulated the expression of ICP0 and gD mRNA, and the effect of RCAE at 6 μg / mL was the best( Figure 9 I-K).

[0163] In summary, in vitro and in vivo experiments can prove that RCAE has antiviral effects, can inactivate the virus or inhibit the adhesion of the virus to cells, and RCAE can inhibit the reactivation of latent viruses.

[0164] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Use of Rhizoma Cibotii or its extract in preparing drugs for preventing and / or inhibiting and / or inactivating HSV-1 virus.

2. The use according to claim 1, characterized in that The extract is selected from water extracts.

3. The use according to claim 1, characterized in that The opening arrow is selected from the group consisting of opening arrow, umbrella-column opening arrow, long-column opening arrow, long-stem opening arrow, Yunnan opening arrow, tube-flower opening arrow, tooth-petal opening arrow, disc-flower opening arrow, Emei opening arrow, long-spike opening arrow, Liangshan opening arrow, sword-leaf opening arrow and Mabian opening arrow; further, the opening arrow is Mabian opening arrow.

4. The use according to claim 2, characterized in that: The medicinal concentration of the water extract of the Aralia ovata is 0.01 to 1000 μg / mL; further, the medicinal concentration of the water extract of the Aralia ovata is 7.5 to 500 μg / mL.

5. The use according to claim 1 or 2, characterized in that: The drug is a drug that regulates the expression of at least one gene or protein among ICP0, VP16, gD, IKKβ, and NF-κB; further, the drug is a drug that inhibits the expression of at least one gene or protein among ICP0, VP16, gD, IKKβ, and NF-κB.

6. An immunomodulator, characterized in that The open-mouthed arrow or its extract as claimed in claim 1 is used as the active substance.

7. The immunomodulator according to claim 6, characterized in that The immunomodulator can increase the number of at least one immune cell among phagocytes, lymphocytes, eosinophils, basophils, natural killer cells, and mast cells; further, the immunomodulator can increase the number of phagocytes and lymphocytes; further, the immunomodulator can increase the number of DC cells, CD4 + 、CD8 + The number of T cells.

8. A pharmaceutical composition, characterized in that The invention comprises the open-mouthed arrow or an extract thereof as claimed in claim 1 and a pharmaceutically acceptable excipient.

9. The pharmaceutical composition according to claim 8, characterized in that The preparation is an oral preparation, an injection or an external preparation; further, the oral preparation is an ointment, a pill, an oral liquid, a powder, a tablet, a granule or a capsule; and / or the external preparation is a solution, a lotion, a liniment, an ointment, a plaster, a paste or a patch; and / or the injection is a solution, a powder or a tablet.

10. A gel for preventing and / or inhibiting and / or inactivating HSV-1 virus, characterized in that: The open-mouthed arrow or its extract as claimed in claim 1 is used as the active substance.

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