Application of triglyceride reducing drug as latent infection herpes virus reactivation inhibitor

Abstract

The invention provides an application of a triglyceride reducing drug as a latent infection blister disease virus reactivation inhibitor. More specifically, the invention provides application of a medicine for reducing triglyceride or an active ingredient of the medicine in preparation of a herpes virus reactivation inhibitor or a medicine composition for preventing or treating herpes virus reactivation related diseases.

Description

Technical Field

[0001] The present invention relates to the field of medicines, and in particular to the use of a triglyceride-lowering drug as a reactivation inhibitor of latently infected herpes viruses. Background Art

[0002] Human herpesviruses (HHVs) are a group of highly contagious viruses that can cause a variety of diseases, ranging from mild skin lesions to severe systemic diseases. Among them, Epstein-Barr Virus (EBV) is a member of the γherpesvirus subfamily and is the first human tumor virus discovered. With a research history of more than 50 years, about 95% of adults worldwide have experienced infection and carry EBV for life. EBV can enter cells through endocytosis or fusion with the plasma membrane. After injecting the viral genome into the nucleus, it can establish a long-term latent state in the host cell and coexist with the host. However, when cells encounter various triggers such as hypoxia, Hippo signaling pathway, inflammasome induction, DNA damage, BCR activation in B cells, and plasma cell differentiation, the virus's early genes BZLF1 and BRLF1 will be activated and initiate transcription of other viral genes, inducing EBV lysis and reactivation, and producing a large number of new viruses. In cultured cells, EBV reactivation can be induced by chemical agents, such as phorbol esters (TPA), calcium ionophores, histone deacetylase inhibitors, and DNA methylase inhibitors, as well as by biological stimuli such as transforming growth factor β (TGF-β), anti-immunoglobulins, hypoxia, reactive oxygen species, and temperature changes.

[0003] It is estimated that more than 200,000 cases of cancer are related to EBV infection each year, and about 1.8% of all deaths caused by cancer are directly related to malignant tumors caused by EBV. Currently known EBV-related diseases include chronic active EBV infection (CAEBV), lymphoproliferative diseases (PTLD), diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma (HL), Burkitt lymphoma (BL), nasopharyngeal carcinoma (NPC), gastric cancer (GC), NK / T cell lymphoma (NKTCL), hemophagocytic syndrome (HPS), etc. Among them, HPS is a common fatal complication of NKTCL. EBV plays an important role in the development of NKTCL into HPS, so inhibiting EBV lysis and reactivation is a very important treatment strategy for NKTCL and HPS.

[0004] Currently, there are no drugs specifically targeting EBV in clinical practice. Finding effective and safe drugs that can effectively inhibit EBV reactivation is of great significance for the treatment of EBV reactivation-related diseases such as NKTCL and HPS. Summary of the invention

[0005] The purpose of the present invention is to provide a safe and effective herpes virus reactivation inhibitor.

[0006] In a first aspect, the present invention provides use of a triglyceride-lowering drug or an active ingredient thereof in the preparation of a herpes virus reactivation inhibitor.

[0007] In another preferred embodiment, the herpes virus is selected from the following group: Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpes virus (KSHV), or a combination thereof.

[0008] In another preferred embodiment, the triglyceride-lowering drug is selected from: fibrate drugs such as fenofibrate, gemfibrozil, bezafibrate; niacin drugs such as niacin and acipimox.

[0009] In a second aspect, the present invention provides use of a triglyceride-lowering drug or an active ingredient thereof in the preparation of a pharmaceutical composition for preventing or treating diseases associated with herpes virus reactivation.

[0010] In another preferred embodiment, the triglyceride-lowering drug is selected from: fibrate drugs such as fenofibrate, gemfibrozil, bezafibrate; niacin drugs such as niacin and acipimox.

[0011] In another preferred embodiment, the disease is selected from the following group: chronic active Epstein-Barr virus infection (CAEBV), lymphoproliferative disease (PTLD), diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma (HL), Burkitt lymphoma (BL), nasopharyngeal carcinoma (NPC), gastric cancer (GC), NK / T cell lymphoma (NKTCL), hemophagocytic syndrome (HPS), or a combination thereof.

[0012] In another preferred embodiment, the above-mentioned cancer is a cancer and / or cell latently infected with herpes virus.

[0013] In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0014] In another preferred embodiment, the dosage form of the pharmaceutical composition is a liquid dosage form or a solid dosage form.

[0015] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: tablets, capsules, pills, granules, injections, powder injections, patches, and liposomes.

[0016] In a third aspect, the present invention provides a method for inhibiting the reactivation of herpes virus in cells, comprising the steps of contacting and culturing cells latently infected with herpes virus with a triglyceride-lowering drug or its active ingredient, thereby inhibiting the reactivation of herpes virus in cells.

[0017] In another preferred embodiment, the above method is in vitro non-therapeutic and non-diagnostic.

[0018] In a fourth aspect, the present invention provides a method for preventing or treating a disease associated with herpes virus reactivation, comprising the steps of administering a triglyceride-lowering drug or its active ingredient to a subject in need, thereby preventing or treating a disease associated with herpes virus reactivation.

[0019] In another preferred embodiment, the subject is a subject latently infected with herpes virus.

[0020] In another preferred embodiment, the subject is a human or a mammal, such as a rat, a mouse, or a monkey.

[0021] In another preferred embodiment, the triglyceride-lowering drug or its active ingredient can be administered preventively before herpes virus reactivation or therapeutically after herpes virus reactivation occurs, preferably before herpes virus reactivation.

[0022] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It shows that fenofibrate and acipimox can reduce the triglyceride content in SNK-1 cells; (A) IC50 test was performed on SNK-1 cells to ensure that the selected drug dose would not cause massive cell death; (B) SNK-1 was stimulated with different doses of acipimox (100μM, 0.5mM) (left) and fenofibrate (50μM, 100μM) (right) for 24h, and then the cells were stained with 2μM BODIPY dye and observed using a fluorescence microscope. Figure 1 Green fluorescence in B indicates lipid droplets storing triglycerides.

[0024] Figure 2 The results showed that fenofibrate and acipimox can inhibit EBV reactivation in SNK-1 and SNT-8 cells; (A) SNK-1 and SNT-8 cells were stimulated with different doses of fenofibrate (50μM, 100μM) and acipimox (0.5mM, 1mM) for 30min, respectively, and then treated with 20ng / ml TPA for 24h or 48h, the cell pellets were collected, WB experiments were performed, and immunoblotting was performed with the antibodies shown in the figure; (B) SNK-1 and SNT-8 cells were stimulated with 100μM fenofibrate and 1mM acipimox for 30min, respectively, and then treated with 20ng / ml TPA for 24h, the cell pellets were collected, total RNA was extracted, reverse transcribed to synthesize cDNA, and the expression of viral genes in the samples was detected by qPCR, and normalized with the expression of GAPDH.

[0025] Figure 3 It shows that fenofibrate and acipimox can inhibit EBV reactivation in Akata, LCL, and Raji. Akata, LCL, and Raji cells were stimulated with the doses of fenofibrate and acipimox indicated in the figure for 30 minutes, followed by the addition of 10ug / ml Ig (G+M) for 24 hours, and the cell pellets were collected for WB experiments and immunoblotting was performed with the antibodies indicated in the figure. DETAILED DESCRIPTION

[0026] The present inventor has conducted extensive and in-depth research, and through a large number of screenings and tests, provided the use of a triglyceride-lowering drug as a latently infected herpes virus reactivation inhibitor. The present invention unexpectedly discovered for the first time that reducing triglycerides in cells can inhibit the reactivation of latently infected herpes viruses in cells, thereby preventing and treating diseases related to herpes virus reactivation, and further provided the use of a triglyceride-lowering drug as a latently infected herpes virus reactivation inhibitor. The present invention was completed on this basis.

[0027] the term

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0029] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0030] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0031] As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4-40°C, preferably, 25±5°C.

[0032] Herpes virus reactivation-related diseases

[0033] Human herpesviruses (HHVs) are a group of highly contagious viruses that can cause a variety of diseases, ranging from mild skin lesions to severe systemic diseases. There are currently eight different herpesviruses known to infect humans, each with its own specific clinical manifestations and transmission routes. Human herpesviruses include: herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus type 6 (HHV-6), human herpesvirus type 7 (HHV-7), Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8).

[0034] Herpes virus reactivation is the reactivation of a previously latent virus, resulting in viral replication and the production of new viral particles. This reactivation can cause a variety of diseases, depending on the type of virus and the host's immune status.

[0035] More preferably, the present invention is more concerned with Epstein-Barr virus (EBV) Kaposi's sarcoma-associated herpes virus (KSHV), because the reactivation of these two viruses can lead to serious cancer diseases, such as (but not limited to) chronic active EB virus infection (CAEBV), lymphoproliferative diseases (PTLD), diffuse large B-cell lymphoma (DLBCL), Hodgkin's lymphoma (HL), Burkitt's lymphoma (BL), nasopharyngeal carcinoma (NPC), gastric cancer (GC), NK / T cell lymphoma (NKTCL), hemophagocytic syndrome (HPS), or a combination thereof. It is understood by those skilled in the art that the cancer of the present invention refers particularly to cancers in which herpes virus latent infection exists in those cells. There is currently no clinically controlled drug related to EBV reactivation, and there is no report of a triglyceride-lowering drug for reducing EBV load and treating EBV-related diseases.

[0036] Active ingredients

[0037] The present invention unexpectedly discovered for the first time that a triglyceride-lowering drug or its active ingredient can inhibit the reactivation of latently infected herpes viruses in cells, thereby providing the use of a triglyceride-lowering drug as the active ingredient of the present application in the preparation of a herpes virus reactivation inhibitor, and the use of the triglyceride-lowering drug in the preparation of a pharmaceutical composition for preventing or treating diseases related to herpes virus reactivation.

[0038] In clinical practice, drugs such as fibrates and nicotinic acid are often used to lower triglycerides and treat hypertriglyceridemia. Typically, fibrates include (but are not limited to) fenofibrate, gemfibrozil, and bezafibrate; nicotinic acid drugs include (but are not limited to) nicotinic acid and acipimox.

[0039] It should be noted that in the inhibitor or pharmaceutical composition of the present invention, the active ingredient for inhibiting herpes virus reactivation refers to the active ingredient for lowering triglycerides in the above-mentioned drugs, and there is no need to limit its formulation form. It can be prepared as a new formulation or use its known formulation form under appropriate circumstances, which can be selected as needed.

[0040] The active ingredients of triglyceride-lowering drugs are known to those skilled in the art, especially those drugs that are already on the market, and their active ingredients can be used in the present invention.

[0041] Typically, the active ingredient in fenofibrate is:

[0042] The active ingredients in gemfibrozil are:

[0043] The active ingredient in bezafibrate is:

[0044] niacin:

[0045] The active ingredient in acipimox is:

[0046] The present invention is also intended to include the pharmaceutically acceptable salts of the above active compounds, or any other known pharmaceutically acceptable forms.

[0047] Pharmaceutical compositions and methods of administration

[0048] The present invention provides for the first time the use of a triglyceride-lowering drug or its active ingredient as the active ingredient of the present application in preparing a herpes virus reactivation inhibitor composition, and in preparing a pharmaceutical composition for preventing or treating a herpes virus reactivation-related disease.

[0049] The pharmaceutical composition of the present invention comprises the active ingredient of the present invention within the safe and effective amount range and a pharmacologically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, and more preferably, contains 10-500 mg of the compound of the present invention per dose. Preferably, the "one dose" is a capsule or tablet.

[0050] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0051] There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administrations include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0052] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0053] Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

[0054] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0055] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0056] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances, and the like.

[0057] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0058] Dosage forms for topical administration of the compounds of the invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0059] The compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds. In another preferred embodiment, the pharmaceutical composition further comprises one or more anticancer agents, immunosuppressants, antiviral agents, or a combination thereof.

[0060] The general range of the therapeutically effective dose of the active ingredient of the present invention will be: about 1-2000mg / day, about 10-about 1000mg / day, about 10-about 500mg / day, about 10-about 250mg / day, about 10-about 100mg / day, or about 10-about 50mg / day. The therapeutically effective dose will be given with one or more doses. However, it should be understood that the specific dose of the compounds of the present invention for any particular patient will depend on multiple factors, for example, the age, sex, body weight, general health, diet, individual response of the patient to be treated, the activity, dosage form, application mode and concomitant drug of the specific compound of the time, the severity of the disease to be treated, the administration. The therapeutically effective amount of a given situation can be determined with routine experiments, and within the scope of the clinician or physician's ability and judgment. In any case, the compound or composition will be given with multiple doses based on the individual situation of the patient and in a manner that allows the delivery of a therapeutically effective amount.

[0061] The main advantages of the present invention include:

[0062] 1. The present invention provides for the first time a drug capable of inhibiting the reactivation of herpes viruses represented by EBV.

[0063] 2. The present invention first discovered that triglyceride-lowering drugs represented by fenofibrate and acipimox can inhibit the reactivation of latently infected herpes viruses (such as EBV and KSHV) in tumor cells, thereby providing their use in preventing or treating diseases related to herpes virus reactivation.

[0064] The present invention will be further described below in conjunction with specific implementation. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0065] Example 1

[0066] Materials and methods

[0067] 1 Experimental Materials

[0068] 1.1 Cell lines

[0069] EBV-positive B lymphoma cell lines Akata and Raji cells were cultured in RPMI1640 medium containing 10% fetal bovine serum (FBS) and 1% double antibodies (penicillin and streptomycin). EBV-positive B lymphoma cell line LCL was cultured in imported RPMI1640 medium containing 10% imported fetal bovine serum (Gibico-FBS) and 1% double antibodies (penicillin and streptomycin). EBV-positive NK / T lymphoma cell line SNT-8 / SNK-1 cells were cultured in AIM-V medium containing 10% human serum (Human Serum).

[0070] 1.2 Reagents and Antibodies

[0071] Antibodies against ZTA (Santa Cruz, sc-53904) and GAPDH (Santa Cruz, sc-32233) were used for western blotting experiments.

[0072] Bodipy493 / 503 (Invitrogen, D3922) is a lipid droplet-marking dye that can pass through the cell membrane into the cell interior and perform specific staining by localizing on polar lipids such as triglycerides in the cell.

[0073] The DNA methyltransferase inhibitor 12-O-tetradecanoylphorbol acetate-13 (TPA, CST, 4174S) was used to reactivate EBV in SNK-1 and SNT-8 cells.

[0074] F(ab')2Fragment Goat Anti-Human IgG+IgM(H+L) (Ig(G+M), Jackson ImmunoResearch, 109-006-127) was used to reactivate EBV in Akata, LCL, and Raji cells.

[0075] Acipimox (selleck, S1806) is a niacin derivative and fenofibrate (selleck, S1794) is a fibrate compound used to lower triglycerides in cells.

[0076] 2 Experimental methods

[0077] 2.1 Observation of lipid droplet levels in cells under fluorescence microscopy

[0078] After 4×105 SNK-1 cells were plated in a six-well plate, 100 μM, 500 μM acipimox or 50 μM, 100 μM fenofibrate were added. After 24 h, the cells were stained with 2 μM BODIPY at 37°C and fixed with 500 μL 4% PFA after 30 min. The cells were observed under a fluorescence microscope, and the green fluorescence indicated the lipid droplets in the cells.

[0079] 2.2 Western Blot

[0080] 4×105 SNT-8 and SNK-1 cells were plated in six-well plates, and 0.5mM, 1mM acipimox or 50μM, 100μM fenofibrate were added and cultured for 30min. Then, 20ng / ml TPA was added to induce EBV lysis and reactivation in the cells, and the cell pellets were collected after 24h or 48h.

[0081] 5×105 Akata cells were plated in six-well plates, 0.5mM, 1mM acipimox or 10μM, 20μM fenofibrate were added and cultured for 30min, then 10ug / ml Ig(G+M) was added to induce EBV lysis and reactivation in the cells, and the cell pellets were collected after 24h.

[0082] 5×105 LCL cells were plated in a six-well plate, and 1mM, 1.5mM acipimox or 10μM, 20μM fenofibrate were added and cultured for 30min. Then, 10ug / ml Ig(G+M) was added to induce EBV lysis and reactivation in the cells, and the cell pellets were collected after 24h.

[0083] 5×105 Raji cells were plated in a six-well plate, and 0.5mM, 1mM acipimox or 50μM, 100μM fenofibrate were added and cultured for 30min. Then, 10ug / ml Ig(G+M) was added to induce EBV lysis and reactivation in the cells, and the cell pellets were collected after 24h.

[0084] (1) SDS-polyacrylamide gel (SDS-PAGE) preparation: 10% ExpressCast PAGE Color Gel Rapid Kit (New Cycle, P2012) was used according to the product instructions.

[0085] (2) Sample preparation: Add 1 ml of PBS to the above cell samples and wash once, and collect the cell pellet. Add an appropriate volume of 1x SDS Loading Buffer, mix well by pipetting, and place on ice. Use an ultrasonic disruptor (VCX 130, Vibracell) to lyse the cells, sonicate three times with 50% Ampl for 5 seconds each time, and cool on ice in between. After the sonication, boil the sample in a 100°C metal bath for 5 minutes, and finally place the sample on ice.

[0086] (3) Sample loading: 100 ml 10x Running Buffer (30.2 g Tris-base, 188 g Glycine, 10 g SDS, add ddH2O to 1 L, stir and mix) add 900 ml ddH2O to prepare 1x Running Buffer. Assemble the electrophoresis tank, fix the gel plate in the electrophoresis tank, and add 1x Running Buffer. Carefully remove the comb, place the samples in the sample wells in order, and add protein marker (Thermo, 26616).

[0087] (4) Electrophoresis: Turn on the power supply and start electrophoresis at 150V for 1 hour. After completion, remove the gel plate.

[0088] (5) Wet transfer: ii100ml 10xTransfer Buffer (30.3g Tris-base, 144.1g Glycine, add ddH2O to 1L, stir and mix) add 700ml ddH2O and 200ml methanol to prepare 1xTransfer Buffer, and place it in a 4℃ refrigerator to pre-cool. Carefully pry open the glass plates on both sides of the gel, take out the gel and place it in 1xTransfer Buffer. Take out the PVDF membrane and soak it in methanol for a while. Make a transfer "sandwich" and assemble it from bottom to top in the order of negative electrode-sponge-filter paper-gel-PVDF membrane-filter paper-sponge-positive electrode, and be careful not to have bubbles. Transfer the "sandwich" gel layer to the wet transfer tank, add 1xTransfer Buffer, connect the power supply to start the transfer, 100V, 1.5h. To prevent the temperature from rising during the transfer process, place the entire transfer tank in an ice bath.

[0089] (6) Blocking: After the transfer, remove the PVDF membrane and soak it in 1×TBST buffer containing 5% skimmed milk powder for 1 hour at room temperature.

[0090] (7) Primary antibody incubation: Discard the milk, dilute ZTA at a ratio of 1:1000 and GAPDH at a ratio of 1:3000 with 5% skim milk, and incubate at 4°C with shaking overnight.

[0091] (8) Secondary antibody incubation: discard the primary antibody, wash the membrane three times with TBST buffer, shaking on a shaker for 8 min each time, add mouse secondary antibody diluted 1:10,000 with skim milk, and incubate at room temperature with shaking for 1 h.

[0092] (9) Development: discard the secondary antibody and wash the membrane three times with TBST buffer. The developer is Immobilon Western Chemiluminscent HRP Substrate (P90720, Millipore), which is prepared in a 1:1 ratio and used immediately. Use a chemiluminescent imager (Tanon, 500) for development. Take out the membrane, dry the surface solution with absorbent paper, spread it flat on the imager development tray, and add developer until it just covers the membrane surface. Close the developer door, focus on the correct plane, adjust the appropriate exposure time, and record the image.

[0093] 2.3 RNA extraction and cDNA synthesis

[0094] 4×105 SNT-8 and SNK-1 cells were plated in six-well plates, 1 mM acipimox or 100 μM fenofibrate was added and cultured for 30 min, then 20 ng / ml TPA was added to induce EBV lysis and reactivation in the cells. After 24 h, the cell pellets were collected for RNA extraction.

[0095] RNA was extracted using a cell / tissue total RNA rapid extraction kit (New Cycle, M5105) according to the product instructions. RNA concentration was tested and used to synthesize cDNA.

[0096] cDNA was synthesized using ReverTra Ace qPCR RT Master Mix (TOYOBO, FSQ-201).

[0097] (1) Removal of gDNA:

[0098] RNA 1 μg

[0099] 4×DN Master Mix 4μl

[0100] RNase-Free ddH2O Make up the system to 16μl

[0101] Incubate at 37°C for 5 min and place on ice;

[0102] (2) Reverse transcription reaction:

[0103] Add 4 μl 5x RT Master Mix to the reaction mixture in step (1).

[0104]

[0105] The obtained cDNA product can be used directly in subsequent experiments or stored at -20℃.

[0106] 2.4 Quantitative Real-time PCR

[0107] The qPCR reagent used was KOD-SYBR qPCR Mix (TOYOBO, QKD-201), and the plate used was LightCycler 480 Multiwell Plate 96 (Roche, 4729692001). The instrument used was Roche 96. The template cDNA was diluted ten-fold. Three replicates and technical replicates were performed for each sample.

[0108] The reaction system is:

[0109]

[0110] The reaction conditions are:

[0111]

[0112] The Cq value was read and normalized to the expression level of the internal reference gene GAPDH, and the relative expression level of the gene was calculated using the 2-ΔΔCt method.

[0113] The primers used for real-time fluorescence quantitative PCR are as follows:

[0114] EBV-ZTA:

[0115] F: 5'-CCGGGCCAAGTTTAAGCAAC-3' (SEQ ID No: 1)

[0116] R: 5'-AGTAAACGAGGCGTGAAGCA-3' (SEQ ID No: 2)

[0117] GAPDH:

[0118] F: 5'-AGAAGGCTGGGCTCATTTG-3' (SEQ ID No: 3)

[0119] R: 5'-AGGGGCCATCCACAGTCTTC-3' (SEQ ID No: 4)

[0120] 3.2.5 Data Statistical Analysis

[0121] Data were analyzed using GraphPad Prism 9, and two-tailed t-test was used to determine whether there were significant differences between samples. *p<0.05, **p<0.01, ***p<0.001.

[0122] 3. Experimental results

[0123] 3.1 Fenofibrate and acipimox reduce triglyceride levels in SNK-1

[0124] This application first analyzes the cytotoxicity of fenofibrate and acipimox ( Figure 1 A), confirmed that non-toxic concentrations can reduce the triglyceride content in the EBV-positive NKTCL-derived tumor cell line SNK-1. SNK-1 cells were stimulated with different doses of fenofibrate and acipimox, and then stained with BODIPY dye. Under a fluorescence microscope, it was observed that acipimox at 0.5mM could inhibit the lipid droplets storing triglycerides in cells. Fenofibrate was more effective, and its effect of inhibiting intracellular lipid droplets at 100uM was very significant, indicating that both fenofibrate and acipimox can reduce the triglyceride content in SNK-1 ( Figure 1 B).

[0125] 3.2 Fenofibrate and acipimox inhibit EBV reactivation in SNK-1 and SNT-8

[0126] Subsequently, fenofibrate and acipimox were used to stimulate SNK-1 and SNT-8 to reduce the triglyceride content in them, and then TPA was used to induce the reactivation of EBV in SNK-1 and SNT-8. Protein immunoblotting and real-time quantitative PCR showed that under the action of triglyceride-lowering drugs, the protein expression of ZTA (Z transcriptional activator) in SNK-1 and SNT-8 was decreased. Figure 2 A) and mRNA levels ( Figure 2 B) were significantly inhibited, indicating that triglyceride-lowering drugs can inhibit EBV reactivation in SNK-1 and SNT-8, and the effect of fenofibrate is very significant.

[0127] 3.3.3 Fenofibrate and acipimox inhibit EBV reactivation in EBV-positive B cells

[0128] In addition to the NKTCL-derived tumor cell lines SNK-1 and SNT-8, the effects of triglyceride-lowering drugs on EBV-positive (latently infected) B cell lines were also tested. Fenofibrate and acipimox were used to stimulate Akata, LCL, and Raji, and then Ig (G+M) was used to induce EBV reactivation. Protein immunoblotting showed that under the action of triglyceride-lowering drugs, the protein expression level of ZTA in EBV-positive B cell lines was significantly inhibited ( Figure 3 ), indicating that triglyceride-lowering drugs can inhibit EBV reactivation in EBV-positive B cell lines, which means that the effect of triglyceride-lowering drugs in inhibiting EBV reactivation is broad-spectrum and can be used as adjuvant therapy for more EBV reactivation-related diseases in clinical practice.

[0129] 4. Conclusion

[0130] Fenofibrate and acipimox reduced the triglyceride content in SNK-1 and inhibited EBV reactivation in NKTCL-derived tumor cell lines SNK-1, SNT-8 and EBV-positive (latently infected) B cells Akata, LCL, and Raji.

[0131] In summary, triglyceride-lowering drugs can inhibit the lysis and reactivation of latent EBV in EBV-positive NK / T cells, B cells and other tumor cell lines, and are excellent candidate drugs for the treatment of diseases related to EBV reactivation, such as NK / T cell lymphoma (NKTCL) and hemophagocytic syndrome (HPS).

[0132] Based on the certain conservatism in the mechanism of herpes virus latent infection to reactivation, triglyceride-lowering drugs represented by fenofibrate and acipimox can also treat diseases related to reactivation of other herpes viruses such as KSHV.

[0133] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. Use of a triglyceride-lowering drug or its active ingredient in the preparation of a herpes virus reactivation inhibitor.

2. The use according to claim 1, characterized in that The herpes virus is selected from the group consisting of Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpes virus (KSHV), or a combination thereof.

3. The use according to claim 1, characterized in that The triglyceride-lowering drug is selected from: fibrate drugs such as fenofibrate, gemfibrozil, bezafibrate; niacin drugs such as niacin and acipimox.

4. Use of a triglyceride-lowering drug or its active ingredient in the preparation of a pharmaceutical composition for preventing or treating diseases associated with herpes virus reactivation.

5. The use according to claim 4, characterized in that The herpes virus is selected from the group consisting of Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpes virus (KSHV), or a combination thereof.

6. The use according to claim 4, characterized in that The triglyceride-lowering drug is selected from: fibrate drugs such as fenofibrate, gemfibrozil, bezafibrate; niacin drugs such as niacin and acipimox.

7. The use according to claim 4, characterized in that The disease is selected from the group consisting of chronic active Epstein-Barr virus infection (CAEBV), percutaneous lymphoproliferative disorder (PTLD), diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma (HL), Burkitt lymphoma (BL), nasopharyngeal carcinoma (NPC), gastric cancer (GC), NK / T cell lymphoma (NKTCL), hemophagocytic syndrome (HPS), or a combination thereof.

8. The use according to claim 4, characterized in that The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

9. The use according to claim 4, characterized in that The dosage form of the pharmaceutical composition is a liquid dosage form or a solid dosage form.

10. A method for inhibiting the reactivation of herpes virus in cells, comprising the steps of contacting and culturing cells latently infected with herpes virus with a triglyceride-lowering drug or its active ingredient, thereby inhibiting the reactivation of herpes virus in cells.

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