Application of all-trans retinoic acid in preparation of medicine for preventing and / or treating novel bunyavirus infectious diseases

Abstract

The invention discloses application of all-trans retinoic acid in preparation of a medicine for preventing and / or treating novel bunyavirus infectious diseases, and belongs to the field of medicines. Experiments prove that the all-trans retinoic acid can reduce the mouse death proportion caused by novel bunyavirus infection and reduce mouse tissue damage. In addition, in-vivo and in-vitro experiments find that all-trans retinoic acid reduces expression of inflammatory factors through PPAR gamma protein. Therefore, the all-trans retinoic acid can be used for preparing the medicine for resisting the novel bunyavirus and / or the medicine for preventing or treating the novel bunyavirus infectious diseases and / or the medicine for inhibiting the inflammation of the novel bunyavirus infectious diseases.

Description

Technical Field

[0001] This application relates to the pharmaceutical field, specifically to the use of all-trans retinoic acid in the preparation of medicaments for the prevention and / or treatment of novel Bunyavirus infectious diseases, namely, the use of all-trans retinoic acid in the preparation of medicaments against novel Bunyavirus. Background Technology

[0002] Retinoic acid (RA) is a metabolite of vitamin A in the body, primarily affecting bone growth and promoting epithelial cell proliferation, differentiation, and keratinization. At least six isomers of RA exist in the human body, with all-trans retinoic acid (ATRA) generally considered the most prevalent biological form. ATRA plays a crucial role in growth, development, and maturation, but animals cannot synthesize vitamin A compounds themselves and must obtain them from food. Extensive previous research has shown that the vitamin A metabolite RA can act on different cells of the innate and adaptive immune systems, and is best known for its effects on inducing intestinal homing molecules in T cells and B cells, inducing regulatory T cells (Tregs), and promoting immune tolerance. RA exerts local effects at the mucosal site (mainly the intestinal mucosa) and plays a key role in maintaining immune homeostasis during inflammatory responses.

[0003] Severe fever with thrombocytopenia syndrome (SFTS) was first reported in 2009. The novel Bunyavirus is transmitted through the bite of infected ticks (primarily Haemaphysalis longicornis). The virus can also be transmitted through direct contact with the blood or contaminated objects of infected individuals, leading to infection among healthcare workers, visitors, and those involved in mortuary services. The general population is susceptible to this disease. Those involved in agricultural activities, tea picking, farmers living in rural hilly or mountainous areas, and tourists visiting such areas are at high risk of SFTSV infection, and the incidence rate increases with age. Clinical symptoms of SFTSV infection include decreased platelet count, decreased white blood cell count, fever, fatigue, malaise, muscle aches, and gastrointestinal symptoms such as loss of appetite, nausea, vomiting, and diarrhea. Critically ill patients may also experience skin ecchymosis, involuntary movements of the jaw and limbs accompanied by increased muscle tone, and neurological symptoms such as drowsiness, confusion, or stupor. Complications may include pulmonary infection, bleeding from the digestive tract, lungs, uterus, and other cavities, and even multiple organ failure leading to death. In a mouse model of novel Bunyavirus infection, early histopathological changes primarily occur in the spleen and bone marrow, with an increase in monocytes and an initial decrease in lymphocytes, which gradually recovers after two weeks. In the later stages of infection, extensive degeneration and necrosis of liver and kidney cells occur, exhibiting pathological changes and damage.

[0004] Currently, there are no effective oral medications against the novel Bunyavirus. Clinical treatment mainly focuses on symptomatic support and treatment of complications.

[0005] As an important vitamin A metabolite, all-trans retinoic acid has not been reported to have any application in combating novel Bunyavirus infection. Summary of the Invention

[0006] The purpose of this invention is to provide a new pharmaceutical use for all-trans retinoic acid.

[0007] The novel pharmaceutical use of all-trans retinoic acid provided by this invention is its application in the preparation of the following products:

[0008] 1) Drugs for treating novel Bunyaviruses;

[0009] 2) Drugs for the prevention and / or treatment of novel Bunyavirus infectious diseases;

[0010] 3) Drugs that inhibit inflammation caused by novel Bunyavirus infectious diseases.

[0011] Specifically, the application is: the use of all-trans retinoic acid or its pharmaceutically acceptable salts, solvates or hydrates in the preparation of any one of the following drugs a)-d):

[0012] a) Drugs that improve the survival rate of receptors after infection with novel Bunyavirus;

[0013] b) Drugs that reduce the expression of inflammatory factors in receptor tissues after infection with novel Bunyavirus;

[0014] c) Drugs that reduce receptor tissue damage after infection with novel Bunyavirus;

[0015] d) Drugs that increase the ATRA content in the serum of receptors infected with novel Bunyaviruses.

[0016] In b), all-trans retinoic acid or its pharmaceutically acceptable salts, solvates or hydrates reduce the expression of inflammatory factors via PPARγ protein.

[0017] The novel Bunyavirus is a fever with thrombocytopenia syndrome virus (SFTSV).

[0018] The structural formula of the all-trans retinoic acid is shown in formula (Ⅰ):

[0019]

[0020] This invention experimentally demonstrates that all-trans retinoic acid (ATA) can reduce the mortality rate and tissue damage in mice infected with novel Bunyavirus. Furthermore, in vitro and in vivo experiments revealed that ATA reduces the expression of inflammatory factors through PPARγ protein. Therefore, ATA can be used to prepare drugs against novel Bunyavirus and / or drugs for the prevention or treatment of novel Bunyavirus infectious diseases and / or drugs for inhibiting inflammation caused by novel Bunyavirus infectious diseases. Attached Figure Description

[0021] Figure 1 This is a diagram showing the experimental results of how all-trans retinoic acid improved the survival rate of mice infected with a novel Bunyavirus in Example 1 of this invention.

[0022] Figure 2 The figure shows the experimental results of the decrease in all-trans retinoic acid (ATA) content in the serum of mice infected with the novel Bunyavirus in Example 2 of this invention, and how increasing the endogenous ATA content can reduce inflammation in the serum of mice infected with the novel Bunyavirus.

[0023] Figure 3 This is a diagram showing the experimental results of all-trans retinoic acid reducing tissue damage and inflammation in mice infected with a novel Bunyavirus in Example 3 of this invention.

[0024] Figure 4 This is a diagram showing the experimental results of all-trans retinoic acid (ATA) reducing inflammation induced by a novel Bunyavirus in THP-1 cells via PPARγ in Example 4 of this invention. In the diagram, Nc represents the group transfected with empty siRNA, RA represents the group treated with trans retinoic acid alone, siPPARγ represents the PPARγ gene knockdown, SFTSV represents the group infected with SFTSV, SFTSV+Nc represents the group transfected with empty siRNA and infected with SFTSV, SFTSV+RA represents the group infected with SFTSV and treated with trans retinoic acid, SFTSV+siPPARγ represents the group knocked down the PPARγ gene and infected with SFTSV, and SFTSV+siPPARγ+RA represents the group knocked down the PPARγ gene, treated with trans retinoic acid, and infected with SFTSV. Detailed Implementation

[0025] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0027] In the quantitative experiments described below, three replicate experiments were conducted, and the average value of the results was taken.

[0028] The all-trans-retinoic acid used in the following examples was purchased from MedChemExpress under the trade name Retinoic acid (Synonyms: Vitamin A acid; Vitamin A acid; all-trans-Retinoic acid; ATRA).

[0029] Example 1: All-trans retinoic acid improves the survival rate of mice infected with a novel Bunyavirus.

[0030] Six-week-old, sex-matched C57BL / 6 mice were housed in a specific pathogen-free (SPF) facility with controlled temperature and humidity (22±2℃, 50±10%). After four weeks of feeding with a vitamin A-deficient diet (VDD, 0 IU / g), the mice were administered 300 μg / mouse of all-trans retinoic acid via gavage for five consecutive days. On day 4 of gavage, 1.5 mg / mouse of anti-IFNAR1 IgG antibody was injected intraperitoneally. On day 5, 60,000 PFU / mouse of SFTSV virus was injected intraperitoneally. The experimental results are as follows: Figure 1 As shown, the survival rate of mice was significantly increased, indicating that all-trans retinoic acid has a preventive effect against the novel Bunyavirus. VDD represented the vitamin A-deficient group, and VDD+ATRA represented the all-trans retinoic acid group.

[0031] Example 2: Endogenous enhancement of all-trans retinoic acid can reduce the level of inflammation in the serum of mice infected with a novel Bunyavirus.

[0032] Six-week-old, sex-matched C57BL / 6 mice were housed in a specific pathogen-free (SPF) facility under controlled temperature and humidity (22±2℃, 50±10%). Three days prior to SFTSV infection, different groups of mice were intraperitoneally injected with either 2.5 mg / kg of Talarozole (an inhibitor of ATRA-degrading enzyme CYP26A1; CAS: 201410-53-9; TA) or 50 mg / kg of 4-Diethylaminobenzaldehyde (an inhibitor of ATRA synthase ALDH1A1; CAS: 120-21-8; DEAB) until the day of SFTSV infection, at which point drug administration was discontinued. Mice were also treated intraperitoneally with 1.5 mg / mouse of anti-IFNAR1 IgG antibody for one day, and intraperitoneally injected with 60,000 PFU / mouse of SFTSV virus. Three days after infection, serum samples were collected to measure the levels of all-trans retinoic acid (ATRA), interleukin-6 (IL-6), and interleukin-1β (IL-1β). Experimental results are as follows Figure 2 As shown, 4-Diethylaminobenzaldehyde (DEAB) and Talarozole (TA) can affect the level of endogenous ATRA and increase or decrease serum inflammation levels. Based on this, it is demonstrated that endogenous enhancement of all-trans retinoic acid can reduce serum inflammation levels in mice infected with a novel Bunyavirus.

[0033] Example 3: All-trans retinoic acid reduces tissue damage and inflammation in mice infected with a novel Bunyavirus.

[0034] Six-week-old, sex-matched C57BL / 6 mice were selected and housed in a specific pathogen-free (SPF) facility with controlled temperature and humidity (22°C). + 2℃, 50 +(10%). Mice were administered all-trans retinoic acid (300 μg / mouse) via gavage for 5 consecutive days. On day 4, mice were injected intraperitoneally with 1.5 mg / mouse of anti-IFNAR1 IgG antibody. On day 5, mice were injected intraperitoneally with 60,000 PFU / mouse of SFTSV virus. On day 8, mice were euthanized with carbon dioxide. After dissection, lungs, liver, and spleen were removed, fixed overnight in 4% paraformaldehyde solution, dehydrated, embedded in paraffin, and cut into 4 μm thick sections. HE staining was performed according to standard procedures. Spleen sections were blocked with 10% normal goat serum for 30 min and incubated overnight at 4°C with IL6 (Proteintech) polyclonal antibody. Sections were incubated with FITC-conjugated rabbit anti-secondary antibody at room temperature for 55 min, microwaved, and then NP stained with anti-SFTSV NP polyclonal antibody (4°C overnight) and CY3-conjugated rabbit anti-secondary antibody (RT, 55 min). Finally, sections were incubated with DAPI solution at room temperature for 10 min. Microscopic observation and fluorescence microscopy were used to collect images. After weighing, the lungs, liver, and spleen were homogenized, and total RNA was extracted. Real-time quantitative PCR (qPCR) was used to detect the expression of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNFα) genes. Experimental results are as follows: Figure 3 As shown, the expression of pro-inflammatory cytokines in the lungs, liver, and spleen of mice in the all-trans retinoic acid group was significantly reduced, and the pathological changes in the lung, liver, and spleen tissues were significantly improved compared with those in Abx or FMT-D mice, indicating that all-trans retinoic acid can reduce tissue damage and inflammation in mice.

[0035] Example 4: All-trans retinoic acid reduces inflammation induced by novel Bunyavirus in THP-1 cells via PPARγ.

[0036] Human monocytes (THP-1) were cultured at 37°C and 5% CO2 in RPPMI-1640 medium containing 10% fetal bovine serum. THP-1 cells in logarithmic growth phase were centrifuged at 100g for 3 min, resuspended in RPMI-1640 medium, and phorbol ester (PMA) was added. Cell counts were performed under a microscope to adjust the THP-1 cell confluence to 80-90%. The final PMA concentration was 100 ng / mL, and the cells were cultured for 12-24 h. PPARγ siRNA was transfected into THP-1 cells using Lipo3000 transfection reagent. The cells were then treated with 100 nM all-trans retinoic acid (ATRA) and infected with 1 MOI of SFTSV. Total RNA was extracted 48 h after infection. Real-time quantitative PCR (qPCR) was used to detect interleukin-6 (IL-6) gene expression. The experimental results are as follows: Figure 4As shown, the expression of IL-6 in the SFTSV group with added ATRA was significantly lower than that in the SFTSV group alone. However, after the PPARγ gene was knocked out, the expression of IL-6 increased, indicating that all-trans retinoic acid reduces inflammation caused by the novel Bunyavirus through PPARγ.

[0037] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Use of all-trans retinoic acid or a pharmaceutically acceptable salt, solvate or hydrate thereof in the manufacture of a medicament for combating a novel bunyavirus.

2. Use of all-trans retinoic acid or a pharmaceutically acceptable salt, solvate or hydrate thereof in the manufacture of a medicament for preventing and / or treating a novel bunyavirus infectious disease.

3. Use of all-trans retinoic acid or a pharmaceutically acceptable salt, solvate or hydrate thereof in the manufacture of a medicament for inhibiting inflammation of a novel bunyavirus infectious disease.

4. Use according to any one of claims 1 to 3, characterized in that, The use is: use of all-trans retinoic acid or a pharmaceutically acceptable salt, solvate or hydrate thereof in the manufacture of any one of a)-d) below: a) a medicament for increasing the survival rate of a subject infected with a novel bunyavirus; b) a medicament for reducing the expression of inflammatory factors in the tissues of a subject infected with a novel bunyavirus; c) a medicament for reducing tissue damage in a subject infected with a novel bunyavirus; d) a medicament for increasing the content of ATRA in the serum of a subject infected with a novel bunyavirus.

5. Use according to claim 4, characterized in that, In the b), all-trans retinoic acid or a pharmaceutically acceptable salt, solvate or hydrate thereof reduces the expression of inflammatory factors through PPARγ protein.

6. Use according to any one of claims 1 to 5, characterized in that, The structural formula of the all-trans retinoic acid is shown in formula (I):

Images