Application of dehydrotumuloic acid and / or salt thereof in preparation of medicine for resisting coxsackie virus
The treatment problem of Coxsackie virus infection is solved by inhibiting the mRNA expression, viral protein expression and MAPK signaling pathway of dehydrogenated titanium acid and/or its salt, and effective inhibition and disease treatment of Coxsackie virus are achieved.
Patent Information
- Application Number
- CN202510691022.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-27
- Publication Date
- 2025-08-08
AI Technical Summary
The prior art lacks effective treatment methods to deal with the infection of Coxsach virus, especially the various diseases caused by Coxsach virus A and B viruses, and the virus has many serotypes and rapid mutations, which lead to treatment difficulties.
Dehydrogenated timolic acid and/or its salts are used to inhibit the mRNA expression, viral protein expression and plaque formation of Coxsackie virus, thereby inhibiting the viral infection capacity, and inhibiting viral replication by inhibiting the phosphorylation of key proteins in the MAPK signaling pathway.
It significantly inhibits the infection ability of Coxsackie virus, reduces the infection of the virus into cells, provides an effective treatment plan, and can serve as a leading compound for anti-Coxsackie virus.
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Figure CN120437138A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of medical technology, and more particularly to the use of dehydrogenated taurine and / or its salt in the preparation of anti-coxsackie virus drugs. Background Art
[0002] Coxsackievirus is a non-enveloped, single-stranded, positive-stranded RNA virus belonging to the Picornaviridae family. It was first discovered by American scientists in Coxsackieville, New York, hence its name. Based on differences in pathogenicity, coxsackieviruses are divided into two main groups, A and B. Group A viruses (CVA) primarily invade nerves and muscles and cause systemic infection, leading to respiratory infections, meningitis, and herpangina, among other conditions. They are the primary cause of hand, foot, and mouth disease. Group B viruses (CVB) primarily affect the heart, brain, and other organs, causing inflammatory diseases such as myocarditis, pericarditis, and viral encephalitis. These diseases trigger myocarditis through direct damage to myocardial cells and complex immune mechanisms (innate and adaptive immunity, mitochondrial autophagy, etc.). The transition from acute to chronic myocarditis is associated with persistent viral presence and immune imbalance.
[0003] While most coxsackievirus infections do not cause severe illness, epidemics have occurred in various regions of the world, primarily affecting immunocompromised newborns, the elderly, and areas with poor sanitation. Its harm should not be underestimated. However, there is currently no specific treatment. Due to the virus's diverse serotypes and rapid mutation rates, developing treatments remains challenging. Summary of the Invention
[0004] The purpose of the present invention is to overcome the problem that the above-mentioned existing anti-coxsackievirus drugs are still relatively scarce, and to provide the use of dehydrogenated taurine and / or its salts in the preparation of anti-coxsackievirus drugs. Dehydrogenated taurine and / or its salts have extremely significant inhibitory effects on the mRNA expression, viral protein expression and plaque formation of coxsackievirus, can reduce the infectivity of coxsackievirus, inhibit the infection of coxsackievirus to cells, and thus have good anti-coxsackievirus activity. Further studies have shown that dehydrogenated taurine and / or its salts effectively inhibit the activation of key protein phosphorylation in the MAPK signaling pathway caused by coxsackievirus infection, and inhibit viral replication. The good anti-coxsackievirus activity of dehydrogenated taurine and / or its salts provides an effective solution for the treatment of coxsackievirus infection and the diseases caused by it, and can also be used as an effective anti-coxsackievirus lead compound.
[0005] The above-mentioned purpose of the present invention is achieved through the following technical solutions: Dehydrotumulosic acid (structural formula: ) and / or its salts in the preparation of anti-Coxsackie virus drugs.
[0006] Existing studies have reported that dehydrogenated taurine is one of the main components of Poria cocos extract. Poria cocos is the dried sclerotium of the Polyporaceae fungus Poria cocos, which has pharmacological effects such as anti-inflammatory, anti-tumor and regulation of intestinal flora.
[0007] The present invention discovered that dehydrogenated taurine and / or its salts have extremely significant inhibitory effects on coxsackievirus mRNA expression, viral protein expression, and plaque formation, reducing the infectivity of coxsackievirus and inhibiting coxsackievirus infection of cells, thereby exhibiting excellent anti-coxsackievirus activity. Further studies have shown that dehydrogenated taurine and / or its salts effectively inhibit the activation of key protein phosphorylation in the MAPK signaling pathway caused by coxsackievirus infection and inhibit viral replication. The excellent anti-coxsackievirus activity of dehydrogenated taurine and / or its salts provides an effective solution for the treatment of coxsackievirus infection and the diseases caused by it, and can also serve as an effective anti-coxsackievirus lead compound.
[0008] Preferably, the Coxsackievirus is at least one of Coxsackie A virus or Coxsackie B virus.
[0009] More preferably, the anti-Coxsackie A virus serotype is at least one of CVA6 or CVA16.
[0010] More preferably, the drug is a drug for preventing and / or treating respiratory tract infections, meningitis and herpangina induced by Coxsackie A virus.
[0011] More preferably, the anti-Coxsackie B virus serotype is at least one of CVB1, CVB2 or CVB3.
[0012] More preferably, the drug is a drug for preventing and / or treating myocarditis, pericarditis and viral encephalitis induced by Coxsackie B virus.
[0013] Preferably, the drug is a drug for resisting coxsackievirus infection mediated by the MAPK signaling pathway.
[0014] Preferably, the drug is a drug that inhibits the expression of Coxsackie virus mRNA.
[0015] More preferably, the mRNA is VP4 mRNA.
[0016] Preferably, the drug is a drug that inhibits the expression of viral proteins of Coxsackievirus.
[0017] More preferably, the viral protein is VP1 protein.
[0018] Preferably, the dosage form of the drug is injection, capsule, tablet, pill or granule.
[0019] Preferably, the medicament comprises pharmaceutically acceptable excipients.
[0020] More preferably, the pharmaceutically acceptable excipient is at least one of a lubricant, a filler, a binder, a disintegrant, a surfactant, an antioxidant or a pH regulator.
[0021] More preferably, the pharmaceutically acceptable excipient is an excipient.
[0022] Preferably, the salt of dehydrogenated tert-butyl ester is a medically acceptable salt, including at least one of a sodium salt or a potassium salt.
[0023] Compared with the prior art, the present invention has the following beneficial effects: The present invention discovered that dehydrogenated taurine and / or its salts have extremely significant inhibitory effects on coxsackievirus mRNA expression, viral protein expression, and plaque formation, reducing the infectivity of coxsackievirus and inhibiting coxsackievirus infection of cells, thereby exhibiting excellent anti-coxsackievirus activity. Further studies have shown that dehydrogenated taurine and / or its salts effectively inhibit the activation of key protein phosphorylation in the MAPK signaling pathway caused by coxsackievirus infection and inhibit viral replication. The excellent anti-coxsackievirus activity of dehydrogenated taurine and / or its salts provides an effective solution for the treatment of coxsackievirus infection and the diseases caused by it, and can also serve as an effective anti-coxsackievirus lead compound. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 This is an inhibition curve diagram of dehydrogenated thiamethoxam in Example 1 inhibiting the cytopathic effect of CVB3 virus.
[0025] Figure 2 This is a graph showing the inhibition of CVB3 virus plaque formation by dehydrogenated thiamoyl alcohol in Example 1 and the statistical results of plaque size.
[0026] Figure 3 This is a statistical graph showing that dehydrogenated thiamin inhibits CVB3 virus mRNA expression in Example 2.
[0027] Figure 4 This is a graph showing the protein expression of CVB3 virus inhibited by dehydrogenated thiamethoxam in Example 2 and the statistical results.
[0028] Figure 5 The figure shows the situation and statistical results of dehydrogenated taurine inhibiting the expression of key proteins (Raf, Mek and Erk) of the MAPK signaling pathway and viral protein (VP1) of CVB3 virus in Example 2.
[0029] Figure 6 3 is a graph showing the inhibition of dehydrogenated thiamethoxam acid on the cytopathic effects of different coxsackie viruses in Example 3. DETAILED DESCRIPTION
[0030] In order to more clearly and completely describe the technical solution of the present invention, the present invention is further described in detail through specific embodiments below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Various changes can be made within the scope of the rights of the present invention.
[0031] Example 1 In vitro anti-CVB3 activity of dehydrogenated taurine 1.1 CPE experiment of dehydrogenated tert-butyl ether against CVB3 Vero (1.5×10 4 Cells were seeded into 96-well culture plates (100 cells / well) and incubated overnight at 37°C with 5% CO2. A mixture of different titers of Coxsackievirus CVB3 and different concentrations of dehydrogenated taurine (DA) was added (100 μl / well) and incubated. After three days of incubation at 37°C with 5% CO2, viable cell counts were detected using Alamar Blue at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. The minimum concentration of compound required to inhibit CPE by 50% was determined and defined as the IC2. 50 Virus inhibition rate (%) = 1-(fluorescence value of the experimental group wells - mean fluorescence value of the cell control group) / (mean fluorescence value of the virus control group - mean fluorescence value of the cell control group) × 100%; IC 50 The viral inhibition rates were imported into GraphPad Prism 8.0 software and analyzed by nonlinear retrospective (curve fitting) analysis of XY.
[0032] The inhibition curves and corresponding IC50 values of dehydrogenated thiamethoxam on Vero cells infected with different titers of CVB3 are shown in Figure 5. Figure 1 As shown. Figure 1 It can be seen that dehydrogenated taurine inhibits the infection of Coxsackie virus CVB3 to cells in a concentration-dependent manner and has good anti-Coxsackie virus activity.
[0033] 1.2 Dehydrogenated thiamethoxam acid anti-CVB3 plaque assay Vero cells (2 × 10 5 Vero cells (100 cells / well) were seeded into 24-well plates and cultured overnight at 37°C, 5% CO2, until they reached a monolayer. Vero cells were treated with a 10,000-fold diluted CVB3 virus solution for 2 hours. The virus solution was discarded, and overlay medium containing serially diluted dehydrogenated taurine was added for 72 hours. After 72 hours, the overlay medium was discarded, and 0.5 ml of formalin fixative was added per well for 10 minutes. The fixative was discarded, and 0.3 ml of crystal violet stain was added per well for 30 minutes. After removing the stain and rinsing with running water, plaques were photographed, and plaque area was calculated using Image J software.
[0034] The statistical results of the plaque formation and plaque size of dehydrogenated thiamethoxam anti-CVB3 are shown in Figure 2 , Figure 2 The results further showed that dehydrogenated taurine could inhibit the ability of coxsackievirus to infect cells.
[0035] Example 2 Mechanism of action of dehydrogenated thiamethoxamic acid against CVB3 activity 2.1 Dehydrogenated thiamethoxam acid anti-CVB3-VP4 mRNA experiment Vero (5×10 5 Cells were seeded into 12-well culture plates (100 cells / well) and incubated overnight at 37°C in the presence of 5% CO2. Virus was co-cultured with various concentrations of dehydrogenated tert-butyl ether (20 μM and 50 μM) and Vero cells at 37°C in the presence of 5% CO2 for 16 hours. The cells were washed with PBS, trypsinized in the 12-well plates, transferred to a centrifuge tube, and centrifuged at 1000 rpm for 5 minutes. The culture medium was aspirated, and RL lysis buffer was added. After pipetting until no clumps were observed, the cells were transferred to an RNase-free EP tube. Total RNA was extracted using the TaKaRa MiniBEST Universal RNA Extraction Kit and reverse-transcribed into cDNA using the TaKaRa PrimeScript™ RT Master Mix (Perfect Real Time). Primers for target and reference genes were used for amplification. Primer sequences are shown in Table 1.
[0036] Table 1
[0037] The expression of viral mRNA in Vero cells infected with CVB3 treated with dehydrogenated taurine Figure 3 As shown. Figure 3 It was found that dehydrogenated taurine significantly inhibited the expression of VP4 mRNA of CVB3 virus.
[0038] 2.2 Experimental study on the expression of key proteins in the MAPK signaling pathway and CVB3-VP1 protein induced by dehydrogenated tacrolimus Vero cells (1 × 10 6Cells (100 cells / well) were seeded into 6-well plates and cultured overnight at 37°C under 5% CO2. Virus was added at an MOI of 0.1 and various concentrations of dehydrogenated taurine (20 μM and 50 μM) and co-cultured with Vero cells at 37°C under 5% CO2 for 16 hours. After washing with PBS, cells were lysed on ice for 20 minutes in RIPA lysis buffer. Cells were harvested with a cell scraper into an EP tube, sonicated, and centrifuged. The supernatant was collected and used to prepare a standard curve and determine protein concentration using a Bradford protein assay kit. After adding 5X Roading Buffer and heating at 95°C for 10 minutes for denaturation, the treated protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis and transferred to a polyvinyl difluoride (PVDF) membrane. The membrane was blocked with 5% skim milk for 2 hours and washed three times with 1X Tween-20 buffered saline (TBST) for 10 minutes each. The membrane was then incubated with a specific primary antibody overnight at 4°C. The next day, the membrane was washed three times with 1X TBST (10 minutes each time). The PVDF membrane was incubated with a horseradish peroxidase (HRP)-conjugated rabbit secondary antibody for 1 hour. After incubation, the membrane was washed three times with 1X TBST (10 minutes each time). The membrane was then soaked in chemiluminescent solution (ECL) for several minutes before development. Antibodies used included GAPDH, β-Actin, pc-Raf, c-Raf, p-MEK1 / 2, MEK1 / 2, p-p44 / 42 MAPK (Erk1 / 2), p44 / 42 MAPK (Erk1 / 2), pPLA2G4A / PLA2G4A (Cell Signaling Technology), p-GR / GR (ABClonal), and CVB-VP1. The horseradish peroxidase (HRP)-conjugated rabbit secondary antibody was from Immunoway.
[0039] The protein expression and statistical results of the virus in Vero infected by CVB3 treated with dehydrogenated thiamethoxam Figure 4 As shown. Figure 4 It can be seen that dehydrogenated taurine significantly inhibits the expression of CVB3 viral protein.
[0040] The expression and statistical results of key proteins (Raf, Mek and Erk) of MAPK signaling pathway and viral protein (VP1) after CVB3 was treated with dehydrotumulosic acid (DA) are shown in Figure 2. Figure 5 As shown. Figure 5It can be seen that dehydrogenated taurine not only inhibits viral replication but also inhibits the phosphorylation of the MAPK signaling pathway caused by viral CVB3 infection. Raf inhibitors, an upstream protein of the MAPK signaling pathway, also inhibit the replication of viral CVB3 while inhibiting the MAPK signaling pathway, indicating that dehydrogenated taurine exerts its antiviral activity by inhibiting the MAPK signaling pathway.
[0041] Example 3 Broad-spectrum activity of dehydrogenated taurine against coxsackieviruses The experiment was performed according to section 1.1 of Example 1, except that the coxsackievirus CVB3 was replaced by CVB1, CVB2, CVB3, CVA6 or CVA16.
[0042] The inhibition curves and corresponding IC50 results of dehydrogenated thiamethoxam on Vero cells infected by different coxsackie viruses (CVB1, CVB2, CVB3, CVA6 and CVA16) are shown in the figure. Figure 6 As shown. Figure 6 It can be seen that dehydrogenated taurine significantly inhibits the infection of different coxsackie viruses to cells and has good broad-spectrum activity against coxsackie viruses.
[0043] In summary, dehydrogenated taurine and / or its salts have extremely significant inhibitory effects on coxsackievirus mRNA expression, viral protein expression, and plaque formation, reducing the infectivity of coxsackievirus and inhibiting coxsackievirus infection of cells, thereby exhibiting excellent anti-coxsackievirus activity. Further studies have shown that dehydrogenated taurine and / or its salts effectively inhibit the activation of key protein phosphorylation in the MAPK signaling pathway caused by coxsackievirus infection, thereby inhibiting viral replication. The excellent anti-coxsackievirus activity of dehydrogenated taurine and / or its salts provides an effective solution for the treatment of coxsackievirus infection and the diseases caused by it, and can also serve as an effective anti-coxsackievirus lead compound.
[0044] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.
Claims
1. Use of dehydrogenated thiamoic acid and / or its salt in the preparation of anti-coxsackie virus drugs.
2. The application according to claim 1, characterized in that The Coxsackie virus is at least one of Coxsackie A virus and Coxsackie B virus.
3. The application according to claim 2, characterized in that: The anti-Coxsackie A virus serotype is at least one of CVA6 or CVA16.
4. The application according to claim 2, characterized in that: The anti-Coxsackie B virus serotype is at least one of CVB1, CVB2 or CVB3.
5. The application according to claim 1, characterized in that: The drug is a drug for resisting coxsackie virus infection mediated by the MAPK signaling pathway.
6. The application according to claim 1, characterized in that: The drug is a drug for inhibiting the expression of Coxsackie virus mRNA.
7. The use according to claim 1, characterized in that The drug is a drug for inhibiting the expression of viral proteins of Coxsackie virus.
8. The application according to claim 1, characterized in that: The dosage form of the medicine is injection, capsule, tablet, pill or granule.
9. The use according to claim 1, characterized in that: The drug includes pharmaceutically acceptable excipients.
10. The application according to claim 1, characterized in that: The salt of dehydrogenated tert-butyl ester comprises at least one of a sodium salt or a potassium salt.