Application of calycosin in preparation of anti-herpes virus infection medicine and medicine

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of calycosin in preparation of a medicine for resisting herpes virus infection and the medicine. It is found through a large number of studies that calycosin has a remarkable inhibiting effect on herpes viruses, and the IC50 of the calycosin for resisting KSHV viruses on HUVEC cells is 5.965 micrograms / mL; the IC50 (half maximal inhibitory concentration) of the anti-KSHV virus on an iSLK.219 cell is 3.984 [mu] g / mL; and the IC50 (half maximal inhibitory concentration) of the anti-KSHV virus on the BCBL-1 cell is 3.023 mu g / mL. The calycosin is a low-toxicity and high-efficiency anti-herpes virus substance, can be used for preparing drugs for resisting herpes viruses and preventing and treating diseases caused by the herpes viruses, provides a safe and effective natural drug with small toxic and side effects for clinical treatment of the herpes virus diseases, and has very high clinical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and in particular relates to an application of calycosin in the preparation of an anti-herpes virus infection drug and the drug. Background Art

[0002] Human herpesviruses (HHVs) belong to the family Herpesviridae, which are enveloped viruses with double-stranded DNA as their genetic material. They can be divided into three subfamilies: the alphaherpesvirus subfamily, which includes herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) and varicella zoster virus (VZV or HHV-3); the betaherpesvirus subfamily, which includes human cytomegalovirus (HCMV or HHV-5), HHV-6 and HHV-7; and the gammaherpesvirus subfamily, which consists of two tumorigenic viruses, Epstein–Barr virus (EBV or HHV-4) and Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8). The life cycle of herpesvirus infection includes two states: lytic infection and latent infection. After the virus infects the cell for the first time, both the lytic and latent genes are expressed, the virus replicates and produces multiple progeny virus particles, but after several rounds of replication, the expression of the lytic genes is turned off, and then the latent infection is quickly established. During this period, only a small amount of viral protein is expressed, and no viral particles are produced. However, this latency is reversible. When stimulated by external factors such as chemical, physical, or pathological factors, the latently infected virus is induced to enter the lytic phase and latent reactivation occurs. Herpes virus infection can cause a variety of diseases, including herpes, nervous system infections, and tumors. For example, KSHV is the direct pathogen that causes three human malignancies, including Kaposi Sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). These malignancies are common in immunosuppressed patients, such as those infected with human immunodeficiency virus (HIV), and are one of the main causes of death in the latter. Studies have shown that the lytic replication of the virus is crucial to the development and maintenance of its related diseases. Currently, effective drugs against herpes viruses are mainly based on acyclovir and other nucleoside analogs that target viral DNA polymerase. These drugs can reduce the severity of symptoms to a certain extent, but cannot completely eliminate the infected herpes virus, and the overuse or misuse of these drugs leads to the development of drug resistance, making the treatment ineffective. Therefore, there is an urgent need to find new sources, such as natural products, that can inhibit herpes viruses at different stages of the life cycle without toxicity and drug resistance. Summary of the invention

[0003] The technical problem to be solved by the present invention is to overcome the shortcomings of existing anti-herpes virus drugs, provide a new use of a plant-derived active ingredient, calycosin, in anti-herpes virus, provide a new choice for anti-herpes virus drugs, and provide a safe, effective, and low-toxic and side-effect natural drug for the treatment of clinical herpes virus diseases.

[0004] The technical solution adopted by the present invention to solve the technical problem is:

[0005] Application of calycosin in the preparation of drugs for preventing herpes virus infection.

[0006] Furthermore, the anti-herpes virus infection drug includes one of the following drugs: a drug for treating and / or preventing diseases caused by herpes virus replication, and a drug for treating and / or preventing diseases caused by herpes virus gene expression.

[0007] Furthermore, the disease is herpes, nervous system infection and tumor.

[0008] Furthermore, the herpes virus is of the γ-herpesvirus subfamily.

[0009] Furthermore, the herpes virus is Kaposi's sarcoma-associated herpes virus.

[0010] Furthermore, the half inhibition concentration of calycosin for inhibiting the replication of herpes virus is 3-6 μg / mL.

[0011] Preferably, the IC of calycosin isoflavones in inhibiting KSHV virus replication in HUVEC cells is 50 5.965 μg / mL;

[0012] Preferably, the IC of calycosin isoflavones in inhibiting KSHV viral replication in iSLK.219 cells is 50 3.984 μg / mL;

[0013] Preferably, the IC of calycosin isoflavones for inhibiting KSHV virus replication in BCBL-1 cells is 50 It is 3.023μg / mL.

[0014] Further, the median cytotoxic concentration of calycosin to HUVEC cells is 272.9 μg / mL; or

[0015] Further, the 50% cytotoxic concentration of calycosin to iSLK.219 cells is 138.8 μg / mL; or

[0016] Furthermore, the median cytotoxic concentration of calycosin to BCBL-1 cells was 82.63 μg / mL.

[0017] Furthermore, the calycosin isoflavones inhibit the nucleic acid replication and viral protein expression of herpes virus in cells.

[0018] Furthermore, the calycosin inhibits the replication of herpes virus by down-regulating the expression of early growth response factor 1 (EGR1).

[0019] The purpose of the present invention can also be achieved through the following technical solutions.

[0020] The invention discloses a drug for preventing herpes virus infection, wherein the active ingredient of the drug is calycosin.

[0021] Furthermore, the form of the calycosin includes a salt form thereof or a form in which the calycosin isoflavone is combined with a pharmaceutically acceptable carrier.

[0022] The calycosin isoflavone accounts for 1 to 99% of the total weight of the medicine.

[0023] In addition, based on the combined use of antiviral drugs, in practice, calycosin can also be used in combination with other anti-herpes virus ingredients to enhance the anti-herpes virus and preventive and therapeutic effects. Therefore, the drug can also contain other anti-herpes virus ingredients.

[0024] Furthermore, the drug against herpes virus infection includes one of the following drugs: a drug that inhibits the replication of herpes virus, and a drug that inhibits the gene expression of herpes virus.

[0025] The source of the calycosin, as an optional implementation scheme, can be purchased from the market, chemically synthesized, or extracted from natural plants. The calycosin (CA) is one of the main active ingredients of the traditional Chinese medicinal material Astragalus (Radix Astragali) in my country, and belongs to the flavonoids. In addition to Astragalus, CA can also be extracted from Chinese medicinal materials such as Licorice, Millettia reticulata, and Radix schizonepetae, but its content is highest in Mongolian Astragalus. The present invention discovered a new activity of CA, namely, significantly inhibiting the transcription and replication of KSHV at a non-cytotoxic concentration, and inhibiting the production of KSHV virus particles in a dose-dependent manner. Based on this activity, it can be further developed into a safe, efficient, and low-toxic drug for the treatment of herpes virus infections, which has broad application prospects.

[0026] By means of the above technical solution, the present invention has at least the following advantages:

[0027] 1) The present invention found that the transcription and replication of KSHV was significantly inhibited at the in vitro cell level and had anti-herpes virus activity.

[0028] 2) The toxicity of the isoflavone is low. The toxicity of isoflavone on HUVEC cells, iSLK.219 cells and BCBL-1 cells is 50They are 272.9μg / mL, 138.8μg / mL and 82.63μg / mL respectively.

[0029] 3) Calycosin inhibited KSHV virus replication and the production of virus particles in a dose-dependent manner in HUVEC cells, iSLK.219 cells and BCBL-1 cells. IC 50 The IC50 of anti-KSHV virus on iSLK.219 cells was 3.984 μg / mL; the IC50 of anti-KSHV virus on BCBL-1 cells was 5.965 μg / mL; the IC50 of anti-KSHV virus on iSLK.219 cells was 3.984 μg / mL; the IC 50 It is 3.023μg / mL.

[0030] 4) CC of calycosin provided by the present invention 50 Value than its IC 50 The value is much larger, indicating that it can inhibit viral infection in a wider concentration range without causing great toxicity to cells.

[0031] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The cytotoxicity curves of the calycosin of Example 1 of the present invention on HUVEC (Figure A), iSLK.219 (Figure B), and BCBL-1 (Figure C);

[0033] Figure 2 This is a bar graph and a curve graph showing that calycosin inhibits the transcription and replication of KSHV in HUVEC cells in Example 2 of the present invention;

[0034] Figure 3 The fluorescence graph, bar graph and curve graph of Example 3 of the present invention showing that calycosin inhibits the production of KSHV virus particles;

[0035] Figure 4 This is a bar graph showing that calycosin down-regulates the expression of EGR1 in Example 4 of the present invention. DETAILED DESCRIPTION

[0036] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation methods, structures, features and effects of the present invention are described in detail below in conjunction with the accompanying drawings and preferred embodiments. Unless otherwise specified, the reagents and materials used in the following examples are all reagents and materials that can be obtained from commercial channels.

[0037] Embodiment 1:

[0038] Toxicity experiments of calycosin on normal human umbilical vein endothelial cells (HUVEC), iSLK.219 cells and BCBL-1 cells.

[0039] Experimental reagents: calycosin isoflavone was purchased from Baoji Chenguang Biotechnology Co., Ltd.; ECM endothelial cell culture medium was purchased from Zhejiang Meisen Cell Technology Co., Ltd.; DMEM (high glucose) culture medium and RPMI1640 culture medium were purchased from Shenggong Biotechnology Co., Ltd.; penicillin-streptomycin was purchased from Nanjing Shenghang Biotechnology Co., Ltd., and fetal bovine serum (FBS) was purchased from ZETAlife; 0.25% trypsin (containing EDTA) was purchased from Suzhou Xinsaimei Biotechnology Co., Ltd.; doxycycline (DOX, catalog number: D3072) and sodium butyrate (NaB, catalog number: B5887) were purchased from SIGMA Reagent Company; 12-O-tetradecanoylphorbol acetate-13 (TPA, catalog number: HY-18739) was purchased from MCE Company. Puromycin was purchased from InvivoGen Company; DMSO, geneticin (G418) and hygromycin were purchased from Beyotime Biotechnology Co., Ltd. HUVEC cells (Cat. No.: CTCC-0804-PC) were purchased from Zhejiang Meisen Cell Technology Co., Ltd.; BCBL-1 cells (KSHV latently infected cell line) were donated by Associate Professor Li Dongmei from the School of Medicine of Shihezi University; iSLK.219 cells (this cell line contains latently infected iKSHV219, which is latently activated under the induction of DOX) were donated by Researcher Deng Hongyu from the Institute of Biophysics, Chinese Academy of Sciences.

[0040] Experimental method: HUVEC cells were plated on a 96-well cell culture plate and cultured overnight at 37°C in a cell culture incubator containing 5% CO2. ECM endothelial cell-specific culture medium containing 5% fetal bovine serum (FBS) was used. When the cells proliferated to 80%, the diluted calycosin (6.25μg / mL-400μg / mL) was added to the cell wells and cultured at 37°C in a cell culture incubator containing 5% CO2 for 72h. CCK-8 was used to detect cell activity, that is, 10μl was added to each well, and the cells were placed in a cell culture incubator at 37°C and 5% CO2 for 1.5h, and then the OD value at a wavelength of 450nm was measured using an enzyme marker. Three replicate wells were set for each concentration; a culture medium blank control and a cell control were also set.

[0041] iSLK.219 cells were plated in a 96-well cell culture plate and cultured in DMEM complete medium containing 10% FBS and 1% penicillin-streptomycin, to which 4μg / mL puromycin, 100μg / mL G418 and 100μg / mL hygromycin were added. When the cells proliferated to 80%, DOX (1μg / mL) and NaB (1.2mM) were added to the cell wells and cultured in a cell culture incubator at 37°C with 5% CO2 for 3h, the cell supernatant was discarded, and the DMEM complete medium was replaced, which contained a multiple dilution of calycosin (3.125μg / mL-200μg / mL), and cultured in a cell culture incubator at 37°C with 5% CO2 for 72h. CCK-8 was used to detect cell activity, and the method was the same as above. Three replicate wells were set for each concentration; a blank control of the culture medium and a cell control were also set.

[0042] BCBL-1 cells were cultured in RPMI-1640 complete medium containing 10% FBS and 1% penicillin-streptomycin. During the logarithmic growth phase, TPA (20 ng / ml) was used to induce 3 h in a cell culture incubator at 37 ° C and 5% CO2, and then evenly plated on a 96-well cell culture plate, and a dilution of calycosin (1.6 μg / mL-400 μg / mL) was added to the cell wells, and the cells were cultured in a cell culture incubator for 72 h. CCK-8 was used to detect cell activity, and the method was the same as above. Three replicate wells were set for each concentration; at the same time, a culture medium blank control (culture medium only) and a cell control (cells without drug treatment) were set.

[0043] Cell survival rate of drug-treated group (%) = (OD 450nm - OD of blank control well 450nm / cell control well OD 450nm - OD of blank control well 450nm )×100%. The mean and standard deviation of the results were calculated by GraphPad Prism 5 software, and the half cytotoxic concentration (CC 50 ).

[0044] Experimental results: Effect of calycosin on CC of HUVEC cells, iSLK.219 cells and BCBL-1 cells 50 They were 272.9 μg / mL, 138.8 μg / mL, and 82.63 μg / mL ( Figure 1 ).

[0045] Embodiment 2:

[0046] Calycosin inhibits the transcription and replication of KSHV in HUVEC cells.

[0047] Experimental reagents: calycosin was purchased from Baoji Chenguang Biotechnology Co., Ltd.; DMSO was purchased from Biyuntian Biotechnology Co., Ltd.; ECM endothelial cell culture medium was purchased from Zhejiang Meisen Cell Technology Co., Ltd.; 0.25% trypsin (containing EDTA) was purchased from Suzhou Xinsaimei Biotechnology Co., Ltd. TRIzol reagent was purchased from Invitrogen; blood / cell / tissue genomic DNA extraction kit and DNaseⅠ were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; reverse transcription kit HiScript IIIRT SuperMix for qPCR (+gDNAwiper) was purchased from Novozyme Biotechnology Co., Ltd. 2×RealStarFast SYBR qPCR Mix was purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd. The plasmid expressing KSHV ORF K9 (referred to as K9 plasmid) was constructed by this laboratory.

[0048] Experimental method: HUVEC cells were plated on a 24-well cell culture plate overnight, and when the cells proliferated to about 90%, the KSHV virus induced from iSLK.219 cells was infected with HUVEC cells (MOI = 5) by centrifugal infection, i.e., 2500rpm, 30°C centrifugal infection for 2 hours, and then the supernatant was removed, washed twice with PBS, and then diluted calycosin (1μg / mL, 5μg / mL, 10μg / mL, 25μg / mL, 50μg / mL) was added to the cell wells, and continued to be cultured for 24h in a cell culture incubator at 37°C with 5% CO2, and then the cells and cell supernatant were collected. For intracellular viral DNA, a blood / cell / tissue genomic DNA extraction kit was used, and then relative quantification was performed by quantitative PCR (qPCR), and the relative change of the KSHV K9 gene was evaluated using intracellular GAPDH as an internal reference control gene. The viral DNA content in the cell supernatant is used to measure the level of viral particles produced. The same blood / cell / tissue genomic DNA extraction kit is used for this purpose. However, before extraction, DNase I is added and treated at room temperature for 10 minutes. Then, the stop solution is added and placed in a 70°C water bath for 10 minutes to remove free DNA and reduce experimental errors. After obtaining the DNA in the supernatant, absolute quantitative PCR is performed. First, a K9 plasmid standard is prepared. The K9 plasmid is diluted 10 9 / μl, and then dilute 10 times to get 10 8 -10 1A total of 8 concentration gradient standards were used, and then qPCR was performed. The standards and extracted DNA samples were used as templates and performed simultaneously. Finally, a standard curve was made, and the copy number of K9 in the sample was calculated based on this standard curve to characterize the copy number of the KSHV genome. For intracellular RNA, TRIzol reagent was used to extract the RNA, and after reverse transcription into cDNA, qPCR was performed to detect the transcription level of different viral genes in the cell. The experiment was repeated three times, and the mean and standard deviation of the results were calculated using GraphPadPrism 5 software, and the half inhibitory concentration (IC 50 ). The qPCR primer sequences used are as follows:

[0049] GAPDH upstream primer: 5'-ATCCCATCACCATCTTCCAGG-3'

[0050] GAPDH downstream primer: 5'-CCTTCTCCATGGTGGTGAAGAC-3'

[0051] K9 upstream primer: 5'-GTCTCTGCGCCATTCAAAAC-3'

[0052] K9 downstream primer: 5'-CCGGACACGACAACTAAGAA-3'

[0053] RTA upstream primer: 5'-CACAAAAATGGCGCAAGATGA-3'

[0054] RTA downstream primer: 5'-TGGTAGAGTTGGGCCTTCAGTT-3'

[0055] K8.1 upstream primer: 5'-AAAGCGTCCAGGCCACCACAGA-3'

[0056] K8.1 downstream primer: 5'-GGCAGAAAATGGCACACGGTTAC-3'

[0057] ORF46 upstream primer: 5'-CTGGGATTGGTTCACGAGTT-3'

[0058] ORF46 downstream primer: 5'-TGAGCGGAGTTCTGTCAATG-3'

[0059] Experimental results: Calycosin has anti-KSHV activity in HUVEC cells and reduces the transcription and replication of KSHV in cells in a dose-dependent manner. The IC value of calycosin for inhibiting KSHV replication was calculated based on the viral DNA copy number in the supernatant and the multiple relative to the DMSO-treated group. 505.965 μg / mL ( Figure 2 ).

[0060] in Figure 2 In the figure, Figure A shows the relative transcription levels of different KSHV genes after treatment with calycosin, with GAPDH as the internal reference gene; Figure B shows the relative change level of KSHV DNA in HUVEC cells, with GAPDH as the internal reference gene; Figure C shows the relative change fold of KSHV viral genome copy number in the cell supernatant corresponding to Figure B. The data were compared with the relative fold obtained by comparing with the infection group without drug addition.

[0061] Embodiment 3:

[0062] The experiment of inhibiting KSHV latent reactivation by calycosin in iSLK.219 cells and BCBL-1 cells.

[0063] Experimental reagents: calycosin isoflavone was purchased from Baoji Chenguang Biotechnology Co., Ltd.; DMSO was purchased from Biyuntian Biotechnology Co., Ltd.; DMEM (high glucose) and RPMI-1640 medium were purchased from Shenggong Biotechnology Co., Ltd.; penicillin-streptomycin was purchased from Nanjing Shenghang Biotechnology Co., Ltd., fetal bovine serum (FBS) was purchased from ZETAlife; 0.25% trypsin (containing EDTA) was purchased from Suzhou Xinsaimei Biotechnology Co., Ltd. Doxycycline (DOX, catalog number: D3072) and sodium butyrate (NaB, catalog number: B5887) were purchased from SIGMA Reagent Company; 12-O-tetradecanoylphorbol acetate-13 (TPA, catalog number: HY-18739) was purchased from MCE Company.

[0064] Experimental method: iSLK.219 cells were plated on a 24-well cell culture plate overnight. After the cells proliferated to 90%, inducers DOX (1 μg / mL) and NaB (1.2 mM) were added to the cell wells and cultured in a cell culture incubator at 37°C with 5% CO2 for 3 h. The cell supernatant was discarded and replaced with fresh culture medium containing the above inducers and different concentrations of calycosin (1 μg / mL, 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL). The culture was continued. After 24 h, the fluorescence was observed under a fluorescence microscope, and then the cells and supernatant were collected to extract the DNA in the cells and supernatant. The experimental method in Example 2, i.e., quantitative PCR, was used to detect the level of viral particles produced. The experiment was repeated three times. At the same time, a cell control well without drug and a control well induced by DOX and NaB were set.

[0065] TPA (20 ng / ml) and corresponding concentrations of calycosin (1 μg / mL, 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL) were added to BCBL-1 cells, and the cells and supernatant were collected after culturing at 37°C in a cell culture incubator containing 5% CO2 for 24 hours, and DNA in the cells and supernatant was extracted. The experimental method in Example 2, i.e., quantitative PCR, was used to detect the level of viral particles produced. The experiment was repeated three times. At the same time, a cell control well without drug and a control well induced by TPA were set.

[0066] Experimental results: Calycosin significantly inhibited the latent reactivation of KSHV in iSLK.219 cells induced by DOX and NaB, as shown by the significant decrease in red fluorescence in iSLK.219 cells with the increase in concentration; the expression level of viral DNA in the cells was significantly decreased, and the copy number of viral DNA in the cell supernatant (representing the induced viral particles) was also significantly reduced ( Figure 3 AC). Similarly, calycosin effectively inhibited the latent reactivation of KSHV in BCBL-1 cells induced by TPA in a concentration-dependent manner ( Figure 3 DE). According to the changes in the number of KSHV K9 gene copies in the supernatant, the IC of calycosin for inhibiting the production of viral particles in iSLK.219 cells and BCBL-1 cells was calculated. 50 They are 3.984μg / mL and 3.023μg / mL respectively.

[0067] Figure 3 Figures AC show the results of treating iSLK.219 cells. After iSLK.219 cells were induced by DOX and NaB, the recombinant KSHV virus in them entered lytic replication and produced red fluorescence. Figure A shows the fluorescence image of iSLK.219 cells treated with calycosin; Figure B shows the relative changes in the level of KSHV DNA in iSLK.219 cells that entered lytic replication after treatment with calycosin, GAPDH was used as the internal reference gene, and the data were normalized to the uninduced cell group; Figure C shows the relative fold change of the KSHV genome copy number in the cell supernatant corresponding to Figure B, and the relative fold obtained by comparison with the induced but drug-free group as the control. Figure DE shows the results of treating BCBL-1 cells. After BCBL-1 cells were induced by TPA (20ng / mL), the KSHV virus in them entered lytic replication. Figure D shows the relative change of KSHV DNA level in TPA-induced BCBL-1 cells after treatment with calycosin; Figure B shows the relative fold change of KSHV genome copy number in the cell supernatant corresponding to Figure A, compared with the relative fold obtained by comparing with the induced but no drug group.

[0068] Embodiment 4:

[0069] Calycosin inhibits KSHV replication by downregulating the expression of Early Growth Response Factor-1 (EGR1).

[0070] Experimental reagents: calycosin isoflavone was purchased from Baoji Chenguang Biotechnology Co., Ltd.; ECM endothelial cell culture medium was purchased from Zhejiang Meisen Cell Technology Co., Ltd.; DMEM (high glucose) culture medium and RPMI1640 culture medium were purchased from Shenggong Biotechnology Co., Ltd.; penicillin-streptomycin was purchased from Nanjing Shenghang Biotechnology Co., Ltd., and fetal bovine serum (FBS) was purchased from ZETAlife; 0.25% trypsin (containing EDTA) was purchased from Suzhou Xinsaimei Biotechnology Co., Ltd.; doxycycline (DOX, catalog number: D3072) and sodium butyrate (NaB, catalog number: B5887) were purchased from SIGMA Reagent Company; 12-O-tetradecanoylphorbol acetate-13 (TPA, catalog number: HY-18739) was purchased from MCE Company. Puromycin was purchased from InvivoGen Company; DMSO, geneticin (G418) and hygromycin were purchased from Beyotime Biotechnology Co., Ltd. HUVEC cells (Cat. No.: CTCC-0804-PC) were purchased from Zhejiang Meisen Cell Technology Co., Ltd.; BCBL-1 cells (KSHV latently infected cell line) were donated by Associate Professor Li Dongmei from the School of Medicine of Shihezi University; iSLK.219 cells (this cell line contains latently infected iKSHV219, which is latently activated under the induction of DOX) were donated by Researcher Deng Hongyu from the Institute of Biophysics, Chinese Academy of Sciences.

[0071] Experimental methods: (1) HUVEC cells were plated on a 24-well cell culture plate overnight. When the cells proliferated to about 90%, the KSHV virus induced from iSLK.219 cells was infected with HUVEC cells (MOI=5) by centrifugation infection, and the supernatant was removed. After washing twice with PBS, 50 μg / mL of calycosin was added to the cell wells, and the cells were cultured in a cell culture incubator at 37°C with 5% CO2 for 24 hours, and then the cells were collected. Cell control and calycosin treatment control were set at the same time. (2) For BCBL-1 cells, they were treated with TPA (20 ng / ml) and 50 μg / mL of calycosin, and the cells were cultured in a cell culture incubator at 37°C with 5% CO2 for 24 hours, and then the cells were collected. (3) For iSLK.219 cells, they were plated on a 24-well cell culture plate overnight. After the cells proliferated to 90%, inducers DOX (1 μg / mL) and NaB (1.2 mM) were added to the cell wells and cultured in a cell culture incubator at 37°C with 5% CO2 for 3 h. The cell supernatant was discarded and replaced with fresh culture medium containing the above inducers and different concentrations of calycosin (5 μg / mL, 25 μg / mL, 50 μg / mL). The culture was continued and the cells were collected after 24 h. TRIzol reagent was used to extract RNA in the cells, and after reverse transcription of RNA into cDNA, qPCR was performed to detect the expression of EGR1 in the cells. The qPCR primer sequences used are as follows:

[0072] GAPDH upstream primer: 5'-ATCCCATCACCATCTTCCAGG-3'

[0073] GAPDH downstream primer: 5'-CCTTCTCCATGGTGGTGAAGAC-3'

[0074] ERG1 upstream primer: 5'-CCACGCCGAACACTGACATT-3'

[0075] ERG1 downstream primer: 5'-GAGGGGTTAGCGAAGGCTG-3'

[0076] Experimental results: Calycosin has no significant effect on the background level of EGR1 expression in HUVEC cells, but after KSHV infection and then treated with Calycosin, the expression level of EGR1 was significantly downregulated ( Figure 4 A). The same result was obtained in BCBL-1 cells, that is, calycosin significantly reduced the level of EGR1 induced by TPA ( Figure 4 B). Moreover, after KSHV latent activation in iSLK.219 cells, calycosin reduced the expression of EGR1 in a dose-dependent manner ( Figure 4C). EGR is a transcriptional regulator of the key KSHV gene RTA (a molecular switch that initiates replication from latent phase to lytic phase). It directly binds to the promoter region of RTA to promote the transcription of the RTA gene, thereby promoting the replication and latent reactivation of KSHV. Combined with the above results, it is shown that calycosin inhibits the transcriptional replication and latent reactivation of KSHV by downregulating EGR1.

[0077] Figure 4 Expression of AEGR1 in HUVEC cells. The mRNA expression level of EGR1 in HUVECs treated with or without calycosin after infection with KSHV. GAPDH was used as the internal reference gene, and the uninfected group without calycosin treatment was used as the control for normalization. Figure 4 Expression of BEGR1 in BCBL-1 cells. The expression of EGR1 gene changes after BCBL-1 cells were induced by TPA (20 ng / mL) and treated or not with calycosin. GAPDH was used as the internal reference gene, and the uninduced group without calycosin treatment was used as the control for normalization. Figure 4 The expression level of EGR1 in CiSLK.219 cells. After iSLK.219 cells were induced by DOX and NaB, the recombinant KSHV virus in them entered lytic replication, and then the expression changes of EGR1 after being treated with different concentrations of calycosin, GAPDH was used as the internal reference gene, and the relative change fold of the gene was calculated with the group without calycosin as the control.

[0078] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention still falls within the scope of the technical solution of the present invention.

Claims

1. Application of calycosin in the preparation of drugs against herpes virus infection.

2. The application according to claim 1, characterized in that: The anti-herpes virus infection drug includes one of the following drugs: a drug for treating and / or preventing diseases caused by herpes virus replication, and a drug for treating and / or preventing diseases caused by herpes virus gene expression.

3. The application according to claim 2, characterized in that: The diseases are herpes, nervous system infections and tumors.

4. The use according to claim 1, characterized in that: The half inhibitory concentration of calycosin isoflavones in inhibiting herpes virus replication is 3-6 μg / mL.

5. The use according to claim 1, characterized in that: The herpes virus is of the γ-herpesvirus subfamily, Preferably, the herpes virus is Kaposi's sarcoma-associated herpes virus.

6. The use according to claim 1, characterized in that: The calycosin isoflavones inhibit the nucleic acid replication and viral protein expression of herpes virus in cells.

7. The use according to claim 6, characterized in that: The calycosin isoflavones inhibit herpes virus replication by down-regulating the expression of early growth response factor 1 (EGR1).

8. A drug for resisting herpes virus infection, characterized in that: The active ingredient of the medicine is calycosin.

9. The drug according to claim 8, characterized in that The forms of the calycosin include the form of its salt or the form of the calycosin in combination with a pharmaceutically acceptable carrier.

10. The drug according to claim 8, characterized in that The drug for resisting herpes virus infection includes one of the following drugs: a drug for inhibiting the replication of herpes virus and a drug for inhibiting the gene expression of herpes virus.

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