Therapy for treating hand-foot-mouth after-healing demethylation caused by CV-A10

Abstract

The invention provides a therapy for treating hand-foot-mouth accompanied post-healing demethylation caused by coxsackie virus A10, and based on research on pathogenesis of coxsackie virus A10 infection, rescue treatment can be realized by regulating and controlling a Wnt / beta-catenin signal channel in a virus infection period. Particularly, research finds that the demethylation phenomenon caused by the coxsackie virus A10 infection can be effectively relieved or prevented by smearing the Wnt agonist in a vaseline cream form. The treatment method provided by the invention provides an effective treatment means for the hand-foot-and-mouth after-healing demethylation caused by the coxsackie virus A10, and has a remarkable clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for treating post-healing onychomycosis of hand, foot and mouth disease caused by Coxsackievirus A10 (CV-A10). Background Art

[0002] The phenomenon of children suffering from abnormal nail development leading to onychomycosis after recovery from hand, foot and mouth disease has existed for many years. However, due to the complexity of the interaction between the virus and the host, the analysis and prevention of this complication have been slow.

[0003] Coxsackievirus A10 (CV-A10, also known as CVA10) is the pathogen that causes hand, foot and mouth disease. In 2000, an epidemiological study found that nail development abnormalities, such as Beau's lines or onychomycosis, occurred 3-8 weeks after hand, foot and mouth disease. In a few cases, some treatments were used to relieve symptoms, including topical administration of keratolytics, antibiotics or fibroblast growth factors, and in some cases, nail surgery, such as nail plate removal or scraping of purulent granulomas.

[0004] However, there is still no clear etiology, so the treatment methods are like trying all kinds of medicines in a hurry and fail to cure the root cause. Summary of the invention

[0005] The inventors started with the characteristic that the Coxsackievirus A10 (CV-A10) receptor KREMEN1 (KRM1) that causes hand, foot and mouth disease also has the function of regulating the Wnt / β-catenin pathway, focusing on the scientific issue of how the virus simulates the signal molecules in the body to complete the invasion and cause occasional onychomycosis after recovery from hand, foot and mouth disease. First, a comprehensive early literature survey was carried out. Epidemiological surveys showed that many CV-A10 and CV-A6 infections showed onychomycosis after recovery. Both viruses use KRM1 as a receptor to invade cells. At the same time, KRM1 interacts with Dickkopf-related protein 1 (DKK1) and low-density lipoprotein receptor-related protein 6 (LRP6) on the cell surface to regulate the Wnt / β-catenin signaling pathway. Therefore, the inventors speculate that the virus binding to KRM1 may affect its interaction with DKK1 and LRP6, thereby interfering with downstream signal transduction and causing abnormal tissue development. Through structural analysis, it was found that KRM1 interacts with DKK1 and CV-A10 using a highly similar interface, suggesting that the virus may simulate DKK1 to bind to KRM1. Subsequently, it was confirmed at the cellular level and in a mouse model that CV-A10 infection inhibits the Wnt / β-catenin signaling pathway by interacting with KRM1, thereby affecting the differentiation of nail stem cells, which may lead to the occurrence of onychomycosis. Importantly, the present invention discovered for the first time that mice also experienced toenail shedding after being infected with CV-A10, and explored the use of the signaling pathway agonist CHIR99021 (CHIR) to treat the disease. Therefore, the present invention uses cell biology, structural biology, bioinformatics and other multidisciplinary cross-integration to explain how Coxsackievirus with KRM1 as a receptor affects the signaling pathway and causes onychomycosis from six perspectives: epidemiological statistics, structural basis, signaling pathway, pathological analysis, animal model, and recovery verification. A model for collaborative research on the virus's use of host receptor invasion mechanism and receptor signaling pathway feedback is initially established, thereby providing a corresponding treatment plan.

[0006] The present invention provides a therapy for treating post-healing onychomycosis of hand, foot and mouth disease caused by CV-A10. Based on the study of the pathogenic mechanism of CV-A10 infection, it is found that rescue treatment can be achieved by regulating the Wnt / β-catenin signaling pathway during viral infection. Further studies have shown that the use of Wnt agonist CHIR in the form of vaseline cream can effectively alleviate or prevent the onychomycosis caused by CV-A10 infection.

[0007] Therefore, the present invention first provides a substance that resists the inhibition of the Wnt / β-catenin signaling pathway for use in the preparation of a drug for treating or preventing post-healing onychomycosis caused by CV-A10. The substance that resists the inhibition of the Wnt / β-catenin signaling pathway is used to replenish and activate the Wnt / β-catenin signaling pathway during CV-A10 infection.

[0008] Specifically, the substance is a Wnt / β-catenin signaling pathway agonist, specifically CHIR99021 or Dishevelled 2.

[0009] Preferably, the agonist is CHIR99021 (abbreviated as CHIR).

[0010] More preferably, the drug is in a dosage form suitable for application, specifically formulated with vaseline into an application agent;

[0011] The concentration of the active ingredient during application is 0.5-10 mM (preferably 1-5 mM, more preferably 2-3 mM, such as 2.5 mM), and it is applied once a day for more than 5 consecutive days, such as 6-30 days.

[0012] The present invention also provides a drug for treating post-healing onychomycosis of hand, foot and mouth disease caused by CV-A10, which contains a substance that resists the inhibition of the Wnt / β-catenin signaling pathway as an active ingredient, and the drug is suitable for application in a dosage form, specifically, it is formulated with vaseline into an ointment; further, it is suitable for application as an ointment on the fingertips of patients.

[0013] Specifically, the substance is a Wnt / β-catenin signaling pathway agonist, specifically CHIR99021 or Dishevelled 2.

[0014] Preferably, the concentration of the active ingredient in vaseline is 0.5-10 mM (preferably 1-5 mM, more preferably 2-3 mM, such as 2.5 mM).

[0015] More specifically, the preparation method is as follows: use DMSO to prepare the mother solution of Wnt / β-catenin signaling pathway agonist, take vaseline ointment and heat it to melt, take the Wnt / β-catenin signaling pathway agonist dissolved in the vaseline according to proportion and mix well, and wait for cooling.

[0016] Preferably, the preparation method is to prepare a mother solution of CHIR99021 with a concentration of 200 mM using DMSO, heat vaseline ointment at 65°C to melt, dissolve CHIR99021 in vaseline according to a certain proportion, vortex mix, and cool. Therefore, the present invention also provides a preparation method of the drug.

[0017] The present invention has found through research that, compared with common administration methods such as intragastric administration and in situ injection, in situ vaseline dissolving and applying the drug has the best effect, while it is difficult to obtain effective results through injection. In summary, the treatment method of the present invention provides an effective treatment approach for post-healing onychomycosis of hand, foot and mouth caused by CV-A10, and has significant clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1A It was shown that the inhibitory effect of CV-A10 on Wnt / β-catenin pathway signaling in RD cells was restored after the addition of CHIR.

[0019] Figure 1B It was shown that the inhibitory effect of CV-A10 on Wnt / β-catenin pathway signaling in HEK293T cells was restored after the addition of CHIR.

[0020] Figure 2 The effects of two administration methods, namely, application of Vaseline ointment to the toes and injection of CHIR in situ during the acute stage of infection, are shown. A shows the enrichment comparison of Wnt-related genes between the CHIR application group and the Vaseline application group (Wnt-related genes were significantly upregulated in the treatment group); B shows the enrichment comparison of Wnt-related genes between the CHIR injection treatment group and the solvent injection group (no significant difference).

[0021] Figure 3 It is shown that CHIR application can effectively restore the transcription level of Wnt-related genes at the animal model level during the acute phase of infection. Among them, A shows the enrichment comparison of Wnt-related genes between the PBS control group and the CV-A10 infection group (Wnt-related genes were significantly downregulated in the CV-A10 group); B shows the enrichment comparison of Wnt-related genes between the treatment group and the CV-A10 infection group (Wnt-related genes were significantly upregulated in the treatment group); C shows the enrichment comparison of Wnt-related genes between the PBS control group and the treatment group (no significant difference).

[0022] Figure 4 It shows that CHIR application is effective in treating the protein level of tissue cells in animal models during the acute stage of infection. A shows the immunofluorescence staining of nail stem cell markers Lgr6 (green) and K17 (red); B shows the area statistics of the positive areas of nail stem cell markers Lgr6 and K17; C shows the immunohistochemical analysis of cell proliferation marker Ki67 (purple) and nail differentiation marker AE13 (brown); D shows the area statistics of Ki67 and AE13 positive areas.

[0023] Figure 5 It was shown that CHIR can treat nail loss in mice caused by CV-A10 infection. DETAILED DESCRIPTION

[0024] The present invention is further described below through specific implementation modes in order to better understand the present invention, but it does not constitute a limitation of the present invention.

[0025] Example 1: CHIR can rescue the abnormal Wnt / β-catenin pathway caused by CV-A10 infection at the cellular level

[0026] CV-A10 uses its receptor KRM1 to complete viral invasion. KRM1 plays a vital role as a switch of the Wnt / β-catenin pathway in cells. Therefore, the present invention uses a luciferase reporter gene experiment to first detect the effect of viral infection on the Wnt / β-catenin signaling pathway at the cellular level. It was found that viral infection can significantly inhibit the activation of the Wnt / β-catenin pathway. Therefore, the applicant used Dvl2, which promotes LRP6 phosphorylation, and CHIR, an inhibitor of GSK-3β, to perform a rescue experiment on the Wnt / β-catenin signaling pathway.

[0027] 293T or RD cells were cultured in a 24-well cell culture plate, and the reporter gene plasmid Top-Flash and the internal reference control pRenilla-TK plasmid were transfected into the cells. The Dvl2 group was added and the plasmid encoding the human Dvl2 gene was transfected at the same time. The transfection system is as follows: 90μl HBS solution, 70μl PEI (0.1mg / ml) and 2μg plasmid were prepared for every 3 wells of the 24-well plate. The DMEM medium was replaced 6 hours after transfection. 12 hours after transfection, Wnt3a Condition Medium, CV-A10 (MOI=1) or CHIR-99021 (CHIR) were added to the culture medium respectively. After 24 hours of treatment, the cells were lysed for luciferase activity detection. Figure 1A and Figure 1B As shown in the results, CV-A10 has a significant inhibitory effect on the Wnt / β-catenin signaling pathway, and this effect disappears when Dvl 2 or CHIR (1 μM) is added to stimulate the cell. Therefore, Dvl 2 and CHIR can serve as a remedy for CV-A10 to inhibit the Wnt / β-catenin signaling pathway in vitro.

[0028] Example 2: Applying CHIR to the toe skin can significantly increase the transcription level of Wnt-related genes

[0029] The inventors studied different treatment methods and compared their effects. Specifically, based on the fact that CHIR can rescue the inhibitory effect of viral infection on the Wnt / β-catenin signaling pathway at the cellular level, the inventors further analyzed the drug effects at the transcriptional and protein levels on the toe tip tissues of mice during the acute phase of viral infection in vivo, and studied the effects of two modes of administration: in situ toe tip petroleum jelly ointment application and injection.

[0030] In this example, 9-day-old BALB / c mice were intraperitoneally challenged with 1×10 4 TCID 50 / mL) 10μL, four groups of mice were intraperitoneally challenged with the following:

[0031] Group 1: Inject 5 μL of 10 mg / kg (10 mg / mL) CHIR dissolved in 10% DMSO and 30% PEG300 into the palm; Group 2: Inject 5 μL of 10% DMSO and 30% PEG300 solvent without CHIR into the palm; Group 3: Apply 2.5 mM CHIR vaseline cream on the toes; Group 4: Apply vaseline cream without CHIR on the toes.

[0032] Subsequent research observations found that the toe tips of the injected mice (i.e., Groups 1 and 2) were swollen after the injection of drugs or solvents, and there were signs of abnormal bites recognized by the mother mice, while no abnormalities were found in the Vaseline application group. After 6 consecutive days of treatment, the mice were euthanized, and RNA was extracted from the toe tips for RNA-seq transcriptome sequencing. The inventors performed GSEA enrichment analysis (NES: normalized value of enrichment score) on the expression profiles of 403 Wnt-related genes in the two treatment groups, and the results are as follows Figure 2 As shown. Among them, A shows the enrichment comparison of Wnt-related genes between the CHIR treatment group and the vaseline group (Wnt-related genes were significantly upregulated in the treatment group); B shows the enrichment comparison of Wnt-related genes between the CHIR injection treatment group and the solvent injection group (no significant difference). It can be seen that the Wnt-related genes in the CHIR group were significantly upregulated compared with the Vaseline ointment group alone, while in the injection group, there was no significant difference between CHIR and the control solvent after injection. It shows that the administration of CHIR in the form of vaseline cream can rescue the inhibition of CV-A10 infection on the transcriptional level of Wnt-related genes in mice.

[0033] We further added a PBS control group that was not challenged with the virus to compare the differences in Wnt gene transcription levels in the toes of mice after CHIR treatment and those without the virus. 4 TCID 50 / mL) 10μL, and to eliminate errors, the control group mice were injected with the same volume of PBS. A total of 3 groups of experiments were set up, PBS control group, CV-A10 infection group, CV-A10 infection + treatment group. On the day of infection, the treatment group applied vaseline ointment containing 2.5mM CHIR on the toes of the mice, and applied it once a day for 6 consecutive days. The other two groups used an equal amount of DMSO to prepare vaseline ointment. After the mice were euthanized 6 days after the infection, the left and right fingers were taken and stored in the fixative for subsequent immunohistochemical staining. RNA was extracted from the remaining toes, and RNA-seq transcriptome sequencing was performed. GSEA enrichment analysis was performed on the expression profiles of 403 Wnt-related genes between each group, and the results are as follows Figure 3 As shown. Among them, A shows the enrichment comparison of Wnt-related genes between the PBS control group and the CV-A10 infection group (Wnt-related genes were significantly downregulated in the CV-A10 group); B shows the enrichment comparison of Wnt-related genes between the treatment group and the CV-A10 infection group (Wnt-related genes were significantly upregulated in the treatment group); C shows the enrichment comparison of Wnt-related genes between the PBS control group and the treatment group (no significant difference). It can be seen that Wnt-related genes were significantly downregulated in the CV-A10 virus infection group, but there was no significant difference with the control group after applying CHIR vaseline cream. It shows that the administration of CHIR can rescue the inhibition of CV-A10 infection on the transcriptional level of Wnt-related genes in mice.

[0034] Example 3: Immunostaining confirms the therapeutic efficacy of CHIR at the protein level in mouse toe tissue cells

[0035] Furthermore, the present invention confirms the therapeutic effect of CHIR at the protein level of mouse toe tissue cells through immunostaining. Lgr6 (Leucine-rich repeat-containing G-protein coupled receptor 6) is a stem cell marker that was originally discovered in skin stem cell research. Later, researchers tracked Lgr6-positive cells to understand the growth and regeneration mechanism of nails, as well as the repair process of nails in injured or diseased states. At the same time, the epidermal stem cell marker keratin 17 (K17) was used to mark nail stem cells for support. In addition, the cell proliferation marker Ki67 and the keratinization marker AE13 were added to show the level of proliferation and differentiation of the toe tip.

[0036] The results are as follows Figure 4As shown. Among them, A shows the immunofluorescence staining of nail stem cell markers Lgr6 (green) and K17 (red); B shows the area statistics of the positive areas of nail stem cell markers Lgr6 and K17; C shows the immunohistochemical analysis of cell proliferation marker Ki67 (purple) and nail differentiation marker AE13 (brown); D shows the area statistics of Ki67 and AE13 positive areas. The results showed that positive cells expressing Lgr6 were localized in the ventral matrix of the PBS group, i.e. the control group, and viral infection caused Lgr6-positive and K17-positive nail stem cells to extend to the nail bed. CHIR alleviated the abnormal localization of nail stem cells caused by the virus. Consistent with this, Ki67-labeled proliferating cells and AE13-labeled keratinocytes also showed no statistical difference from the PBS group under the action of CHIR.

[0037] Based on the results of Examples 2 and 3 above, the therapeutic effect of CHIR on the toes of mice during the acute phase of viral infection in mice was demonstrated at the transcriptional and protein levels.

[0038] Furthermore, the inventors observed the loss of nails after recovery from viral infection in an animal model through long-term drug administration.

[0039] Example 4: CHIR can treat toenail loss in mice caused by CV-A10 infection

[0040] The inventors found that viral infection can cause the proliferation and differentiation of nail stem cells to be blocked, so the development of nails is temporarily inhibited during the acute stage of hand, foot and mouth disease. In previous studies, the inventors unexpectedly found that when suckling mice were challenged with viruses for multiple times, each time with an interval of one week, that is, the virus infection cycle was extended, the mice would develop symptoms of nail loss 5-8 weeks after the first challenge. In order to explore the therapeutic effect of the drug of the present invention on nail loss, Wnt agonist vaseline ointment was applied to the toes of mice during the challenge cycle. Specifically, 9-day-old BALB / c mice were challenged with viruses by intraperitoneal injection 4 times (the first 1×10 4 TCID 50 / mL, the next three times 2×10 4 TCID 50 / mL). From the first day of the virus challenge, petroleum jelly containing 2.5mM CHIR was applied to the toes of the mice once a day for 31 consecutive days. The other two groups were treated with an equal amount of DMSO to prepare petroleum jelly. During the 12 weeks, each nail of the hind toes of the mice was carefully examined and recorded every week. The results are shown in Figure 5 Application of CHIR to the toes during viral infection phenotypically rescued mice from the risk of developing onychomycosis.

[0041] In summary, these results indicate that CHIR application therapy can alleviate the inhibition of Wnt / β-catenin signaling pathway induced by viral infection, restore the normal proliferation and differentiation of nail stem cells, and is expected to become a potential drug for the treatment of onychomycosis.

Claims

1. Use of a substance that resists the inhibition of the Wnt / β-catenin signaling pathway in the preparation of a drug for treating or preventing post-healing onychomycosis caused by Coxsackievirus A10.

2. The use according to claim 1, characterized in that The substance is a Wnt / β-catenin signaling pathway agonist, specifically CHIR99021 or Dishevelled 2.

3. The use according to claim 1 or 2, characterized in that: The agonist is CHIR99021.

4. The use according to claim 3, characterized in that The medicine is in a dosage form suitable for application, specifically formulated with vaseline into an application agent.

5. The use according to claim 4, characterized in that The concentration of the active ingredient during application is 0.5-10 mM (preferably 1-5 mM, more preferably 2-3 mM, such as 2.5 mM), and it is applied once a day for more than 5 consecutive days, such as 6-30 days.

6. A drug for treating or preventing post-healing onychomycosis caused by Coxsackievirus A10, characterized in that: The active ingredient of the drug is a substance that resists the inhibition of the Wnt / β-catenin signaling pathway, and the drug application method is suitable for a dosage form, specifically, it is formulated with vaseline into an ointment; further, it is suitable for being applied to the fingertips of patients.

7. The drug according to claim 6, characterized in that The substance is a Wnt / β-catenin signaling pathway agonist, specifically CHIR99021 or Dishevelled 2.

8. The drug according to claim 7, characterized in that The concentration of the active ingredient in vaseline is 0.5-10 mM (preferably 1-5 mM, more preferably 2-3 mM, such as 2.5 mM).

9. The drug according to claim 8, characterized in that The preparation method is as follows: prepare the mother solution of CHIR99021 with DMSO, take vaseline ointment and heat it to melt, dissolve the Wnt / β-catenin signaling pathway agonist in the vaseline according to proportion, mix well, and wait for cooling.

10. The drug according to claim 9, characterized in that The specific preparation method is as follows: use DMSO to prepare a mother solution with a CHIR99021 concentration of 200 mM, take vaseline ointment and heat it to 65°C to melt it, dissolve CHIR99021 in the vaseline according to the proportion, vortex mix, and wait for cooling.

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