Use of isoflavones in the preparation of a medicament for the treatment of feline infectious peritonitis

Abstract

The present disclosure mainly relates to the application of isoliquiritigenin in the preparation of a drug for treating feline infectious peritonitis. The present disclosure introduces isoliquiritigenin into the application of inhibiting feline infectious peritonitis virus, which can effectively inhibit the replication of FIPV on CRFK cells, and the inhibitory effect on feline infectious peritonitis virus is significantly enhanced with the increase of drug concentration, which has high practical application significance.

Description

Technical Field

[0001] This disclosure relates to the field of biomedical technology, specifically to the application of isoliquiritigenin in the preparation of drugs for treating feline infectious peritonitis, the application of isoliquiritigenin in the preparation of drugs for inhibiting feline infectious peritonitis virus, a drug for treating feline infectious peritonitis, and a drug for inhibiting feline infectious peritonitis virus. Background Technology

[0002] Feline infectious peritonitis (FIP) is a systemic, fatal disease caused by the feline infectious peritonitis virus (FIPV). Clinically, FIP is characterized by fibrinous and granulomatous serositis, protein-rich serous effusions, or purulent granulomatous lesions. It has become a leading cause of death in cats, accounting for approximately 5% of deaths in domestic cats. The disease is widely believed to be caused by a mutation of the feline enteric coronavirus (FECV), which is prevalent in cat populations, and this pathogenic mutation has persisted as cats have become increasingly common as pets. To date, despite decades of research into the etiology, pathogenesis, transmission, and prevention of FIP, it remains the most common and fatal infectious disease in cats, and there is still no effective treatment.

[0003] While vaccines are the primary means of preventing and controlling viral diseases, no truly effective vaccine has yet been approved for market release. Currently, antiviral drugs serve as a crucial line of defense in the treatment of these diseases and are one of the most effective ways to replace and supplement the ineffectiveness of vaccines. Although the emergence of the nucleoside analogue GS-441524 has brought a turning point in the treatment of this disease, its long treatment cycle, significant side effects, and high cost hinder its widespread application in veterinary clinical practice. Therefore, developing a novel, safe, and highly effective drug for the treatment of FIP and anti-FIPV has become one of the urgent problems to be solved. Summary of the Invention

[0004] The purpose of this disclosure is to provide the application of isoliquiritigenin in the preparation of drugs for treating feline infectious peritonitis (FIPV), the application of isoliquiritigenin in the preparation of drugs for inhibiting FIPV, a drug for treating feline infectious peritonitis, and a drug for inhibiting FIPV. This drug can effectively inhibit FIPV replication in feline kidney cells (CRFK), and the inhibitory effect on FIPV significantly increases with increasing drug concentration, demonstrating high practical application value in clinical practice.

[0005] To achieve the above objectives, this disclosure provides, on the one hand, the use of isoliquiritigenin in the preparation of a medicament for treating feline infectious peritonitis.

[0006] On the other hand, this disclosure provides the use of isoliquiritigenin in the preparation of drugs that inhibit feline infectious peritonitis virus.

[0007] On the other hand, this disclosure provides a medicament for treating feline infectious peritonitis, wherein the medicament comprises an effective amount of isoliquiritigenin.

[0008] Optionally, the content of isoliquiritin in the drug is 1-50 μM.

[0009] On the other hand, this disclosure provides a medicament for inhibiting feline infectious peritonitis virus, the medicament comprising an effective amount of isoliquiritigenin.

[0010] Optionally, the content of isoliquiritin in the drug is 1-50 μM.

[0011] Through the above technical solution, this disclosure provides the application of isoliquiritigenin in the preparation of drugs for treating feline infectious peritonitis. This drug can effectively inhibit the replication of FIPV on CRFK cells, and the inhibitory effect on feline infectious peritonitis virus is significantly enhanced with increasing drug concentration, which has high practical application significance.

[0012] Other features and advantages of this disclosure will be described in detail in the following detailed description section. Attached Figure Description

[0013] The accompanying drawings are provided to further illustrate the present disclosure and form part of the specification. They are used together with the following detailed description to explain the present disclosure, but do not constitute a limitation thereof. In the drawings:

[0014] Figure 1 To detect the toxicity of isoliquiritigenin to CRFK cells using the MTT assay.

[0015] Figure 2 To detect the effect of isoliquiritigenin on the inhibition of FIPV virus replication in CRFK cells by Western blot.

[0016] Figure 3 To indirectly detect the effect of isoliquiritigenin on the inhibition of FIPV virus replication in CRFK cells using immunofluorescence.

[0017] Figure 4 For TCID 50 To investigate the effect of isoliquiritigenin on inhibiting FIPV virus replication in CRFK cells.

[0018] Figure 5 For TCID50 The effects of different concentrations of isoliquiritigenin on the inhibition of FIPV virus replication in CRFK cells were investigated. Detailed Implementation

[0019] The following provides a detailed description of specific embodiments of this disclosure. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit this disclosure.

[0020] On the one hand, this disclosure provides the use of isoliquiritigenin in the preparation of drugs for treating feline infectious peritonitis.

[0021] Isoliquiritigenin (ISL) used in this disclosure is a flavonoid compound containing a chalcone structure found in plants such as licorice. It is characterized by convenient extraction, low price, and green safety, and is an important pharmaceutically active ingredient in medical research, possessing various pharmacological activities, including anti-inflammatory, antioxidant, antibacterial growth inhibition, and antiplatelet aggregation effects. It has shown great potential, especially in treating intestinal inflammation and reducing inflammatory responses. Currently, isoliquiritigenin, as a lead compound, has not been used in drug research for the treatment of FIP, and its anti-FIPV activity is unclear. Through extensive in vitro experiments, the inventors of this disclosure have discovered that isoliquiritigenin can serve as an effective ingredient in both FIPV inhibitory drugs and FIP treatments, effectively inhibiting FIPV replication.

[0022] On the other hand, this disclosure provides the use of isoliquiritigenin in the preparation of drugs that inhibit feline infectious peritonitis virus.

[0023] On the other hand, this disclosure provides a medicament for treating feline infectious peritonitis, the medicament comprising an effective amount of isoliquiritigenin.

[0024] Optionally, the content of isoliquiritin in the drug is 1-50 μM.

[0025] On the other hand, this disclosure provides a medicament for inhibiting feline infectious peritonitis virus, the medicament comprising an effective amount of isoliquiritigenin.

[0026] Optionally, the content of isoliquiritin in the drug is 1-50 μM.

[0027] The present disclosure is further described in detail below through examples. All raw materials used in the examples are commercially available.

[0028] Virus, cells, serum and proteins: FIPV DQ2023 strain (GenBank: OR711916) was stored in our laboratory at -80℃; FIPVN protein polyclonal antibody was stored in our laboratory at -40℃; cat kidney cells (CRFK cells) were stored in liquid nitrogen in our laboratory.

[0029] Main reagents: MTT assay kit and SDS-PAGE gel preparation kit were purchased from Beijing Solarbio Science & Technology Co., Ltd.; high-glucose DMEM medium was purchased from Haiclone Biotechnology Co., Ltd.; 1×PBS buffer and cell lysis buffer were purchased from Beyotime Biotechnology Co., Ltd.; isoliquiritigenin was purchased from Shanghai Dingrui Chemical (Bio) Co., Ltd., CAS No.: 961-29-5, with the following structural formula:

[0030]

[0031] Example 1

[0032] Cytotoxicity assay of isoliquiritigenin on CRFK cells: CRFK cells were cultured in DMEM medium containing 10% fetal bovine serum at a concentration of 1×10⁻⁶ mg / L. 5 Cells were inoculated at a rate of 200 μL / mL into 96-well cell culture plates and cultured at 37°C in a 5% CO2 incubator. Once the cells reached a confluent monolayer, the culture medium was discarded. Isoliquiritin was diluted with DMEM to the following concentrations: 0 μM, 20 μM, 40 μM, 60 μM, 80 μM, 100 μM, 120 μM, 140 μM, 160 μM, 180 μM, and 200 μM, with three replicates for each concentration. CRFK cells were treated with 100 μL / well of DMSO control and diluted isoliquiritin for 24 h in 96-well cell culture plates. The supernatant was removed, and 90 μL of DMEM medium was added, followed by 10 μL of MTT solution. The cells were then cultured at 37°C in a 5% CO2 incubator for another 4 h. The supernatant was then removed, and 100 μL of Formazan dissolving solution was added to each well. The plates were then shaken at low speed for 10 min to fully dissolve the crystals. The absorbance of each well was measured at 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader to determine the maximum non-toxic dose (NDF) and half-maximal inhibitory concentration (IC50) of isoliquiritigenin for CRFK cells. 50 The result is as follows Figure 1 As shown, when isoliquiritigenin concentrations ranged from 0 to 80 μM, cell viability remained above 95%, and this concentration did not affect the growth of CRFK cells, indicating that 80 μM is the maximum non-toxic dose of isoliquiritigenin. Simultaneously, the half-maximal inhibitory concentration (IC50) of isoliquiritigenin on CRFK cells was calculated. 50 The value is 160.10 μM.

[0033] Example 2

[0034] Western blot analysis of the effect of isoliquiritigenin on FIPV replication in CRFK cells: Vero E6 cell suspension was diluted to 1×10⁻⁶. 5 Cells were inoculated at a density of 100 cells / mL into 6-well cell culture plates, 2 mL of cell suspension per well. After the cells reached a confluent monolayer, the culture medium was aspirated, and FIPV virus was inoculated. The cells were incubated in a 5% CO2, 37°C cell culture incubator for 2 h. The liquid was gently aspirated, and virus maintenance medium containing 80 μM isoglycyrrhizin was added. A virus control group was also set up. The cells were incubated in a 5% CO2, 37°C cell culture incubator for another 24 h. The samples were collected into EP tubes and lysed with an appropriate amount of protease lysis buffer at 4°C for 30 min. Subsequently, the collected cell lysates were centrifuged at 10,000×g for 5 min, the supernatant was collected, and an appropriate amount of 5× protein loading buffer was added. After thorough mixing, the mixture was boiled for 15 min. The prepared protein samples were subjected to SDS-PAGE (SDS polyacrylamide gel electrophoresis) and then transferred to a membrane. 5% skim milk was added and the membrane was blocked at room temperature for 2 h. Subsequently, the membrane was washed five times with PBST (phosphate-Tween buffer) for 10 min each time, then incubated with the corresponding antibody at 37°C for 2 h. After washing five times with PBST for 10 min each time, the membrane was incubated with the corresponding secondary antibody at room temperature in the dark for 1 h. Following incubation, the membrane was washed five times with PBST for 10 min each time. Finally, the membrane was scanned using a LI-COROdyssey infrared imaging system.

[0035] Western blot results are as follows Figure 2 As shown, after 24 hours of infection of CRFK cells with the FIPV DQ2023 strain, the expression level of N protein of the FIPV DQ2023 strain in the isoliquiritigenin group (ISL+FIPV) was significantly lower than that in the FIPV-infected control group (DMEM+FIPV) (P<0.01). This result indicates that isoliquiritigenin can inhibit the replication of the FIPV DQ2023 strain at the protein level.

[0036] Example 3

[0037] Indirect immunofluorescence assay of the effect of isoliquiritigenin on FIPV replication in CRFK cells: Cells were seeded in 6-well cell culture plates. When the cell density reached 100%, the supernatant was discarded, and the cells were washed three times with PBS. 500 μL of DMEM virus maintenance medium containing 0.25% trypsin was added, and FIPV cells were inoculated at 0.1 MOI. The DQ2023 strain was cultured in a cell culture incubator for 2 hours, washed three times with PBS, and 2 mL of virus maintenance medium containing 80 μM isoglycyrrhizin was added. A virus control group was also set up. When 60% of the cells showed cytopathic effects, the culture medium was discarded, the cells were washed three times with PBS, and fixed with 4% paraformaldehyde at room temperature for 10 minutes. The waste liquid was discarded, the cells were washed twice with PBS, and then blocking buffer containing 1% BSA in PBST was added. The cells were incubated at 37°C for 1 hour. The blocking buffer was discarded, the cells were washed twice with PBST, and then diluted primary antibody (FIPVN protein polyclonal antibody) was added. The cells were incubated at 37°C at room temperature for 1 hour. After washing five times with PBST, secondary antibody was added, and the cells were incubated at 37°C in the dark for 1 hour. The secondary antibody was discarded, the cells were washed four times with PBS, and the cells were observed and photographed under a fluorescence microscope.

[0038] Indirect immunofluorescence results as follows Figure 3 The results showed that no fluorescence was detected in the blank cell control group, while the fluorescence intensity of CRFK cells pretreated with isoliquiritigenin was significantly lower than that in the virus control group. This indicates that ISL can effectively inhibit the replication of the FIPV DQ2023 strain at the protein level.

[0039] Example 4

[0040] TCID 50 To determine the effect of isoliquiritigenin on FIPV replication in CRFK cells: Cells were seeded in 6-well plates. When the cell density reached 100%, the supernatant was discarded, and the cells were washed three times with PBS. 500 μL of DMEM virus maintenance medium containing 0.25% trypsin was added, followed by inoculation with FIPV DQ2023 strain at 0.1 MOI. Cells were cultured in a cell culture incubator for 2 h, washed three times with PBS, and 2 mL of virus maintenance medium containing 80 μM isoliquiritigenin was added. A virus control group was also included. Virus titer was detected according to the Reed-Muench method. Alternatively, cells were seeded in 96-well plates. When the cell density reached 100%, the supernatant was discarded, and the cells were washed three times with PBS. 100 μL of serially diluted (10... -1 ~10 -10 The virus sample to be tested was added to the cells. Cytopathic effects were observed and viral titers were calculated 60–72 hours post-infection.

[0041] The results are as follows Figure 4As shown, the virus titer of FIPV DQ2023 strain in the isoliquiritigenin pretreatment group was significantly lower than that in the control group (p<0.05). This result indicates that ISL can significantly inhibit the proliferation of FIPV DQ2023 strain virions at the cellular level.

[0042] Example 5

[0043] TCID 50 Detection of the effect of different concentrations of isoliquiritigenin on FIPV replication in CRFK cells: seed the cells in a 6-well plate, when the cell density reaches 100%, discard the supernatant, rinse 3 times with PBS, add 500 μL of DMEM virus maintenance medium containing 0.25% trypsin, then inoculate FIPV DQ2023 strain at 0.1 MOI and culture in a cell incubator for 2 h, rinse 3 times with PBS, add 2 mL of virus maintenance medium containing 0 μM, 5 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM isoliquiritigenin respectively, and set up a virus control group at the same time. The detection of virus titer refers to the method of Reed-Muench. After seeding the cells in a 96-well plate, when the cell density reaches 100%, discard the supernatant and rinse 3 times with PBS. Take 100 μL of serially diluted (10 -1 -10 -10 ) test virus sample and add it to the cells. At 60-72 h after infection, observe the cytopathic effect, detect the effect of different concentrations of isoliquiritigenin on virus titer, and calculate the inhibition rate of the drug, half maximal effective concentration (EC 50 ) and selective index SI (SI=IC 50 / EC 50 ).

[0044] The results are shown in Figure 5 : as the concentration of isoliquiritigenin increases, the ability of isoliquiritigenin to inhibit FIPV replication is enhanced with the increase of concentration, showing a certain concentration gradient dependence. The half maximal effective concentration is 6.425 μM, and the SI value of isoliquiritigenin for inhibiting FIPV replication is calculated as 24.92, indicating that isoliquiritigenin has obvious anti-FIPV effect and application prospect for the treatment of feline infectious peritonitis.

[0045] All the above experiments were independently repeated 3 times. Statistical analysis of the data was performed using GraphPad Prism 8.0 software (GraphPad Software, Inc., La Jolla, CA, USA). P>0.05 indicates no significant difference (ns); 0.01<P<0.05 indicates a significant difference, which is marked as * ; P<0.01 indicates an extremely significant difference, which is marked as ** .

[0046] The isoliquiritigenin disclosed herein has a significant anti-FIPV effect in CRFK cells, and this effect is concentration gradient dependent.

[0047] The preferred embodiments of this disclosure have been described in detail above. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure.

[0048] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0049] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

Claims

1. Application of isoliquiritigenin in the preparation of drugs for treating feline infectious peritonitis.

2. Application of isoliquiritigenin in the preparation of drugs that inhibit feline infectious peritonitis virus.

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