阅读说明：本技术 利福霉素的新用途 (New application of rifamycin ) 是由 杜飞 于 2020-03-31 设计创作，主要内容包括：本申请公开了利福霉素的新用途,将利福霉素用于制备预防和治疗乙型脑炎药物的应用。本申请发现利福霉素在乙型脑炎病毒的感染中发挥重要的作用,利福霉素具有抑制乙型脑炎病毒感染的特性,进一步发现利福霉素可通过抑制乙型脑炎病毒在侵入的靶细胞中进行复制的作用,进而抑制所述病毒的感染。本发明提供了利福霉素在治疗乙型脑炎病毒感染中的应用,为乙型脑炎病毒的预防和治疗提供了新的药物,具有良好的市场价值和临床应用前景。解决了现有技术中一直未有预防和治疗乙型脑炎特效药物的问题。(The application discloses a new application of rifamycin, in particular to an application of rifamycin in preparing medicines for preventing and treating Japanese encephalitis. The application discovers that rifamycin plays an important role in infection of Japanese encephalitis virus, rifamycin has the characteristic of inhibiting infection of Japanese encephalitis virus, and further discovers that rifamycin can inhibit infection of Japanese encephalitis virus by inhibiting replication of Japanese encephalitis virus in invaded target cells. The invention provides the application of rifamycin in treating Japanese encephalitis virus infection, provides a new medicine for preventing and treating Japanese encephalitis virus, and has good market value and clinical application prospect. Solves the problem that no specific medicine for preventing and treating Japanese encephalitis exists in the prior art.)

1. The new application of rifamycin is characterized by that it can be used for preparing medicine for preventing and curing Japanese encephalitis.

2. The new use as claimed in claim 1, wherein the rifamycin salt has inhibitory effect on Japanese encephalitis virus when used in the preparation of a medicament for preventing and treating Japanese encephalitis.

3. The new use as claimed in claim 2, wherein rifamycin salt has the effect of inhibiting replication of Japanese encephalitis virus in invading target cells, thereby inhibiting infection by said virus.

4. The new use as claimed in any one of claims 1 to 3, wherein the rifamycin is used for the preparation of a medicament for preventing and treating Japanese encephalitis, and the effects of the rifamycin on preventing and treating and inhibiting the Japanese encephalitis virus are positively correlated with the concentration of rifamycin.

5. The novel use according to claim 4, wherein the rifamycin is present at a concentration of 10-40 μ M.

Technical Field

The application relates to the technical field of medicines, in particular to a new application of rifamycin.

Background

JEV is a pathogen of epidemic encephalitis B, is mainly transmitted by mosquitoes, can cause serious acute nervous system symptoms such as encephalitis and meningitis after infection, and can cause death of severe infectors, and JEV and West Nile Virus (WNV), Yellow Fever Virus (Yellow Fever Virus, YFV), dengue Virus (DengueVirus, DENV) are classified as members belonging to Flaviviridae (Flaviviridae) Flavivirus genus (Flavivirus) in the process of assembling JEV particles with diameters of about 50nm, the whole genome length of about 11kb, mainly encode 3 structural proteins (core protein C, membrane protein M, envelope protein E) and 7 non-structural proteins (NS 5, NS2A, 2, B, NS3, Controla 674, a 5, a protein C, a protein M, an envelope protein E) and 7 non-structural proteins (NS 8, NSE) involved in the process of Virus surface invasion of CNS 19, CNS, and HIV infection [ 1. A. JEV is also involved in the process of assembling of HIV, S.S.S.S.S.S.7. A. A. is also relates to the immune host protein of Japanese encephalitis Virus with the immune host protein of Japanese encephalitis Virus, a Virus with the Virus, a Virus with the immune host protein of Japanese encephalitis Virus, a Virus with the immune host protein of CNS, a Japanese encephalitis Virus, a Virus with the immune host protein of Japanese encephalitis Virus, a Virus of a Virus, a Virus of Japanese encephalitis Virus, a Virus of Japanese encephalitis Virus of Japanese.

To date, there is no specific drug or specific method for treating encephalitis b, mainly broad-spectrum antiviral therapy and symptomatic therapy. Broad-spectrum antiviral nucleic acid analogues (such as ribavirin) and cytokines (such as interferon) are mainly used in antiviral drugs, and nucleic acid targeted therapeutic agents, flavonoid compounds and the like are also reported to be used for treatment. At present, although there are some reports on antiviral drugs, vaccines, RNA and the like aiming at the replication of the Japanese encephalitis virus, most of them are still in the development stage. In order to find an effective treatment method, researchers at home and abroad are always researching and developing drugs for targeting encephalitis B virus infection, and mainly focus on several research directions: firstly, the encephalitis B is treated by searching the existing medicines with better antiviral effect; secondly, designing and synthesizing derivatives of the existing antiviral drugs according to the molecular biology characteristics of the JEV; thirdly, designing and screening new anti-JEV drugs. However, the evaluation of the anti-JEV effect achieved today mainly stays at the stage of animal model, cell and molecular level, and whether it can be applied to clinical treatment or not is yet to be studied intensively.

Rifamycin (Rifamycin) drugs are antibiotics produced by Streptomyces mediterranei, have broad-spectrum antibacterial action and have strong inhibitory action on gram-positive bacteria such as tubercle bacillus and staphylococcus aureus, among which Rifampicin, rifapentine and rifabutin are the drugs which are clinically used at present, not only have wide antibacterial spectrum and can be used for various bacterial infectious diseases, but also have outstanding curative effect on tuberculosis, and are the first line drugs for treating tuberculosis [ Rothstem DM. rifaxins, Alone and combination. Cold Spring harbor Perspecific medicine.2016; 6(7): a011. purified 2016 J.1. doi: 10.1101/cshpoper. 027. 011] in vitro experiments, and show that Rifampicin can be proved to inhibit replication of porcine virus (antibiotic) viruses [ Acribe, virus.

At present, no document reports the effect of rifamycin drugs in resisting Japanese encephalitis virus infection and the application of rifamycin drugs in treating Japanese encephalitis.

Disclosure of Invention

The main purpose of the application is to provide a new application of rifamycin to solve the problem that no specific drug for Japanese encephalitis virus exists in the related art.

In order to achieve the above objects, in a first aspect, the present application provides a novel use of rifamycin.

The new use of the full dress mycin according to the application: the application of rifamycin in preparing medicine for preventing and treating Japanese encephalitis is disclosed.

Furthermore, when the rifamycin salt is used for preparing the medicine for preventing and treating the Japanese encephalitis, the rifamycin has an inhibitory effect on the Japanese encephalitis virus.

In particular, rifamycin salts have the effect of inhibiting replication of Japanese encephalitis virus in invading target cells, thereby inhibiting infection by the virus.

On the other hand, the application discovers that when the rifamycin is used for preparing the medicine for preventing and treating the Japanese encephalitis, the preventing and treating effects of the rifamycin and the effect of inhibiting the Japanese encephalitis virus are positively correlated with the concentration of the rifamycin. For example, the rifamycin concentration is 10-40. mu.M.

By combining the technical scheme, the technical problem that no specific medicine for preventing and treating encephalitis B is found in the prior art is solved, a new medicine is provided for preventing and treating encephalitis B virus, and the medicine has good market value and clinical application prospect.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, serve to provide a further understanding of the application and to enable other features, objects, and advantages of the application to be more apparent. The drawings and their description illustrate the embodiments of the invention and do not limit it. In the drawings:

FIG. 1 is a graph showing the effect of rifampicin on viral infection, with drug concentration on the abscissa and JEV viral gene amount on the ordinate;

FIG. 2 is a graph of the effect of rifampicin at different concentrations on cytotoxicity, drug concentration on the abscissa and cell viability on the ordinate;

FIG. 3 is a photograph of immunofluorescence assays for the effect of rifampicin on viral infectivity;

FIG. 4 is a graph of the statistical analysis of fluorescence detection pictures of the effect of rifampicin on viral infectivity;

FIG. 5 shows the effect of rifampicin on the amount of virus invasion, with drug concentration on the abscissa and viral gene amount on the ordinate;

FIG. 6 is a graph of the amount of replication of a virus after rifampicin treatment, with drug concentration on the abscissa and viral gene copy amount on the ordinate;

FIG. 7 is a graph showing the effect of rifampicin on the amount of virus in brain tissue of JEV-infected mice, in which the abscissa represents the grouping and the ordinate represents the amount of virus gene copy in brain tissue.

Detailed Description

In order to make the technical solutions better understood by those skilled in the art, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only partial embodiments of the present application, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

It should be noted that the terms "first," "second," and the like in the description and claims of this application and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It should be understood that the data so used may be interchanged under appropriate circumstances such that embodiments of the application described herein may be used. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

In this application, the terms "upper", "lower", "left", "right", "front", "rear", "top", "bottom", "inner", "outer", "middle", "vertical", "horizontal", "lateral", "longitudinal", and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings. These terms are used primarily to better describe the present application and its embodiments, and are not intended to limit the indicated devices, components or elements to a particular orientation or to be constructed and operated in a particular orientation.

Moreover, some of the above terms may be used to indicate other meanings besides the orientation or positional relationship, for example, the term "on" may also be used to indicate some kind of attachment or connection relationship in some cases. The specific meaning of these terms in this application will be understood by those of ordinary skill in the art as appropriate.

In addition, the term "plurality" shall mean two as well as more than two.

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments with reference to the attached drawings.

The new application of the rifamycin discovered by the application is the application of the rifamycin in preparing the medicines for preventing and treating Japanese encephalitis. When the rifamycin salt is applied to preparing the medicines for preventing and treating the Japanese encephalitis, the rifamycin has an inhibitory effect on the Japanese encephalitis virus.

The inhibition is that the rifamycin salt has the function of inhibiting the Japanese encephalitis virus from replicating in invaded target cells, thereby inhibiting the infection of the virus.

Meanwhile, the application also finds that when the rifamycin is applied to preparing the medicament for preventing and treating the Japanese encephalitis, the preventing and treating effects of the rifamycin and the effect of inhibiting the Japanese encephalitis virus are positively correlated with the concentration of the rifamycin. For example, the rifamycin concentration is 10-40. mu.M.

The following are the experiments in the present application to demonstrate the preventive and therapeutic effects of rifamycin on brain type b: rifampin (Rifampicin) is a representative of rifamycin class of drugs. It should be noted that other rifamycin drugs, such as rifapentine and rifabutin, have the same inhibitory effect and inhibitory mechanism against encephalitis b virus and rifampin, and will not be repeated for saving space. The human neuroblastoma line SK-N-SH used in the application is purchased from ATCC, and the accession number is as follows: ATCCHTB-11.

1JEV infects SK-N-SH cells

(1) SK-N-SH cells (purchased from ATCC, accession number: ATCC HTB-11) were plated on 24-well cell culture plates and cultured for 12-16h so that the cell density at the time of the experiment was about 70% -80% of the well area.

(2) Removing the culture solution during a virus infection experiment, cleaning the virus with 37 ℃ pre-heated PBS for 3 times, inoculating the JEV virus strain SA14, diluting the virus with a full culture solution, wherein the inoculation dose is MOI (equal to 0.5), incubating the virus in an incubator at 37 ℃ for 1h after inoculation, removing the virus solution, rinsing the virus solution with the pre-heated PBS for 3 times, adding a fresh culture medium, and continuously culturing the virus solution for virus detection.

2 real-time fluorescent quantitative PCR (RT-PCR) for detecting JEV virus amount

(1) Extracting total RNA of tissues or cells of each group by using a TRIzol lysis method, and comprises the following steps:

after cell treatment, culture supernatant was removed, washed with PBS 3 times, 1ml of TRIzol was added to the cells, and the cells were lysed by mixing well for 3-5min at room temperature. After animal brain tissue treatment, 100mg of each sample is weighed, after liquid nitrogen is fully ground, 2ml of TRIzol is added, and cells are fully mixed and lysed at room temperature for 5-10 minutes. 1/5 volumes of chloroform were added to each sample, mixed vigorously by hand, and centrifuged at 12,000 rpm for 15min at 4 ℃. After the centrifugation is finished, the upper aqueous phase is carefully sucked up and transferred to a new EP tube, after adding equal volume of isopropanol, the mixture is fully mixed and is placed at room temperature for precipitation for 10 min. Centrifuging at 4 deg.C for 10min at 12,000 rpm, discarding supernatant, and adding 1ml of 75% ethanol pre-cooled at 4 deg.C into each tube. And (3) fully washing the precipitate, centrifuging at 12,000 ℃ for 5min at 4 ℃, discarding the supernatant, opening an EP tube cover, drying the RNA precipitate at room temperature, and adding equal-volume DEPC (diethylpyrocarbonate) to dissolve the precipitate to obtain the total RNA.

(2) The cDNA in each group of samples was extracted using a reverse transcription kit (Takara) according to the following steps:

a reaction system was arranged in a PCR tube,

mixing the above materials by a pipette, placing in a 37 deg.C water bath for reaction for 15min, and heating in 85 deg.C water for 5s to inactivate reverse transcriptase in the system.

(3) Gene expression detection by real-time fluorescent quantitative PCR (RT-PCR)

The reaction system was prepared using SYBR Premix Ex Taq kit (Takara) as follows,

two-step amplification was performed using a Rotor Gene 3000A instrument with pre-denaturation at 95 ℃ for 2min and PCR for 40 cycles of 95 ℃ for 5s and 60 ℃ for 30 s.

3 detection of JEV antigen by immunofluorescence

SK-N-SH cells are treated and then continuously cultured in an incubator for 48h, and the expression of virus antigens is detected by adopting an immunofluorescence method, and the steps are as follows:

(1) fixing the cells: removing culture medium from 96-well plate, adding PBS to wash cells for 3 times, adding 100 μ l precooled methanol into each well, fixing at-20 deg.C for 30min, removing methanol, and washing cells with precooled PBS for 3 times.

(2) Membrane permeation: after the fixed cells were reduced to room temperature, 100. mu.l of 0.1% TritonX-100 was added to each well, incubated in a horizontal shaker at room temperature for 15min, and washed 3 times with PBS.

(3) And (3) sealing: mu.l of 3% BSA was added to each well and incubated for 1h at room temperature on a horizontal shaker.

(4) Incubating the primary antibody: mu.l of mouse-derived monoclonal antibody (1:2000 dilution) specific to JEV envelope protein E was added to each well, incubated at room temperature for 1 hour, and after the incubation was completed, the primary antibody was removed and washed 3 times with PBS.

(5) Incubation of secondary antibody: mu.l of green fluorescence labeled anti-mouse IgG (1:1000 dilution) was added to each well, incubated at room temperature for 1h in the dark, and after the incubation, the secondary antibody was removed, and washed 3 times in the dark with PBS.

(6) Marking cell nucleus: nuclear fluorescent dye DAPI (1:10000 dilution) was added to each well, incubated at room temperature in the dark for 15min, and washed 3 times with pre-cooled PBS in the dark.

(7) And adjusting the fluorescence microscope to a 488nm wavelength fluorescence channel, and detecting and counting the clone number of the green fluorescence positive cells.

4 rifampicin drug action

(1) Grouping: the experiment is divided into a general infection group and a rifampicin drug treatment group, each group is provided with 3 multiple wells for each drug concentration, and the experiment is independently repeated three times.

(2) SK-N-SH cells are laid on a 24-hole cell culture plate to be cultured for 12-16h, so that the cell density is about 70% -80% of the hole area when experiments are carried out. The culture supernatant was aspirated, rinsed 3 times with 37 ℃ pre-warmed PBS, 500. mu.l of each well was added with different concentrations of the drug, incubated at 37 ℃ for 1h, and the same amount of whole culture medium was added to the general infected group for incubation.

(3) The chemical solution was discarded, an equal amount of virus diluent (MOI ═ 0.5) was added to each well, and the wells were incubated at 37 ℃ for 1 hour for virus infection, after which the virus solution was discarded and pre-warmed PBS rinsed 3 times. 500 mul of cell culture medium containing drugs with different concentration gradients is added into each hole respectively, and the cells are cultured for 48 hours continuously and then used for virus detection. The JEV virus amount detection method is the same as the step 2, and the JEV antigen immunofluorescence detection is general in the step 3.

5 cytotoxicity assay

The CCK-8 method is adopted to detect the influence of rifampicin on the cell activity, and comprises the following steps:

collecting cells in logarithmic growth phase, inoculating the cells into a 96-well plate, inoculating 3000 cells in each well, after the cells are cultured for 12 hours in an adherent manner, changing the culture solution into a culture solution containing rifampicin drugs with different concentrations, continuously culturing for 48 hours, detecting the cell proliferation condition, discarding the original culture medium, adding a fresh culture medium containing 10 mu L CCK-8 into each well, 110 mu L, incubating for 3 hours, stopping incubation, detecting the absorbance value of each well by using an enzyme-labeling instrument at the wavelength of 450nm, independently repeating the experiment for 3 times, and calculating the average value.

6 Virus invasion assay

(1) SK-N-SH cells are inoculated in a 96-well plate or a 24-well plate and cultured in an incubator for 12-16 h. The experiment is provided with a drug treatment group and a general infection group, each group is provided with 3 multiple wells for each drug concentration, and the experiment is independently repeated for 3 times.

(2) Rifampicin with different concentration gradients is added into each well and then incubated for 1h at 37 ℃, and the same amount of whole culture solution is added into a general infected group. The JEV virus solution containing the drug was added to the cells, incubated at 4 ℃ for 1h, and then transferred to 37 ℃ for infection for 1 h.

(3) Washing with pre-cooled PBS to remove unbound virus, and adding 400. mu.l proteinase K solution (1mg/ml) per well to remove virus bound to cells but not into cells. Incubate at 4 ℃ for 30min, discard the supernatant, rinse 3 times with pre-cooled PBS. The JEV virus amount detection method is the same as the step 2.

7 Virus replication experiments

(1) Grouping: the experiment is divided into a general infection group and a chemical drug interference group, each group is provided with 3 compound holes for each drug concentration, and the experiment is independently repeated three times.

(2) SK-N-SH cells are laid on a 24-hole cell culture plate to be cultured for 12-16h, so that the cell density is about 70% -80% of the hole area when experiments are carried out. The culture supernatant was aspirated, an equal amount of virus diluent (MOI ═ 0.5) was added to each well, incubation was performed at 37 ℃ for 1 hour for virus infection, and after completion, the virus solution was discarded, and pre-warmed PBS was rinsed 3 times. Adding 500 mul of cell whole culture solution of rifampicin medicine with final concentration of 30 muM into each well, culturing, and collecting each group of cells for virus detection at 2h, 12h and 24h after culture. The JEV detection method is the same as that in step 2.

8 animal experiments

(1) 54C 57B L/6 female mice, 3 weeks old, were randomly divided into 3 groups, a control group, a JEV-infected group + a rifampicin group, and 18 mice per group.

(2) JEV strain SA14 was diluted with DMEM, and the virus was intraperitoneally injected into the JEV-infected group, the JEV-infected group and rifampicin group at a dose of 100. mu.l/mouse (2L D50), and the control group was intraperitoneally injected with an equivalent amount of DMEM to 100. mu.l/mouse, after inoculation, the mice were raised under SPF-grade conditions.

(3) After virus inoculation, the JEV-infected group + rifampicin group was injected with a rifampicin dilution at a dose of 20mg/kg tail vein once a day. The control group and the JEV infected group were injected with an equal volume of PBS solution in tail vein. The mice were observed and recorded daily, and 6 mice were randomly selected from each group at 2, 4, 6 days after virus infection, brain tissue was collected after sacrifice from anesthesia and frozen at-80 ℃. After the experiment is finished, extracting the genome of each group of brain tissues for detecting the virus amount. The detection method is the same as the step 2.

The experimental results are as follows:

rifampicin with different concentration gradients is adopted to act on SK-N-SH cells, and the influence of the rifampicin on JEV infected target cells after the action of the medicament is detected. As a result, it was found that the viral load of JEV gradually decreased with increasing drug concentration (fig. 1), suggesting that rifampicin has an inhibitory effect on JEV infection and exhibits concentration dependence. In order to eliminate the toxic effect of the drugs on the cells, the proliferation activity of the target cells after the drugs with different concentrations act is detected by a CCK8 method, and the result is shown in figure 2, the rifampicin with different concentrations does not produce obvious cytotoxicity, which indicates that the inhibition effect of the rifampicin on the virus infection is not caused by the cytotoxicity. Further, after rifampicin at different concentrations, the influence on JEV infection was analyzed by detecting viral antigen by immunofluorescence assay, and it was found that the inhibition of JEV infection was enhanced with increasing rifampicin concentration, having gradient dependence, consistent with the results of viral RNA detection (fig. 3 and fig. 4). The above results indicate that rifampicin can inhibit JEV infection and that antiviral ability is positively correlated with drug concentration.

Whether the rifampicin acts on the invasion link of the virus is analyzed by adopting a virus invasion experiment, and the result shows that the rifampicin with different concentrations does not influence the JEV to enter the SK-N-SH cells (figure 5), which indicates that the rifampicin does not act on the invasion link of the virus. Further analysis of the effect of rifampicin on viral replication revealed that rifampicin was able to significantly inhibit the copy number of JEV after its action (fig. 6), indicating that rifampicin significantly inhibited viral replication. The effect of rifampicin on JEV replication in vivo is analyzed by using a C57 mouse virus infection animal model, and the effect of rifampicin on JEV in brain tissue of mice can be obviously inhibited after the rifampicin acts compared with a non-administration group (figure 7), so that the effect of rifampicin on JEV infection is further proved.

The above experimental results prove that: the invention finds that rifampicin can obviously inhibit infection of JEV in SK-N-SH cells, and further finds that rifamycin influences infection of encephalitis B virus by inhibiting replication of encephalitis B virus in cells, thereby playing a role in resisting virus. The invention provides a new medicine for preventing and treating the Japanese encephalitis virus, and has better market value and clinical application prospect.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.